Xeno Transplantation

D.K.C. Cooper E. Kemp K.
Reemtsma
D.J.G. White (Eds.)
Xeno-
transplantation
The Transplantation of Organs
and Tissues Between Species
Springer-Verlag
Berlin Heidelberg New York
London Paris Tokyo
Hong Kong Barcelona
Budapest
D.K.C.COOPERM.D. PH. D.
Oklahoma Transplantation Institute, Baptist Medical Center,
3300 N. W. Expressway, Oklahoma City, OK 73112, USA
EJVIND KEMP M. D.
Department of Nephrology, Odense University Hospital,
DK-SOOO Odense, Denmark
KEITH REEMTSMA M. D.
Department of Surgery, Columbia-Presbyterian Medical Center,
622 West 168th Street, New Yark, NY 10032, USA
D.l.G. WHITE PH. D.,MRCPATH.

Department of Surgery, Addenbrooke's Hospital, Hills Road,
Cambridge CB2 2QQ, United Kingdom
ISBN-13: 978-3-642-97325-3 e-ISBN-13: 978-3-642-97323-9

DOl: 10.1007/978-3-642-97323-9
This work is subject to copyright. All rights are reserved, whether the whole or part of the
material is concerned, specifically the rights of translation, reprinting, re-use of illustra-
tions, recitation, broadcasting, reproduction on microfilms or in other ways, and storage
in data banks, Duplication of this publication or parts thereof is only permitted under the
provisions of the German Copyright Law of September 9, 1965, in its current version,
and a copyright fee must always be paid. Violations fall under the prosecution act of the
German Copyright Law,
<D Springer-Verlag Berlin Heidelberg 1991
Softcover reprint of the hardcover 1st edition 1991
The use of general descriptive names, registered names, trademarks, etc, in this publica-
tion does not imply, even in the absence of a specific statement, that such names arc ex-
empt from the relevant protective laws and regulations and therefore free for gcneral
use..
Product Liability: The publisher can give no guarantee for information about drug
dosage and application thereof contained in this book, In every individual case the re-
spective user must check its accuracy by consulting other pharmaceutical literature.
Typesetting: Appl. Wemding
23/3145/543210- Printed on acid-free paper
Normal macroscopic appearance at autopsy of chimpanzee kidneys that had
functioned well for a period of almost 9 months in a 23-year-old woman who
had undergone renal xenotransplantation in 1963. This operation was one of a
small series of kidney xenotransplants performed by Keith Reemtsma and his
colleagues at Tulane University in New Orleans (Chaps. 2, 33)
Foreword
The cover of this book depicts a Lamassu, one of the "fabulous"

beasts of mythology [1]. Like many similar creatures, such as the
Chimera, Griffon, Hippocamp, and Cockatrice, the body of the
Lamassu was clearly a combination of structures derived from sev-
eral different species - in other words, it provides a highly success-
ful example of xenotransplantation. But in selecting a symbol of
xenotransplantation to grace the cover of this volume, why choose
the Lamassu in preference to the other ancient beasts? The reason
is that the Lamassu appears to have been endowed with a much
Fig.I. Homer described the Chimera as consisting of a lion's foreparts , a goat

in the middle, and a serpent's hind parts
VIII Foreword
Fig. 2. The Griffon had the foreparts of an eagle, and the re ar, tail , and
hindlegs of a lion. Its eagle-like head had pointed, upstanding ears like those of
an ass. Feathers grew upon its head, nec k and chest, and the rest of its body was
covered in leonine fur
more benign and desirable character than many of its mythologi-

cal associates.
For example, reliable reports state that the Chimera (Fig. 1) -
hitherto the animal most commonly selected to symbolize
xenografting - killed everyone who came within range of its fiery
breath. Perhaps not surprisingly, therefore , the Chimera is vari-
ously described as one of the "largest monsters ever born," a "sav-
age creature," and a "symbol of complex evil." With these descrip-
tions in mind , it certainly does not seem to be the ideal choice to
represent a field of surgery and science that is intended to be whol-
ly beneficial to the human race!
A second possible choice as a symbol of xenogeneic transplan-
tation is the Griffon (or Gryphon) (Fig. 2). The Griffons, said to be
guardians of hidden treasures, were, however, described as "fear-
some, greedy, and rapacious animals" - surely enough to disquali-
fy them also as representatives of our field or surgical interest,
even though we are told that "the blending of two solar creatures,
Foreword IX
the lion and the eagle, shows that the Griffon is a beneficient char-
acter."
Lamassu, on the other hand, were considered to be kindly
figures, specifically acting as guardian divinities of cities.
Accordingly, the same name has been given to the pairs of monu-
mental carved figures placed at the gates of cities and palaces; fur-
thermore, these guardians have a ritual title of Kerub, a derivative
of a word meaning to pray or to bless. The Lamassu can be com-
pared with the Egyptian Sphinx, as it has a human head set on an
animal's body - sometimes that of a lion, but more often that of a
bull- with wings that are believed to represent spiritual elevation.
Its elegantly bearded head wears a headdress shaped like a crown,
similar to those worn by the images of the Gods, denoting the
divine character of this "Great Winged Bull of Babylon."
Surely, we, the potential xenotransplanters, would prefer our
aims and efforts to be associated with an animal of "kindly" dispo-
sition and "divine character" rather than with the various alterna-
tive ancient mythological beasts. Yes, the Lamassu would definite-
ly seem to be our choice.
Oklahoma, May 1991 DAVID K.c. COOPER
Reference
1. Hargreaves, J. Bestiary. Gothic Image Publications, Glastonbury, U. K.,

1990.
Preface
There was considerable clinical and experimental activity in the

realm of xenotransplantation, particularly with regard to the kid-
ney, in the mid-1960s. By the mid-1970s, however, the relatively
poor results that had followed clinical xenotransplantation led to
diminishing interest in this area, and little experimental work was
carried out during the following 1a-year period.
With the introduction of cyclosporine, the steadily improving
long-term results of organ allografting have led to an increasing
number of patients being referred for these procedures, and this
has greatly exacerbated the shortage of suitable human donors.
This lack of donors, together with the introduction of certain new
drugs that have recently become available to the experimental
transplanter, has stimulated a rapidly expanding interest in xeno-
transplantation once again.
During the mid-1980s, a handful of groups initiated new en-
deavors in the experimental aspects ofxenotransplantation. In the
spring of 1987, following a symposium held in Philadelphia on allo-
transplantation across the ABO blood group barrier [1] - itself a
situation akin to xenotransplantation in that it relates to antibody-
mediated rejection - two of the editors of this volume, Ejvind
Kemp and myself, discussed the possibility of putting together a
book that reviewed the current status of xenotransplantation.
Ejvind Kemp had indeed already written a small book on this topic
some years previously, following his own research into this area
[2], but this clearly required updating. An initial group of con-
tributing authors was slowly put together from those who had
shown an experimental interest in the topic.
By the time of the XII International Congress of the
Transplantation Society held in Sydney, Australia, in August 1988,
there seemed to be sufficient activity to form a group of those in-
terested in xenotransplantation. The initial suggestion for such a
group came from David White, and we were fortunate to gain the
support of Keith Reemtsma (Fig. 1), arguably the major early clin-
ical pioneer in this field.
At an informal meeting held in Syndey about 50 or so interested
scientists and physicians met and formed a group to promote re-
XII Preface
Fig. I. Keith Reemtsma headed the group at Tulane University in New

Orleans, which was the first to explore xenotransplantation in a scientific man-
ner. Their clinical work, carried out between 1963 and 1965, on kidney trans-
plantation using chimpanzees as donors (described in Chaps. 2 and 33) stimu-
lated many other experimental and clinical studies during the subsequent
decade
search in xenotransplantation. Such promotion seemed to us to be

necessary in view of the lack of time allocated for papers and dis-
cussion on this topic at the major transplantation meetings. At
Keith Reemtsma's suggestion, the group called itself "ClubXeno"
which gave it what he believed to be a suitably " underworld" (and
possibly reprobate) character. Its intended roles were to facilitate
the organization of subsidiary symposia at the major transplanta-
tion meetings at which papers on xenotransplantation could be
presented, and to encourage and enhance informal communica-
tion between those working in this field. The first such symposium
was planned to coincide with the IV Congress of the European
Society for Organ Transplantation in Barcelona, Spain, in
November 1989.
Before this symposium took place, interest in xenotransplanta-
tion was stimulated further by "Xenograft 25 ", an international
congress organized by Mark Hardy to mark the 25th anniver-
sary of Keith Reemtsma's first chimpanzee-to-human kidney
xenograft in 1963. This small gathering of invited surgeons and sci-
entists, held in the beautiful setting of Arden House in Harriman,
New York, in November 1988, was invaluable in updating those
attending on the various approaches to xenotransplantation that
Preface )(111
had been, and were currently being, investigated. The proceedings
of this congress were subsequently published, and provide the
most wide-ranging review ofthis field available to date [3].
When the symposium on xenografting was held in Barcelona
1 year later, interest in the topic was clearly increasing as approxi-
mately 200 people were present and 18 papers were submitted and
presented [4]. These papers covered all aspects of xenotransplan-
tation from in vitro cellular studies to nonhuman primate organ
transplantation.
By the time plans were being made for the )(111 International
Congress of the Transplantation Society, to be held in San
Francisco in August 1990, only 9 months later, it was clear that in-
terest had expanded immensely. In view of the clear acceptance of
the subject by the organizers, as evidenced by the allocation of sev-
eral sessions to it in the main congress, the Club)(eno members did
not feel that the organization of a subsidiary symposium was nec-
essary.
In San Francisco it become clear that interest in xenotransplan-
tation was already at a high, and rapidly increasing, level; all of the
xenotransplant sessions were well attended. It was agreed, there-
fore, that, as the original major purpose for Club)(eno - namely to
stimulate interest in xenotransplantation - no longer existed, this
informal group should be disbanded. I must admit to being sur-
prised that it should so rapidly become unnecessary - but such is
the speed of scientific progress today.
The optimism of the members that xenotransplantation, at
least as an experimental topic in immunology, was here to stay was
confirmed by plans to hold a First International Congress on
)(enotransplantation, in Minneapolis in August, 1991.
Thus, over a period of less than 5 years, xenotransplantation as
a subject for research and discussion has expanded from one that
interested a handful of immunologists and transplant surgeons to
one that has clearly become of widespread interest in the trans-
plant community.
This volume, originally planned by Ejvind Kemp and myself,
and subsequently edited and expanded in collaboration with Keith
Reemtsma and David White, therefore reflects this increasing in-
terest. We have attempted to cover the subject ofxenotransplanta-
tion comprehensively, and in this regard we have been fortunate in
receiving contributions from a large number ofthe world's experts
on a wide variety of related topics.
The reader will note that not all of the contributors to this vol-
ume are in agreement with each other even over key points such
as, for example, the pathogenesis of xenograft rejection; such dis-
crepancies and controversies merely illustrate the primitive state
of our knowledge at the present time.
)(1\1 Preface
Our knowledge of the subject, however, is expanding rapidly

and much of the information included in these covers may soon be
outdated. Nevertheless. we hope that the historical aspects and
many of the principles of the subject will remain of interest for
many years. The book attempts - and we believe succeeds - to pro-
vide the reader with a wide basis of information gleaned from pre-
vious and current work on the subject. On this base of knowledge.
we may all build for the future.
Oklahoma. May 1991 DAVID K.C. COOPER
References
1. Bannett, A. D .. Brynger. H .. McAlack. R. F.. Breimer, M. E .. Samuelson.

B.E. (cds.) ABO-incompatibility in transplantation. Transplant. Pmc. 19.
6.4397-4642. 19~7.
2. Kemp, E. Xenografting - the Future of Organ Transplantation. Odense
University Press; Odense. 1978.
3. Hardy. M. A. Xenograft 25. Elsevier; Amsterdam, New York, Oxford, 1989.
4. Cooper. D. K. C. Reemtsma. K. (eds.) Xenotransplantation. Tramplant.
Pmc. 22.1057-1092.1990.
Acknowledgements
The edi tors wish to thank N azih Zuhdi, M. D., Medical Director of
the Oklahoma Transplantation Institute, and Stanley Hupfeld,
President of the Oklahoma HealthCare Corporation, for freely
making available to us excellent secretarial and medical illustra-
tion facilities at Baptist Medical Center in Oklahoma City. We are
also indebted to Francisca Neethling M. Sc (Med) for considerable
help in the checking of proofs and preparation of the index.
The entire manuscript was typed or re-typed most efficiently
and accurately by Crystal Taylor, to whom we are greatly indebt-
ed. Further valuable secretarial help was given by Kelly Ward and
Sue Colliver. Much photographic work was contributed by John
Philbin of the Department of ProGraphics of Baptist Medical
Center. We gratefully acknowledge their respective skills.
Finally, we wish to express our appreciation to our many co-
authors, who have contributed their knowledge, experience, and
time in the preparation of this volume.
May1991 DAVID K.c. COOPER

EJVINDKEMP
KEITH REEMTSMA
DAVID IG. WHITE
Contents
Section I
Introduction and Historical Perspective . . . . . . . . . . . . . . . . . . 1
Chapter 1
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
D. K C. COOPER, E. KEMP, K REEMTSMA, and D. J. G. WHITE
Chapter 2
Xenotransplantation - A Brief History of Clinical Experiences:
1900-1965. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
KREEMTSMA
Chapter 3
The Scientific Study ofXenografting: 1964-1988. . . . . . . . . .. 23
H. AUCHINCLOSS JR.
Section II
Immunobiology of Xenograft Rejection. . . . . . . . . . . . . . . . .. 45
Chapter 4
Mechanism of Humoral Xenograft Rejection. . . . . . . . . . . . .. 47
L.C.PAUL
Chapter 5
Mechanism of Tissue Injury in
Hyperacute Xenograft Rejection. . . . . . . . . . . . . . . . . . . . . . .. 69
J. L. PLATT and F. H. BACH
Chapter 6
Accomodation - The Role of Natural Antibody
and Complement in Discordant Xenograft Rejection. . . . . .. 81
F. H. BACH, J. PLATT, and D. K C. COOPER
XVIII Contents
Chapter 7
Mechanism of Cellular Xenograft Rejection . . . . . . . . . . . . .. 101
R. D. MOSES and H. AUCHINCLOSS JR.
Chapter 8
Xenotolerance Through Bone Marrow Transplantation 121
M. SYKES, I. AKSENTIJEVICH, Y. SHARABI, and D. H. SACHS
Chapter 9
Immunobiology of Xenografting in Rodents . . . . . . . . . . . . .. 139
F. T. THOMAS, C. W. MARCHMAN, A CAROBBI, R. DEMASI,
D. R. ARANEDA, T. N. PATSELAS, E. W. LARKIN, K. PITTMAN,
M. E. ALQAISI, C. HArSCH, and J. M. THOMAS
Chapter 10
Immunosuppression in Xenotransplantation . . . . . . . . . . . . .. 161
J.B. VAN den BOGAERDE, andD.J.G. WHITE
Section III
Histopathology of Xenograft Rejection .................. 179
Chapter 11
Histopathology of Kidney Xenograft Rejection ............ 181
S. LARSEN and H. ST ARKLINT
Chapter 12
Histopathological, Immunofluorescent, and
Electron-Microscopic Features of Hyperacute Rejection in
Discordant Renal Xenotransplantation . . . . . . . . . . . . . . . . .. 207
I. R. MARINO, S. CELLI, G. FERLA, A C. STIEBER,
N. MAGGIANO, and P. MUSIANI
Chapter 13
Histopathology of Cardiac Xenograft Rejection. . . . . . . . . .. 231
AG.RoSE
Chapter 14
Ultrastructure of Hyperacute Rejection
in Cardiac Xenografts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 243
A G. ROSE and D. K. C. COOPER
Chapter 15
Histopathology of Liver Xenograft Rejection . . . . . . . . . . . .. 253
D. G. D. WIGHT
Contents XIX
Section IV
Experimental Xenotransplantation ...................... 273
Chapter 16
Isolated Pancreas Islet Xenografting ..................... 275
F.T.THOMAS
Chapter 17
Experimental Xenotransplantation of Encapsulated Cells. .. 297
R P. LANZA and P. SOON-SHIONG
Chapter 18
Experimental Xenotransplantation in Rodents -
I: Plasma Exchange and Splenectomy .................... 313
RREDING
Chapter 19
Experimental Xenotransplantation in Rodents-
II: Skin Versus Heart Grafts ............................ 323
E.BOUWMAN, M.C.J.WOLVEKAMP, RW. F.DE BRUIN,
J. JEEKEL, and R L. MARQUET
Chapter 20
III: Total Lymphoid Irradiation, Cyclosporine,
and Monoclonal Antibodies ............................ 331
D. A. STEINBRUCHEL, B. NIELSEN, and E. KEMP
Chapter 21
Experimental Xenotransplantation Between
Closely Related Nonprimate Species ..................... 339
C.HAMMER
Chapter 22
Closely Related Primate Species . . . . . . . . . . . . . . . . . . . . . . .. 365
J. A. SANCHEZ, R E. MICHLER, E. A. ROSE and D. K. C. COOPER
Chapter 23
Distantly Related Nonprimate Species ................... 377
E.KEMP
XX Contents
Chapter 24
Experimental Xenotransplantation in Nonhuman
Primates Using Distantly Related Donor Species . . . . . . . . .. 389
Y. YE and D. K. C. COOPER
Chapter 25
Ex Vivo Organ Perfusion Studies in Xenograft Research. . .. 395
K. E. OTTE, D. STEINBRUCHEL, and E. KEMP
Chapter 26
Effects of Formed Elements on Xenograft
Rejection in an Ex Vivo Organ Perfusion Model ........... 405
B. A. BRYAN, M. L. HENRY, L. K. HAN, D. D. SEDMAK,
and R. M. FERG USON
Section V
Aspects of Xenotransplantation in Man . . . . . . . . . . . . . . . . .. 427
Chapter 27
Evolutionary, Physiological, and Immunological
Considerations in Defining a Suitable Donor for Man ....... 429
C.HAMMER
Chapter 28
Nonhuman Primate Blood Group Serology:
Some Implications for Xenotransplantation. . . . . . . . . . . . . .. 439
W. W. SOCHA and J. MOOR-JANKOWSKI
Chapter 29
The Nonhuman Primate as Potential Organ
Donor for Man: Virological Considerations. . . . . . . . . . . . . .. 457
S.S.KALTER
Chapter 30
The Pig as Potential Organ Donor for Man. . . . . . . . . . . . . . .. 481
D. K C. COOPER, Y. YE, L. L. ROLF JR., and N. ZUHDI
Chapter 31
Human Antibodies to Pig Determinants and Their
Association with Hyperacute Rejection of Xenografts ...... 501
K. I. WELSH, D. H. TAUBE, M. THICK, A. PALMER,
N. STEVENS, and R. M. BINNS
Contents XXI
Chapter 32
An Ethical Framework for Considering the Development
of Xenotransplantation in Man . . . . . . . . . . . . . . . . . . . . . . . .. 511
R. A. WRIGHT
Section VI
Clinical Experience. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 529
Chapter 33
Experience with Clinical Kidney Xenotransplantation ...... 531
K. REEMTSMA and A. I. BENVENISTY
Chapter 34
Experience with Clinical Heart Xenotransplantation . . . . . .. 541
D. K. C. COOPER and Y. YE
Chapter 35
Liver Xenotransplantation: Clinical Experience
and Future Considerations. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 559
D. V. CRAMER, L. SHER, and L. MAKOWKA
Subject Index ........................................ 575

Contributors
AKSENTIJEVICH I., M. D.
Fellow, Biotechnology, Immunology Branch, National Cancer
Institute, National Institutes of Health. Bethesda, Maryland.
USA
ALQAISI M. E., M. D.
Assistant Professor, Department of Radiation Oncology,
East Carolina University School of Medicine, Greenville,
North Carolina, USA
ARANEDA D. R, B. S.
Research Technician, Department of Surgery, East Carolina
University School of Medicine, Greenville, North Carolina, USA
AUCHINCLOSS H. Jr., M. D.
Associate Professor of Surgery, Department of Surgery, Harvard
Medical School, Massachusetts General Hospital, Boston,
Massachusetts, USA.
BACH F. H., M. D.
Director, Immunobiology Research Center, Department of
Laboratory Medicine and Pathology, University of Minnesota
School of Medicine, Minneapolis, Minnesota, USA
BENVENISTY AI.,M.D.
Assistant Professor of Surgery, Department of Surgery,
Columbia University College of Physicians and Surgeons,
NewYork,NewYork, USA
BINNS R M., M. A, Vet. M. B., Ph. D., M. R C. V. S.

Senior Principal Scientific Officer and Deputy Head, Department
of Immunology, AFRC Institute of Animal Physiology and
Genetics Research, Cambridge Research Station, Babraham,
Cambridge, UK.
BOUWMAN E., M. D.
Laboratory of Experimental Surgery, Department of Surgery,
Erasmus University, Rotterdam, The Netherlands.
XXIV Contributors
BRYAN B. A., M. D.
Resident, Department of Surgery, The Ohio State University
College of Medicine, Columbus, Ohio, USA.
CAROBBI A., M. D.
Fellow, Department of Surgery, East Carolina University School
of Medicine, Greenville, North Carolina, USA.
CELLI S., M. D.
Fellow, Department of Surgery, Pediatric Surgery Division,
Catholic University, Rome, Italy.
COOPER D. K. c., M. A., M. D., Ph. D., F. R. C. S.
Cardiothoracic Surgeon and Director of Research and Education,
Oklahoma Transplantation Institute, Baptist Medical Center,
Oklahoma City, Oklahoma, USA.
CRAMER D. V., D. V. M., Ph. D.
Director, Transplantation Biology Laboratory, Department of
Surgery and Transplantation Services, Cedars-Sinai Medical
Center, Los Angeles, California, USA.
DE BRUIN, R. W. F., B. S.
Department of Surgery, Erasmus University, Rotterdam,
The Netherlands.
DEMASIR.,M.D.
Resident, Department of Surgery, East Carolina University
School of Medicine, Greenville, North Carolina, USA.
FERGUSON R. M., M. D., Ph. D.
Professor of Surgery and Chief, Division of Transplantation,
Department of Surgery, The Ohio State University, Columbus,
Ohio, USA.
FERLAG.,M.D.
Associate Professor of Surgery, Department of Surgery,
University of Milan, Milan, Italy.
HAISCHC.,M.D.
Assistant Professor, Department of Transplantation Surgery,
University of Vermont, Burlington, Vermont, USA.
HAMMER c., Dr. med., Dr. med. vet.
Professor, Institute for Surgical Research, University of Munich,
Munich, Germany.
HAN L. K., M. D.
Surgical Research Fellow, Department of Surgery,
The Ohio State University, Columbus, Ohio, USA.
Contributors XXV
HENRY M. L., M. D.
Assistant Professor of Surgery, Division of Transplantation,
Department of Surgery, The Ohio State University, Columbus,
Ohio, USA.
JEEKEL J., M. D.
Professor and Head, Department of Surgery,
Erasmus University, Rotterdam, The Netherlands.
KALTER S. S., Ph. D.
President, Virus Reference Laboratory Inc., San Antonio, Texas,
USA.
KEMpE.,M.D.
Professor and Head, Department of Nephrology, Odense
University Hospital, Odense, Denmark.
LANZARP.,M.D.
Senior Scientist, BioHybrid Technologies, Inc., Shrewsbury,
Massachusetts, and Research Associate in Surgery, Harvard
Medical School, Boston, Massachusetts, USA. Formerly, Mary K.
Iacocca Transplantation Fellow, Department of Medicine,
UCLA School of Medicine, Los Angeles, California, USA.
LARKINE.W., M.D.
Associate Professor, Department of Pathology and
Laboratory Medicine, East Carolina University School of
Medicine, Greenville, North Carolina, USA.
LARSENS.,M.D.
Professor and Head, Department of Pathology, Herlev Hospital,
University of Copenhagen, Herlev, Denmark.
MAGGIANoN.,Ph.D.
Assistant Professor, Department of Pathology, Catholic
University, Rome, Italy.
MAKowKA L., M. D., Ph. D., FRCS (C)
Director of Surgery and Transplantation Services, Department of
Surgery, Cedars-Sinai Medical Center, and Professor of Surgery,
UCLA School of Medicine, Los Angeles, California, USA.
MARCHMANC. W.,M.D.
MARINO LR, M.D.

Transplantation Division, University of Pittsburgh, Pittsburgh,
Pennsylvania, USA.
XXVI Contributors
MARQUET R. L., Ph. D.

The Netherlands.
MICHLER R. E., M. D.
Assistant Professor of Surgery, Division of Cardiothoracic
Surgery, Department of Surgery, Columbia-Presbyterian Medical
Center, New York, New York, USA.
MOOR-JANKowSKI J., M. D.
Research Professor and Director, Laboratory for Experimental
Medicine and Surgery in Primates, New York University School
of Medicine, New York, New York, USA, and W. H. O.
Collaborating Centre for Haematology of Primate Animals.
MOSES R. D., M. D.
Research Fellow, Department of Surgery, Harvard Medical
School, Massachusetts General Hospital, Boston, Massachusetts,
USA.
MUSIANI P., M. D.
Professor of Pathology, Department of Pathology, University of
Chieti, Chieti, Italy.
NIELSEN B., M. D.
Consultant in Cardiac Pathology, Institute of Pathology,
Odense University Hospital, Odense, Denmark.
OTTE K., M.D.
Registrar, Laboratory of Nephropathology, Department of
Nephrology, Odense University Hospital, Odense, Denmark.
PALMERA.,M.B.,B. S.
Senior Registrar, Renal Unit, St. Mary's Hospital, London, UK.
PATSELAS T. N., M. D.
PAULL.C.,M.D.,Ph.D.,FRCP(C)
Professor and Head, Division of Renal Medicine, Foothills
Hospital, University of Calgary, Calgary, Alberta, Canada.
PITTMAN K., B. S.
Research Technician, Department of Surgery, East Carolina
University School of Medicine, Greenville, North Carolina, USA.
PLATTJ.L.,M.D.
Associate Professor, Departments of Pediatrics, Cell Biology and
Neuroanatomy, Immunobiology Research Center, University of
Minnesota School of Medicine, Minneapolis, Minnesota, USA.
Contributors XXVII
REDING R, M. D.
Department of Pediatric Surgery and Liver Transplantation,
St-Luc University Clinics, Brussels, Belgium.
REEMTSMA K., M. D.
Professor and Chairman, Department of Surgery, Columbia
University College of Physicians and Surgeons, New York,
New York, USA.
ROLF L. L. Jr., Ph. D., D. V. M.
Clinical Assistant Professor, Department of Pathology, Division
of Animal Resources, University of Oklahoma Health Sciences
Center, Oklahoma City, Oklahoma, USA.
ROSE A. G., M. D., M Med (Path), MRCPath
Professor and Head, Department of Pathology, University of
Cape Town Medical School, Cape Town, South Africa.
RosEE.A.,M.D.
Associate Professor and Director, Division of Cardiothoracic
Surgery, Department of Surgery, Columbia University
College of Physicians and Surgeons, New York,
New York, USA.
SACHS D. H., M. D.
Professor of Surgery (Immunology), Harvard Medical School,
and Director, Transplantation Biology Research Center,
Massachusetts General Hospital, Boston, Massachusetts, USA.
Formerly, Chief, Transplantation Biology Section, Immunology
Branch, National Cancer Institute, National Institutes of Health,
Bethesda, Maryland, USA.
SANCHEzJ.A.,M.D.
Post -Doctoral Research Scientist, Cardiac Transplantation
Research Laboratory, Department of Surgery, Columbia-
Presbyterian Medical Center, New York, New York, USA.
SEDMAKD.D.,M.D.
Assistant Professor, Department of Pathology, The Ohio State
University, Columbus, Ohio, USA.
SHARABI Y., M. D.
Visiting Fellow, Transplantation Biology Section,
Immunobiology Branch, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland, USA.
SHERL.,M.D.
Assistant Director, Liver Transplantation, Department of
Surgery and Transplantation Services, Cedars-Sinai Medical
Center, Los Angeles, California, USA.
XXVIII Contributors
SOCHA W.W.,M.D.
Research Professor, Laboratory for Experimental Medicine and
Surgery in Primates, New York University School of Medicine,
New York, New York, USA, and W. H. O. Collaborating Centre
for Haematology of Primate Animals.
SOON-SHIONG P., M. D., FRCS (C)

Assistant Clinical Professor, UCLA School of Medicine,
and Director, Islet Transplant Center, Century City Hospital,
Los Angeles, California, USA.
STARKLINT H., M. D.
Assistant Professor and Head, Laboratory of Nephropathology,
Institute of Pathology, Odense University Hospital, Odense,
Denmark.
STEINBRUCHEL D. A, M. D.
Senior Registrar, Department of Thoracic and Cardiovascular
Surgery, Skejby Sygehus - Aarhus University Hospital, Aarhus,
Denmark. Formerly, Research Assistant, Laboratory of
Nephropathology, Odense University Hospital, Odense,
Denmark.
STEVENS N., M. D.
Registrar, Department of Medicine, Hammersmith Hospital,
London, UK.
STIEBER A C, M. D.
Transplantation Division, University of Pittsburgh, Pittsburgh,
Pennsylvania, USA
SYKES M., M. D.
Assistant Professor of Surgery (Immunology), Harvard Medical
School, and Senior Investigator, Transplantation Biology
Research Center, Massachusetts General Hospital, Boston,
Massachusetts, USA Formerly, Senior Staff Fellow,
Transplantation Biology Section, Immunology Branch,
National Cancer Institute, National Institutes of Health,
Bethesda, Maryland, USA
TAUBE D. H., M. A, M. B., FRCP

Consultant Physician, Renal Unit, St. Mary's Hospital, London,
UK.
THICK M. E., M. A, M. B., B. Ch., FRCS
Consultant Transplant Surgeon, Renal Unit, St. Mary's Hospital,
London, UK.
Contributors XXIX
THOMAS F. T., M. D.
Professor of Surgery, Department of Surgery, Division of
Transplantation, East Carolina University School of Medicine,
Greenville, North Carolina, USA.
THOMAS1.M.,M.D.
Professor of Surgery, Adjunct Professor, Departments of
Microbiology and Immunology, East Carolina University School
of Medicine, Greenville, North Carolina, USA.
VAN DEN BOGAERDE 1. B., M. B., B. Ch., Ph. D.

Research Fellow, Department of Surgery, University of
Cambridge, Cambridge, UK.
WELSH K.I., Ph. D.

Senior Lecturer in Immunogenetics, Department of Immunology,
Guy's Hospital, London, UK.
WHITE D. 1. G., M. A., Ph. D., MRCPath

Immunologist and Lecturer, Department of Surgery, University
of Cambridge, Cambridge, UK.
WIGHT, D. G. D., M. D., MRCPath

Consultant Pathologist, Department of Pathology,
Addenbrooke's Hospital, Cambridge, UK.
WOLVEKAMPM.C.l.,M.D.
The Netherlands.
WRIGHT R. A., Ph. D.

Director, Biomedical and Health Care Ethics Program,
University of Oklahoma Health Sciences Center, Oklahoma City,
Oklahoma, USA.
YEY.,M.D.
Research Fellow, Oklahoma Transplantation Institute, Baptist
Medical Center, Oklahoma City, Oklahoma, USA.
ZUHDlN.,M.D.
Director and Chief Transplant Surgeon, Oklahoma
Transplantation Institute, Baptist Medical Center, Oklahoma
City, Oklahoma, USA
Section I
Introduction and
Historical Perspective
Chapter 1
Introduction
D.K.C. COOPER, E. KEMP, K. REEMTSMA, AND D.l.G. WHITE
In 1969 (when the terms heterograft and homograft were still being used to
describe the xenograft and allograft, respectively), no less an authority than Sir
Peter Medawar made the following prophetic remarks: "A new solution is
therefore called for: the use of heterografts - that is to say, of grafts trans-
planted from lower animals into man. Of the use of heterografts I can say only
this: that in the laboratory we are achieving greater success with grafts
between species today than we achieved with grafts within 15 years ago. We
shall solve the problem by using heterografts one day if we try hard enough,
and maybe in less than 15 years." [1].
Unfortunately, over 20 years later, his optimism has not yet been fulfilled,
and there remains a major shortage of donor organs worldwide. The success of
allotransplantation has encouraged physicians to refer an ever-increasing
number of patients to the transplant units for consideration for organ replace-
ment, and this has steadily exacerbated the inadequacy of suitable donor
organs. This shortage has been clearly documented by a number of authors
and we do not need to emphasize it further here.
Great efforts have been expended in recent years to address this problem,
in particular by education of both the public and the medical profession to
ensure that suitable donors are not lost to the transplanters, and by the organi-
zation of efficient organ procurement networks both within countries and
across international borders, and yet the problem remains and, if anything,
steadily becomes more serious.
In some fields of medicine, the patient with end-stage organ disease can be
supported, at least temporarily, by the use of artificial organs such as the dialy-
sis machine and the artificial heart or ventricular assist device. In the case of
dialysis, this support can often be long-term, though with regard to heart
support the techniques currently available have relatively severe limitations.
Progress in the development of permanently replaceable artificial organs,
particularly in the realm of the heart, has been taking place, but to date has not
achieved the success one would have hoped, particularly when one considers
the extensive resources that have been applied in an attempt to find a solution
to this problem.
It is in the field of xenotransplantation, however, that many feel the poten-
tial resolution of the problem of organ supply is situated. The ability to use a
commonly available, inexpensive animal, such as the pig or sheep, as a donor
of all organs would obviously solve the donor shortage problem conclusively.
4 Introduction
In regard to organs such as the liver, whose complexity is unlikely to be dupli-

cated by a mechanical device in the foreseeable future, xenografts would
appear to be perhaps the only solution.
The use of animals such as the pig or sheep as donors forman, however, has to
date been prevented by the occurrence of rapid and violent rejection that can be
almost immediate. Current evidence suggests that this is related to the presence
of natural preformed antibodies in man against these species, and, as yet,
methods of removal of such antibodies have not been totally successful. Much
ofthis volume relates to the resolution of this problem.
More closely related animals to man, such as the nonhuman primates,
would not be rejected in such a vigorous and rapid manner, but are almost
certainly rejected by the slower development of antibodies, possibly in associ-
ation with cellular mechanisms. Although cellular rejection is likely to be
controlled by the pharmacologic agents presently available - or in various
stages of experimental and clinical trial - the development of antibodies has
proven to be a difficult problem to resolve to date.
Even if this difficulty could be surmounted, the use of nonhuman primates
as organ donors for man might be greatly restricted both by the lack of ready
availability of these animals worldwide and by opposition from some
members of the general public. Such public opposition would be expected to
be far more muted if an animal that is commonly slaughtered for food for
human consumption could be used as the donor, and thus the pig or sheep
become most likely candidates.
Experimental work, however, is progressing in both of these areas, namely
with regard to widely disparate animal organ donors for man (e.g., the pig) and
to closely related animal donors (e.g., the nonhuman primate). Several experi-
mental models where the disparity between donor and recipient is comparable
to that between the pig and man (e.g., guinea pig-to-rat, pig-to-dog, pig-to-
baboon) or to that between the nonhuman primate and man (hamster-to-rat,
wolf-to-dog, vervet monkey-to-baboon) are currently under investigation and
are described in this volume. This book, therefore, concerns itself with all
aspects ofxenotransplantation and with many potential species of organ donor.
Today, transplantation between widely disparate species is commonly
termed discordant xenotransplantation and that between closely related
species concordant. These terms were originally coined in 1970 by Caine [2],
however, to identify and describe the type of rejection that occurred when
transplantation was performed between different species. At that time, for
example, transplantation between closely related species was thought to result
in acute cellular rejection such as occurs during first-set allograft rejection
(and was termed concordant). Rejection between widely disparate species
was thought to be antibody-mediated, such as occurs during second-set
allograft rejection in a sensitized host (discordant).
Though these terms have proved to be helpful, our increasing knowledge
suggests that whether the donor is from a closely or distantly related species
rejection is frequently, at least in part, antibody-mediated. In the concordant
relationship, there may be an initial extremely low level or absence of antibod-
Introduction 5
ies, but they develop steadily over the first few days following transplantation
and playa role (often together with cellular mechanisms) in bringing about
graft rejection. In transplantation between discordant species, the antibodies
are clearly preformed and the antibody level is so high initially that rejection is
extremely rapid, often within a few minutes.
It is not only the above terms that may prove confusing. As noted above,
antibody-mediated rejection may be almost immediate, in which case it has
been termed hyperacute, or it may be more gradual and take place over sever-
al days or even several weeks, in which case it is frequently termed vascular or
humoral rejection. The chronic rejection of an allografted organ - for
example, the accelerated atherosclerosis that may develop over months or
years in a cardiac allograft - may also be antibody-mediated, though this
remains uncertain at the present time. In transplantation between closely
related species, acute cellular rejection may predominate, either in isolation or
in association with an antibody-mediated response. When these two forms of
rejection occur together, then the histopathological appearances have been
described as demonstrating mixed rejection.
In this volume we have taken care to try to provide consistent use of all of
the above terms. In general, the contributing authors have used the term
"concordant" to mean closely related and "discordant" to mean widely
disparate (species). These terms have, therefore, not been used as originally
suggested by CaIne, namely to describe the type of rejection (cellular or
antibody mediated) that occurs.
It has already been pointed out that the major problem facing the trans-
plant team with regard to transplantation between widely disparate species is
that of pre-existing natural antibodies against the donor species. These must
clearly be removed and, furthermore, their production suppressed, at least
temporarily, if such transplantation is to prove successful. There is evidence
that the cellular response following transplantation between widely disparate
species may be rather weak, and there is optimism that the currently available
pharmacologic immunosuppressive agents should be able to prevent this form
of rejection from developing.
In transplantation between more closely related species, although the
original level of antibodies may not be high enough to lead to hyperacute
rejection, the subsequent increase in the level of antibodies may result in
vascular rejection occurring within a few days. There may also be a greater or
lesser cellular response. Both the antibody-mediated and the cellular response
will have to be controlled by the various techniques and drugs that are being
explored at the present time.
Such pharmacologic agents include FK-506, rapamycin, and RS 61443, a
form of mycophenolic acid. Whether these drugs will enable us to overcome the
hurdles that are presented by xenografting remains uncertain, though some of
them, or combinations of them, would appear to offer encouragement.
If these early rejection problems can be overcome, the long-term survival
of xenografted organs remains uncertain. Whether they will be even more
susceptible than allografts to chronic rejection, which mayor may not be
6 Introduction
antibody mediated, remains largely unknown, though experience in the realm

of transplantation across the ABO blood group barrier [3] or where there are
initial high levels of HLA antibodies [4] - both situations in which antibodies
playa major role - suggest that this will not be so.
Once clinical xenotransplantation has become a reality, we could be faced
by a new spectrum of infectious complications in the transplanted patient. The
animal species that are most likely to be used as donors carry different spectra
of infectious agents than does man, and transfer of such organisms could prove
a major problem. One solution would be the use of gnotobiotic animals that
would be microorganism-free. Indeed, such donors would be an improvement
on the human donors that are currently available, some of whom carry
microorganisms such as cytomegalovirus, hepatitis viruses, and toxoplasma
that may prevent the use of their organs or may be transferred to the recipient.
A final area which to date remains largely unexplored is the function of an
organ of one species in another. Although clinical observations have been
made of relatively long-term function of nonhuman primate kidneys in man,
and also of short-term function of nonhuman primate and pig livers in man,
our knowledge in this area is extremely limited. Whether a pig liver, for
example, can function satisfactorily in the milieu of the human body remains
uncertain, though there is optimism in this regard [5]. A whole new science
may be born when xenografting between widely disparate species becomes a
reality.
Successful clinical xenotransplantation will change the outlook and activi-
ties of the transplanters in many major ways. With the easy availability of
donor organs, selection of recipients will almost certainly become less restric-
tive. Less than ideal candidates, who today would be denied allografting, may
be given an opportunity to undergo organ transplantation if death seems
otherwise inevitable. The international network of organ sharing agencies
may become a feature of the past, to be replaced by a new network of suppli-
ers of gnotobiotic animals. No longer will midnight flights by the donor
procurement team to distant places be necessary.
The pretransplant stay of very sick patients in the hospital, particularly in
those with end-stage heart or liver disease, will be greatly reduced. No longer
will a patient in heart failure need to be hospitalized for several weeks or
months whilst a suitable donor is sought. No longer will a patient supported by
a ventricular assist device require to remain bedridden for the same reason. A
patient will need to be hospitalized only until his clinical status has been
improved and stabilized sufficiently for him to undergo the transplant proce-
dure. Transplantation will be carried out electively at the most optimal time.
Transplant operations will, therefore, be performed at 8:00 a.m. on the routine
operating list rather than in the middle of the night or at the weekend, as is
most commonly the case today.
The combination of these changes should lead to a considerable reduction
in the cost of the care of the transplant patient. The length of time some
patients are confined to hospital, awaiting a suitable donor, is a major expense,
particularly in those awaiting heart transplantation. The costs incurred by the
References 7
donor heart and liver teams, flying by private jet to the donor center and
returning with the organ, will no longer be necessary.
If, however, despite short-term success, the long-term results ofxenografts
prove to be poor, it may be that xenotransplantation will be carried out as an
emergency procedure only in those patients who would clearly not survive
until an allograft becomes available. The xenograft would become a "bridg-
ing" device, be it of heart, liver or other vital organ. Or, if long-term function
is guarded, the xenograft will be chosen for the older patient and the allograft
reserved for the younger patient who has a longer natural lifespan to face. In
the realm of kidney transplantation, the living related donor may remain the
optimum choice for the very best long-term result.
We will undoubtedly see much opposition from animal rights groups
against the use of animals for the purposes of organ donation. Hopefully, in
the case of animals such as the pig or sheep, this opposition will be muted and
temporary. It would seem churlish for activists to criticize the use of such
animals for organ donation when they are killed in very large numbers on a
daily basis as a source of food for humans.
With regard to more closely related species - the nonhuman primate - it is
likely that transplantation on a large scale may be prohibited by the public
response. Already such animals as the chimpanzee are endangered species
and, by common consent, have been excluded as donors for man. Certain
smaller species of nonhuman primate, however, such as the African baboon,
still exist in large enough numbers to make them - theoretically, at least - a
possible source of donor organs, particularly perhaps for children. The phylo-
genetic closeness of the nonhuman primates to man, however, will ensure that
the animal protection groups will be far more vociferous in opposing their use
than they will be in regard to the common farm animals.
There will clearly be much discussion and controversy, but it is hoped that
opposition will be overcome, just as it has been in the field of heart valve
surgery, where pig valves are used in large numbers to replace those defective
in man. Of one thing we can be certain - the next decade will prove one of great
challenge and, hopefully, great rewards to those involved in organ transplan-
tation. It is our hope that this volume will inspire and facilitate future research
and progress in the exciting field ofxenotransplantation.
References
1. Medawar, P. Quoted by Reemtsma, K. Heterotransplantation. Transplant. Proc. 1,251,
1969.
2. Caine, RY. Organ transplantation between widely disparate species. Transplant. Proc. 2,
550,1970.
3. Alexandre, G.P.J., Squifflet, J.P., De Bruyere, M., Latinne, D., Reding, R., Gianello, P.,
Carlier, M., Pirson, Y. Present experience in a series of 26 ABO-incompatible living
donor renal allografts. Transplant. Proc. 19,4538,1987.
4. Palmer, A., Welsh, K., Gjorstrup, P., Taube, D., Bewick, M., Thick, M. Removal of anti-
HLA antibodies by extracorporeal immunoadsorption to enable renal transplantation.
Lancet. 1,10,1989.
5. Auchincloss, H. Jr. Xenogeneic transplantation. Transplantation. 46, 1, 1988.
Chapter 2
Xenotransplantation - A Brief History

of Clinical Experiences: 1900-1965
K.REEMTSMA
The Mythology of Transplantation
The idea of transplanting organs from animals to humans has intrigued man
for as long as he has recorded his myths and his history. Daedelus, who grafted
bird feathers to his arms, was perhaps the first to transplant across the species
barrier successfully. He escaped from his island prison in Crete and flew to the
mainland of Greece. A similar experiment by his son, Icarus, ended in acute
graft rejection, attributed to a thermolabile adhesive. After flying too close to
the sun he plunged into the water which is now called, in his honor, the Icaran
Sea [1].
Early Attempts at Renal Xenografting
Early in the twentieth century reports on cross-species grafting (then called

heterotransplantation) appeared in the scientific literature. In 1905,
Princeteau [2], in France, inserted slices of rabbit kidney into a nephrotomy in
a child with renal insufficiency (Fig. 2.1). "The immediate results were excel-
lent, " he wrote, "The volume of urine increased; vomiting stopped ... On the
16th day the child died of pulmonary congestion ... ".
In the following year, 1906, Jaboulay [3], also in France, on two occasions
attempted renal heterotransplantation into man using vascular anastomoses
(Fig. 2.2). The xenografts, one from a pig and another from a goat, were insert-
ed into the antecubital space. Neither graft functioned, and failure was at-
tributed to vascular thromboses.
In 1910, Unger [4], in Germany, described his attempt at transplantation of
kidneys from a nonhuman primate into man (Fig. 2.3). The patient died 32 h
after transplantation, and autopsy showed venous thromboses.
In 1923, Neuhof [5], in New York, attempted treatment of a patient with
mercury bichloride poisoning by renal heterotransplantation (Fig. 2.4). When
he was unable to obtain a human kidney, he transplanted the kidney of a lamb
into the patient. The patient died 9 days later, but Neuhof was not totally dis-
couraged. He wrote "[this case] proves, however, that a heterografted kidney
in a human being does not necessarily become gangrenous and the procedure
is, therefore, not necessarily a dangerous one, as had been supposed. It also
10 Xenotransplantation - A Brief History of Clinica l Experiences: 1900- 1965
Y. Greffe rGnale. ~c..O\J"!.J -;, .. J ! J..........(

)L PRI:<CI~·rEAU presento lea deux reins ,)"un enfant ,Ie
son service et fait pnrt I!. laSocielC do l"obscrvntion sllivallte:·
Un enf:\IIt atteint d·oste~yelite de l"cxlremite snphillnrc
du f~mur droit ayllnt fuse <IrIIlS I'os iliaque entre II rh&pilal
des Enrants dans un Hat trca grave, nynnt de l"nlbumino
dun91es urines et un ana.ollrque tr~ marque. Immediatc-
ment it fit une grllnde incision, nettoya Ie foyer principal
et IIpres qUlltre interventions succe8sivea l"elat de I·enfant
a·elait amelior'. Snbiwment, en fin noilt, cet enfnnt fit de Ja
paralysie facillie ct un wdeme gen~tn1ise, vomiS/l."\nt tnut ce
qll·i\ prenait et urinan! II peine 20 grammes de Iiquide par
jour.
Devant de si graves aymplomes, M. Princetollu fit uno
llephrotomic II gauche et inclut dnns Ie rein deux tranches
de reiD d·un InpiD. Lea resultats immediats furent exceJlents,
l"urine augmenta, les vomissements cepserent; qUiDze jour,;
apres !"intervention III quantite d·urine elait de un litro var
jour. Le Keizieme jour, I"enfllnt 8uccomDait A Ulle conge~lion
pulmonllire.
A )" autopsie, on trollvll les deux reins gr09 et blllucs, lin
foie IIlliscaue et la rate cxtrcmement mmol\ie. Qur.nt nUl(
inclusions de trRnches do rein de lapin, alles pnrais8:licllt
grefTees, mais un examen histologique est necessnire pour
montrer 111 nlltu re de I"adh~rence . J. M.
Fig. 2.1. Princeteau's original description of inserting slices o[rabbit kidney into a nephroto-
my in a child with renal insufficiency
demonstrates that thrombosis or hemorrhage at the anastomosis is not in-

evitable. I believe that this case report should turn attention anew ... ".
However, scientific interest in transplantation declined when the immuno-
logical basis of the rejection process was established. With the demonstration
of effectiveness of immunosuppressive drugs, there was renewed interest in
transplantation. An accelerated effort in renal allotransplantation was accom-
panied by problems in procuring organs. Ethical considerations posed difficult
problems, particularly in the use of volunteer human donors. The use of or-
gans harvested from human cadavers depended on rapid transfer or preserva-
tion and imposed restrictions of supply, selection and scheduling.
The Tulane University Chimpanzee-to-Man Renal Xenograft

Experience
In our renal allografting program at Tulane University in New Orleans, we had

increasing difficulty obtaining donor organs. Attempts to use cadaveric kidneys
were inadequate. We were reluctant to press the use of volunteer humans for
ethical, scientific, and legal reasons. Chronic dialysis was not available.
The Tulane University Chimpanzee-to-Man Renal Xenograft Experience 11
I17U.ETIN.
BULLETIN DU LYON MEDICAL
(;R£"" DF. RF.IN!I At: PI.I DU COt;D" PAR 1I0UDURU

AIITERIELLES ET VI:INI:Un;~,
J'ai pratique I" greffd r~n .. le pllr luturea valcn).irct, dellE

foi., I., :l4 jaDvier 8t I" 0 avril de cette annee; j., mo propo-
JAil d'Hablir UDI! suppleancc (unctiunn"lIe pour la l~cr'tiOIl
urinaire, en in.'nlhlnl UII r"in 'tuDger mail ~.in, pou"aDt
'l'eoir eo .. ide aux urifllne. Dutllrel~ qui 6taient aUeiotl de
mal .. diel iDcurable.,
La pr"miilre foia c'et.it chez une ("mme de 48 alii,
brightique, pre.cntllnt une furte h'ypcrl~Dlioo, AVec dpha.
lalgie, diminutioD dllla vue lit d. I'uur", at qui o"mettail
par juur que flOO CC, en'l'iroll d'uDe uriae albumiaoule at DI
ConteaaDt que 4 gr, d'ur'e, Un r"in de pore qui avait 6t6
Iue troi~ heuree 1I,'aDt, mil immedi.tement aprb lOll utra~
liuD dlln. du I~rum artiSei .. ) tiilde, Cut alll1"omOI' par MI
"aiunux aul niuellllX du pli du eoudl de III mal,d.,
C'ttait Ie rein gauebe et Ie pH du coude gauche, Ulle
incitiOD longitudiDIIle, C.ile luiTIID! III directioD de I'.rtll"
hUlQeral~ • ce Di'l'uu, mit i\ IIU d'.bord I. niDI m'di.lle
cepl,.liquil qui fut di5Sequc!~, pui. pruruDd~meDt rart.ra
_Vllnt d" bifllrcliliuD, Un~ bllDd" d'E.m.rch .".il 616 plac6 •
• la racino du bUI, UDe ligillure Ilyaal 61' min .ur la
purtiun d~. vKi.aeau, qui d~vail iltro leur bOllt p~riph~rlqu ••
ceul-ci {urcDI .e~liolln~ .. , montraDt leur buu! c6Dlrai .idl
<lc >IIDg, Alon I~ rein Cut 616 dan. cetta plaia, la race
"nt~riellro eD avant ~t nun recuUVerte, I'liretll,. oeCup'Dt la
plRn profilnd ct dirigil vera Ie burd iDterae du pli da couda,
L'lIftere r~nlllM futl"u,lt~u au bouI ceDtral del'artera hum6r,la,
I. v.iDe reDale .u boul c"Dtral de I, veiDe m'di .. na c6pbali-
~u~, d81. fdcon Iui.lnte: ehaelln do. v.ill."u" dela m,l .. d,
et.it introduit dltn~ la IlitIlicre d'un. virol" milt,llique, d.
~.m"n~ion'ltpprupriec~, plli~ r.tourn~e a l'utr6lait6 de cetta
"~rol" iu.qu'. lind rij{ul. cireul.irl! oil il I!tltit tid par UII 81
CItC"IKlr,,; I.. I'ouui .ulurno dll Yai,aCIiU. cnd.rter" uu eado-
'l'oin., oI.YOIuuil lIinai ntertl8 CI pUUYllit 4trOl coaplea ...ec I,
Fig, 2,2, Report of Jaboulay's attempts at renal heterotransplantation
As this impasse was developing, we decided to explore the use of nonhu-

man sources for clinical renal transplantation, This decision was prompted, in
part, by clinical urgency, Additionally, a regional primate center in this vicini-
ty brought scientists experienced in primatology, Furthermore, an active pro-
gram in transplantation immunology had been developed to give an added
base to the study,
12 Xenotransplantation - A Brief History of Clinical Experiences: 1900- \965
:\w; del' exp(:ril!lclltell-l,iologischen AI,teilllng des

l\ijni;.d, 1,,,tholo!!isdll:n Instituts dcl' Uni\'ersitat Berlin
IIlId alls tl CI' Pl'i,';otklinik DI', El'nst rnger,
Nierentransplantationen.
(I I. lliltei l uog.)
Dr. Era., Cng.r· B.,lio.

(""b ',in.", an. ~. ~I ,,, 191" in der B.rli".r medizioiscben Gtsel hcbaft
gtkllttDelJ Y(frtragt.)
.. ~1. H.: 1m Apri l l~(Y.t batte icb mir erl aub!, Ihnen iiber
:\tereUlransplantationen IU bericlo 'en. Carrel uod Gu t hrie ge-
Fig. 2.3. Original report of the work of Ernst Unger who attempted kidney transplantation
[rom a nonhuman primate to man
Our basic conjecture was that kidneys from nonhuman sources closely re-
lated to man would respond similarly to human kidneys following transplanta-
tion into man. The problem of presumably more strenuous immune suppres-
sion was balanced against the advantages in the use of nonhuman donors.
In practice, all patients were terminal uremics, maintained on dialysis, who
were presented with the following alternatives:
(i) supportive treatment only, (ii) an allograft from a relative, (iii) a cadaveric
allograft, if and when available, or (iv) a heterograft (xenograft),
The risks, the uncertainties, and the experimental nature of the work were
discussed with the patients and their families, If they chose to proceed with
transplantation and had no volunteer donor, a search was made for a cadaver-
ic kidney. If no suitable cadaver kidney became available, a xenograft was
used, with the patient's understanding and consent.
The Chimpanzee as Donor
The chimpanzee was selected as the donor for several reasons, which are dis-
cussed more fully in Chap. 33. They included (i) the chimpanzee's close taxo-
nomic relationship to man, (ii) its range of size which approximates that of
man, a factor which might have significance in the transplantation of other or-
gans in addition to kidneys, (iii) its renal function corresponds closely to that
of man , and (iv) chimpanzees have been found to be of blood types A and 0 ,
thereby offering the possibility of the universal donor from the standpoint of
blood groups.
Between November 5, 1963, and February 10, 1964, six patients received
renal heterotransplants from chimpanzees (see also Chap. 33). All patients
were in terminal uremia necessitating dialysis and all patients received pre-
transplantation treatment with azathioprine, actinomycin C, and steroids,
Selection of the donor was based on body size and blood typing of donor and
recipient. Creatinine clearance was determined in each donor.
THE TRANSPLANTATION
OF TISSUES
BY
JL\ROLD :\EUIIOF, )I.D.
n".T. l'~IIII S' I' l ' . O IU l Y. CuLl, ... I" UlIIIVE "" ITl' . ( 'OLL£aIE 0 ,. ' tl ... .. IC' I ... ". ..
",}I>c,; " 1 - .~ ~0l"'1II : ", IIAQeU TK: fI. \; .O J; O~ TO .KLL~n ' r.:. III Q \ ' }o'l" "" !Ii"',
A S!) . " ,TI1f'lulil ll: MUA"lTA~ : " n I!: IIi DI .... O .... -= ' · . (» Lutl l l.: ... L
. "' .~I:~~ , \."Kl'Il'AAL. "1'010 :rU:U.o LOdl~"L yo.r lT4 "
WITI( Til ..: (,OLL.AHOf( ... T I U~ U t>'
S A)It:~; L 1I1R ~ IIFELD. )1.0.
f;l"ltGICAJ. )[O:-lOGRAPHS
])J:;A~ LEWIS. A.D., )1.1>. Et:GENE H. POOL. A.B.• )[.D.
ARTHlR W. ELTI;\G. A.H .. )I.D.
D. APPLETO~ AND CO~lPA~Y

NEW YORK LOXVO;\'
1923
Fig. 2.4. Title page of Harold Neuhof's work in which he describes his transplantation of a
lamb kidney into a patient
In each instance the donor received general endotracheal anesthesia with

monitoring of blood pressure, electrocardiogram and body temperature. At
moderate hypothermia (about 30°C) the entire renal complex, including both
kidneys and ureters, aorta, and vena cava, was removed en bloc after antico-
agulation and was irrigated. Patients were prepared simultaneously by ex-
traperitoneal exposure of the external iliac artery and vein. In each instance
the aorta and vena cava of the graft were anastomosed to the recipient's ex-
ternal iliac artery and vein, respectively, in an end-to-side fashion (Fig. 2.5).
The periods of ischemia, from the time of vessel clamping in the donor until
14 Xenotransplantation - A Brief History of Clinical Experiences: 1900- 1965
Fig. 2.5. Technique of combined kidney transplantation as carried out in the chimpanzee-ta-
man series at Tulane University
blood flow was restored through the graft in the recipient, varied from 36 to
43 min.
All patients received postoperative azathioprine, actinomycin C, and
steroids, and X-irradiation to the graft. We will summarize two cases.
Casel
A 43-year-old former dock worker with a history of hypertension since 1957

was admitted to the Veterans Administration Hospital, New Orleans, in 1959.
Renal biopsies showed nephrosclerosis and chronic glomerulonephritis. He
was treated with dietary management, including salt restriction. He was read-
mitted in June 1963 because of progressive uremia, hypertension, and conges-
tive heart failure. Laboratory studies included the following: blood urea
nitrogen (BUN) 240 mg%, creatinine 14 mg%, and creatinine clearance
• • ~
::r
I G
~
S
::>
'G"
c:
::>
:;::.
(1)
~.
II _ -<n
::r
§.
"0
:m: / mm m ~ ' m ~__ '"::>
N
(1)
:rl L......... .............. . ~

5"
~
::>
'"
;;0
:y1 (1)
::>
:lIT ~ - -----:----- e:-
X
(1)
::>
wl o
(]Q
...,
:2IIT 1 '";:::>
tTl
><
"0
(1)
...,
c;.
x l ::>
• • • • • !)t tOlK_ ~"l
g
~L~==~====~~~~~~~~~::: J .... .J ..... . . .. .
Fig. 2.6. Flow sheet demonstrating clinical course of patie nt in Case 1. nine (mg%) , (v) creatinine clearance (mUmin ), (vi) wh ite blood count ,.......
VI
From the top the parameters charted are (i) urine volume (mlf24 h), (ii) (cellsfmm 3), (vii) blood pressure (mmHg), a nd dosages of (vii) pred-
uri ne sodium content (mEqf24 h), (iii) BUN (mg%), (iv) serum creati- nisone (mg), (ix) azat hioprine (mg), and (x) actinomycin C (g)
Fig. 2.7. Post-transpla nt intra-

venous urogram in Case I
8 mllmin. There was no improvement with dietary management, and peri-

toneal dialysis was required.
On November 5, 1963, he received a renal xenograft. During the first 14 h
after transplantation, the urinary output was 5700 ml. The BUN, which was
112 mg% on the day of operation, decreased to 39 mg% by the 4th day follow-
ing transplantation. The creatinine, which was 1l.2 mg% on the day of opera-
tion , fell to 1.5 mg% 48 h after transplantation. Four days after transplanta-
tion, rejection occurred, but was reversed following local irradiation to the
graft and increased doses of immunosuppressive drugs (Fig. 2.6). His early
course has been reported previously in detail. Function of the graft was con-
firmed by renograms, scans and intravenous urogram (Fig. 2.7).
Serial renograms (Fig. 2.8) demonstrated a progressive delay in the appear-
ance of the peak of uptake. Changes in the renogram , however, were not cor-
related with biochemical changes in renal function.
Hemagglutination studies (Fig. 2.9) demonstrated a precipitous rise in titer
beginning on the 4th day following transplantation. This titer fell to pretrans-
plantation levels at the end of 1 month and remained at this level throughout
the 2nd month. Data on cytotoxicity studies are shown.
On December 18, he was allowed to leave the hospital because he was
asymptomatic and had normal renal function. He was readmitted on
December 20 with a temperature of 39.4°C, and radiographic evidence of an
infiltrate in the right middle lobe with pleural effusion. Culture of the sputum
JU
5.0
4.5
4.0
3.5
3.0
2.!i
2.0
1.5
1.0
.5
o ~~--~--~--~--~--~--~
A 11- 12-63 8 12-1-63
~~ V'-JV~I\
3.0
2.5
2.0
1.5
1.0
.5
Fig. 2.8. Serial renograms post-transplant in Case 1
1024 " l
r
A
512
z
• xxx x x x
j
0 256
0-
::J
..J
128 xx ~
0
~ 64 'II'
::J
a::
w xx-xx x X xx
Ul 32
IL
0 16
..J
H KII--lI
50 8
a::
Il. 4 "J{
U
w
a:: 2
o 8 16 24 32 40 48 56 64
DAYS FOLLOWING TRANSPLANTATION
Fig. 2.9. Hemagglutination studies post-transplantation in Case 1

18 Xenotransplantation - A Brief History of Clinical Experiences: 1900-1965
Fig. 2.10. Macroscopic appearance of one kidney at autopsy in Case I
Fig. 2.11. Photomicrograph of kidney at autopsy in Case I

revealed Aerobacter aerogenes. The dosage of azathioprine was lowered be-
cause of leukopenia, but renal function continued satisfactorily. The patient's
condition later deteriorated rapidly, and he died 63 days after transplantation
following a period of shock, apparently due to sepsis.
Autopsy showed acute bronchopneumonia (right lower lobe) and acute
tracheobronchitis with resolving abscess (right middle lobe). The transplanted
kidneys showed acute tubular necrosis, consistent with shock; there were no
cellular infiltrates or changes in the blood vessels (Figs. 2.10, 2.11).
Case 2
A 23-year-old school teacher was admitted in November 1963 with chronic

glomerulonephritis and progressive uremia. She had had an episode of acute
glomerulonephritis at age 14, and demonstrated persisting proteinuria. She
had remained asymptomatic until approximately 5 months before admission,
when she noted weakness and dizziness.
On admission her blood pressure was 190/120 mmHg and laboratory studies
included BUN of 184 mg%, creatinine of 40 mg%, and creatinine clearance of
4 ml/min. Rapid deterioration of her condition necessitated peritoneal dialysis.
On January 13, 1964, she received a renal heterotransplant. Diuresis oc-
curred with a urinary output on the day of operation of 71. By the 3rd day fol-
lowing transplantation the BUN had fallen from a pre transplant level of
116 mg% to 12 mg%, and the serum creatinine from a preoperative level of 21
mg% to 0.9 mg% . Creatinine clearance was 50 mllmin. Her blood pressure fell
to normotensive levels (110/70 mmHg). Her subsequent course demonstrated
satisfactory renal function until the 23rd day following operation when threat-
ened rejection was suspected (Fig. 2.12).
Urinary output decreased to 1,000 ml/24 h, BUN and creatinine rose to
28 and 1.9 mg%, respectively. Creatinine clearance fell to 23 ml/min, and uri-
nary sodium content to 11.6 mEq for a 24-h period. Gradual reversal of rejec-
tion occurred during the following two weeks, although unexplained fever
persisted for 3 months. She became asymptomatic and had normal renal func-
tion 8 months after transplantation.
Serial renograms (Fig. 2.13) in this patient demonstrated a delay in peak ac-
tivity which coincided with clinical and biochemical evidence of threatened re-
jection. Following reversal of rejection, the renogram resumed a more normal
pattern. An intravenous urogram 12 weeks after transplantation showed func-
tion of both transplanted kidneys (Fig. 2.14).
Agglutination studies (Fig. 2.15) demonstrated a slight rise in titer at ap-
proximately 3 weeks after transplantation. The agglutination titer subse-
quently returned to previous levels.
This patient died 9 months after transplantation. The cause of death was
thought to be acute electrolyte imbalance. Autopsy showed no other cause of
death. Histology of the transplanted kidneys showed no cellular infiltration,
but subintimal hyperplasia ofthe arterioles [6].
E P
"_I
T, ..."".~
i .I 1000
.~
2r'>OO'200'
+ +
,
+
''''. '>+0'
~~ ~ .
~i ·,'000" ~ ... ,
. ~_.__~
.L-_________<>~~ . •__~__~._____-_ __
. •_____. ________________
~~I··~L----------------------
~~ _==---..:::,.~= __________ __ ""~===::::::::::..
~)~
'001
---
~ l~
~~l
'!DiL
l __---------
_ ----=======::::=::=::==::::::::--------
===::::::====__
~r
~ I
2001
100
•
• •
it·
'~l
ii. ...
;, .0'1
E
~i 20.
,
L 'oD'00 I
~ 0
z , .. "It 1 • II
"011 If I) '4 1'"
"IA.IIIu."" It6.
1""" .1
n 1,"20212223142$ aZl.2t30SI 12: I ..
1111111111111 " ,
~ • '7 • , ' 0 II r.t
'fl.llu.t.,."
I~
11 14
141$ " 11 11 .'10
Fig. 2.12. Flow sheet demonstrating clinical course of patient in Case 2. Columns from above
down as for Fig. 2.6
Subsequent Clinical Studies
Following the initial experience in New Orleans, there was a flurry of activity
in the field of primate-to-man transplantation. In December, 1964, three dis-
tinguished transplant surgeons, Drs. J.D. Hardy, D.M. Hume and T.E. Starzl,
were attending a surgical meeting in New Orleans. At the end of the meeting I
showed these three surgeons the first patient, who was doing well with normal
renal function 7 weeks after transplantation. Each of these three surgeons be-
gan working in clinical xenografting.
Dr. James Hardy [7] reported a few months later the first case of heart
transplantation in man (Chap. 34). He used the heart of a chimpanzee, but was
unsuccessful in this attempt. Dr. David Hume [8] did a chimpanzee-to-man re-
nal transplant, and the patient died the following day of excessive diuresis
(Chap. 33).
Subsequent Clinical Studies 21
EP
5.0
4.5
~
4.0
3.5
3.0
2.5
2.0
1.5 /
1.0
.5
0
A o
5.0
.r-
1\
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
Fig. 2.13. Serial rcnograms .5
post-transplant in Case 2 0 , o
Fig. 2.14. Post-transplant intra-

venous urogram in Case 2
22 Xenotransplantation - A Brief History of Clinical Experiences: 1900-1965
....
(/) 128 x
z
0
....::J 64 • J(
•
-'
0
~
32 • • • • • •
::J
a:
w 16 • • XI
• • "
(/)
lL 8
.
0
-'
4
u
0
a: 2
"-
U
w
a:
0 8 16 24 32 40 48 56 64
DAYS FOLLOWING TRANSPLANTATION
Fig. 2.15. Hemagglutination studies post-transplant in Case 2
Dr. Tom Starzl began a series of baboon-to-man renal transplants which

were studied extensively and have been reported in detail [9] (Chap. 33). The
histopathological studies in our chimpanzee-to-man work and in Starzl's
baboon-to-man cases were all carried out by Dr. Ken Porter of London
(Chap. 33).
By 1965 we had dialysis facilities available at Tulane University, and we had
developed a successful cadaver organ procurement program. For these rea-
sons we discontinued our clinical renal xenotransplantation.
I have subsequently, however, maintained experimental programs in xeno-
transplantation, including transplantation of islet cells in several animal mod-
els, and xenotransplantation of the heart between different species of primate.
The emerging studies, focused on the basic biology of the xenograft re-
sponse, suggest that we shall see a renaissance of interest in this exciting and
promising field.
References
1. Ovid and Apollodorus, quoted by Edith Hamilton. Mythology. Littlc, Brown and Co.,
Boston, 1940.
2. Princeteau, M. Greffe renalc. I. Med. Bordeaux. 26,549,1905.
3. Jaboulay, M. Greffc de reins au pli du coude par soudures arterielles et veineuses. Lyon
Med.I07.575,1906.
4. Unger, E. Nierentransplantationen. Klin. Wschr. 47.573,1910.
5. Neuhof, H. The Transplantation of Tissues. Appleton and Co., New York, 1923, p. 260.
6. Reemtsma, K., McCracken, B.H., Schlegel, J.U., Pearl. M.A .. Pearce, C.W .. DeWitt,
C.W., Smith, P.E., Hewitt, R.L.. Flinner, R.L., Creech, O. Renal heterotransplantation in
man. Ann. Surg. 160, 31l4, 1964.
7. Hardy.J.D., Chavez, C.M., Kurrus, F.D., Neely, W.A .. Eraslan, S., Turner, M.D., Fabian,
L.W., Labecki, T.D. Heart transplantation in man. I. A. M.A. ISIl,I132, 1964.
S. Hume. D.M. Discussion of paper by Reemtsma et al.Ann. Surg. 160,384.1964.
9. Starzl, T.E. Discussion of paper by Rcemtsma et al. Ann. Sltrg. 160.384, 1964.
Chapter 3
The Scientific Study of Xenografting: 1964-1988

H. AUCHINCLOSS
Introduction
The early clinical efforts at xenografting described by Reemtsma in Chap. 1

[1--4] and several additional efforts reported by Hitchcock [5], Starzl [6,7] and
Hardy [8, 9] were prompted by the shortage of human organs for transplanta-
tion at a time when there were few alternatives for treating end-stage organ
failure. While none of these efforts achieved I-year graft or patient survival,
they did come close in several cases. Thus they demonstrated the potential for
successful xenogeneic transplantation. Shortly after these first efforts, the con-
cept of "brain death" became widely accepted, greatly increasing the supply of
human organs for clinical transplantation and removing the principal justifica-
tion for the use of animal organs. Thus only a few sporadic attempts at clinical
xenogeneic transplantation were attempted over the next 25 years. However,
one of the apparent consequences of this early clinical effort was to spark sci-
entific interest in xenogeneic transplantation. Figure 3.1 shows the increased
number of scientific publications involving xenografting which followed
Reemtsma's clinical effort. That interest lasted almost a decade before the
200
..
GI
U
I:
!!
.!! 100
&!
z
o
O~-r----~---.----'---~r----r----~--~--
51·5556·6061·6566·7071·7576·8081-8586-90
Year
Fig. 3.1. Approximate number of scientific papers on the subject of xenogeneic transplanta-
tion during each 5-year period from 1951 to 1990, inclusive. The number of references in
each time period represents the publications entered by the author in a computer database.
Although this database now contains nearly 8oo references, it is presumably still incomplete.
However, those who would like to obtain the database for the Macintosh computer pro-
gram, Pro-Cite (Personal Bibliographic Software, Ann Arbor, MI) should send a blank 3.5"
disk with a stamped return envelope to the author of this chapter
24 The Scientific Study of Xenografting: 1904-1988
frustrations of finding solutions to the problems and the lack of need for ani-
mal donors led to a decline in the research effort.
More recently, human organ transplantation has achieved remarkable suc-
cess and become the accepted form of treatment for many patients with kid-
ney, heart, liver and lung failure. The number of organ transplants performed
each year has increased dramatically, but the number of candidates for these
operations has grown even faster. As a result, the application of human trans-
plantation is again limited, as it was in the early 1960s, by the supply of donor
organs. Responding to this shortage, especially for pediatric recipients, the
"Baby Fae" heart transplant was undertaken in 1984. This effort was again un-
successful, but again it was followed by a remarkable increase in the research
effort in xenografting as shown in Fig. 3.1.
This book is an effort to capture the results of this burst of scientific study.
The purpose of this chapter is to highlight some of the important scientific
work which preceded the recent intense effort and which set the stage for the
studies described in this book.
Antibody-Mediated Rejection
The immune destruction of foreign grafts can occur by humoral and by cellular
mechanisms, and there are probably several different processes of rejection
which can operate within each category. Over the years since the first clinical
effort at xenografting, the roles of these different mechanisms in xenograft de-
struction have been investigated.
Hyperacute Rejection
The phenomenon of hyperacute rejection was first recognized and described

from experience in clinical allogeneic transplantation and not from studies of
xenografting. Several investigators realized that kidney transplants sometimes
suffered almost immediate destruction, usually within minutes of revascular-
ization, and that this event correlated well with the presence in the recipient, be-
fore transplantation, of antibodies specific for donor antigens [6,10,11]. In the
case of allogeneic transplantation, such antibodies were the result of prior expo-
sure to alloantigens by previous transplants, transfusions, or pregnancies, or
they existed without prior exposure when there were blood group mismatches.
Once the phenomenon of hyperacute rejection had been recognized, however,
it was rapidly apparent that it was the fate of many experimental xenografts as
well. In this case, the pre-existing antibodies were "natural" antibodies, which
exist in the serum of members of one species even without prior immunization
and which are specific for antigens of other species,
Not all xenografts suffer hyperacute rejection, as was demonstrated by the
early clinical trials using primate donors for humans. Studies during the mid
Antibody-Mediated Rejection 25
1960s by Perper and Najarian established the principle that transplantation be-
tween distantly related species resulted in hyperacute rejection, while trans-
plantation between more closely related species did not [12, 13]. In 1970, CaIne
suggested that this difference could be described by the terms "discordant"
and "concordant" species [14]. His definition was an operational one, based
on the development or not of hyperacute rejection. Strictly speaking, the
terms have been misused frequently since 1970 by investigators using "discor-
dant" to mean the presence and "concordant" the absence of natural anti-
bodies. There is not, however, an absolute correlation between the two de-
finitions, partly because different organs are not equally susceptible to
hyperacute rejection, and partly because very low levels of natural antibody
may not elicit hyperacute rejection even of susceptible organs. Thus there
have been many semantic battles over questions such as whether hamsters and
rats are concordant or discordant species. Despite confusion in their use, the
terms have proven valuable as short -hand descriptions to define the context of
experimental xenografting. They probably should not be used when the issues
involve subtle outcomes resulting from low levels of preformed antibody or
when early rejection, not quite typical of hyperacute rejection, may nonethe-
less be caused by pre-existing antibodies.
Although xenografts did not provide the first characterization of hyper-
acute rejection, it was quickly recognized that discordant xenografts provided
an excellent model for the study of mechanisms and treatment of this form of
graft destruction. Many of the research studies between 1965 and 1975 in xeno-
geneic transplantation were directed at these issues. The primary purpose of
these studies was generally to find strategies to prevent hyperacute rejection,
such as complement depletion, antibody elimination, or interruption of the
clotting cascade. Dozens of publications resulted from this effort with investi-
gators such as Winn [15, 16], Rosenberg [17-19], Najarian [20-25], Starzl
[26--29], Varco [30--32], Kemp [33-37], and others contributing frequently to
this literature. These studies demonstrated that antibody fixation initiated the
process and that coagulation, complement, and other inflammatory mediators
then participated in causing graft destruction. But beyond offering these in-
sights into mechanisms, the results of these efforts were disappointing from
the point of view of clinical application. They suggested that hyperacute rejec-
tion was always initiated if enough natural antibody was present and that while
graft destruction could be delayed by several forms of treatment, it could not
be prevented in a clinically meaningful way. Many concluded from these ob-
servations that elimination of natural antibody was the key to successful xeno-
geneic transplantation between discordant species. Simple plasmapheresis to
accomplish this purpose was attempted during this early period, but without
success [38]. More recent efforts to expand the use of plasmapheresis or to
control hyperacute rejection by better characterization ofthe natural antibod-
ies and the antigens involved have been attempted [39--42] and are described
later in this book.
Natural Antibody
It was already recognized at the time of Reemtsma's first clinical efforts that
natural antibodies reactive with antigens of other species exist in all but the
most closely related species. Much of this understanding stemmed from the
studies of Landsteiner during the 1930s [43]. His work suggested that natural
antibodies tend to be immunoglobulin M (IgM) in class, to react with glyco-
protein and glycolipid determinants, and to be present without a requirement
for previous antigenic exposure. Natural antibodies were thought to arise by
cross-reactions with determinants expressed on common environmental
pathogens. Landsteiner also demonstrated that the expression of natural anti-
bodies could be used to construct an accurate phylogenic tree of animal
species.
There was little change in our understanding of xenoreactive natural anti-
bodies for more than 50 years after Landsteiner's studies. Modern concepts of
immunology have offered an explanation for the failure to see secondary re-
sponses of these antibodies and conversion to IgG production based on the
idea that carbohydrate determinants cannot produce peptides which can be
presented to helper T cells in the cleft of major histocompatibility complex
(MHC) antigens. But beyond these conceptual insights, almost no research
between 1964 and 1988 attempted to characterize further the natural antibod-
ies themselves or the antigens with which they react. This is an extraordinary
gap in the research of xenogeneic transplantation given the central role these
antibodies play in xenograft rejection. Fortunately, several investigators have
begun to address these issues during the past several years [44-46].
Induced Antibody Responses
In addition to the expression of natural antibodies before antigen exposure,

recipients of xenografts are also induced to form new antibodies after expo-
sure to xenogeneic antigens. Sachs and colleagues [47] and others [48] charac-
terized these induced antibodies in closely related species combinations. They
found that induced xenoantibodies were much like alloantibodies in that they
were generally specific for MHC antigens and were able to distinguish allode-
terminants of MHC molecules. However, two other important features of the
induced antibody response to xenografts were not explored until recently: (a)
whether the antibody response to xenografts was quantitatively stronger than
that to allografts, and (b) whether the induced antibodies playa role in the ear-
ly destruction of xenogeneic organs. The Baby Fae heart transplant was appar-
ently lost to early antibody-mediated rejection. In addition, experimental evi-
dence, some of which is discussed in Chap. 4 of this book, suggests that more
powerful and earlier antibody responses to xenografts can indeed be responsi-
ble for their rejection [49-55]. Thus the evidence now available suggests that
induced antibody responses, as well as preformed natural antibodies. are im-
Induced Antibody Responses 27
portant components of the humoral response to xenografts and that both are
more significant obstacles than in allogeneic transplantation. Overall, the
strength and importance of humoral rejection of xenografts is the dominant
feature which distinguishes xenografting from allografting.
Tissues Resistant to Humoral Rejection
The greater strength of the humoral response to xenografts than allografts and
the difficulty in controlling this response has made the prospects seem bleak
for performing successful xenogeneic transplants. There are, however, some
tissues which appear to be particularly resistant to antibody-mediated rejec-
tion which might, therefore, be candidates for the early application of xeno-
geneic transplantation even before a solution to the humoral problem is
achieved. The liver has been found to resist hyperacute rejection in many cases
for reasons that have not yet been determined [56-58]. Perhaps the large size
of the organ can absorb preformed antibody without producing concentra-
tions sufficient to trigger the mechanisms of hyperacute rejection, or there
may be critical differences in the site of antigen expression or in the character
of liver endothelium. Whatever the mechanism, the liver may be a primarily
vascularized organ which can be transplanted successfully even in discordant
xenogeneic combinations. On the other hand, long-term studies have suggest-
ed that livers transplanted across a positive cross-match or in the face of ABO
incompatibility do not survive as well as other liver transplants [57] and indi-
vidual cases of hyperacute rejection of the liver have been reported [59-61].
Thus even xenogeneic liver transplantation may require new approaches to
control humoral responses.
In addition to the liver, several tissues which require neovascularization by
the recipient appear to be resistant to humoral rejection. Winn and colleagues
studied the rejection of skin grafts by antibody during the 1970s [15, 16,62].
They found that passively transferred hyperimmune serum could destroy skin
grafts, but only after neovascularization had taken place (about 10 days after
grafting) and only when large quantities of antibody were administered over a
short period of time. The susceptibility of skin grafts to transferred antibody
diminished after 2 weeks, perhaps as the endothelial cells of the donor mi-
crovasculature were replaced by recipient cells. Furthermore, long-term sur-
vival of skin grafts was obtained in some cases despite the deposition of large
quantities of antibody on donor cells of the graft [63]. Although frequently
misunderstood since their publication, these studies were interpreted to show:
(a) that skin grafts are probably not susceptible to rejection by natural anti-
body, (b) that they are susceptible to rejection by induced antibody for only a
limited period of time, and (c) that they probably are not actually rejected by
induced antibody in most transplant experiments, given the large quantity of
antibody that seems to be required.
Although transplantation of skin is an interesting experimental model for
studies of xenogeneic transplantation because it avoids the issue of humoral
rejection, it is not likely that there will be a large demand for xenogeneic skin
grafts for cosmetic reconstruction of human patients. Other tissues, however,
appear to behave like skin grafts in their resistance to humoral rejection, in-
cluding pancreatic islets. Pancreatic islets can be destroyed by hyperimmune
sera passively transferred after revascularization (exactly as skin grafts)
[64-67], but successful transplantation of cultured xenogeneic pancreatic
islets has been achieved in species combinations where natural antibody is
known to be present [68-74]. Thus transplantation of cultured xenogeneic
islets might be an important application of xenografting which would avoid
the humoral rejection problem.
The ability of pancreatic islets to resist rejection by natural antibodies sug-
gests that other tissues, particularly those which are not primarily vascularized
at the time of transplantation, might be used successfully for xenogeneic trans-
plantation. One application of xenografting, therefore, might be in the form of
transplantation of xenogeneic cells (as opposed to organs) which either natu-
rally provide some essential function to the recipient or which have been mod-
ified to do so, perhaps by techniques of genetic engineering. Not all cell types
are resistant to humoral rejection, however, and bone marrow cells, in particu-
lar, have been found to bind natural antibody (M. Sykes, personal communica-
tion). Thus the idea of using cells for xenogeneic transplantation requires fur-
ther study to determine which cell types express antigens for natural
antibodies and which are susceptible to rejection by induced antibodies direct-
ed at their MHC antigens. Perhaps xenogeneic donors can be modified not
only to carry essential functions, but also to lose the expression of antigens
which cause their rejection.
Cell-Mediated Xenograft Rejection
The existence of organs and tissues which are relatively resistant to humoral
rejection makes it possible to conceive of successful xenogeneic transplanta-
tion in humans despite the difficulty in overcoming humoral responses. Thus
there have been many studies of the cellular mechanisms of xenograft rejec-
tion both in vivo, using concordant species combinations or tissues which are
resistant to humoral destruction, and in vitro, using standard assays of T cell
function.
In Vivo Studies of Cell-Mediated Xenograft Rejection
Dozens of studies of cell-mediated rejection of xenografts have been per-

formed in vivo since 1963 [75-88]. Many investigators have contributed to this
work, including Brendel, Hammer, and their colleagues [89-93], and Perper,
Najarian, and their colleagues [12, 94, 95], to mention just two of the many
groups involved. Two basic observations have emerged from these studies.
Cell-Mediated Xenograft Rejection 29
First, xenografts are essentially always rejected faster than allografts when
similar types of tissues are transplanted under similar circumstances. Second,
there is almost no known type of immunosuppression which has been shown
to be more effective for xenogeneic transplantation than allogeneic transplan-
tation even when cell-mediated rejection mechanisms are involved. Thus ev-
ery investigator who has witnessed rejection of xenogeneic tissue has been im-
pressed that this is a peculiarly powerful event relative to allograft rejection.
Next to recognition of the great strength of humoral rejection of xenografts,
recognition of the speed of cellular rejection should be the second most impor-
tant feature of xenografting considered by all investigators. Nonetheless,
there are several interesting exceptions to these two basic observations which
should be considered.
In the absence of immunosuppression, a single study has been reported in
which a xenograft survived longer than an allograft. This was the observation
by Hammer et al. [90] that wolf kidneys transplanted into dogs can survive
longer than dog kidneys, an example which obviously involves a very closely
related species combination. In the presence of immunosuppression, howev-
er, Winn and colleagues were also able to demonstrate that transplants of both
heart and skin grafts from rats could survive longer on mice than similar mouse
tissues [96, 97]. This example was particularly interesting because the longer
survival of xenografts than allografts occurred only in certain mouse strains
and not in others, indicating that there is a distinction between high and low re-
sponder mouse strains for some mechanism of cell-mediated xenograft rejec-
tion which persists despite thymectomy plus anti thymocyte globulin (A TG)
treatment. The nature of this mechanism has still not been determined. While
this example again involved a closely related species combination, Pierson and
colleagues have recently shown that depletion of CD4 +T cells can prolong the
survival of widely disparate xenogeneic skin grafts better than the survival of
whole MHC-disparate allogeneic grafts [98]. This finding suggests that the
mechanisms of cell-mediated xenograft rejection are different from those
used for allografts and are weaker than allogeneic responses once the CD4+
popUlation has been eliminated. Finally, Thomas's group has reported im-
pressive survival of xenogeneic grafts in rodents using 15-deoxspergualin plus
splenectomy plus ATG treatment [99]. They have not shown that this combi-
nation is actually better for xenografts than allografts, but their studies have
suggested that 15-deoxyspergualin is more effective for xenogeneic transplan-
tation than other nonspecific pharmacologic immunosuppressive agents
which have been tested. Unfortunately we do not yet know enough about the
mechanism by which 15-deoxyspergualin is immunosuppressive to gain in-
sight into the mechanisms of xenograft rejection from this observation.
One of the problems in evaluating the nature of cellular xenograft rejection
is that some cases of early graft destruction in concordant species may not be
due to T cell effector functions at all. As discussed above, induced antibody re-
sponses may be stronger to xenografts than allografts and may be responsible
for accelerated graft rejection in some of the experiments which have been
thought to involve T cell mechanisms. The clinical experience of Baby Fae
30 The Scientific Study of Xenografting: 1964~ 1988
11 00] and experimental studies reported by Valdavia and by others support

this idea [51, 52, 55]. According to this interpretation, anti-CD4 treatmcnt
might be especially effective for xenografts because it blocks the helper T cell
function necessary for an induced antibody response, and 15-deoxyspergualin
might be a particularly useful pharmacologic agent because of its effect on hu-
moral immunity. Thus the cell-mediated response to xenografts may be truly
weaker than to allografts, but the weakness of these cellular mechanisms may
be obscured by powerful humoral responses even in concordant species com-
binations. This interpretation does not fit perfectly with the information avail-
able on the amount and timing of antibody administration which is required to
reject skin grafts, but it could explain some other cases of early xenograft re-
jection. In the case of skin grafts and other tissues which are resistant to hu-
moral rejection, it still appears that there is some cellular mechanism of
xenograft rejection which we do not yet understand which depends on CD4+
T cells and which is more powerful than for allograft rejection.
Alternative Mechanisms of Xenograft Rejection
Although many investigators have speculated over the years that unusual
mechanisms ofxenograft rejection must exist. the data indicating the nature of
these putative mechanisms is scant. Several investigators have suggested that
natural killer (NK) cells or antibody-dependent cell-mediated cytotoxicity
(ADCC) mechanisms might playa role in xenograft rejection, and others have
suggested that delayed type hypersensitivity reactions might playa larger role
in xenograft than allograft rejection [92,101-106]. If so, the experimental tech-
niques have not been identified which can suppress these mechanisms selec-
tively and render the rejection of xenografts similar to that of allografts.
In Vitro Studies of Cell-Mediated Immunity to Xenografts
A number of techniques have been developed in cellular immunology to mea-

sure T cell functions in vitro, including the mixed lymphocyte reactions
(MLR), cell-mediated lympholysis (CML), and interleukin-2 (IL-2) produc-
tion assays. Many investigators have used these assays to study T cell respon-
ses to xenoantigens [107-121]. While these experiments have the advantage
that they clearly avoid the confounding effects of antibodies, they have the dis-
advantage that they may not accurately reflect the mechanisms of xenograft
rejection in vivo.
Many of the experimental studies of cellular immune responses to
xenoantigens are summarized in Chap. 7. The results of these studies show
that primary xenogeneic cell-mediated helper responses are weak compared
to alloreactive responses, that secondary xenogeneic responses can be ob-
tained in vitro and are powerfuL and that these secondary xenogeneic respon-
ses require processing and presentation of xenogeneic antigens in association
Cell-Mediated Xenograft Rejection 31
with MHC antigens of the responding species. Results such as these have been
obtained by several investigators during the past 5 years [122-126]. In addi-
tion, determination of the precursor frequency of cytotoxic T cells for xeno-
geneic MHC antigens has been performed recently by several groups which
have found that this precursor frequency is lower for xeno- than for allo-MHC
antigens [127-133]. Thus there is now substantial evidence that in vitro xeno-
geneic cell-mediated responses are weaker than allogeneic responses and dif-
ferent in character.
Given the frequency with which this conclusion has been reported recently,
it is important in this chapter to indicate that the studies of xenogeneic cellular
immunity published over the past two decades have not always been so clear
cut. For example, over half of the 20 or so reports of xenogeneic MLRs pub-
lished between 1969 and 1984 suggested that xenogeneic proliferation was as
strong or stronger than the allo-MLR. Furthermore, Swain reported studies in
1983 which suggested that exactly the same T cell subsets were involved in
xenogeneic responses, making use of the same accessory molecules, as in allo-
geneic responses [112]. There is no clear explanation for the different results
which have been published during the late 1980s compared with those from a
decade earlier. In some cases different species combinations have been used
and in others different assay conditions were involved. Still, it is unsettling to
have an issue which seemed so uncertain just 5 years ago seem so clearly re-
solved today. One suspects that the characterization of in vitro cellular re-
sponses to xenoantigens is not as simple as the recent studies suggest, and in-
vestigators should beware that slightly different species combinations and
assay conditions may still produce conflicting results.
To the extent that defects in T cell responses to xenoantigens compared
with alloantigens do exist, a great deal of effort has been directed recently at
defining the causes of this weaker cellular immunity [122, 123, 128-141]. Most
of these studies have concluded that one or another of the accessory
molecules of T cell activation do not function well across species differences.
For example, several investigators have concluded that the interaction of
CD4 or CD8 with their respective ligands on MHC molecules may be weak
when the MHC antigens are from a different species [131-138]. Others have
found that lymphocyte function associated antigen-l (LFA-I) and its ligand
intercellular adhesion molecule (ICAM) may have weak binding across
species differences [139, 140]. Still others have suggested that some of the
lymphokines which are produced by stimulating cells of one species do not
function well with receptors for these lymphokines expressed on T cells of an-
other species [122, 141]. The one potential defect in cell-mediated xenoreac-
tivity which has not been found to playa role in these reduced cellular re-
sponses is the possibility that xeno-MHC antigens may be so different in
structure from the MHC antigens of the responding species that the T cell
repertoire (selected for reactivity with modified self MHC antigens) will not
include receptors capable of directly recognizing xeno-MHC antigens.
Indeed the information obtained from sequencing studies of MHC antigens
of different species suggests that allelic MHC antigens within a species are at
least as different from one another as each one is from particular MHC anti-
gens of a different species [142].
Overall, the studies seeking to identify the nature of defective xenoreactiv-
ity in vitro suggest that multiple defects probably exist, but that not every de-
fect applies in every species combination. Thus the current issue of importance
for clinical xenotransplantation is to identify the particular defects which are
most important in the response by human T cells to stimulation by cells from
different animal species. Some animal species are likely to elicit more defec-
tive human responses than others. Identifying which species these are and
which elements of T cell activation remain intact will likely identify the best
potential donors for human transplantation and the forms of immunosuppres-
sion which may be most useful in these combinations. Many of the chapters in
this book contribute to this undertaking. In addition, the in vitro studies of cel-
lular xenogeneic immunity have not yet revealed why in vivo rejection of
xenografts, even in the absence of humoral rejection, is so powerful. Thus an
important challenge in the study of xenogeneic transplantation is to use the
standard assays of T cell function or to apply new techniques to identify the
mechanisms of cell-mediated xenograft rejection.
Tolerance to Xenoantigens
The effort to accomplish specific nonresponsiveness to foreign antigens is cen-

tral to all research in transplantation. It may be even more important in the ap-
plication of xenogeneic transplantation since (a) the more powerful immune
responses to xenoantigens discussed above may require more complete abro-
gation of that response, and (b) because some of the important potential appli-
cations of xenografting (including pancreatic islet transplantation and cellular
transplants to achieve gene therapy) will only be really attractive if they can be
accomplished without the need for systemic immunosuppression. Efforts to
accomplish tolerance to xenoantigens have been reported over the years but
with less success than for alloantigens [143-166].
The most frequent effort to achieve tolerance to xenoantigens has in-
volved efforts to create xenogeneic bone marrow chimeras in the same way as
has been used to create allospecific tolerance in many animal models
[146-155]. These studies have suggested that much larger numbers of xeno-
geneic bone marrow cells are required to reconstitute an irradiated recipient
than allogeneic cells and that the chimerism achieved by this approach may
not be long-lasting. Furthermore, xenogeneic chimeras have not been suc-
cessfully created across wider species differences than the hamster-to-mouse
combination. Sachs' group has worked with a variant of the whole-body irra-
diation chimera approach, creating mixed xenogeneic chimeras in mice which
have been partly reconstituted by murine cells and partly by rat xenogeneic
cells [143-145]. They have found that addition of anti-NK antibodies in the
preparation of the recipient as well as much larger doses of xenogeneic than
Function in a Xenogeneic Environment 33
allogeneic cells can achieve partial xenochimerism in this closely related

species combination [40].
Another approach to xenogeneic tolerance which has occasionally been at-
tempted is the application of neonatal tolerance. Several studies, even before
Reemtsma's first clinical effort, suggested that neonatal tolerance to xenoanti-
gens can be induced in closely related species [157-166]. However, there has
been a notable lack of such reports since the early 1960s. With the ability today
to diagnose critical birth defects in utero and to accomplish intravenous injec-
tion into the unborn fetus, it is conceptually possible that neonatal tolerance
could be accomplished in humans for a specific animal donor which would
then be used for transplantation after birth. While this idea has occurred to
many investigators, the results of studies of this possibility have not yet been
reported.
The difficulty in achieving tolerance to xenoantigens in models which have
been so successful in allogeneic combinations, plus the lack of any report de-
scribing down-regulation of xenoresponses by pretransplant transfusions or
by other techniques which are often effective in allogeneic combinations, sug-
gests that there is something fundamentally different about the xenogeneic ef-
fort. The finding that severe combined immunodeficiency (SCID) mice can be
reconstituted with human lymphohematopoietic cells suggests that xenogene-
ic chimerism can be achieved if there is even more complete abrogation of the
immune system than is required for allogeneic reconstitution [167, 168]. On
the other hand, the difficulty in achieving long-lasting chimerism in xenogene-
ic combinations suggests that environmental factors may not always support
the survival of xenogeneic cells as well as they do cells from the same species.
Function in a Xenogeneic Environment
Given the difficulty in overcoming the immunological response to xenogeneic

tissues, it has been almost impossible to determine whether tissues or organs
from other animals could function successfully in the environment of another
species if they did survive. This is obviously a critical issue in xenogeneic trans-
plantation, and it is by no means clear that satisfactory function of xenogeneic
organs can always be expected.
There is little firm evidence with which to address this question.
Reemtsma's experience described in Chap. 2 indicated that primate kidneys
can support human life without disturbances in electrolytes or other metabolic
parameters. The clinical experience with liver transplantation was not suffi-
cient to demonstrate satisfactory function of this organ, but a number of pa-
tients with end-stage liver failure were treated during the 1960s with temporary
perfusion of animal livers ex vivo using the patients' blood [169-175]. The re-
sults of this effort suggested that deep hepatic coma could occasionally be tem-
porarily reversed by circulation through an animal liver and that pig livers
could produce bile and manufacture pig serum albumin which appeared in the
34 The Scientific Study of Xenografling: 1964-1988
patients' blood. Furthermore, human beings have been using insulin produced
by animal cells for several decades and pancreatic islets have maintained satis-
factory glucose control for recipients of different species in several animal
models. These examples make it clear that xcnogeneic organs can function
well in some cases.
On the other hand, the existence of defects in cell-mediated xenoreactivity,
as described earlier in this chapter, which apparently occur because of defec-
tive interactions between cellular receptors and their designated ligands ex-
pressed by other species, makes it very likely that similar defects will exist
which affect the physiological functions of transplanted xenogeneic organs.
Given the many enzymatic processes and hormonal interactions which are in-
volved in organ function, it is unlikely that some of these will not also prove de-
fective in xenogeneic combinations. Indeed, one of the fascinating by-prod-
ucts of immunologically successful xenogeneic transplantation will probably
be the insights this provides into the physiological processes of organ function
which we do not now even imagine. Several of the chapters in this book will
consider these issues.
Among the many possible consequences of poor function in a xenogeneic
environment is that stcm cells from a different specics may not survive indefi-
nitely in a new recipient. This would account for the difficulty in achieving last-
ing xenochimerism and might prevent thc achievement of long-term tolerance
in xenogeneic combinations. Such a nonimmunological survival disadvantage
for xenogeneic tissues might limit the effectiveness of cellular xenotransplants
such as pancreatic islets or modified cells used as vectors for "gene therapy".
Comment
This chapter has surveyed some of the important scientific research in xeno-
geneic transplantation which has been performed since Reemtsma's clinical
undertaking. This review emphasizes especially the many unanswered ques-
tions and critical issues which rcmain to be addressed in the field of xenogene-
ic transplantation. What is the character of natural antibody and what can be
done to remove it from recipients before transplantation? What are the anti-
gens of natural antibodies and can they be used for immunoabsorption or
themselves rcmoved from a donor animal? What can be done to prevent the
strong induced antibody responses to xenogeneic grafts? Why is the cell-me-
diated rejection of xenografts so powerful when the in vitro assays of cellular
immunity suggest that it should be weaker? Are there alternative mecha-
nisms of xenograft rejection which we do not yet understand? What can be
done to achieve tolerance to xenogeneic cells or is this impossible in a foreign
environment?
These questions and others will be addressed in the remaining chapters of
this book based on the many new studies ofxenogencic transplantation which
have been performed in the past few years.
References 35
References
1. Reemtsma, K Renal heterotransplantation from nonhuman primates to man. Ann.

N. Y. Acad. Sci.; 162,412;1969.
2. Reemtsma, K; McCracken, B. H.; Schlegel, 1. Y.; Pearl, M. Heterotransplantation of
the kidney: two clinical experiences. Science; 1964; 143: 700.
3. Reemtsma, K.; McCracken, B. H.; Schlegel, 1. U.; Pearl, M. A.; Pearce, e. W.; Dewitt,
e. W.; Smith, P. E.; Hewitt, R L.; Flinner, R L.; Creech, O. lr. Renal heterotransplan-
tation in man. Ann Surg.; 1964; 160: 384.
4. Reemtsma, K; McCracken, B. H.; Schlegel, 1. V.; Pearl, M. A.; Dewitt, e. W.; Creech,
O. lr. Reversal of early graft rejection after renal heterotransplantation in man.
l.A.M.A.; 1964; 187: 691.
5. Hitchcock, e. R; Kiser, 1. e.; Telander, R L.; Seljeskob, E. L. Baboon renal grafts.
l.A.M.A.; 1964; 189: 934.
6. Starzl, T. E. Experiences in Renal Transplantation. Philadelphia: W. B. Saunders; 1964.
7. Starz!, T. E.; Marchioro. T. L.; Peters, G. N.; Kirkpatrick, e. H.; Wilson, W. E. e.;
Porter. K. E.; Rifkind, D.; Ogden, D. A.; Hitchcock, e. R: Waddell, W. R. Renal het-
erotransplantation from baboon to man: experience with six cases. Transplantation;
1964; 2: 752.
8. Hardy, 1. D.; Chavez, e. M.; Kurrus, F. D.; Neely, W. A.; Eraslan, S.; Turner, M. D.;
Fabian, L. W.; Labecki, T. D. Heart transplantation in man: Developmental studies
and report of a casco l.A.M.A.; 1964; 188: 1132.
9. Hardy, 1. D.; Chavez, e. M. Thc first heart transplant in man. Am. l. Cardiol.; 1968; 22:
772.
10. Kissmeyer-Niclsen, F.; Olscn, S.; Petersen, V. P.; Fjeldborg, O. Hyperacute rejection of
kidney allografts associated with pre-existing humoral antibodies against donor cells.
Lancet; 1966; 2: 662.
11. Terasaki, P. 1.; Marchioro, T. H.; Starzl, T. E. Sero-typing of human lymphocyte anti-
gens: preliminary trials on long-term kidney homograft survivors. In: Amos, D. B.,
(ed.) Workshop on Histocompatibility testing. Washington, D. e.: National Research
Council; 1964: 83.
12. Perper, R 1.; Najarian, 1. S. Experimental renal heterotransplantation: II. Closely relat-
ed species. Transplantation; 1966; 4: 700.
13. Perper, R 1.; Najarian, 1. S. Experimental renal heterotransplantation: I. In widely
divergent species. Transplantation; 1966; 4: 377.
14. Caine, R Y. Organ transplantation between widely disparate species. Transplant
Proc.; 1970; 2: 550.
15. Baldamus, e. A.; McKenzie, I. F. e.; Winn, H. 1.; Russell, P. S. Acute destruction by hu-
moral antibody of rat skin grafted to mice.l. Immunol.; 1973; 110: 1532.
16. Winn, H. 1.; Baldamus, e. A.; 100ste, S. V.; Russell, P. S. Acute destruction by humoral
antibody of rat skin grafted to mice: The role of complement and polymorphonuclear
leukocytes. l. Exp. Med.; 1973; 137: 893.
17. Rosenberg, 1. e.; Broersma, R 1.; Bullemer, G.; Mammen, E. F.; Lenaghan, R;
Rosenberg, B. F. Relationship of platelets, blood coagulation, and fibrinolysis to hyper-
acute rejection of renal xenografts. Transplantation; 1969; 8: 152.
18. Rosenberg, 1. e.; Hawkins, E.; Rector, F. Mechanisms of immunological injury during
antibody-mediated hyperacute rejection of renal hetcrografts. Transplantation; 1971;
11: 151.
19. Rosenberg, 1. e.; Hawkins, E.; Mammen, E.; Palutke, M.; Riddle, 1.; Rosenberg, B. F.
Hyperacute rejection of heterografts: Studies of pig to dog renal transplants. In Von
Kaulla, K.; Thomas, e. e.; (ed.) Coagulation Problems in Transplanted Organs; 1971.
20. Shons, A. R; Bier, M.; letzer, T.; Najarian, 1. S. Techniques of in vivo plasma modifica-
tion for the treatment of hyperacute rejection. Surgery; 1973; 73: 28.
21. Shons, A. R; letzer, 1.; Moberg, A. W.; Najarian, 1. S. Prolongation of heterograft sur-
vival by electrophorectic extraction of preformed antibody. Surg. Forum 1970; 21: 263.
36 The Scientific Study of Xenografting: 1964~19RR
22. Shons. A. R.; Najarian, J. S. Xenograft rejection mechanisms in man. Trans. Alii. Soc.
Arlif /nterl1. Organs: 1974: 20: 502.
23. Sho~s. A. R.: Najarian, J. S. Modification of xenograft rejection by aspirin, dextran, and
cinanserin: The importance ofplatelcts in hyperacute rejection. Trallsplant Proc.: 1974:
0: 435.
24. Moberg. A. W.: Shons, A. R.: Gewurz, H.: Mozes, M.: Najarian, J. S. Prolongation ofre-
nal xenografts by the simultaneous sequestration of preformed antibody, inhibition of
complement. coagulation, and antibody synthesis. Transplant Proc.: 1971: 3: 53R.
25. Hawkins, E.: Mammen, E.: Rosenberg, J. C. Effects of heparin and steroids on hypera-
cutely rejected renal xenografts.l. Sllrg. Res.: 1971: 11: 492.
20. Bier, M.; Beavers, C. D.: Merriman, W. G.: Merke!. F. K.: Eiseman. B.: Starz!' T. E.
Selective plasmapharesis in dogs for delay of heterograft response. TrailS. Alii. Soc.
Arli! Intern. Organs: 1970: 10: 325.
27. Kux. M.; Boehmig, H. J.: Amemiya. H.: Torisu. M.: Yokoyama. T.: Launois, B.:
Popovtzer, M.: Wilson, C. B.: Dixon. F. J.: Starz!. T. E. Modification of hyperacute ca-
nine renal homograft and pig-to-dog heterograft rejection by the intra-arterial infusion
of citrate. Sllrxery: 1971; 70: 103.
2R. Makowka, L.; Miller, c.: Chapehap, P.; Podesta. L.; Pan, c.; Pressley. D.: Mazzaferro.
V.; Esquivel, C. 0.; Todo, S.; Banner. B.; Jaffe, R.; Saunders. R.: Starz!' T. E.
Prolongation of pig-to-dog renal xenograft survival by modification of the inllammato-
ry mediator response. Ann. SlUg.: 19R7; 200: 482.
29. Merkel. F. K.: Bier, M.; Beavers. C. D.; Merriman, W. G.: Wilson. c.: Starz!. T. E.
Modification of xenograft response by selective plasmapheresis. Transplant Froc.:
1971:3:534.
3~. Clark, D. S.: Gewurz, H.; Good. R. A.; Varco, R. L. Complement fixation during homo-
graft rejection. Sllrg. Forum 1964: 15: 144.
31. Clark, D. S.: Foker, J. E.; Pickering, R.; Good, R. A.; Vareo, R. L. Evidence for two
platelet populations in xenograft rejection. Slag. Forum; 1966; 17: 264.
32. Gcrwurz, H.: Clark. D. S.: Finstad. J.: Kelley, W. D.: Varco. R. L.: Good, R. A.:
Gabrielson, A. E. Role of C' in graft rejection in experimental animals and man. Ann.
N. y. Acad. Sci.: 1966: 129: 073.
33. Kemp, E.; Kemp, G.; Starklint, H.; Larsen, S. Immunosuppression with cobra venom
factor. anti-platelet aggregator. and cyc1osporin A in renal xcnotransplantation.
Transplant Proc.: 19R2: 14: 110.
34. Kemp. E.: Kemp. G.: Svendsen, P.; Nielsen, E.: Buhl, M. R. Prolongation of xenograft
survival by infusion of heterologous antibodies against recipient serum. Acta Pat/IO/.
Microbio/. Scand.: 1976; 84C: 342.
35. Kemp, E.: Kemp. G.: Abildgaard-J acobsen.l.: Lundborg, C. Prolongation of survival of
renal xenografts by infusion of donor blood. Acta Pat/wI. Microbiol. Scand. (A); 1977:
R5(A): 267.
36. Kemp. E.; Stcinbruche!' D.: Starklint. H.: Larsen. S.: Henriksen, 1.; Dieperink, H. Renal
xenograft rejection: Prolonging effect of captopril. ACE-inhibitors. prostacyclin. and
cobra venom factor. Transplant Pmc.; 1987; 19: 4471.
37. Kemp, E.; White, D.; Dieperink, H.; Larsen, S.: Starklint, H.: Steinbruchel. D. Delayed
rejection of rabbit kidneys transplanted into baby pigs. Transplant Froc.: 19R7:
19: 1143.
3R. Reding, R.; Davies.H. ff. S.: White, D. J. G.: Wright, L. J.: Marbaix. E.: Alexandre, G. P.
J.: Squifflet, J. P.; Caine, R. Y. Effect of plasma exchange on guinea pig-to-rat heart
xenografts. Transplant Proc.; 1989; 21: 534.
39. Fischel. R. J.; Bolman, R. M. Ill; Platt, J. L.; Najarian, J. S.: Bach. F. H.: Matas. A. J.
Removal of IgM anti-endothelial antibodies results in prolonged cardiac xenograft sur-
vival. Transplant Froc.; 1990; 22: 1077.
40. Cooper. D. K.c.; Human, P. A.; Rose, A. G.; Rees.J.: Keraan, M.: DuToit. E.; Orio!' R.
Can cardiac allografts and xenografts be transplanted across the ABO blood group bar-
rier') Transplalll Proc: 19R9; 21: 549.
41. Cooper. D. K. C. Xenograft transplantation. Transplantation; 19R9; 47: 408.
References 37
42. Cooper, D. K. c.; Human, P. A.; Lexer. G .. Rose, A.G., Rees, J., Keraan, M., Du Toit.
E.; Effects of cyclosporine and antibody adsorption on pig cardiac xenograft survival in
the baboon. 1. Heart Transplant.; 1988; 7: 238.
43. Landsteiner, K. Specificity of Serological Reactions. New York: Dover Pub!.; 1962.
44. Platt, J. L.; Lindman, B. J.; Chen, H.; Spitalnik, S. L.; Bach, F. H. Endothelial cell anti-
gens recognized by xenoreactive human natural antibodies. Transplantation; 1990; 50:
817.
45. Platt, J. L.; Turman, M. A.; Noreen, H. J.; Fischel, R J.; Bolman, R M.; Bach, F. H. An
Elisa assay for xenoreactive natural antibodies. Transplantation; 1990; 49: 1000.
46. Edwards, N.; Ott, G.; Berger, c.; He, X.; Tcppler, L; Copey, L.; Smith, c.; Reemtsma,
K.; Rose, E. Incidence of preformed antibodies against potential xenodonors in human
sera. Transplantation: 1990; 49: 1022. .
47. Sachs. D. H.: Winn, H. J.: Russell, P. S. The immunologic response to xenografts: recog-
nition of mouse H-2 histocompatibility antigens by the rat. 1. Immunol.; 1971;
107: 481.
48. Staines. N. A. Relative immunogenicity of H-2 and other membrane components in
xenoimmunization. Transplantation; 1974; 17: 470.
49. Bouwman, E.: de Bruin, R W. F.; Marquet, R L.; Jeekel, J. Prolongation of graft sur-
vival in sensitized xenotransplantation. Transplant Proc.; 1989; 21: 551.
50. Bouwman. E.; de Bruin, R W. F.: Marquet, R L.; JeekeL J. Prolongation of graft sur-
vival in hamster to rat xenografting. Transplant Proc.; 1989: 21: 540-1.
51. Nakajima, K.; Sakamoto, K.; Asano, T.; Isono, K. Effects of 15-deoxyspergualin and
FK506 on the histology and survival of hamster-to-rat cardiac xenotransplantation.
Transplant Proc.; 1989: 21: 546.
52. Monden, M.; Valdivia, L. A.; Gotoh, M.; Kubota, N.; Hasuike, Y.: Nakano, Y.;
Okamura, J.; Mori, T. A crucial effect of splenectomy on prolonging cardiac xenograft
survival in combination with cyclosporine. Surgery 1989; 105: 535.
53. Valdivia, L. A.; Monden, M.; Gotoh, M.; Kubota, N.; Hasuike, Y.; Nakano, Y.;
Okamura, J.; Mori, T. Prolonged cardiac xenograft survival by 15-deoxyspergualin
combined with splenectomy. Transplant Proc.; 1989: 21: 532.
54. Valdivia, L. A.; Monden, M.; Gotoh, M.; Hasuike, Y.; Kubota, N.; Ichikawa, T.:
Okamura, J.; Mori, T. Prolonged survival of hamster-to-rat liver xenografts using
splenectomy and cyclosporine administration. Transplantation; 1987; 44: 759.
55. Valdivia, L.A.: Monden, M.; Gotoh, M.; Nakano, Y.: Tono, T.: Mori, T. Evidence that
15-deoxyspergualin prevents sensitization and first-set cardiac xenograft rejection in
rats by suppression of antibody formation. Transplantation; 1990; 50: 132.
56. Gordon, RD.; Fung, J. J.; Markus, B.: Fox, I.; Iwatsuki, S.; Esquivel, C. 0.; Tzakis, A.;
Todo, S.; Starzl, T. E. The antibody cross match in liver transplantation. Surgery; 1986:
100: 705.
57. Gordon, RD.; Iwatsuki, S.; Esquivel, C. 0.; Tzakis, A.; Todo, S.; Starzl, T. E. Liver
transplantation across ABO blood groups. Surgery: 1986; 100: 342.
58. Settaf, A.; Meriggi, F.; van de Stadt,J.; Gane, P.; Crougneau, S.; Reynes, M.; Rouger, P.;
Houssin, D. Delayed rejection of liver xenografts compared to heart xenografts in rats.
Transplant Proc.; 1987; 19: 1155.
59. Starzl, T. E.; Ishikawa, M.; Putnam, C. W.; Porter, K. A.; Picache, R; Husberg, B. S.;
Balgrimson, C. G.; Schroter, G. Progress in and deterrents to orthotopic liver trans-
plantation, with special reference to survival, resistance to hyperacute rejection, and
biliary duct reconstruction. Transplant Proc.: 1974; 6: 129.
60. Knechtle, S. J.; Kolbeck, P. c.; Tsuchimoto, S.; Coundouriotis, A.: Sanfilippo, F.:
Bollinger, R R Hepatic transplantation into sensitized recipients: demonstration of
hyperacute rejection. Transplantation 1987; 43: 8.
61. Merion, R M.: Colletti. L. M. Hyperacute rejection in porcine liver transplantation. L
Clinical characteristics, histopathology, and disappearance of donor-specific lympho-
cytotoxic antibody from serum. Transplantation; 1990; 49: 861-868.
62. Baldamus, C. A.; Winn, H. J.; Russell, P. S. Acute rejection of skin xenografts in the
mouse after passive transfer of humoral antibody. Fed. Proc.; 1970; 29: 785.
63. Jooste, S. V.; Colvin, R B.; Winn, H. J. The vascular bed as the primary target in the de-
struction of skin grafts by antiserum: II. Loss of sensitivity to antiserum in long-term
xenografts of skin. 1. Exp. Med.; 1981; 154: 1332.
64. Delmonico, F. L.; Chase, C. M.; Russell, P. S. Transplantation of rat islets of
Langerhans into diabetic mice. Transplant Proc.; 1977; 9: 367.
65. Naji, A; Reckard, C. R; Ziegler, M. M.; Barker, C. F. Vulnerability of pancreatic islets
to immune cells and serum. Slag Forum; 1975; 26: 459.
66. Frangipane, L. G.; Poole, T. W.; Barker, C. F.; Silvers, W. K. Vulnerability of allogeneic
and xenogeneic islets to alloantisera. Transplant Proc.; 1977; 9: 371.
67. Perl off, L. J.; Naji, A; Barker, A F. Islet sensitivity to humoral antibody. SlIrg. FOrLlm;
1981; 32: 390.
68. Ricordi, c.; Kneteman, N. M.; Scharp, D. W.; Lacy, P. E. Transplantation of cryo-
preserved human pancreatic islets into diabetic nude mice. World Sllrg; 1988;
12: 861.
69. Ricordi, c.; Lacy, P. E.; Sterbenz, K.; Davie, J. M. Low-temperature culture of human
islets or in vivo treatment with L3T4 antibody produces a marked prolongation of islet
human-to-mouse xenograft survival. Proc. Natl. Acad. Sci. USA; 1987; 84: 8080.
70. Ricordi, c.; Lacy, P. E. Renal subcapsular xenotransplantation of purified porcine
islets. Transplantation; 1987; 44: 721.
71. Ricordi, c.; Scharp, D. W.; Lacy, P. E. Reversal of diabetes in nude mice after trans-
plantation of fresh and 7-day-cultured (24 degrees C) human pancreatic islets.
Transplantation; 1988; 45: 994.
72. Lacy, P. E.; Ricordi, c.; Finke, E. H. Effect of transplantation site and anti alpha L3T4
treatment on survival of rat, hamster, and rabbit islet xenografts in mice.
73. Sullivan, F. P.; Ricordi, c.; Hauptfeld, V.; Lacy, P. E. Effect oflow-temperature culture
and site of transplantation on hamster islet xenograft survival (hamster to mouse).
74. Chabot, J.; Weber, c.; Hardy, M. A; Rivera, S.; Bailey-Braxton, D.; Strausberg, L.;
Wood, M.; Chow, J.; Pi-sunyer, F. X.; Reemtsma, K. Synergy of ALS and UV-B in pro-
longation of primate-to-mouse islet xenograft survival. Transplant Proc.; 1987;
19: 1160.
75. Auchinc\oss, H. Jr. Xenogeneic transplantation. A review. Transplantation; 1988; 46: 1.
76. Manning, D. D.; Reed, N. D.; Shaffer, C. F. Maintenance of skin xenografts of widely di-
vergent phylogenetic origin on congenitally athymic (nude) mice. 1. Exp. Med.; 1973;
138:488.
77. Hullett, D. A.; Falany, J. L.; Love, R B.; Burlingham, W. J.; Pan, M.; Sollinger, H. W.
Human fetal pancreas - A potential source for transplantation. Transplantation; 1987;
43: 18.
78. Monaco, A. P.; Wood, M. L.; Gray, J. G.; Russell, P. S. Studies on heterologous
anti-lymphocyte serum in mice: Effect on the immune response. 1. Immunol.; 1966;
96: 229.
79. Monaco, A P.; Wood, M. L.; Russell, P. S. Studies on heterologous anti-lymphocyte
serum in mice: III. Immunologic tolerance and chimerism produced across the H-210-
cus with adult thymectomy and anti-lymphocyte serum. Ann N. Y. Acad. Sci.; 1966; 129:
190.
80. Lance, E. M.; Medawar, P. B. Survival of skin heterografts under treatment with anti-
lymphocyte serum. Lancet; 1968; 1: 1174.
81. Dieperink, H.; Steinbruchel, D.; Stark lint, H.; Larsen, S.; Kemp, E. Improvement in
hare-to-rabbit kidney transplant survival. Transplant Proc.; 1987; 19: 1140.
82. Kemp, E.; Starklint, H.; Larsen, S.; Dieperink, H. Cyc\osporine in concordant renal
hare-to-rabbit xenotransplantation: prolongation and modification of rejection, and
adverse effects. Transplant Proc.; 1985; 17: 1351.
83. Valdivia, L. A; Monden, M.; Gotoh, M.; Hasuike, Y.; Kubota, N.; Endoh, W.;
Okamura, J.; Mori, J. Hepatic xenografts from hamster to rat. Transplant Proc.; 1987;
19: 1158.
References 39
84. Monden, M.; Valdivia, L. A.; Gotoh, M.; Hasuike, Y.; Kubota, N.; Kanai, T.; Okamura,
J.; Mori, T. Hamster to rat orthotopic liver transplantation. Transplantation; 1987; 43:
745.
85. Michler, R E.; McManus, R P.; Smith, C. R; Sadeghi, A. N.; Marboe, C. c.; Reemtsma,
K.; Rose, E. A. Prolongation of primate cardiac xenograft survival with cyclosporine.
86. Caine, R Y.; White, H. J. 0.; Herbertson, B. M.; Millard, P. R; Davis, D. R; Salaman, J.
R; Samuel, J. R Pig to baboon liver xenografts. Lancet; 1968; 1: 1176.
87. Sollinger, H. W.; Burkholder, P. M.; Rasmus, W. R; Bach, F. H. Prolonged survival of
xenografts after organ culture. Surgery; 1977; 81: 74.
88. Steinmuller, D. Cross-species transplantation in embryonic and neonatal animals.
Transplant Proc.; 1970; 2: 438.
89. Brendel, W.; Duswald, K. H.; von Scheel, J.; Chaussy, c.; Sollinger, H. W.; Hammer, C.
Prolonged survival time of canine xenografts using a new schedule of horse anti-dog
lymphocyte globulin (ALG) therapy. Transplant Proc.; 1978; 9: 379.
90. Hammer, c.; Chaussey, c.; Welter, H.; Weinbacher, J.; Hobel, G.; Brendel, W.
Exceptionally long survival time in xenogeneic organ transplantation. Transplant
Proc.; 1981; 13: 881.
91. Hammer, c.; Chaussy, c.; von Scheel, J.; Pongratz, H.; Roscher, E.; Pielsticker, K.
Survival times of skin and kidney grafts within different canine species in relation to
their genetic markers. Transplant Proc.; 1975; 7: 439.
92. Chaussy, c.; Hammer, c.; Pongratz, H.; von Scheel, J.; Pfeifer, K. J.; Land, W.;
Sollinger, H. W.; Pielsticker, K. Prolongation of graft survival in rats and dogs by a spe-
cific antimacrophage serum (AMS). Transplant Proc.; 1975; 7: 779.
93. Chaussy, c.; Hammer, c.; von Scheel, J.; Pielsticker, K.; Sollinger, H. W.; Pfeifer, K. J.;
Pongratz, H. Xenogeneic skin and kidney transplants in a closely related canine system,
fox-dog. Transplantation; 1975; 20: 150.
94. Perper, R J. Renal heterotransplant rejection: a model for separation of humoral and
cellular mechanisms. Transplantation; 1971; 12: 519.
95. Perper, R J.; Monovich, R E.; Bowersox, B. E. Long-term skin allograft survival elicit-
ed by a finite treatment with anti-lymphocyte serum combined with cytarabine. 1.
Immunol.; 1970; 104: 1063.
96. Burdick, J. F.; Jooste, S. V.; Winn, H. J. Variations in the responses of mouse strains to
rat xenografts. 1. Immunol.; 1979; 123: 954.
97. Jooste, S. V.; Winn, H. J. Strain variation in the responses of mice to xenografts of skin.
Transplant Proc; 1977; 9: 375.
98. Pierson, R N, III; Winn, H. J.; Russell, P. S.; Auchincloss, H. Jr. Xenogeneic skin graft
rejection is especially dependent on CD4+ T cells. 1. Exp. Med.; 1989; 170: 991.
99. Thomas, F. T.; DeMasi, R; Daniel, H.; et al. Comparative immunosuppression for
xenografting. In: Hardy, M. A., ( ed). Xenograft 25. The Netherlands: Elsevier,
Amsterdam, New York, Oxford, 1989, p. 55.
100. Bailey, L. L.; Nehlsen-Cannarella, S. L.; Concepcion, W.; Jolley, W. B. Baboon-to-hu-
man cardiac xenotransplantation in a neonate. l.A.M.A.; 1985; 254: 3321.
101. Sakakibara, N.; Click, R E.; Condie, R M.; Jamieson, S. W. Rejection/acceptance of
xenografts. Transplant Proc.; 1989; 21: 524.
102. Dennert, G. Effector mechanisms of cell-mediated immunity to xenogeneic cell anti-
gens. 1. Immunol.; 1974; 113: 201.
103. Moller, G.; Svehag, S. E. Specificity oflymphocyte-mediated cytotoxicity induced by in
vitro antibody coated target cells. Cell Immunol.; 1972; 4: 1.
104. Harding, B.; MacLennan, 1. C. M. The distribution of antibody and antibody-producing
cells after immunization with xenogeneic cells. Immunology, 1972; 23: 35.
105. Escobar, M. R; Sigel, M. M. Effects oflymphoid cells and serum on target cells in xeno-
geneic cell-culture systems. Proc. Soc. Exp. Bioi. Med.; 1970; 134: 744.
106. Grant, C. K.; Avis, P. J.; Hale, J. G.; Alexander, P. Evidence for the role of antibody in
the cytotoxic action oflymph cells on xenogeneic target cells. Transplantation; 1971; 11:
356.
40 The Scientific Study of Xenografting: 1964~ 191\1\
107. Shons. A. R: Kromrey. c.: Najarian. 1. S. Xenogeneic mixed lymphocyte response. Cell
1I7II7IIInol.: 1973: 6: 420.
I Oil. Woolnough. 1. A.: Misko. I. S.: Lafferty. K . .I. Cytotoxic and proliferative response to al-
logeneic and xenogeneic antigens in vitro. Amt. J. Exp. Bioi. ivIed. Sci.: 1979: 57: 467.
109. Wilson. D. B.: Fox. D. H. Quantitative studies on the mixed lymphocyte interaction in
rats: VI. Reactivity of lymphocytes from conventional and germfree rats to allogeneic
and xenogeneic cell surface antigcns.i. Exp. Med.: 1971: 134: 857.
110. Wilson. D. B.: Nowell. P. C. Quantitative studies on the mixed lymphocyte interaction
in rats: IV. Immunological potentiality of the responding cells. J. Exp. Med.: 1970: 131:
39.
Ill. Yoshizawa. K.: Yano. A. Mouse T lymphocytes proliferative responses for human
MHC products in mouse anti-human xenogeneic MLR. 1. 1II/I7IIII/of.: 191\4: 132: 21\20.
112. Swain. S. L.: Dutton. R. W.: Schwab. R.: Yamamoto. 1. Xenogeneic human anti-mouse
T cell responses arc due to the activity of the same functional T cellsubsets responsihle
for allospecific and major histocompatibility complex-restrictcd responses. 1. Exp.
Mer!.: 191\3: 157: 720.
113. Peck. A. B.: Altcr. B. 1.: Lindahl. K. F. Specificity in Tcell mediated lympholysis: identi-
cal genetic control of the prolifcrative and effector phases of allogeneic and xenogeneic
reactions. Transplant Rev.: 1976: 29: 189.
114. LindahL K. F.: Bach. F. H. Genetic and cellular aspccts of xenogcneic mixed lympho-
cyte culture reaction. 1. Exp. Mell.: 1976: 144: 305.
115. Hirshberg. H.: Rankin. B.: Thorsby. E. Specificity of human lymphocytes proliferating
in rcsponsc to allogencic and xenogcneic cells in vitro. Transplantation 1974: 17: 593.
116. Bach. F. H.: Engstrom. M. A.: Bach. M. L.: Sell. K. W. Histocompatibility matching: VII
Mixed leukocyte cultures between chimpanzee and man. Transplant Proc.: 1972: 4: 97.
117. Nielsen. H. E. Reactivity of lymphocytes from germfree rats in mixed leukocyte culture
and in graft-versus-host reaction. f. Exp. Med.: 1972; 136: 417.
11K Widmer. M. B.: Bach. F. H. Allogeneic and xenogeneic response in mixed leukocyte
cultures..!. Exp. Merl.: 1972: 135: 1204.
119. Widmer. M. B.: Son del. P. M.: Bach. F. H. Responsiveness of germfree mice in mixed
leukocyte culture: Re-examining the nature of the responding unit..I. Exp. Med.: 1972:
136: 398.
120. Harris. 1. E.: Hersh. E. M.: Huggins. S. Human immune response to a xenogeneic nucle-
ated cell. TrallSplal1lation: 1970: 9: 440.
121. Knight. S. c.: Thorbecke. G. 1. Ontogcny of cellular immunity: Development in rat thy-
mocytes of mixed lymphocyte reactivity to allogeneic and xenogeneic cells. Cell
1I7lmunol.: 1971; 2: 91.
122. Alter. B.: Bach. F.H. Cellular basis of the proliferative response of human T cells to
mouse xenoantigens . .I. Exp. Med.: 1990: 171: 333.
123. Lucas. P . .I.: Shearer. G. M.: Neudorf. S.: Gress. R E. The human anti-murine xeno-
geneic cytotoxic response: I. Dependence on responder antigen presenting cells. 1.
lmmunol.: 1990: 144: 4541\
124. Auchincloss. H. lr. Xenografting: a review. Transplant Rev.; 1990: 4: 1.
125. Auchincloss. H. lr.; Pierson. R N. III.: RusselL P. S.: Winn. H . .I. The cell-mediated re-
sponse to xenogeneic antigens is similar to that to minor allogeneic histocompatibility
antigens. Xenograft 25. Hardy. M.A .. (cd.) Elsevier. Amsterdam. New York. Oxford.
1989: 207.
126. Moses. R D.: Pierson. R N. III: Winn. H. 1.: Auchincloss. H. lr. Xenogeneic prolifera-
tion and lymphokine production are dependent on CD4+ helper T cells and self anti-
gen-presenting cells in the mouse.f. Exp. Mal.; 1990; 172: 567.
127. Fischer-LindahL K.; Wilson. D. B. Histocompatibility antigen-activated cytotoxic T
lymphocytes. II. Estimates of the frequency and specificity of precursors . .I. Exp. Med.:
1977; 145: 508.
128. Hammerling. G. 1.; Hunt. T.: Dill. 0.: Moreno. 1. Cytotoxic T lymphocyte recognition
of HLA-A2 antigens in normal and HLA-Cw3-transgenic mice. Ellr. f. lmmllflof.:
1989: 19: 599.
References 41
129. Barra, c.; Perarnau, B.; Gerlingcr, P.; Lemeur, M; Gillet, A; Gibier, P.; LcMonnier,
F. A Analysis of the HLA-Cw3-specific cytotoxic T lymphocyte response of
HLA-B7xhuman Beta-2 Microglobulin double transgenic mice. 1. Immunol.; 1989;
143:3117.
130. Le, A X.; Bernhard, E. 1.; Holterman, M. 1.; Strub, S.; Parham, P.; Lacy, E.; Engelhard,
V. H. Cytotoxic T cell responses in HLA-A2.1 transgenic mice. Recognition ofHLA al-
loantigens and utilization ofHLA-A2.1 as a restriction element. I. Immunol.; 1989; 142:
1366.
131. Kievits. F; Wijffels. 1.; Lokhorst, W.; Ivanyi. P. Rccognition of xeno-(HLA SLA) major
histocompatibility complex antigens by mousc cytotoxic T cells is not H-2 restricted; a
study with transgenic mice. Proc. Natl. Acad. Sci. USA; 1989; 86: 617.
132. Holterman, M. 1.; Engelhard. V. H. HLA antigens expressed on murine cells are prefer-
cntially recognizcd by murine cytotoxic T cells in the context of the H-2 major histo-
compatibility complex. Proc. Natl. A cad. Sci. USA; 1986; 83: 9699.
133. Engelhard. V. H.: Le. A T.; Holterman. M. J. Species-specific structural differences in
the alpha 1 and alpha2 domains determine the frequency of murine cytotoxic T cell pre-
cursors stimulated by human and murine class I molecules. 1. Immunol.; 1988; 141:
1835.
134. Samberg, N. L.; Scarlett, E. c.; Stauss, H. 1. The alpha3 domain of major histocompati-
bility complex class I molecules plays a critical role in cytotoxic T lymphocyte stimula-
tion. Eur. I. lmmunol.; 1989; 19: 2349.
135. Irwin, M. 1.; Heath, W. R; Sherman, L. A Species-restricted interactions between CD8
and the alpha3 domain of class I influence the magnitude of the xenogeneic response. 1.
Exp. Med.; 1989; 170: 109l.
136. Auchincloss. H. lr.; Moses. R D.; Conti, D.; Sundt. T.; Smith, c.; Sachs, D. H.; Winn, H.
1. Rejection of transgenic skin expressing a xeno-class I antigen is CD4-dependent and
CD8-independent. Transplant Proc.; 1990; 22: 1059.
137. Maziarz, R T.; Burakoff. S. 1.; Bluestone, 1. A. The alpha-3 domain of class I MHC pro-
teins influences cytotoxic T lymphocyte recognition of antigenic determinants located
within the alpha-l and alpha-2 domains. I. Immunol.; 1988; 140: 4372.
138. Kalinke. U.; Arnold, B.; Hammerling. G. 1. Strong xenogeneic HLA response in trans-
genic mice after introducing an alpha-3 domain into HLA B27. Nature; 1990; 348: 642.
139. Bernabeu, c.; Maziarz, R; Spits, H.; de Vries. 1.; Burakoff, S. 1.; Terhorst, C.
Co expression of the human HLA-A2 or HLA-B7 heavy chain gene and human B2-mi-
croglobulin gene in L cells. 1. Immunol.; 1984; 133: 3188.
140. Altmann, D. M.; Hogg. N.; Towsdale. 1.; Wilkinson. D. Cotransfection of Icam-l and
HLA-DR reconstitutes human antigen-presenting cell function in mouse L cells.
Nature; 1989; 338: 512.
141. Brunswick, M.; Lake, P. Functional interactions of human and murine lymphoid cells. 1.
Analysis of primary and secondary responses. Cell Immunol.; 1986; 103: 441.
142. Gustafsson, K.; Germana, S.; Hirsch. F.; Pratt. K.: LeGuern, c.; Sachs. D. H. Structure
of miniature swine class II DRB genes: Conscrvation of hypervariable amino acid
residues betwecn distantly related mammalian species.Proc. Natl. A cad. Sci. USA.;
1990; 87: 9798.
143. Ildstad, S. T.; Russell, P. S.; Chase. C. M.: Sachs. D. H. Long-term survival of primarily
vascularized cardiac xenografts in mice repopulated with syngeneic plus xenogeneic
bone marrow. Transplant Proc.; 1985; 17: 535.
144. Ildstad. S. T.; Sachs, D. H. Reconstitution with syngeneic plus allogeneic or xenogeneic
bone marrow leads to specific acceptance of allografts or xenografts. Nature; 1984; 307:
168.
145. Ildstad, S. T.; Wren, S. M.; Sharrow. S. 0.; Stcphany, D.; Sachs, D. H. In vivo and in vit-
ro characterization of specific hyporeactivity to skin xenografts in mixed xenogeneical-
ly reconstituted mice (BI0 + F344 rat to BI0).1. Exp. Med.; 1984; 160: 1820.
146. Muller-Rucholtz, W.; Muller-Hermelink, H. K.; Wotage. H. U. Induction of lasting
hematopoietic chimerism in a xenogeneic (rat to mouse) model. Transplant Proc.;
1979; 11: 517.
42 The Scicntific Study of Xenografting: 1964-191l1l
147. Trentin. J. J.; RauchwergeL J. M.; Ballagher. M. T Genetic resistance to marrow trans-
plantation. Eiomed; 1973; 18: 109.
141l. van Bekkum. D. W. A new heterologous radiation chimera. Nature; 1964; 202: 1311.
149. van Bekkum. D. W. Radiation chimeras. TTi/nsplant Proc.; 1970; 2: 479.
ISO. van Bekkum. D. W.; Vos. O. Immunologic aspects of homo- and heterologous bone
marrow transplantation in irradiated animals..!. Cell Comp. Physiol.; 1957; 50 (Supp I):
139.
151. Gengozian. N. Transplantation of rat bone marrow in irradiated mice: Effect of expo-
sure rate. Science; 1964; 146: 663.
152. Gengozian. N.: Carlson. D. E.: Allen. E. M. Transplantation of allogeneic and xeno-
geneic (rat) marrow in irradiated mice as affected by radiation exposure ratio.
Transplantation: 1969: 7: 259.
153. Gengozian. N.: Makinodan. T Antibody response of lethally x-irradiated mice treated
with rat bone marrow. 1. Immlll/ol.: 1956: 77: 430.
154. Zoalberg. O. B.: Vos. 0.: van Bekkum. D. W. Surviving rat skin grafts in micc. Nature;
1957; 180: 23K
155. Makinodan. T Circulating rat cells in lethally irradiated mice protectcd with rat bone
marrow. Pmc. Soc. Exp. BioI. Meel.: 1956; 92: 174.
156. Gengozian. N.: Makinodan. T; Congdon. C C; Owen. R E. The immune status of
long-term survivors of lethally x-irradiated mice protected with isologous. homolo-
gClUS. or heterologous bone marrow. Pmc. Nml. Acad. Sci. US/I.; 1958; 44: 560.
157. Buchsbaum. H. L Aliminosa. K. V.: Southam. C M. Prolonged survival of human skin
grafts in immunologically tolerant rats. Transplal1lation: 1969: 8: 901.
158. Cerilli. G. J.; Gideon, L. Succcssfullong term inhibition of xcnograft rejection. Surg.
Forum: 1969: 20: 284.
159. Reilly. R W.: Woodruff. M. F. A. Induction of tolerance of rat skin in newborn mice.
Transplant Bull.; 1961: 21l: 39.
160. Hasek. M.; Hort. J. Studies of tolerance after inter-species embryonic parabiosis. In:
Mechanisms of ImmulloloKical Tolerallce. Czech. Acad. Sci.: 1962: 143.
161. Hasek. M.; Hraba, T; Hort. J. Acquired immunological tolerance of heterografts.
Nature; 1959: 11l3: 1199.
162. Mitchison. N. A. The results of transplantation of turkey spleen and blood cells into
chicken embryos. Transplant Bull.: 1958: 5: 66.
163. Egdahl, R H.; Good. R A.: Varco. R L. Studies in homograft and heterograft survival.
Surgery: 1957; 42: 228.
164. Egdahl, R H.; Roller, F. R.; Swanson. R L.: Varco. R. L. Acquired tolerance to homo-
grafts and heterografts in the rat. Anll. N. Y. Acad. Sci: 1958: 73: 1l42.
165. McKenzie. 1. F. C; Stocker. J.: Ting. A.: Morris. P. J. Human Iymphocytotoxic and
hemagglutinating activity against sheep and pig cclls. Lancet: 1968; 2: 386.
166. Simonsen, M. Artificial production of immunological tolerance. Induced tolerance to
heterologous cells and induced susceptibility to virus. Nalllre; 1955: 175: 763.
167. McCune.J.M.; Namikawa, R.; Kaneshima. H.: Shultz. L.D.: Licbcrman. M.: Wcissman,
I.L. The Scid-hu mouse: Murine model for the analysis of human hematolymphoid dif-
ferentiation and function. Science: 1988; 241: 1632.
168. Mosier, D.E.; Gulizia, R.J.; Baird. S.M.; Wilson, D.B. Transfer of a functional human
immune system to mice with severe combined immunodeficiency. Nmure; 1988; 335:
256.
169. Hume, D. M.; Gayle, W. E. Jr.; Williams, G. M. Cross circulation of patients in hepatic
coma with baboon partners having human blood. Surg. Gynec. Obstet: 1969; 128: 495.
170. Ham.J. M.; Pirola. R. c.; Davidson. G. M.: Yarrow, S.; Elmsliz. R G. Pig liver perfusion
for the treatment of acute hepatic coma. Surg. Gynec. Obstet.; 1968; 127: 453.
171. Abouna, G. M.; Fisher, L. M.; Still, W. J.; Hume, D. M. Acute hepatic coma successfully
treated by extracorporeal baboon liver perfusion. Er. Med. 1.: 1972; I: 23.
172. Abouna. G. M.: Ashcroft. T; Muckle. T J.: Skillen. A. W.; Hule. C J.: Kirkley. J. R;
Hodson, A. W. Heterologous extra corporeal hepatic support: hemodynamic. biochem-
ical and immunological observation. Er. 1. SurK.: 1970: 57: 213.
References 43
173. Saunders, S. J.; Bosman, S. C. W.; Walls, R.; Terblanehe, J.; Harrison, G. c.; Hickman,
R.; Biebuyck, J.; Dent, D.; Pearce, S.; Barnard, C. N. Acute hepatic coma treated by
cross-circulation with a baboon and by repeated exchange transfusions. Lancet; 1968; 2:
585.
174. Eiseman, B.; Liem, D. S.; Raffucci, F. Heterologous liver perfusion in treatment ofhep-
atic failure. Ann. Surg.; 1965; 162: 329.
175. Norman, J. c.; Saravis, C. A.; Brown, M. E.; McDermott, W. V. Jr. Immunochemical
observations in clinical heterologous (xenogeneic) liver perfusions. Surgery; 1966; 60:
179.
Section II
Immunobiology of
Xenograft Rejection
Chapter 4
Mechanism of Humoral Xenograft Rejection

L.C. PAUL
Introduction
During the 1960s, as allogeneic clinical organ transplantation became to some

extent successful while the supply of organs was limited, a few clinical xeno-
transplants were performed with organs from subhuman primates [1-3]. The
increased availability of human organs after the introduction of brain death
criteria, together with failure to achieve any long-term success with xenogene-
ic organs, however, dampened enthusiasm for xenotransplantation consider-
ably. The current success rate of allotransplantation together with the wider
acceptance of high-risk patients has, however, caused a tremendous increase
in demands for organs and stimulated a renewed interest in the possibility of
xenotransplantation.
The question has been raised whether the nonacceptance of tissues from
cross-species or xenogeneic donors is caused by immunological mechanisms
similar to those that cause rejection of allografts or whether other mechanisms
underlie the rapid and vigorous destruction that is characteristic for xenogene-
ic grafts. The fact that immunologically incompetent embryos and neonates
readily accept grafts from xenogeneic donors illustrates that cells of different
species are not innately incompatible; on the contrary, in the absence of an im-
mune response, graft and host cells of like histogenicity from widely divergent
species show remarkable affinities. The ability to reject foreign grafts depends
on the maturation of the ability to respond immunologically [4]. The nature
and specificity of the immunological reactions that underlie xenograft rejec-
tion are presently not well defined, and in this chapter the information regard-
ing the possible role and mechanisms of antibody-mediated xenograft rejec-
tion is reviewed.
Based on the severity and pattern ofxenograft rejection, the terms "concor-
dant" and "discordant" xenografts were proposed in 1970 by CaIne [5]. First -set
xenografts which are rejected at a tempo and with the morphological character-
istics similar to first-set allografts are designated concordant xenografts. First-
set xenografts which are rejected hyperacutely are called discordant xenografts
and resemble the rejection pattern of some second-set allografts. Thus, the dis-
tinction of concordant or discordant xenografts is not based on any taxonomic
relationship, but solely on the pattern of graft rejection.
The hyperacute rejection of allogeneic kidney or heart grafts is clinically
and pathologically well defined and has been observed in patients with high
48 Mechanism of Humoral Xenograft Rejection
titers of alloantibodies against donor antigens expressed in high density on the

graft vascular endothelium [6-9]. Based on these observations, many consider
hyperacute rejection the prototype of antibody-mediated graft damage. This
generalization is very likely justifiable in allogeneic combinations, although
allogeneic hyperacute rejection may occasionally occur in the absence of de-
tectable antibodies [10].
Considering the similarities in the pattern of allogeneic and xenogeneic hy-
peracute rejection as well as the presence of naturally occurring cross-species
antibodies in many combinations [11], it has been widely assumed that natu-
rally occurring xenoantibodies mediate discordant xenograft rejection. This
assumption has led to the use of the term discordant to indicate the presence of
antibodies in members from one species against members from other species
or orders. It should, however, be reserved to describe the rejection pattern
since some organs from a specific donor source are rejected hyperacutely
while other organs in that same combination are rejected acutely, like a prima-
ry allograft.
The mechanisms of antibody-mediated graft damage, the susceptibility of
different organs to this form of damage, the role of antibodies in xenogeneic
rejection, and humoral mediators of discordant xenograft rejection other than
antibodies will be discussed in the following sections.
Mechanisms of Antibody-Mediated Graft Destruction
Experimental and clinical studies of allografts have shown that antibodies may
influence the fate of the graft in a positive or negative way. Alloimmune sera
may have the power to greatly prolong or significantly curtail the graft survival
time, depending on the source and quantity of antisera administered, the
species and strain oftest animals, and the kinds of grafts investigated. The prin-
ciple of the protective effect in these situations appears to reside in the ability to
restrict in a specific way the active responses of the recipient to the incompatible
transplantation antigens ofthe graft [12]. In the xenogeneic situation, onlyfrag-
mentary evidence for antibody-mediated graft protection has been reported in
a concordant mouse-to-rat skin graft model, while virtually all available evi-
dence suggest that antibodies have either no effect or cause graft damage.
The interaction between the combining site of an antibody and an antigenic
determinant may result in a number of phenomena such as the formation of
immune complexes, agglutination of cells that carry the relevant antigens, or
the induction of cytolytic or inflammatory reactions. The cytotoxic effect and
the production of inflammatory changes depend on the antibody's abilities to
activate secondary and tertiary mechanisms of tissue injury. The biologic
properties of antibodies, therefore, depend on both antigenic specificity as
well as their ability to activate a variety of biologic systems.
Only in rare circumstances has it been documented that antibodies cause
functional impairment without apparent activation of mediators of tissue
Mechanisms of Antibody-Mediated Graft Destruction 49
damage. This may occur with the use of monoclonal antibodies against certain
cell surface antigens like OKT3 antibodies that modulate the T 3 antigen-recep-
tor complex on T lymphocytes, or with the use of monoclonal antibodies
against biologically active mediator substances. Furthermore, some antibod-
ies against selected structural components of the kidney glomerular basement
membrane or the glomerular epithelial cells may cause increased glomerular
permeability for proteins without participation of any systemic or cellular me-
diator systems [13-15].
In most instances, antibodies mediate tissue injury by activating a variety of
effector mechanisms, chief among which are components of the complement
system, platelets, granulocytes, monocytes and killer cells, as well as the blood
coagulation system. Interactions with secondary effector systems are initiated
when recipient antibody binds to an intrinsic antigen of the transplanted tissue
or an extrinsic antigen, such as a viral antigen, expressed on an infected cell in
the transplant [16].
The type and extent of tissue injury initiated by the antibody depends on
the quantity, distribution, and location of the antigen and the biologic proper-
ties of the antibody bound. For example, a single immunoglobulin M (IgM)
antibody that binds multiple antigenic sites on a cell surface assumes a "spider-
like" configuration and in so doing exposes its constant or Fc fragments to ini-
tiate activation of the classical route of the complement system.
In contrast, if the distribution of the antigens on the cell is such that the IgM
antibodies bind to the cell surface by only one or two antigen-binding frag-
ments, the IgM pentamers may retain "snowflake" shapes and not expose
their Fc regions for complement activation. Similarly, those subclasses of IgG
which are efficient activators of complement (IgG3, 1 and 2) can most effec-
tively activate the classical route of complement when at least two antibodies
are bound to antigens in close proximity so that their Fc regions are adjacent to
one another.
Complement Pathways
Classical complement pathway activation is initiated by the C1q component of

C1, which binds with high affinity to the Fc region of immunoglobulins. C1q
binding is followed by internal activation of C1r and C1s to produce enzymati-
cally active C1qrs, which cleaves C4 and C2 to generate the C4b2a complex,
the classical pathway C3 convertase, which cleaves C3 to C3a and C3b. C3b
binding to this enzyme produces C4b2a3b, a new enzyme that cleaves C5 to
C5a and C5b (Fig. 4.1).
The alternative complement pathway can be activated nonimmunological-
ly by a variety of activator substances including polysaccharides, bacteria,
damaged tissues as well as by IgA and IgE [17-19]. Alternative pathway acti-
vation requires C3b, which is generated randomly from C3 cleavage by nor-
mally present C3 cleaving enzymes or by a C3b-like molecule formed from
water-hydrolyzed C3 [20, 21]. When C3b binds to normal cell membranes, it
C-fixing Ab+C 1qrs

}C4b"~1
Classical
Route A..c3a
C4b2a3b
A
y
C5a - C5 C5b + C6,7 -C5b67
+
membrane
C3bBbP l
~
Alternative 3 membraneC5b67
+
Route C8
Properdin
Factor B l C3b !
Activator surface C3b ----7- C3bBb / membraneC5b678
+
J~3a c9
!
C3bi' + C3~b Amplification loop C5b-9
Factort/H MAC
Enzymes
C3
Fig. 4.1. Schematic representation ofthc complement system showing the classical and alter-
native activation pathways
is rapidly inactivated by the regulatory protein factor H (P],H), which allows

cleavage of C3b by factor I (C3b inactivator) to form inactive C3bi [22].
However, C3b bound to alternative pathway activator surfaces binds prefer-
entially to complement factor B [23] which is then cleaved by factor D to form
the alternative pathway C3 convertase, C3bBb. C3bBb greatly increases C3
cleavage and thereby C3b generation through the C3b amplification.
The alternative pathway C3 convertase is stabilized by properdin, a normal
protein [24] and under pathological conditions by C3 nephritic factor, an IgG
autoantibody to neoantigens on the C3bBb convertase and found in some pa-
tients with renal diseases. As with the classical pathway C3 convertase
(C4b2a), the binding of additional C3b to C3bBb converts the enzyme to a C5
convertase, which cleaves C5 to C5a and C5b. Generation of C5b through ei-
ther pathway initiates self-assembly of the C5b-9 membrane attack complex.
Following complement activation along either the classical or the alterna-
tive pathway, enough of the intermediate complement components may be ac-
tivated to allow C5 through C9 to form tunnel-like structures, "membrane at-
tack complexes", which penetrate the plasma membrane and induce cell death
via mechanisms believed to involve colloid osmotic deregulation. By electron
microscopy a circular doughnut-like structure with a 200 Adiameter electron-
lucent rim and a 100 Aelectron-opaque center is seen. Although one such hole
is enough to cause lysis of an erythrocyte, metabolically more active nucleated
cells are more resistant to cytolysis in part due to their ability to rapidly elimi-
nate the C5b-9 complex, the ability to repair the plasma membrane as well as
the osmoregulatory properties of nucleated cells.
Therefore, although prompt leakage of ions can be demonstrated at low
doses of complement, cell lysis occurs only after the formation of several trans-
membrane channels and an appreciable time delay [26]. Even if the "mem-
brane attack complex" does not cause cytolysis, it may contribute to the devel-
opment of tissue damage through the stimulation of production of potent
inflammatory mediators such as reactive oxygen metabolites, prostaglandins,
and interleukin-1-like cytokines [27-30].
If not enough complement is activated to produce the membrane attack
complex, cleavage products of complement produced during the complement
activation cascade can also initiate tertiary mechanisms of tissue injury.
Granulocytes and monocytes are attracted to sites of complement activation
by the chemotactic factor C5a and the trimolecular C5, 6, 7 complex; C5a also
stimulates the release of leukocyte lysosomal enzymes and may playa role in
the granulocyte-dependent induction of inflammatory edema [31]. The mech-
anisms by which granulocytes may cause tissue damage have recently been re-
viewed [32]. When C3b is bound to the cells of the graft, circulating cells with
C3b receptors can adhere to the graft cells and be activated. In this way C3b
acts as an opsonin for monocytes and polymorphonuclear granulocytes.
Likewise, C3b can cause immune adherence of erythrocytes. In addition, in
experimental animals, but not in man, C3b can cause immune adherence of
platelets which may lead to the release of vasoactive amines and nucleotides
from these platelets. C3a and C5a furthermore stimulate mast cells to release
histamine, contract smooth muscle cells, and increase capillary permeability,
while a fragment of C2 has a kinin activity.
The complement fragments C3a and C5a have been shown to produce ma-
jor biologic effects in, for example, the function of the heart. Activated C3a
can result in in situ release of histamine and cyclooxygenase metabolites in iso-
lated heart preparations perfused with crystalloid solution [33], while intra-
coronary infusion of C5a decreases coronary blood flow, the contractile func-
tion, and produces local leukocyte accumulation. The local production of the
vasoactive eicosanoids thromboxane A2 and peptidoleukotrienes LTC4,
LTD 4, and LTE4 are primarily responsible for the ischemia and contractile
dysfunction [34]. The cells that serve as the sources of the C5a-liberated
eicosanoids are not yet defined.
It was recently reported that naturally occurring antibodies and comple-
ment react with cultured endothelial cells and cause activation of the cell to-
gether with very rapid degradation of the cell membrane-associated heparan
sulfate proteoglycan [35]. The endothelial cell activation, as evidenced by re-
lease of thrombomodulin and elaboration of tissue factor, together with the
loss of the heparan sulfate proteoglycan, may further promote rapid intravas-
cular coagulation which very likely precedes irreversible endothelial cell in-
jury. This loss of endothelial cell anticoagulant activity depends on comple-
ment activation along the classical route [35].
Fc-Receptor-Interaction-Dependent Antibody Effects
In complement-independent antibody-dependent cell-mediated cytotoxicity

(ADCC), antibody which is bound to antigen on the target cell by virtue of its
serologic specificity is further bound by an Fc receptor-bearing cell such as a
monocyte or a killer lymphocyte. which subsequently delivers a lethal "'hit" to
the antibody-coated target cell. Only small amounts of antibody are required
to localize and trigger such effector cells, and such a mechanism may increase
the effectiveness of suboptimal concentrations of antibody. Under these con-
ditions, antibody concentrations that are too low to initiate complement-me-
diated cytotoxicity, and otherwise might cause shedding of surface antigens.
could initiate damage when cells with Fc receptors are available. ADCC
mechanisms have been reported to kill histoincompatible target cells in vitro.
Xenogeneic skin graft rejection in the mouse that is initiated by noncomple-
ment-fixing antibodies may be an example of graft rejection mediated by
ADCC[36].
A pharmacologically potent cell with Fc receptors is the platelet. Platelets
may interact with graft-bound antibodies either through their Fc receptor or
via complement-dependent pathways. Platelets contain potent vasoconstric-
tive, proaggregatory, and proinflammatory substances including platelet-de-
rived growth factor, epidermal growth factor, serotonins, thromboxanes, and
platelet-activating factor (PAF). These compounds are released under a vari-
ety of conditions and may explain many physiological features of hyperacute
rejection such as severe vasoconstriction and organ dysfunction [37-41].
Evidence for in vivo involvement of PAF in hyperacute rejection has been
obtained in an allogeneic rabbit model of hyperacute renal transplant rejec-
tion ; shortly after transplantation, P AF concentration in the venous effluent
of the graft is increased dramatically. Since the appearance of P AF precedes
the intraglomerular agglomeration of platelets or inflammatory cells, the pos-
sibility was raised that antibody and complement interactions with graft en-
dothelial cells rather than platelets cause release of PAF [43,44] or perhaps
other vasoactive substances which may playa role in the pathogenesis of hy-
peracute rejection.
The importance of platelets in the pathogenesis of hyperacute rejection is
not limited to their release of chemotactic, inflammatory, and vasoconstrictor
substances, but they may also serve as activators for the complement system.
Most of the complement components are present on the platelet surface in-
cluding Clq, C2, C4, and C3, and platelets are capable of cleaving C3 into C3a
and C3b. Thus, platelets that are bound to endothelium can secondarily initi-
ate the complement cascade in addition to initiating thrombosis by generating
fibrin from fibrinogen. Thus, even noncomplement-fixing antibodies that bind
platelets through their Fc portion to vessel endothelium can exert tissue dam-
age through platelet-bound complement components.
In Vivo Patterns of Antibody-Mediated Hyperacute Rejection
Hyperacute antibody-mediated rejection of rat skin grafted to immunosup-

pressed mice recipients may occur following passive transfer of antibodies to
recipients with well-healed grafts. Histological studies of such grafts show the
typical features of either an Arthus-like reaction, characterized by early accu-
mulation of polymorphonuclear granulocytes in the vessels and subsequent
intravascular coagulation and eventual necrosis, or a Schwartzman-like pat-
tern characterized by early intravascular coagulation [45,46].
Which of these patterns will develop depends on the amount of antibody
injected: high doses produce the violent Schwartzman-like reaction, whereas
low doses cause the more slowly developing Arthus-like reaction. Depletion
of complement by cobra venom treatment does not prevent acute rejection af-
ter injection of high doses of antisera but changes the reaction pattern from the
Schwartzman-type to the Arthus-type. Conversely, supplementary adminis-
tration of rabbit complement causes a violent Schwartzman-like graft destruc-
tion after injection of low doses of antibody which in complement-normal
mice gave an Arthus-Iike reaction [45].
Thus, in this situation both Schwartzman or Arthus-like reactions are ex-
pressions of an antibody-mediated reaction but of different intensity, depen-
dent on the antigen-antibody-complement interaction. Administration of
F( ab h fragments in this model cause only mild tissue inflammation that invari-
ably subsides [47].
Are Antibodies Involved in Chronic Vascular Rejection?
As pointed out by several authors, no effective therapy is currently available

to prevent or treat hyperacute xenograft rejection. If such a therapy becomes
available and hyperacute as well as acute rejection can be avoided, the next
question that emerges is whether antibodies playa role in the pathogenesis of
chronic vascular rejection, a problem that is receiving increasing attention [48,
49]. At present, the immunology and pathophysiology of this syndrome have
not been characterized extensively, but it is thought by many investigators that
this form of graft damage results from previous or persistent low-grade im-
munological damage of the graft vessel wall, followed by repair mechanisms
that lead to vessel wall remodeling, as has been proposed in the "response-to-
injury" hypothesis to explain atherosclerosis following endothelial cell injury
in general [53,54].
According to this hypothesis, injury of vascular endothelial cells leads to
accumulation of platelets and macrophages at injured sites followed by local
production of growth-promoting peptides, such as platelet-derived growth
factor, by the cells that have invaded the vessel wall.
One of the major themes that has emerged from recent advances in basic
vascular biology is a wider appreciation of the functional potentials of vessel
wall cells. Both endothelial and smooth muscle cells have the capability to pro-
duce cytokines with a wide range of vasoactive, proaggregatory, proinflamma-

tory, and mitogenic properties [52,53]. The mitogenic properties of cytokines
like platelet-derived growth factor, produced by either inflammatory cells or
vessel walls cells, very likely induce biochemical and morphological "pheno-
typic-modulation" of smooth muscle cells, followed by proliferation and mi-
gration of some of these cells from the media into the intima where they under-
go further proliferation and produce extracellular matrix material [48]. Based
on the histological resemblance of these lesions and atherosclerosis, the for-
mer is often referred to as graft atherosclerosis.
Several differences do, however, exist. Vessels from organs that undergo
chronic vascular rejection have more extensive infiltration of the vessel wall
with immune and inflammatory cells, and more extensive and concentric inti-
ma proliferation that develops more rapidly than normally occurring
atherosclerotic lesions. It would, nevertheless, appear that chronic vascular
rejection may share many pathogenic features with normally occurring
atherosclerosis.
Since vessel wall lesions similar to those observed in chronic rejection have
been produced in carotid arteries of experimental animals after repeated local
injections of allo- or xenoantisera that contained antibodies against histocom-
patibility antigens of the recipient [54,55], it has been widely assumed that an-
tibodies are responsible for the chronic rejection lesions in transplanted or-
gans. Although there is clinical precedent for an association of antiendothelial
cell antibodies and atherosclerosis [56, 57], it is important to note that the
atherosclerotic reaction pattern is not specific for antibody-mediated injury
and that any type of endothelial cell or vessel wall injury can potentially initi-
ate an atherosclerotic reaction pattern. It, therefore, remains to be established
whether antibodies are responsible for the development of chronic vascular
rejection.
Susceptibility of Different Organs to Antibody-Mediated Damage
Several experiments in the early part of this century established the in vivo
toxicity of antisera against isolated cells and tissues. Cell culture techniques
have, furthermore, been used to document the cytotoxicity of normal human
sera for xenogeneic cells [58]. Baumann and Witebsky described the reaction
of a 2-day-old chicken embryo exposed to xenogeneic Forssman antisera and
complement; the vascular network of the embryo shrank and sank into the
yolk, and the embryo died when fresh Forssman antisera or even fresh normal
rabbit serum containing natural Forssman antibodies was placed on the em-
bryo [59].
Many experimental and clinical observations have shown that kidney and
heart allografts are susceptible to antibody-mediated tissue damage and, there
are no a priori reasons to suggest that this does not also apply to xenogeneic or-
gans. Pig kidneys placed into dog recipients are usually rejected within a few
Susceptibility of Different Organs to Antibody-Mediated Damage 55
minutes [60-65]. Shortly following transplantation, the graft shows a rapid dif-
fuse blanching, indicating a vasospastic response, followed by irregular dark
venous mottling. Biopsies taken as early as a few minutes after the reconnec-
tion of the graft show platelet and fibrin aggregates in small arteries, arterioles,
and glomeruli while the platelets seem often attached to the endothelium. The
endothelial lining may be swollen and separated from the remainder of the
vessel wall and micro hemorrhages occur in the tissue space. About 20 min af-
ter revascularization, extensive congestion and interstitial hemorrhages are
observed, and after 24 h the classic pattern of necrosis and neutrophilic inva-
sion is present [61]. This histological pattern resembles that of hyperacute re-
nal allograft rejection in recipients with preformed donor-specific alloanti-
bodies.
Discordant cardiac grafts are also rejected within minutes to hours. White
Landrace pig hearts grafted into Chacma baboon recipients, for example, are
rejected within 1.5 h and the histopathology of such grafts shows interstitial
hemorrhages and edema, fibrin thrombi, and scanty or absent mononuclear cell
infiltration together with contraction bands and myocardial cell necrosis [66].
Similar vascular lesions have been described in the guinea pig-to-rat heart
model [67, 68] and resemble the changes found in allogeneic heart transplants
placed into recipients with high titers of donor-directed alloantibodies [69].
It is less evident whether other organs such as pancreas, skin or liver trans-
plants are susceptible to antibody-mediated graft rejection. Although pancre-
atic islet grafts injected into the portal circulation of neonatally tolerant rats
can be induced to reject with passive transfer of humoral antibodies, vascular-
ized pancreas grafts seem resistant to antibody-mediated damage [70, 71].
There is, furthermore, not much support for the hypothesis that xenogeneic
pancreas islets grafted beneath the splenic capsule are susceptible to damage
by naturally occurring xenoantibodies [72].
Skin allotransplants placed on a recipient who has received repeated
donor-specific antigenic stimuli can be rejected in an almost hyperacute fash-
ion, classically known as "white" graft failure. Such white grafts are very likely
caused by cellular immune mechanisms since skin grafts are resistant to the ef-
fects of antibodies in the early post-transplant period. This apparent resis-
tance can be explained by the lack of revascularization of the skin graft in the
first few days following transplantation [47, 73]. Only during a few weeks'
time-window does administration of donor-specific antibody induce hyper-
acute rejection [73]. The fact that such skin grafts are only susceptible to anti-
body-mediated damage during a certain time interval illustrates the impor-
tance of donor-derived graft endothelium as the target structure for
antibodies. In the first week after transplantation, skin grafts are not suscepti-
ble to antibody-mediated damage because the graft is not yet vascularized,
while later on the graft endothelium is replaced by recipient-derived endothe-
lium.
Xenogeneic liver grafts appear less susceptible to rejection than other vas-
cularized organs [74,75], a feature that is well known from liver allografts [76].
This relative resistance of liver grafts to rejection cannot be accounted for by
differences in the nature of the criteria to diagnose rejection. Several experi-

ments suggest that liver grafts activate immunological mechanisms that facili-
tate graft acceptance more readily than, for example, kidney or heart grafts.
Although this does not explain the apparent resistance to immediate destruc-
tion, liver grafts may be relatively resistant to immediate antibody-mediated
rejection because of the venous nature ofthe portal system and its termination
in a sinusoid structure which may not permit vasospasm and complete throm-
bosis as easily as in an arterial system. However, this does not completely pre-
clude immediate graft damage by humoral antibodies [77].
Antibodies in Xenograft Rejection
Cross-species or xenogeneic antibodies may arise under a variety of circum-

stances. The best -known examples of xenogeneic antibodies in man are the so-
called heterophile antibodies such as the Paul-Bunnell and rhesus monkey
erythrocyte antibodies produced by patients with infectious mononucleosis
[78]. The immunogeneic stimuli for the formation of such antibodies are not
clear, and it has been postulated that the viral infection causes expression of
the Paul-Bunnell antigen as a neoantigen on the patient's tissues, which subse-
quently stimulates formation of antibodies [70].
Hanganutziu reported in 1924 that patients who received therapeutic injec-
tions of horse antisera formed antibodies that agglutinated sheep erythro-
cytes. Independently, Deicher studied sera from patients who had received
horse or sheep antisera, and both investigators found that the sera from these
patients reacted with erythrocytes of many animal species. Many organ trans-
plant recipients who have received heterologous antilymphocyte sera to avoid
or treat transplant rejection may have Hanganutziu-Deicher antibodies that
react with a variety ofxenogeneic tissues [79]. It has, furthermore, been estab-
lished that rejection of an allogeneic kidney transplant [80], and perhaps allo-
geneic pregnancies [78], may produce antibodies that react with allogeneic
and xenogeneic tissues as well as various gram-negative bacteria [81].
Xenogeneic antibodies may, furthermore, develop following intentional
xenogeneic immunizations that result in the production of antibodies that re-
act with the immunizing material as well as with cells from a variety of other
species. This cross-reactivity is due to sharing of antigens or antigenic determi-
nants among various species. The first report to describe cross-reacting anti-
bodies was that of Ehrlich and Morgenroth in 1900 [82], while the best known
antibodies in this category are the Forssman antibodies (reviewed in [78]).
Many animals have circulating antibodies that react with cells and tissues
from other, totally unrelated species without any obvious previous exposure
to xenogeneic tissues and are called naturally occurring antibodies [11]. The
nature and specificity of these antibodies is virtually unknown.
We have recently investigated some aspects of naturally occurring antibod-
ies in rats against guinea pig tissues. At birth, most rats have low titers of IgG
Antibodies in Xenograft Rejection 57
antibodies against guinea pig cells, possibly as a consequence of transplacental

transfer of maternal xenoantibodies ; xenogeneic IgM antibodies appear a few
days later [83]. Although immunizations of adult animals with guinea pig cells
results in xenoantisera that contain multiple antibody specificities that were
not present at birth, neonatal injections of xenogeneic lymphocytes have
failed to produce tolerance. On the other hand, neonatal tolerance can be
readily produced for alloantigens [84, 85].
Although more experiments are needed to address this issue, these observa-
tions are compatible with the hypothesis that, in certain combinations, natural-
ly occurring xenoantibodies are immunoglobulins against a variety of environ-
mental and self antigens with low-affinity cross-reactivity with a number of
xenogeneic cell surfaces [83]. This does not exclude, however, that in other com-
binations high-affinity antibodies emerge as a consequence of immunizations
with environmental antigens with a high degree of cross-reactivity with certain
xenoantigens, comparable perhaps to the ABO blood group antibodies.
As alluded to, many experiments have suggested that naturally occurring
antibodies participate in discordant xenograft rejection. The observations that
hyperacute rejection does not occur in xenogeneic combinations that have no
naturally occurring xenoantibodies [86, 87] provide circumstantial evidence in
favor of this hypothesis. More direct evidence comes from studies that have
shown a significant decline in antibody titers between the arterial inflow and
venous outflow of a discordant xenograft together with immunohistological
documentation of recipient-type immunoglobulin deposition within the graft
[62]. It was recently reported that ex vivo perfusion of guinea pig heart tissue
with IgM F( ab h fragments results in a delay of the hyperacute rejection fol-
lowing transplantation into a discordant rat recipient [63], suggesting that such
fragments produce specific blocking of binding sites for naturally occurring
xenoantibodies on the graft vessel endothelium.
Many physiological and histopathological features of hyperacute rejection
can be reproduced by ex vivo perfusion of an isolated organ with cell-free
xenogeneic serum. Ex vivo perfusion of isolated pig kidneys with dog serum,
for example, produces an immediate rise in the resistance to flow as well as
histopathological evidence of rejection [88], although it is not clear which com-
ponents in the serum are responsible for these changes.
The efficacy of interventions to delay discordant xenograft rejection ap-
pears to correlate with the effectiveness to remove anti donor antibodies from
the recipient circulation [64,89]. Although such experiments seem to support
the hypothesis that antibodies playa central role in hyperacute xenograft re-
jection, it is difficult to exclude that such procedures may partly exert their
beneficial effect through depletion of, for example, complement proteins. The
role of naturally occurring xenoantibodies in discordant xenograft rejection
can only be investigated reliably once methods become available to produce
xenoantigen-specific tolerance or specific removal of antibodies without any
change in secondary or tertiary mediators of tissue injury.
Xenografts that are not rejected in a hyperacute fashion are usually reject-
ed acutely by mechanisms that are very likely similar, although not necessarily
identical, to those that produce acute allograft rejection [90, 91]. The elabora-
tion of serum antibodies in association with acute concordant graft rejection as
well as immunoglobulin deposition within the graft [74,87] lend support to the
possibility that antibodies contribute to this form of acute graft rejection albeit
that suppression of humoral immunity does not prevent the acute rejection re-
action [74].
Xenoantigens
Interaction of the recipient's immune system with incompatible transplanta-

tion antigens of the graft constitutes the core event in the rejection of foreign
tissues. It is, therefore, timely to discuss the state of knowledge regarding the
antigens that are involved in xenogeneic interactions by antibodies. Contrary
to the extensive amount of knowledge available regarding the structure, func-
tion, and genetics of the antigens involved in allogeneic interactions, our
knowledge of xenogeneic antigens is limited.
The serologic and biochemical characterization of the "classical" xenoanti-
gens, such as the Forssman and other "heterophile" systems, was mainly car-
ried out in the early part of the current century. The Paul-Bunnell antisera
from patients with infectious mononucleosis have been shown to react with
the red blood cells of a variety of species including cattle, sheep, and horse as
well as a variety of tissues including aortic intima and kidney cells [92, 93].
Based on immunoprecipitation of red blood cell stromata from different
species, the Paul-Bunnell antigen consists of at least two separate antigens,
very likely glycoproteins [78]. The Hanganutziu-Deicher antigen is present in
a ganglioside fraction of bovine erythrocyte membranes and is, therefore,
most likely a glycolipid while in the serum it is present in the albumin fraction
[78,94].
The Forssman antigen consists of a ceramide pentasaccharide hapten por-
tion which is loosely bound to a protein that is necessary for its immunogenici-
ty [95]. The antigen has been localized by immunofluorescence in the form of
droplets in the endothelium and adventitial connective tissues in most organs
of Forssman-positive species [96]. Similar intracytoplasmic localization of the
antigen was found by some investigators in cultured cells originating from
guinea pig kidneys, while other investigators demonstrated the Forssman anti-
gen on membranes of various guinea pig cells using a mixed agglutination test.
With the exception of smooth muscles and epithelium of jejunum, cells of all
organs studied, such as heart, kidney, liver, spleen, lung and testis, contained
the antigen on their surface [97], and various nucleated cells of guinea pig,
hamster, and mouse are susceptible to cytolysis by Forssman antibodies and
complement [92]. The presence of antigen on vascular endothelium and
perivascular connective tissues would account for the well-known toxicity of
anti-Forssman sera upon intravenous injection into guinea pig or the "carotid
syndrome" induced by injection into the common carotid artery [98].
Xenoantigcns 59
In the 1960s, investigators studied xenogeneic serologic similarities to the

human leukocyte antigen (HLA) system by testing human alloantisera with
cells from individual donors of various primate species. The first study on the
reactions of human alloantisera with chimpanzee cells used leukocyte-aggluti-
nating sera which were not known to be monospecific [99]; at that time the
current HLA designations were not used. This early work, however, did estab-
lish that human leukocyte alloantisera could also be used to detect polymor-
phism in a primate species. Later studies showed that chimpanzee alloantisera
may contain antibodies of sufficient specificity to be used as typing reagents
for certain human leukocyte groups [100, 101]. Based on panel analysis, ab-
sorptions, and absorption-elutions, it was subsequently shown that many
shared antigens are associated with the HLA [100-102]. Later studies have
confirmed the existence of extensive serologic cross-reactivity of major histo-
compatibility complex (MHC) antigens between different species [103].
Following intentional xenoimmunizations, many species produce antibod-
ies against the MHC of the immunizing species [104, 105]. For example, immu-
nizations of rabbits with insoluble mouse spleen cell membranes results in the
formation of antibodies that react with mouse MHC molecules [105]. Thus, in
Fig. 4.2. Indirect immunofluorescence micrograph of a guinea pig liver section that was incu-
bated with normal serum from a Munich-Wistar strain rat and FITC-labeled goat anti rat
IgG; there is specific staining of vascular endothelial cells, cells lining the sinusoids. and the
liver parenchymal cells
the xenogeneic situation the MHC appears similarly immunogenie as in the al-
logeneic situation but in the latter condition a variety of other systems serve as
equally strong immunogens.
Although it has been suggested that normal rabbit serum may contain natu-
rally occurring Forssman antibodies [59]. there is virtually no information
availahle regarding the nature of the antigens that are recognized hy naturally
occurring antibodies. Perper and Najarian reported that dog serum reacts in
vitro with antigens prepared hy sonic disruption of pig kidney cells and that ah-
sorption of the sera with these cells failed to remove the hemolytic antibody
activity. suggesting that not all erythrocyte antigens recognized hy naturally
occurring antibodies arc present in the kidney [60].
We have recently shown that rat sera contain naturally occurring antihod-
ies of both IgG and IgM class against sUbpopulations of guinea pig erythro-
cytes. leukocytes. and a variety of tissue structures. including vascular en-
dothelial and organ parenchymal cells (Fig. 4.2). Although the antihody titers
were low. a few absorption experiments were performed and showed that ah-
sorptions of the sera with erythrocytes did not suhstantially affect the tissue
staining. suggesting that the sera contained at least two different antihody
specificities.
Recent studies of human sera and monoclonal antihodies derived from
CD5+ B cells have shown that human xenoreactive natural antibodies react
with glycoproteins on pig endothelial cells but not the erythrocytes or lympho-
cytes [106]. Irrespective of the fine serologic specificities. in most or all donor-
recipient combinations investigated. the endothelium appears to constitute
the common in vivo target structure for naturally occurring xenoantibodies
[65] which may explain the predominantly vascular lesions ohserved in reject-
ed discordant xenografts.
Complement and Hyperacute Xenograft Graft Rejection
The participation of the complement system as an important secondary or ter-

tiary mediator of tissue injury in the pathogenesis of antihody-mediated hy-
peracute rejection has been reviewed in a previous section. As discussed. com-
plement may participate after activation through antihodies along the classical
route or after activation through platelet-bound complement components.
The low or undetectahle levels of naturally occurring xenoantibodies in some
discordant combinations have raised questions on the contribution of the anti-
bodies to the pathogenesis of hyperacute xenograft rejection [83. 107].
Observations of allogeneic transplants performed in the presence of donor-di-
rected antibodies suggest that complement-fixing antibodies are mostly effec-
tive in mediating tissue damage if they are present in high titers so that comple-
ment activation along the classical route can occur [6. 7]. As discussed. low
levels of antibodies or noncomplement fixing antihodies may initiate tissue
damage through an ADCC mechanism. but the speed of such a reaction may
Complement and Hyperacute Xenograft Graft Rejection 61
be too slow to fully explain the vigorous reaction pattern that occurs in some
discordant xenografts.
Thus, the possibility has to be considered that mechanisms other than anti-
bodies are the primary initiators of hyperacute xenograft rejection, at least in
some donor-recipient combinations. In line with this possibility are immuno-
histological studies of hyperacutely rejected xenografts that have failed to
show immunoglobulin deposits in the rejected tissues [108]. Furthermore, per-
fusion of rabbit kidneys with heparinized human blood produces a rapid and
extensive deposition of C3 to graft endothelium, while the binding of human
IgG is weak and focally distributed without any binding of Clq and weak and
inconsistent binding C4, suggesting complement participation through alter-
native pathway complement activation (Fig. 4.1) and independent of IgG or
IgM antibodies [65].
Miyagawa et al. showed that the hyperacute rejection of guinea pig hearts
placed into unmodified rat recipients is associated with more or less normal
levels of C2 and C4, but low levels of C3 [107]. The authors showed, further-
more, that xenogeneic red blood cells can be lysed by normal rat sera through
alternative route complement activation. It was suggested that the C3b that is
formed on xenogeneic cell surfaces is not inactivated because of functional in-
compatibility between the deposited recipient C3b and the donor tissue regu-
latory proteins, i.e., factor I and/or H C3b inactivator (Fig. 4.1).
In the allogeneic situation, deposited C3b is normally inactivated by graft
tissue regulatory proteins. Thus, it is conceivable that direct complement acti-
vation occurs in certain discordant xenogeneic situations without participa-
tion of antibodies. Antibody-independent complement activation and cytoly-
sis does not appear to be a problem in in vitro complement-dependent
lymphocytotoxicity assays in which selected xenogeneic serum is added as a
complement source to lymphocyte suspensions and antisera. The sera have
been screened for the absence of toxicity which is usually attributed to natural-
ly occurring antibodies. It is, however, difficult to translate such artificial and
selected in vitro systems to the actual in vivo situation.
Pretreatment of the recipient with injections of cobra venom has been used
to assess the relevance of the complement system in vivo. Cobra venom factor
is an analog of mammalian C3c, which binds to factor B and is cleaved by fac-
tor D to form a C3 convertase that cannot be regulated by factors H (~l,H) and
I (C3b in activator) and, therefore, allows C3 activation to proceed to exhaus-
tion [109, 110]. Most studies concur that such treatments prolong the survival
of discordant vascularized grafts significantly, sometimes up to a few days [68],
but the ultimate histopathology of the graft after such a treatment is usually
not different from unmodified hyperacute rejection.
Although it can be argued that the treatment protocols may have caused in-
sufficient degrees of long-lasting complement depletion, it was shown to be ef-
fective to convert hyperacute rejection of a concordant xenograft transplanted
into a presensitized recipient to an acute rejection reaction, while it was unable
to achieve such conversion in an established discordant combination [111].
This suggests that in addition to antibody and complement, other yet unde-
fined mechanisms may be operative in discordant xenograft rejection. One

possibility which needs further study is that platelets interact directly with
xenogeneic graft endothelium and produce a variety of functional and mor-
phological changes.
Comment
In this chapter the humoral mechanisms that may produce hyperacute rejec-
tion of allo- and xenografts are discussed. Antibodies may cause tissue damage
through activation of the complement system along the classical route as well
as through complement-independent mechanisms, although complement
may still participate in the latter condition through platelet-bound comple-
ment components. The in vivo pattern of antibody-mediated tissue damage
depends on the antigen-antibody-complement interaction. Antibody-comple-
ment interaction results in severe vasoconstriction as well as the recruitment
of neutrophils or platelets and damage to the vascular endothelium and its
extracellular matrix substance, resulting in inflammation, thrombosis, and
necrosis.
The evidence that supports a role for antibody-mediated tissue damage in
at least some combinations of discordant graft rejection is reviewed, as well as
recently published data which suggest that in at least some xenogeneic donor-
recipient combinations, complement is activated via the alternative route.
Taken together it would appear that classical antibody and complement -medi-
ated rejection as well as direct complement-mediated tissue damage may oc-
cur, dependent on the model and conditions studied. The available data leave
room for both possibilities as well as the possibility that yet other mechanisms
playa pathogenetic role.
Acknowledgements. The author was supported by a Scientist Award from the

Alberta Heritage Foundation for Medical Research and grants from the
Canadian Heart Foundation and The Kidney Foundation of Canada. Dr. John
Klassen is thanked for discussions regarding the manuscript and Ms. Charlene
Norris for secretarial assistance.
References
1. Reemtsma K, McCracken BH, SchlegelJU, Pearl MA, Pearce CW, De Witt CW, Smith
PE, Hewitt RL, Flinner RL, Creech Jr. O. Renal heterotransplantation in man. Ann.
Surgery 160: 384-410, 1964.
2. Starzl TE, Marchioro TL, Peters GN, Kirkpatrick CH, Wilson WEC, Porter KA,
Rifkind D, Ogden DA, Hitchcock CR, Waddell WR. Renal heterotransplantation
from baboon to man: experience with six cases. Transplantation 2: 752-776,1964.
3. Hardy JD, Chavez CM. The first heart transplant in man. Developmental animal inves-
References 63
tigations with analysis of the 1964 case in the light of current clinical experience. Am. 1.
Cardiol. 22: 772-781,1968.
4. Steinmuller D. Cross-species transplantation in embryonic and neonatal animals.
Transplant. Proc. 2: 438-445,1970.
5. Caine RY. Organ transplantation between widely disparate species. Transplant. Proc.
2: 550-553, 1970.
6. Kissmeyer-Nielsen F, Olsen, S, Petersen VP, Fjeldborg O. Hyperacute rejection of kid-
ney allografts associated with pre-existing humoral antibodies against donor cells.
Lancetl:662-665,1966.
7. Paul LC, Baldwin WM III, van Es LA. Vascular endothelial alloantigens in renal trans-
plantation. Transplantation 40: 117-123, 1985.
8. Baldwin WM III, Paul LC, Claas FH1, Daha MR. Antibody-mediated events in trans-
plantation: challengcs to past dogma and hopes for the future. In: Progress in
Transplantation, Vol. 3. Morris Pl, Tilney NL (eds), Churchill Livingstonc; Edinburgh,
London, Melbourne, New York, 1986,pp. 85-114.
9. Trento A, Hardesty RL. Griffith P, Zerbe T, Kormos RL. Bahnson HT. Role of anti-
body to vascular endothelial cells in hyperacute rejection in patients undergoing car-
diac transplantation. 1. Thorac. Cardiovasc. Surg. 95: 37-41, 1988.
10. Eichwald El, Shelby 1. Is delayed-type hyper-sensitivity the pathway of hyperacute re-
jection in transplanted mouse hearts? Transplant. Proc. 19: 426-427,1987.
11. Hammer C, Chaussy CH, Brendel W. Preformed natural antibodies in animals and
man. Outlook on xenotransplantation. Europ. Surg. Res. 5: 162-166, 1973.
12. Tilney NL, Kupiec-Weglinski lW, Heideche CD, Lear PA, Strom TB. Mechanisms of
rejection and prolongation of vascularized organ allografts. Immuno!. Rev. 77:
185-216,1987.
13. Couser WG, Darby C, Salant Dl, Adler S, Stilmant MM, Lowenstein LM. Anti-GBM
antibody-induced proteinuria in isolated perfused rat kidney. Am. 1. Physiol. 249:
F241-F250, 1985.
14. Boyce NW, Holdsworth SR. Direct anti-GMB antibody-induced alterations in
glomerular permse!ectivity. Kidney Int. 30: 666--672, 1986.
15. Mendrick DL, Rennke HG. 1. Induction of proteinuria in the rat by a monoclonal anti-
body against SGF-115/l07. Kidney Int. 33: 818-830, 1988.
16. Porter KA. The effects of antibodies on human renal allografts. Transplant. Proc. 8:
189-197,1976.
17. Baker Pl, Osofsky SG. Activation of human complement by heat-killed, human kidney
cells grown in cell culture. 1. Immunol. 124: 81-86, 1980.
18. Reid KBM. Proteins involved in the activation and control of the two pathways of hu-
man complement. Biochem. Soc. Trans. 11: 1-12, 1983.
19. Minta 10, Movat HZ. The complement system and inflammation. Curro Top. Path. 68:
135-178,1979.
20. Fearon DT, Austen KF. Initiation of C3 cleavage in the alternative complement path-
way. 1. Immunol. 115: 1357-1361, 1975.
21. Fishelson Z, Pangburn MK, Muller-Eberhard Hl. Characterization of the initial C3
convertase of the alternative pathway of human complement. 1. Immuno!. 132:
1430-1434,1989.
22. Whaley K, Ruddy S. Modulation of the alternative complement pathway by BIH.
JExp.A1ed.144:1147-1163,1976.
23. Kazatchkine MD, Fearon DT, Austen KF. Human alternative pathway membrane as-
sociated sialic acid regulates the competition between Band BIH for cell bound C3b.
1. Immunol. 122: 75-81, 1979.
24. Fearon DT, Austen KF. Properdin: initiation of alternative complement pathway.
Proc. Nat!. Acad. Sci. USA 72: 3220-3224,1975.
25. Biesecker G. Membrane attack complex of complement as a pathologic mediator. Lab.
Invest. 49: 237-249, 1983.
26. Kim S-H, Carney DF, Hammer CH, Shin ML. Nucleated cell killing by complement: ef-
fects of C5b-9 channel size and extracellular Ca 2+ on the lytic process. 1. Immunol. 138:
1530-1536,1987.
27. Lovett D, Hansch G, Resch K, Gemsa D. Activation of glomerular mesangial cells by

terminal complement components. Stimulation of prostanoid and interleukin-I like
factor release. Immunology 168: 34-35.1984.
28. Polley Ml, Nachman RL, Weksler BB. Human complement in the arachidonic acid
transformation pathway in platelets. 1. Exp. Med. 153: 257-268, 1981.
29. Hansch GM, Seitz M, Marinotti G. Betz M. Rauterberg EW. Gemsa D. Macrophages
release arachidonic acid. prostaglandin E z, and thromboxane in response to late com-
plement components.l.lmmunol. 133: 2145-2150,1984.
30. Adler S. Baker Pl.lohnson Rl, Ochi RF, Pritzl P, Couser WG. Complement membrane
attack complex stimulates production of reactive oxygen metabolites by cultured rat
mesangial cells.l. Clin. Invest. 77: 762-767, 1986.
31. Wedmore CV, Williams n. Control of vascular permeability by polymorphonuclear
leukocytes in inflammation. Nature 289: 646-650, 1981.
32. Weiss Sl. Tissue destruction by neutrophils. N. Engl. 1. Med. 320: 365-376.1989.
33. Del BaIza UH, Levi R, Polley MI. Cardiac dysfunction caused by purified human C3a
anaphylatoxin. Proc. Natl. Acad. Sci. USA 82: 886-890,1985.
34. Ito BR, Roth DM, Engler RL. Thromboxane A2 and peptidoleukotrienes contribute to
the myocardial ischemia and contractile dysfunction in response to intracoronary infu-
sion of complement C5a in pigs. Circ. Res. 66: 596-607, 1990.
35. Platt lL, Key NS, Dalmasso AP, Vercellotti GM, Lindman Bl, Bach FH. Complement
mediated loss of endothelial cell proteoglycans. Kidney. Int. 37: 599,1990 (Abstract).
36. Berden IH, Bogman Ml, Hageman FH, Tamboer WP, Koene RA. Complement-de-
pendent and independent mechanisms in acute antibody-mediated rejection of skin
xenografts in the mouse. Transplantation 32: 265-270, 1981.
37. Berk BC, Alexander RW, Brock T A. Gimbrone lr. MA, Webb RC. Vasoconstriction:
a new activity for platelet-derived growth factor. Science 232: 87-90,1986.
38. Schwertschlag US, Dennis VW, Tucker lA, Camussi G. Non-immunological alter-
ations of glomerular filtration by s-PAF in the rat kidney. Kidney Int. 34: 779-785,1988.
39. Armstead WM, Pourcyrous M, Mirro R, Leffler CW, Busija DW. Platelet activating
factor: a potent constrictor of cerebral arterioles in newborn pigs. Circ. Res. 62: 1-7,
1988.
40. Camussi G, Tetta C, Coda R, Segolini GP, Vercellone A. Platelet-activating factor-in-
duced loss of glomerular anionic charges. Kidney Int. 25: 73-81,1984.
41. Benveniste 1, Boullet C, Brink C, Labat C. The actions of P AF -acether (platelet -activat-
ingfactor) on guinea-pig isolated heart preparations. Br. 1. Pharmacol. 80: 81-83, 1983.
42. Ito S, Camussi G, Tetta C, Milgrom F, Andres G. Hyperacute renal allograft rejection
in the rabbit: the role of platelet-activating factor and of cationic proteins derived from
polymorphonuclear leukocytes and from platelets. Lab. Invest. 51: 148-161, 1984.
43. Whatley RE, Zimmerman GA, McIntyre TM, Prescott M. Endothelium from diverse
vascular sources synthesizes platelet-activating factor. Arteriosclerosis 8: 321-331,
1988.
44. Camussi G, Aglietta M, Malavasi F, Tetta C, Piacibello W, Sanavio F, Bussolino F.
The release of platelet-activating factor from human endothelial cells in culture.
1. ImmzlIlol. 131: 2397-2403, 1983.
45. Bogman, Ml, Berden IH, Hagemann FH, Maass CN, Koene RA. Patterns of vascular
damage in the antibody-mediated rejection of skin xenografts in the mouse. Am. 1.
Pathol. 100: 727-738, 1980.
46. Bogman Ml, Berden IH, Cornelissen 1M, Maass CN, Koene RA. The role of comple-
ment in the induction of acute antibody-mediated vasculitis of rat skin grafts in the
mouse. Am. 1. Pathol. 109: 97-106, 1982.
47. Winn HJ, Baldamus CA, Jooste SV, Russell PS. Acute destruction by humoral anti-
body of rat skin grafted to mice. The role of complement and polymorphonuclear
leukocytes. 1. Exp. Med. 137: 893-910. 1973.
48. Paul LC, Solez K. Chronic rejection of vascularized organ allografts. In: Organ
Transplantation: Long Term Results. Paul LC, Solez K (eds.). Marcel Dekker Inc., New
York (in press).
References 65
49. Reemtsma K, Pierson III RN, Marboe CC, Michler RE, Smith CR, Rose EA, Fenoglio
11 Jr. Will atherosclerosis limit clinical xenografting? Transplant. Proc. 19 (supp!. 5):
108-118,1987.
50. Ross R. The pathogenesis of atherosclerosis - an update. N. Engl. 1. Med. 314: 489-500,
1986.
51. Davies PF. Vascular cell interactions with special reference to the pathogenesis of
atherosclerosis. Lab. Invest. 55: 5-24, 1986.
52. Di Corleto PE, Bowen-Pope DF. Cultured endothelial cells produce a platelet-derived
growth factor-like protein. Proc. Natl. Acad. Sci. USA 80: 1919-1928, 1983.
53. Nilsson J, Sjolund M, PalmbergJ, ThybergJ, Heldin C-H. Arterial smooth muscle cells
in primary culture produce a platelet-derived growth factor-like protein. Proc. Nat!.
Acad. Sci. USA 82: 4418-4422, 1985.
54. O'Connell TX, Mowbray JF. Arterial intimal thickening produced by alloantibody and
xenoantibody. Transplantation 15: 262-263, 1973.
55. Friedman RJ, Moore S, Singal DP. Repeated endothelial injury and induction of
atherosclerosis in normolipemic rabbits by human serum. Lab. Invest. 30: 404 415,1975.
56. Leung DYM, Collins T, Lapierre LA, Geha RS, Pober JS. Immunoglobulin M antibod-
ies present in the acute phase of Kawasaki syndrome lyse cultured vascular endothelial
cells stimulated by gamma interferon. 1. Clin. Invest. 77: 1428-1435, 1986.
57. Leung DYM, Geha RS, Newburger JW, Burns JC, Fiers W, Lapierre LA, Pober JS.
Two mono kines, interleukin 1 and tumor necrosis factor, render cultured vascular en-
dothelial cells susceptible to lysis by antibodies circulating during Kawasaki syndrome.
~Exp.Med. 164:1958-1972,1986.
58. Fedoroff S, Webb SJ. Natural cytotoxic antibodies in human blood sera which react
with mammalian cells and bacteria. Nature 193: 80-81,1962.
59. Baumann A, Witebsky F. Un nouveau phenomene pour la demonstration de la toxicite
du serum. Ann. Inst. Pasteur 53: 282-289,1934.
60. Perper RJ, Najarian JS. Experimental renal heterotransplantation. I. In widely diver-
gent species. Transplantation 4: 377-388, 1966.
61. Rosenburg JC, Broersma RJ, Bullemer G, Mannen EF, Lenaghan R, Rosenberg BF.
Relationship of platelets, blood coagulation and fibrinolysis to hyperacute rejection of
renal xenografts. Transplantation 8: 152-161, 1969.
62. Rosenberg JC, Hawkins E, Rector F. Mechanisms of immunological injury during anti-
body-mediated hyperacute rejection of renal heterografts. Transplantation 11:
151-157,1971.
63. Gambiez L, Wei! BJ, Chereau Ch, Calmus Y, Houssin D. The hyperacute rejection of
guinea pig to rat heart xenografts is mediated by preformed IgM. Transplant. Proc. 22:
1058,1990.
64. Moberg A W, Shons AR, Gewurz H, Mozes M, Najarian JS. Prolongation of renal
xenografts by the simultaneous sequestration of preformed antibody, inhibition of
complement, coagulation and antibody synthesis. Transplant. Proc. 3: 538-541, 1971.
65. Rossman P, Matousovic K. The production oflesions in the rabbit kidney by xenoperfu-
sion with fresh human blood. Virchows Arch. fA]. 382: 95-111, 1979.
66. Lexer G, Cooper DKC, Wicomb WN, Rose AG, Rees J, Keraan M, Reichart B, Du Toit
E. Cardiac transplantation using discordant xenografts in a nonhuman primate mode!.
Transplant. Proc. 19: 1153-1154, 1987.
67. Jamieson SW. Xenograft hyperacute rejection. A new mode!. Transplantation 17:
533-534,1977.
68. Adachi H, Rosengard BR, Hutchins GM, Hall TS, Baumgartner WA, Borkon AM,
Reitz BA. Effects of cyclosporine, aspirin, and cobra venom factor on discordant car-
diac xenograft survival in rats. Transplant. Proc. 19: 1145-1148, 1987.
69. Forbes RDC, Guttmann RD. Mechanisms of humoral-mediated cardiac allograft rejec-
tion in vivo: controlled, comparative morphological studies utilizing inbred rat models.
Heart Transplantation 1: 196--202, 1982.
70. Frangipane LG, Poole TW, Barker CF, Silvers WK. Vulnerability of allogeneic and
xenogeneic pancreatic islets to alloantisera. Transplant. Proc. 9: 371-373, 1977.
71. Pcrloff LJ. Naji A. Barkcr CF. Islet sensitivity to humoral antibody. SlIrg. Forum 32:
390-391, 1981.
72. Chabot J. Weber C. Hardy MA. Rivera S, Bailey-Braxton D. Strausberg L, Wood M.
Chow J, Pi-Sunyer FX, Reemtsma K. Synergy ofALS and UV-B in prolongation ofpri-
mate-to-mouse islet xenograft survival. Transplant. Proc. 19: 1160-1165, 1987.
73. Baldamus CA. McKenzie IFe. Winn HJ, Russell PS. Acute destruction by humoral an-
tibody of rat skin grafted to mice. 1. Immmllnol. 110: 1532-1541, 1973.
74. Valdivia LA. Monden M, Gotoh M. Hasuike Y. Kubota N. Endoh W. Okamura J, Mori
T. Hepatic xenografts from hamster to rat. Transplant. Pmc. 19: 1158-1159. 1987.
75. Settaf A. Meriggi F, Van de Stadt J. Gane P. Crougneau S. Reynes M. Rouger P,
Houssin D. Delayed rejection of liver xenografts compared to heart xenografts in the
rat. Transplant. Proc. 19: 1155-1157.1987.
76. lwatsuki S, lwaki Y, Kano T, K1intmalm G. Koep LJ. Weil R. Starzl TE. Successful
liver transplantation from crossmatch-positive donors. Transplant. Proc. 13: 286-290.
1981.
77. Knechtle SJ. Kolbeck Pc. Tsuchimoto S, Coundouriotis A. Sanfilippo F, Bollinger RR.
Hepatic transplantation into sensitized recipients. Demonstration of hyperacute rejec-
tion. Transplantation 43: 8-12,1987.
78. Kano K, Milgrom F. Heterophile antigens and antibodies in medicine. ClIr. Top.
Microbiol. lmmllnol. 77: 43-69,1977.
79. Kashiwagi N. Brantigan CO. Brettschneider L. Groth CS. Starzl TE. Clinical reactions
and serologic changes after the administration of heterologous antilymphocyte globu-
lin to human recipients ofrenal homografts. Anl1. Intern. Med. 68: 275-286,1968.
80. McDonald Jc. A heterophile system in human renal transplantation. II. Relationship
to clinical renal transplantation and the HLA system. Transplantation 15: 123-126.
1973.
81. McDonald Jc. A heterophile system in human renal transplantation. l. Distribution of
antigens and reactivity of the antibodies. Transplantation 15: 116-121. 1973.
82. Ehrlich P. Morgenroth l. Ehrlich Gesammelte Arbeiten. Vol. 2. Berlin-Heidelberg:
Springer 1957.
83. Paul LC. Benediktsson H. Kingma l, Green BJ, Davidoff A W. Naturally-occurring and
induced rat anti-guinea pig antibodies: tissue reactivity and specificity. Transplant.
Proc.22:1057.1990.
84. Nossal GlV. The induction of immunological tolerance in rats to foreign erythrocytes.
Allstral. 1. Exp. Biol. 36: 235-244, 1958.
85. Egdahl RH. Roller FR. Swanson DL. Varco RL. Acquired tolerance to homografts and
heterografts in the rat. Ann. NY Acad. Sci. 73: 842-847,1958.
86. Michler RE, McManus RP. Smith CR. Sadeghi AN. Marboe Cc. Reemtsma K, Rose
EA. Prolongation of primate cardiac xenograft survival with cyclosporine. Trans-
plantation 44: 632-636,1987.
87. Valdivia LA, Monden M. Gotoh M. Hasuike Y, Kubota N. Ichikawa T, Nakano Y.
Okamura J, Mori T. An important role of the spleen in rejection of hamster-to-rat
xenografts. Tran~plant. Pmc. 20: 329-331,1988.
88. Linn BS, Jensen lA, Pardo V. Davies D, Franklin L. Relationship between structural
and functional changes in rejecting renal xenograft. Tramplant. Proc. 3: 527-530, 1971.
89. Van de Stadt J, Vendeville B, Weill B, Crougneau S, Michel A, Filipponi F. Icard P.
Renoux M. Lovel A. Houssin D. Discordant heart xenografts in the rat. Additional ef-
fect of plasma exchange and cyclosporine, cyclophosphamide, or splenectomy in delay-
ing hyperacute rejection. Transplantation 45: 514-518,1988.
90. Valdivia LA, Monden M, Gotoh M, Hasuike Y, Kubota N, Ichikawa T. Okamura J.
Mori T. Prolonged survival of hamster-to-rat liver xenografts using splenectomy and
cyclosporine administration. Transplantation 44: 759-763.1987.
91. Pierson III RN, Winn HJ, Russell PS, Auchincloss Jr H. CD4+ lymphocytes playa
dominant role in murine xenograft rejection. Transplant. Proc. 21: 519. 1989.
92. lugi T. Kano K. Milgrom F. Antibody induced lysis of nucleated cells in agar gel. Int.
Arch. Allergy 41: 739-753,1971.
References 67
93. Mori S, Kano K, Milgrom F. Heterophile antigen detectable by infectious mononucleo-
sis sera on bone marrow cells. Int. Arch. Allergy 51: 214-225,1976.
94. Kasukawa R, Kano K, Bloom M, Milgrom F. Heterophile antibodies in pathologic hu-
man sera resembling antibodies stimulated by foreign species sera. Clin. Exp.
Immunol. 25: 122-132, 1976.
95. Siddiqui B, Hakomori S. A revised structure for the Forssman glycolipid hapten. I. Bioi.
Chem. 246: 5766-5769, 1971.
96. Tanuka N, Leduc EH. A study of the cellular distribution of Forssman antigen in vari-
ous species. I. Immunol. 77: 198--212,1956.
97. Hawes MD, Coombs RRA. Cellular distribution of Forssman membrane antigen in the
guinea pig determined by the mixed agglutination reaction. I. Immunol. 84: 586-589,
1960.
98. Leibowitz S, Morgan RS, Berkinshaw-Smith EMl, Payling-Wright G. Cerebral vascu-
lar damage in guinea pigs induced by various heterophile antisera injected by the
Forssman intracarotid technique. Brit. I. Exp. Path. 42: 455-463, 1961.
99. Metzgar RS, Zmijewski CM. Species distribution of human tissue isoantigens. I.
Detection of human tissue isoantigens in chimpanzees. Transplantation 4: 84-93, 1966.
100. Bainer H, Van Leeuwen A, Dersjant H, Van Rood JJ. Defined leukocyte antigens of
chimpanzees: use of chimpanzee isoantisera for leukocyte typing in man.
Transplantation 5: 624-642,1967.
101. Bainer H, Van Leeuwen A, Van Vreeswijk W, Dersjant H, Van Rood JJ. Leukocyte
antigens of chimpanzees and their relation to human HL-A antigens. Transplant. Proc.
2: 454-462, 1970.
102. Dorf ME, Eguro SY, Amos DB. Cross-reactions of HL-A antibodies. IV. Absorptions
and elutions with primate platelets. Transplant. Proc. 4: 71-75, 1972.
103. Gotze D (ed). The Major Histocompatibility System in Man and Animals. Springer-
Verlag, Berlin, Heidelberg, New York, 1977.
104. Staines NA. Relative immunogenicity of H-2 and other membrane components in
xenoimmunization. Transplantation 17: 470-476,1974.
105. Sachs DH, Cone JL. A mouse B-cell alloantigen determined by gene(s) linked to the
major histocompatibility complex. I. Exp. Med. 138: 1289-1304, 1977.
106. Turman MA, Casali P, N otkins AL, Fischel RJ, Lindman BJ, Vercellotti G M, Bach FH,
Platt JL. Human monoclonal and polyclonal xenoreactive natural antibodies (XNA).
Kidney Int. 37: 601, 1990 (Abstract).
107. Miyagawa S, Hirose H, Shirakura R, Naka Y, Nakata S, Kawashima Y. Seya T,
Matsumoto M. Uenaka A, Kitamura H. The mechanism of discordant xenograft rejec-
tion. Transplantation 46: 825-830, 1988.
108. Larsen S, Starklint H, Dieperink H, Kemp E. Immunofluorescence microscopy in ex-
perimental renal allo- and xenografts. Transplant. Proc. 22: 1061-1062, 1990.
109. Cochrane CG, Muller-Eberhard HJ, Aikin BS. Depletion of plasma complement in
vivo by a protein of cobra-venom: its effect on various immunologic reactions.
I. Immunol. 105: 55-69, 1970.
110. Vogel CW, Smith CA, Muller-Eberhard HJ. Cobra venom factor: structural homology
with the third component of human complement. I. Immunol. 133: 3235-3241, 1984.
111. Rosengard BR, Hall TS, Imagawa DK, Ueda K, Baumgartner WA, Borkon M, Smith
RRL, Hutchins GM, Reitz BA. Hyperacute rejection of cardiac xenograts : two new
models showing differences in pathogenesis. Surg. Forum 36: 361-365, 1985.
Chapter 5
Mechanism of Tissue Injury

in Hyperacute Xenograft Rejection
J.L. PLATT AND F.H. BACH
Introduction
For nearly three-quarters of a century, it has been known that the transplanta-
tion of an organ from an individual of one species into an individual of a phylo-
genetically distant species leads rapidly and inexorably to a violent form of
rejection that culminates in the demise of the organ graft [1-5]. This phe-
nomenon of hyperacute xenograft rejection poses what appears to be an abso-
lute barrier to clinical xenotransplantation [4].
Perper and Najarian [2] made the critical observation that antibody from
the recipient was deposited in the rejected organ, and Linn et al. [6] that deple-
tion of natural antibody by absorption would enable prolonged survival of a
vascularized xenograft. Sir Roy CaIne suggested a paradigm wherein suscepti-
bility to hyperacute rejection of cross-species grafts might be predicted by the
presence in the serum of the recipient animal of natural antibodies reactive
with hemopoietic cells of a would-be discordant donor [7]. While natural anti-
bodies demarcated the barrier to xenotransplantation, other investigators
suggested that the activation of complement [8-11], formation of platelet or
fibrin thrombi [12, 13], adherence of neutrophils to vessel walls, or vasocon-
striction [12-15] might in various combinations playa predominant role in the
pathogenesis of hyperacute xenograft rejection. Thus, there remains some
doubt concerning which factors initiate hyperacute rejection in certain models
and how the aforementioned pathophysiological phenomena connect the in-
citing events with the common pathological picture seen in the rejecting organ
[4].
In this chapter we shall summarize recent observations from our laborato-
ries which we believe provide a conceptual framework for understanding the
cellular and molecular pathogenesis of hyperacute xenograft rejection [16].
This model takes as its premise that the reaction of natural antibodies with
donor endothelial cells sparks a series of events that lead to tissue injury.
These events, we hypothesize, reflect metabolic and structural changes by en-
dothelial cells which together are commonly termed endothelial cell activa-
tion. In sum, we view the endothelial cell as both the target and the instrument
of xenograft destruction. It must be emphasized that this current synthesis is a
model that may be useful for testing certain hypotheses but which should, like
all models, be modified and if necessary abandoned as new observations
emerge.
70 Mechanism of Tissue Injury in Hyperacute Xenograft Rejection
Natural Antibodies and B Cells
Despite the predominant role of humoral immunity in the rejection of vascu-

larized xenografts, there are few published data concerning the distinguishing
properties, if any, of xenoreactive natural antibodies or of the cells that syn-
thesize them. This information might be used in a variety of ways to develop
strategies for depletion of natural antibodies.
Our observations [16-18], and those of others [19,20] suggest that if anti-
graft antibodies are temporarily depleted from the circulation of a recipient of
an ABO-incompatible allograft, with anti-human leukocyte antigen (HLA)
antibodies directed at the donor organ or at a discordant xenograft, the organ
may survive despite the eventual return of antigraft antibodies to the circula-
tion. This phenomenon, which we have called "accommodation" [16] and
have discussed in detail elsewhere in this volume (Chap. 6), offers some
promise that the existence of preformed antibodies against a donor organ may
not pose an absolute barrier to transplantation.
All mammalian species have circulating alloreactive, natural antibodies di-
rected against blood group antigens [21-23]. Since these natural antibodies
are capable of causing hyperacute rejection of renal or cardiac allografts [24,
25], it has been hypothesized that similar antibodies, which agglutinate xeno-
geneic red blood cells, may contribute to the hyperacute rejection of organ
xenografts [26]. Moreover, the structural properties of certain blood group
antigens and observations deriving from the transplantation of organs across
blood group barriers may provide a very useful model [16, 22, 26].
Most blood group antigens consist of carbohydrate structures associated
with protein or lipid cores [27,28]. The antibodies that recognize such deter-
minants are often of the immnunoglobulin M (IgM) isotype. The carbohy-
drate epitopes on blood group antigens may be shared by a number of differ-
ent glycoconjugates. For example, the blood group A antigen may be linked to
anyone of several core carbohydrate structures attached to a variety of pro-
tein or lipid cores [27]. Thus, a limited number of determinants may be associ-
ated with a far greater variety of macromolecules.
Natural antibody titers are commonly assayed by hemagglutination or lym-
phocytotoxicity [2,29,30]. Such assays will predict the outcome of an organ
xenograft to the extent that endothelial cells and hemopoietic cells express
similar or cross-reactive determinants. However, if the determinants are locat-
ed on complex carbohydrate structures as discussed above, there is no assur-
ance that the antigens (recognized by natural antibodies) on blood cells would
have the same structure and proportionate representation as the antigens on
endothelial cells. Indeed, we have recently demonstrated that human and rhe-
sus monkey natural antibodies recognize different macromolecules on
porcine erythrocytes and lymphocytes than they do on porcine endothelial
cells [31]. Since we view blood vessels as the major target of natural antibodies
in hyperacute xenograft rejection, we have emphasized the use of endothelial
cells for the assay of natural antibodies [32] and for the characterization of nat-
ural antibody targets.
Endothelial Cell Antigens Recognized by Natural Antibodies 71
In contrast to elicited antibodies, at least some naturally occurring antibod-

ies are manifestly polyreactive [33,34] ; for example, natural antibodies which
recognize fetuin also react with tubulin [33]. Polyreactive natural antibodies
derive exclusively from a population of B cells that expresses the CDS glyco-
protein on the cell surface [3S]. Autoantibodies made by CDS+ (Lyl+) B cells
include low-affinity, highly cross-reactive, IgM rheumatoid factors and appear
to be encoded by a restricted set of light and heavy chain V region genes [36].
Other autoantibodies synthesized by CDS+ B cells bind to phosphatidyl-
choline (exposed by protease treatment) in biologic membranes [37].
Polyreactive autoantibodies are thought to provide initial protection
against invasive microorganisms or prevent autosensitization [21, 36, 38].
Additionally, since some polyreactive antibodies recognize cells after prote-
olytic cleavage of cell surface moieties, it is possible the antibodies might help
to clear damaged or infected cells from the circulation [39,40].
Turman, in our laboratories, has recently shown that human serum con-
tains xenoreactive natural antibodies that can be partially blocked with com-
pounds commonly recognized by polyreactive antibodies and that polyreac-
tive human monoclonal antibodies, presumably from CDS+ B cell lines, show
specificity toward xenogeneic cell membrane components [41,42]. Our stud-
ies thus suggest that CDS+ B cells may provide a component of the human nat-
ural antibody repertoire and as such might participate in the rejection of an or-
gan xenograft.
The permanent engraftment of a discordant xenograft would likely require
at least temporary depletion of circulating xenoreactive natural antibodies
and/or of the cells that synthesize them. If xenoreactive natural antibodies,
which potentially recognize a highly diverse group of macromolecules, have
properties similar to polyreactive natural antibodies or to antiblood group an-
tibodies, as suggested above, then it is possible that such antibodies might be
immunoabsorbed with a relatively limited number of ligands.
Endothelial Cell Antigens Recognized by Natural Antibodies
The identity of the target antigens recognized by human natural antibodies

has been a vexing question in the field of xenotransplantation. Whether the
antigens play an active role in the pathogenesis of vascular lesions or serve
merely as a passive target is a salient question. Further, there is a need to iden-
tify the targets of natural antibodies in order to measure precisely natural anti-
body concentrations and develop the means specifically to remove natural an-
tibodies from the circulation.
We carried out a series of experiments directed at identifying the compo-
nents of porcine endothelial cells that would be recognized by natural antibod-
ies if a porcine organ were transplanted into a human or rhesus monkey [31].
Such antigens might include glycolipids, proteoglycans, or glycoproteins.
Extracts derived from porcine endothelial cells and containing endothelial cell
72 Mechanism ofTissuc Injury in Hyperacute Xenograft Rejection
200-
135
-../125
116 -- -./
_115
95--
567
65--
29--
2 3 4
Fig. 5.1. Porcine endothelial cell glycoproteins recognized by human natural antibodies. Of
the numerous cell membrane glycoproteins resolved by electrophoresis (lane I), human
serum is demonstrated in lane 2 to react predominantly with moieties at 115-135 kDa (bands
at65 kDa and 80 kDa are artifactual). Human serum reacts weakly with cytoplasmic compo-
nents (lane 3). The predominant antigens recognized by human natural antibodies comi-
grate with cell surface glycoconjugates labeled with 'H (lane 4) and appear as three discrete
bands after H PLC (lanes 5-7). Lane I, protein stain of glycoproteins isolated from porcine
endothelial cell membranes, resolved in an 8% - 18'Yo polyacrylamide gel and transferred to
nitrocellulose; lane 2, glycoproteins resolved as in lane l, reacted with human serum and
stained for human IgM ; lane 3, endothelial cell cytosol stained with human serum; lane4, au-
toradiogram of extract from endothelial cells labelled with ['H]NaBH4 after treatment with
neuraminidase and galactose oxidase; Inset (lanes 5-7), reactivity of human serum with
porcine endothelial cell glycoproteins partially separated by reverse phase HPLC. (From
[311)
lipids or endothelial cell proteoglycans did not react substantially with human
serum, whereas extracts containing endothelial cell membrane glycoproteins
contained a number of immunoreactive components.
Western blot analysis demonstrated that of the numerous porcine aortic
endothelial cell membrane glycoproteins detected by natural antibodies in
human serum, the most intensely reactive components migrated in gels at
115-135 kDa (gp115/135) (Fig. 5.1). Separation by reversed phase high-per-
formance liquid chromatography (HPLC) revealed that the complex consist-
ed of 115 kDa, 125 kDa, and 135 kDa components (Fig. 5.1. inset). Recent
studies have shown that the gp115/135 triad is expressed by porcine microvas-
cular endothelial cells, the 135 kDa moiety prominently so.
To test the potential biologic relevance of the antigens recognized by hu-
man natural antibodies, we first showed that rhesus antibodies recognize anti-
Complement 73
Fig. 5.2. Reactivity with components of porcine aortic endothelial cell

membranes of rhesus monkey serum obtained before (lane I) and
after (lane 2) in vivo absorption of natural antibodies by perfusion of
porcine kidneys. (From [31]) 2
gens which comigrate in single dimensional studies, and in the case of gp135 in
two-dimensional gels, with the antigens recognized by human sera. Further,
rhesus serum obtained after perfusion of two porcine kidneys was no longer
reactive with gp115/135 complex in western blots (Fig. 5.2). These results sug-
gest that the gp115/135 moieties are expressed on porcine endothelial cell sur-
faces and can be recognized by natural antibodies in the circulation. At a later
time, when the cardiac xenograft was rejected, antibodies reactive with cul-
tured porcine endothelial cells and with gp 115/135 appeared in the circulation.
Human natural antibody binding was abrogated after digestion of porcine
endothelial cell membrane with a specific series of exoglycosidases and with
certain endoglycosidases but not by treatment with proteinases. These studies
suggest that human natural antibodies recognize carbohydrate determinants
located on oligosaccharide substitutions. Moreover, human and rhesus natu-
ral antibodies recognize the same immunodominant sugars associated with
the same glyoproteins suggesting the epitopes recognized by human and rhe-
sus are similar.
Complement
A number of in vivo data suggest that complement activation is a critical event

in the pathogenesis of hyperacute rejection [8-11]. Complement may be acti-
vated by the classical pathway when Clq binds to aggregates of IgG or to indi-
vidual molecules of IgM on cell surfaces. Independent of antibody binding,
C3b, which is formed by spontaneous cleavage of C3, may complex with factor
B to yield C3b-factor B complex. Cleavage of factor B yields C3bBb, the alter-

native pathway convertase which cleaves C3 to form additional C3b.
Formation of C3 convertase by the classical or alternative pathway gener-
ates biologically active fragments of C3 (C3a) and C5 (C5a) and causes the for-
mation of the C5b-9 complex which leads to polymerization of C9 to form the
membrane attack complex (MAC). The reader is referred to recent reviews
for more detailed discussion [43,44].
Several complement products may play significant roles in vascular injury.
C3a and C5a may mediate vasoactive phenomena by activating inflammatory
cells. Our colleagues G. Vercellotti and A. Dalmasso, with us, have shown that
the presence of C3b on the endothelial cell surface promotes adherence of
neutrophils and promotes formation of the classical or alternative pathway C3
convertase which initiates reactions leading to assembly of the terminal com-
ponents and polymerization of C9 as described above. The MAC forms poten-
tially lethal pores on endothelial surfaces and exposes the prothombinase
complex [45]. As will be discussed below, the activation of complement also
activates or exposes an enzyme which mediates cleavage of endothelial cell-
associated heparan sulfate, a glycosaminoglycan that is structurally and
biosynthetically related to heparin.
A Model of Hyperacute Xenograft Rejection
The model of hyperacute rejection we have found most useful focuses on the
endothelial cell as the initial target of natural antibodies and as a cell the aber-
rant function of which might directly cause the pathological picture of hyper-
acute rejection [16] (Fig. 5.3). Normal, resting endothelial cells function ac-
tively to provide a barrier to the egress of protein and cells from blood vessels
and to inhibit intravascular thrombus formation. The surface of normal en-
dothelial cells is not adherent for platelets or neutrophils. When, however, en-
dothelial cells are perturbed by noxious stimuli or by cytokines their function-
al properties change dramatically. These changes are termed endothelial cell
activation [46-51].
Endothelial cell activation is associated with conversion of the endothelial
cell surface from anticoagulant to procoagulant through the elaboration of
substances such as tissue factor and plasminogen activator inhibitor and
through the loss of thrombomodulin, a cell surface protein which together
with thrombin activates the circulating anticoagulant protein C. Activated en-
dothelial cells elaborate platelet-activating factor (PAF), which promotes ad-
hesion and aggregation of platelets, and cell adhesion molecules such as
GMP140 and endothelial-leukocyte adhesion molecules (ELAM), which pro-
mote adherence of inflammatory cells [4H, 52J.
In addition to the aforementioned phenomena, we have recently shown
that the action of natural antibodies and complement on endothelial cells
causes the rapid cleavage and loss of heparan sulfate from cultured endothe-
A Model of Hyperacute Xenograft Rej ection 75
Natural Antibody and Complement

~
Endothelial Cell Activation
- ~
platelet activating faclor platelet aggregation. vasoconstrictio n
thrombomodulin loss fibrin
plasminogen activator inhibition fibrin
tissue factor synthesis fibrin
heparan sulfate loss fibrin, interstitial edema & hemorrahage
cell adhesion molecules PMN adhesion
Fig. 5.3. A model for the pathogenesis of hyperacute rejection. The pathological findings of
hyperacute rejection which include platelet aggregation, intravascular coagulation, intersti-
tial edema and hemorrhage, and neutrophil adhesion are hypothesized to derive from en-
dothelial cell activation (see [16] for review). Endothelial cell activation, which is associated
with heparan sulfate release [53] and the other phenomena listed [46-52] may be caused by
fixation of natural antibodies and activation of complement. (From [16])
lial cells (Fig. 5.4) [53]. Heparan sulfate proteoglycan is thought to promote
the integrity of blood vessels by inhibiting the loss of blood cells and plasma
proteins through blood vessel walls, and by promoting the adherence of en-
dothelial cells to extracellular matrix. Heparan sulfate mediates attachment of
endothelial surfaces and activation of antithrombin Ill, a major anticoagulant
[54, 55] , and superoxide dismutase, which clears potentially injurious free oxy-
gen radicals [56, 57] . Thus, the loss of heparan sulfate proteoglycan from en-
dothelium may well contribute significantly to many of the pathological fea-
tures of hyperacute rejection.
Other consequences of complement activation on endothelial surfaces
might affect the course of hyperacute rejection. The formation of C5b-9 com-
plexes on endothelial cell surfaces causes vesiculation of endothelial cells [45].
60
50
Fig. 5.4. The rapid loss of heparan

sulfate from cultured porcinc
.
::
40
aortic endothelial cells is trig-
.
.!!
a: 30
gered by the binding of human
natural antibodies and the activa-
C tion of complement. Endothelial
"~ cell proteoglycans were labelled
"
Q.
20 biosynthetically with [' OS I sulfate.
washed. and then incubated in
25% human serum . The percent-
10 age of '5S-macromolecules re-
leased into culture medium was
determined. The released macro-
0 molecules were characterized as
8 15 30 60 120 240
heparan sulfate. as detailed in
Minutes 153J
The vesicles released from endothelial cells express binding sites for activated
coagulation factors Xa and Va. These vesicles might thereby provide a nidus
for thrombus formation. (Conversely, such vesicles may provide mechanisms
for removal of potentially lytic complement components from the cell surface
and may, since they spare thrombomodulin. preserve the antithrombotic sur-
face of the affected cell.) Vercellotti, Dalmasso. we [58] and others [59] have
observed that complement activation promotes the adherence to endothelial
cells of neutrophils via complement (C3bi) receptors (CR3). Adherent neu-
trophils may further damage endothelium and cause the cleavage of endothe-
lial cell proteoglycans [60].
Because the vascular lesions of hyperacute rejection develop in minutes to
a few hours. it would appear most appropriate to focus on the loss of heparan
sulfate. loss of thrombomodulin, complement-mediated vesiculation. and ad-
herence of neutrophils to CR3 as potentially critical events in the pathogenesis
of hyperacute rejection. The model proposed herein suggests a number of
questions which might be addressed by future investigations. Such questions
include the specific roles of endothelial cell surface antigens and of comple-
ment in triggering activation; the identification of which of the many pheno-
mena associated with endothelial cell activation actually contributes to graft
destruction and the development of agents which might inhibit or reverse
these phenomena. Surely. if this model is valid. it is likely that the successful
treatment of the consequences of endothelial cell activation will require the
use of multiple agents and modalities to address the multifaceted phenomena
associated with hyperacute rejection.
Acknowledgements. The work described in this chapter derives from a pro-

grammatic effort involving the Departments of Laboratory Medicine and
References 77
Pathology, Surgery, Pediatrics and Medicine at the University of Minnesota.
The authors gratefully acknowledge the manifold contributions of their senior
co-workers, Drs. Bolman, Dalmasso, Matas, Najarian and Vercellotti. This
work was supported in part by grants from the NIH (AI17687 and DK13083),
the March of Dimes Birth Defects Foundation, and the Minnesota Medical
Foundation. Jeffrey L. Platt is an Established Investigator of the American
Heart Association; Fritz H Bach holds the Harry Kay Chair in
Immunobiology. This is paper #549 from the Immunobiology Research
Center.
References
1. Reemtsma, K., McCracken, B.H., Schlegel, J.U., Pearl, M.A, Pearce, C.W., DeWitt,
C.W., Smith, P.E., Hewitt, RL., Flinner, RL., Creech, O. Renal heterotransplantation
in man. Ann. Surg. 160,384, 1964.
2. Perper, RJ., Najarian, J.S. Experimental renal heterotransplantation. 1. In widely diver-
gent species. Transplantation. 4,337,1966.
3. Giles, G.R, Boehmig, H.J., Lilly, J., Amemiya, H., Takagi, H., Coburg, AJ., Hathaway,
W.E., Wilson, C.B., Dixon, F.J., Starzl, T.E. Mechanism and modification of rejection of
heterografts between divergent species. Transplant. Proc. 2,522, 1970.
4. Auchincloss, H. Xenogeneic transplantation. Transplantation. 46, 1, 1988.
5. Reemtsma, K. Xenotransplantation : a personal history. In Xenograft 25. M.A. Hardy
(Ed.), Elsevier Science Publishers, New York, 1989, p. 7.
6. Linn, B.S., Jensen, J.A, Portal, P., Snyder, G.B. Renal xenograft prolongation by sup-
pression of natural antibody.f. Surg. Res. 8,211,1968.
7. Caine, RY. Organ transplantation between widely disparate species. Transplant. Proc.
2, 550, 1970.
8. Gewurz, H., Clark, D.S., Cooper, M.D., Varco, RL., Good, RA Effect of cobra venom-
induced inhibition of complement activity on allograft and xenograft rejection reactions.
Transplantation. 5,1296,1967.
9. Mejia-Laguna, J.E., Martinez-Palomo, A, Biro, C.E., Chavez, B., Lopez-Soriano, F.,
Garcia-Cornejo, M. Morphologic study of the participation of the complement system in
hyperacute rejection of renal xenotransplantation. Am. f. Pathol. 69,71,1972.
10. Kemp, E., Steinbruchel, D., Starklint, H., Larsen, S., Henriksen, 1., Dieperink, H. Renal
xenograft rejection: prolonging effect of captopril, ACE-inhibitors, prostacyclin and
cobra venom factor. Transplant. Proc. 19,4471,1987.
11. Miyagawa, S., Hirose, H., Shirakura, R, Naka, Y., Nakata, S., Kawashima, Y., Seya, T.,
Matsumoto, M., Uenaka, A, Kitamura, H. The mechanism of discordant xenograft re-
jection. Transplantation. 46,825,1988.
12. Rosenberg, J.c., Hawkins, E., Rector, F. Mechanisms of immunological injury during
antibody-mediated hyperacute rejection of renal heterografts. Transplantation. 11, 151,
1971.
13. Colman, RB., Habal, M., Hollenberg, N.K., Birtch, AG., Busch, G.J. Hyperacute renal
allograft rejection in the primate. Am. f. Pathol. 82,25, 1976.
14. Busch, G.J., Martins, A.C.P., Hollenberg, N.K., Moretz. RC., Wilson, RE., Colman,
RW. Successful short-term modification of hyperacute renal allograft rejection in the
primate. Am. f. Pathol. 82,43, 1976.
15. Marceau, J.P., Hallenbeck, G.A., Zollman, P.E., Butler, H.C., Shorter, RG. A compari-
son of autoplastic, allogeneic and xenogeneic perfusion of isolated kidneys. f. Surg. Res.
5,492,1965.
16. Platt, J.L., Vercellotti, G.M., Dalmasso, AP., Matas, AJ., Bolman, RM., Najarian, I.S.,
Bach. F.H. Transplantation of discordant xenografts : a review of progress. Immunol.

Today. 11,450. 1990.
17. Fischel, Rl., Bolman, RM., Platt, 1.L.. Najarian, 1.S .. Bach, F.H., Matas, A.l. Removal
of IgM anti-endothelial antibodies results in prolonged cardiac xenograft survival.
Transplant. Proc. 22, 1077, 1990
18. Chopek, M.W., Simmons, RL.. Platt.l.L. ABO incompatible renal transplantation: ini-
tial immunopathologic evaluation. Transplant. Proc. 19,4553,1987.
19. Palmer, A, Welsh. K., Gjorstrup, P., Taube, D., Bewick, M., Thick, M. Removal of anti-
Lancet. 1.10,1989.
20. Alexandre, G.P.J., Gianello, P., Latinne, D., Carlier, M., Dewaele, A, Van Obbergh. L.,
Moriau, M., Marbaix, E .. Lambotte, 1.L., Lambotte, L.. Squifflet, 1.P. Plasmapheresis
and splenectomy in experimental renal xenotransplantation. In Xenograft 25. M.A
Hardy (Ed.), Elsevier Science Publishers, New York, 1989, p. 259.
21. Boyden, S.V. Natural antibodies and the immune response. Adv. Immunol. 5, 1, 1964.
22. Samuelsson, B.E., Breimer, M.E. ABH antigens: some basic aspects. Transplant. Proc.
19,4401,1987.
23. Oriol, R, Le Pendu, 1., Mollicone, R Genetics of ABO, H, Lewis, X and related anti-
gens. Vox. Sang. 51, 161, 1986.
24. Paul, L.c., Van Es, LP., de la Riviere, G.B., Eernisse, G., de Graeff, 1. Blood group B
antigen on renal endothelium as the target for rejection in an ABO-incompatible recipi-
ent. Transplantation. 26,268,1978.
25. Pikul, F.l .. Bolman, RM., Saffitz, 1.E., Chaplin, H. Anti-B-mediated rejection of an
ABO-incompatible cardiac allograft despite aggressive plasma exchange transfusion.
Transplant. Proc. 19,4601,1987.
26. Hammer, C. Isohemagglutinins and preformed natural antibodies in xenogeneic organ
transplantation. Transplant. Proc. 19,4443,1987.
27. Clausen, H., Hakomori, S. ABH and related histo-blood group antigens; immunochem-
ical differences in carrier isotypes and their distribution. Vox. Sang. 56, 1,1989.
28. Anstee, D.l. Blood group-active surface molecules of the human red blood cell. Vox.
Sang. 58, 1, 1990.
29. Deodhar, S.D. Review of xenografts in organ transplantation. Transplant. Proc. 18,83,
1986.
30. Hammer, C. Preformed natural antibodies (Pnab) and possibilities of modulation of hy-
peracute xenogeneic rejection (Hxar). Transplant. Proc. 21,522,1989.
31. Platt, 1.L., Lindman, B.l., Chen, H., Spitalnik, S.L., Bach, F.H. Endothelial cell antigens
recognized by xenoreactive human natural antibodies. Transplantation. 50,817, 1990.
32. Platt, 1.L., Turman, M.A, Noreen, H., Fischel, Rl., Bolman, RM., Bach, F.H. An Elisa
assay for xenoreactive natural antibodies. Transplantation. 49, 1000, 1990.
33. Sela, B.A, Wang, 1.L., Edelman, G.M. Antibodies reactive with cell surface carbohy-
drates. Proc. Nat. Acad. Sci. USA. 72,1127,1975.
34. Guilbert, B., Dighiero, G., Avrameas, S. Naturally occurring antibodies against nine
common antigens in human sera. I. Detection, isolation and characterization. f.
Immunol. 128,2779,1982.
35. Casali, P., Notkins, AL. Probing the human B-cell repertoire with EBV: polyreactive
antibodies and CD5+ B lymphocytes. Ann. Rev. Immunol. 7,513,1989.
36. Casali, P., Notkins, AL. CD5+ B lymphocytes, polyreactive antibodies and the human
B-cell repertoire. Immunol. Today. 10,364,1989.
37. Mercolino, T.l., Locke, AL., Afshari, A, Sasser, D., Travis, W.W., Arnold, L.W.,
Haughton, G. Restricted immunoglobulin variable region gene usage by normal Ly-I
(CD5+) B cells that recognize phosphatidyl choline.f. Exp. Med. 169,1869,1989.
38. Fong, S., Chen, P.P., FOX, RI., Goldfien, RO., Radoux, V., Silverman, G.l., Crowley,
1.1., Roudier, 1., Carson, D.A. The diversity and idiotype patterns of human rheumatoid
factors in disease. Concepts Immunopathology. 5, 168, 1988.
39. Cunningham, A.l. Large numbers of cells in normal mice produce antibody components
of isologous erythrocytes. Nature. 252,749, 1974.
References 79
40. Galili, U., Flechner, I., Knyszynski, A., Danon, D., Rachmilewitz, E.A. The natural anti-
galactosyl IgG on human normal senescent red blood cells. Br. 1. Haernatol. 62,317,
1986.
41. Turman, M.A., Casali, P., Notkins, A.L., Fischel, R.J., Lindman, BJ., Vercellotti, G.M.,
Bach, F.H., Platt, J.L. Human monoclonal and polyclonal xenoreactive natural antibod-
ies. (Abstract) Kidney Int. 37,601,1990.
42. Turman, M.A., Bach, F.H., Casali, P., Platt, J.L. Polyreactive antibodies from CD5+ B
cells may constitute a component of the repertoire of xenoreactive natural antibodies
(XNA). (Abstract) Fasebl. In press.
43. Muller-Eberhard, HJ. Molecular organization and function of the complement system.
Ann. Rev. Biochern. 57,321,1988.
44. Dalmasso, A.P. CRC complement in the pathophysiology and diagnosis of human dis-
eases. Crit. Rev. C/in. Lab. Sci. 24, 123, 1986.
45. Hamilton, K.K., Hattori, R., Esmon, C.T., Sims, P.l. Complement proteins C5b-9 induce
vesiculation of the endothelial plasma membrane and expose catalytic surface for assem-
bly of the prothrombinase enzyme complex. f. Bioi. Chern. 265,3809,1990.
46. Nawroth, P.P., Stern, O.M. Modulation of endothelial cell hemostatic properties by tu-
mor necrosis factor. 1. Exp. Med. 163,740,1986.
47. Ryan, U.S. The endothelial surface and responses to injury. Fed. Proc. 45, 101, 1986.
48. Bevilacqua, M.P., Pober 1.S., Mendrick, D.L., Cotran, R.S., Gimbrone, M.A. JR
Identification of an inducible endothelial-leukocyte adhesion molecule. Proc. Natl.
Acad. Sci. USA. 84,9238,1987.
49. Pober, J .S. Cytokine-mediated activation of vascular endothelium. Arn. 1. Patho!. 133,
426,1988.
50. Schleef, R.R., Bevilacqua, M.P., Sawdey, M., Gimbrone, M.A. JR., Loskutoff, OJ.
Cytokine activation of vascular endothelium. Effects on tissue-type plasminogen activa-
tor and type 1 plasminogen activator inhibitor. 1. Bio!. Chern. 263,5797, 1988.
51. Brett, J., Gerlach, H., Nawroth, P., Steinberg, S., Godman, G., Stern, D. Tumor necrosis
factor/cachectin increases permeability of endothelial cell monolayers by a mechanism
involving regulatory G proteins. 1. Exp. Med. 169,1977,1989.
52. Geng, J.G., Bevilacqua, M.P., Moore, K.L., McIntyre, T.M., Prescott, S.M., Kim, 1.M.,
Bliss, G.A., Zimmerman, G.A., McEver, R.P. Rapid neutrophil adhesion to activated
endothelium mediated by GMP-140. Nature. 343,757,1990.
53. Platt, 1.L., Vercellotti, G.M., Lindman, G.J., Oegema, T.R., Bach, F.H., Oalmasso, A.P.
Release of hepar an sulfate from endothelial cells. 1. Exp. Med. 171,1363,1990.
54. Marcum, J.A., Reilly, C.F., Rosenberg, R.D. Heparan sulfate species and blood vessel
wall function. In Biology of Proteoglycans. T.N. Wright and R.P. Mecham (Eds.),
Academic Press, Orlando, 1987. P 301.
55. Marcum, J.A., Atha, D.H., Fritze, L.M.S., Nawroth, P., Stern, D., Rosenberg, R.D.
Cloned bovine aortic endothelial cells synthesize anticoagulantly active heparan sulfate
proteoglycan.l. BioI. Chern. 261,7507,1986.
56. Karlsson, K., Marklund, S.L. Heparin-induced release of extracellular superoxide dis-
mutase to human blood plasma. Biochern. 1. 242,55,1987.
57. Karlsson, K., Marklund, S.L. Plasma clearance of human extracellular superoxide dis-
mutase C in rabbits. 1. Clin. Invest. 82,762, 1988.
58. Vereellotti, G.M., Platt, 1.L., Bach, F.H., Dalmasso, A.P. Enhanced neutrophil adhesion
to xenogeneic endothelium via C3bi. Submitted for publication.
59. Marks, R.M., Todd, R.F. Ill, Ward, P.A. Rapid induction of neutrophil-endothelial ad-
hesion by endothelial complement fixation. Nature. 339,314,1989.
60. Vercellotti, G.M., Key, N.S., Platt, 1.L., Jacob, H.S. Neutrophil proteases cleave en-
dothelial proteoglycan eore protein: potential role in inflammatory thrombosis.
(Abstract)l. Cell Biochern. SuppI.14E,217, 1990.
Chapter 6
Accommodation -
The Role of Natural Antibody and Complement
in Discordant Xenograft Rejection
F.H. BACH, 1.L. PLATT, AND D.K.C. COOPER
Introduction
With the increasing problem of donor organ shortages, the potential value of
achieving successful discordant xenografting takes on special importance.
Formidable problems must be solved before discordant xenografting can be-
come a clinical reality. Nevertheless, the availability of new agents that may
well ameliorate certain of the undesirable processes involved in the rejection
of xenografts and the power of molecular biology, which may allow the "de-
sign" of immunologically compatible organs, encourage an effort in this re-
gard.
The most vexing problem associated with discordant xenografting is hyper-
acute rejection which, in turn, appears to involve the action of natural antibod-
ies and complement of the recipient on donor endothelium [1]. The binding of
natural antibody to blood vessels in the graft and the activation of complement
ultimately cause the loss of vascular integrity, the formation of platelet and fib-
rin thrombi, and the infiltration of neutrophils through graft microvascula-
ture.
While the pathogenesis of hyperacute rejection at the cellular and molecu-
lar level has not been elucidated, we are currently testing the hypothesis that
the reaction of antibody and complement with blood vessels causes activation
of endothelial cells. (The implications of endothelial cell activation in this con-
text are discussed in detail in Chap. 5.) As a consequence of endothelial cell ac-
tivation, the normal anticoagulant function of the endothelium is abrogated;
in fact, the endothelial cells are changed in a manner that actively promotes
coagulation, i.e., the endothelium becomes procoagulant. Also lost is the abil-
ity of small blood vessels to serve as an impermeable conduit of plasma and
blood cells.
Because endothelial cell activation embodies so many changes in the func-
tion and metabolism of endothelium [2-8], it seems unlikely that any given
agent which might reverse one change, e.g., an anticoagulant, would effective-
ly counter the manifold consequences. Indeed, while xenograft survival has
been prolonged by various drugs such as anticoagulants, platelet inhibitors,
vasodilators, etc., enduring function of organ xenografts has not been
achieved [9-14]. We have, therefore, focused on the removal of natural anti-
bodies and the inhibition of complement with the aim of inhibiting the devel-
opment of endothelial cell activation.
82 Accommodation
Clearly, one of the major problems confronting those in this field is whether
it will be necessary to remove natural antibodies and/or inhibit complement
permanently in order to avoid hyperacute rejection? Such an approach, while
not totally inconceivable (at least in terms of permanent depletion of xenore-
active natural antibodies) is daunting at best. On the other hand, the ability of
ABO- or human leukocyte antigen (HLA)-ineompatible allografts to survive
in recipients who have circulating antigraft antibodies [15-19J has encouraged
us to formulate an alternative working model.
This is as follows. If xenoreactive natural antibodies are depleted, with or
without manipulation of the complement system. before the graft is implanted
and are maintained in a depleted state for an as yet undefined period of time,
then when the natural antibodies are allowed to return to normal (or higher)
levels, survival of the graft will continue despite the fact that the graft endothe-
lium continues to express the target antigens for the natural antibodies. We
hypothesize that "accommodation" will take place between the natural anti-
bodies and complement of the recipient and the vascular endothelium of the
donor [20, 21]. The model can be applied to any system in which antibodies ex-
ist that are potentially reactive with antigens on the endothelium of a primari-
ly vascularized organ graft.
We present this model in the context of several areas of current investiga-
tion. First, we discuss in detail the work that has been done in other laborato-
ries as well as in our own, in which accommodation has been observed after the
temporary depletion of antigraft antibodies and possibly of other factors.
Secondly, we discuss certain new approaches that might be applied toward
achieving accommodation through the inhibition of complement. Thirdly, we
discuss in detail the various methods which have been used for the depletion of
xenoreactive natural antibody and which might be applied in an effort to pro-
mote accommodation of discordant grafts.
Accommodation in Other Systems
The most complete information regarding accommodation comes from stud-

ies of patients who have received kidneys from ABO-incompatible donors.
Ordinarily, the presence of the isohemagglutinins against donor Al or B anti-
gen leads to accelerated or hyperacute rejection of a renal or cardiac graft [19,
22,23]. Hyperacute rejection is associated with the deposition in the graft of
recipient anti-A or anti-B antibodies [23].
It can be averted by removal of the potentially offending antibodies (by
plasmapheresis or adsorption on columns loaded with the specific ligand of
the antibodies) before transplantation and maintenance of low levels of those
antibodies for a period of time after the transplant is in place. Studies by Paul
et al. [23] and by Chopek et al. [15] have demonstrated that the B or A antigen
in question was still present on the endothelial cells of the donor organ and, de-
spite the presence of circulating antibodies directed against these antigens,
vascular rejection did not occur.
Accommodation in Other Systems 83
Very revealing is a series of immunopathology studies attempting to under-
stand the relationship between the antibodies in the serum (after they re-
turned to the circulation) and the antigens on the endothelium. We have ob-
served two immunopathological concomitants of accommodation.
In several cases, recipients of ABO-incompatible renal grafts continued to
express A or B antigen on graft endothelium and yet had no evidence of anti-
body attached to the endothelium despite detectable circulating isohemagglu-
tinins. It is possible that in these cases the antigen expressed on the endothelial
cells could not be recognized by the circulating antibody due to a change in th~
antibody repertoire or the core structure of the antigen (the blood group A or
B oligosaccharides may be associated with a variety of core structures [24]). In
one case a graft biopsy revealed recipient immunoglobulin M (lgM) bound to
endothelium, but very little evidence of complement activation. In this case it
is possible that the ability of complement to become activated was impaired,
perhaps through the increased expression of cell membrane-associated com-
plement inhibitors (see discussion below).
Less well studied are the cases of patients who had high-titer antidonor
HLA class I antibodies and high percentage panel reactivity [17]. In a number
of these patients the antibodies have been removed on a protein A column
(which removes IgG and to some extent IgM) after which a renal transplant
was carried out. Accommodation appears to have developed in that signifi-
cant graft survival occurred. One must assume that the donor antigens are pre-
sent on the endothelium of those grafts in the presence of the returning anti-
HLA antibodies. It is uncertain whether the antibodies which returned
became deposited in the grafts. This area needs further investigation.
There exists some evidence for the accommodation of vascularized
xenografts. Alexandre and colleagues in Brussels performed a series of swine-
to-baboon renal xenografts [25]. The recipients were prepared for the proce-
dure by plasmapheresis and were given immunosuppressive therapy. At least
three recipients achieved graft survival exceeding 2 weeks in the face of circu-
lating antigraft antibodies. At the University of Minnesota, Bolman and col-
leagues in our own group engrafted a swine heart into a rhesus monkey that
had been subjected to plasma exchange and organ perfusion as well as im-
munosuppression [26]. The xenograft continued to function for 8 days until
the recipient was sacrificed for reasons unrelated to the graft. In this single
case antiswine antibody was in the circulation and deposited on the blood ves-
sels of the graft.
There was very little evidence of complement activation in the graft despite
periodically normal circulating complement levels. We believe this case repre-
sents accommodation, and the potential mechanisms allowing graft survival
are discussed below.
84 Accommodation
Possible Explanations for Accommodation
Explanations for accommodation can be grouped into three categories.

I. It may be that the antibodies that return to the circulation after the trans-
plant is in place are different in isotype, affinity and/or specificity, from the
antibodies that were originally present. Changes in anyone of these param-
eters could account for the inability of the antibodies that return to initiate
vascular rejection.
We have noted that the antibodies that are deposited in swine-to-rhesus
monkey xenografts consist predominantly of IgM. whereas the xenoreac-
tive antibodies that are present following depiction of natural antibodies by
organ (kidney) perfusion include both IgM (as was originally present) as
well as IgG. Whether the IgG antibodies represent the products of the same
cells making the original natural antibodies (after isotype switching) is not
known. It will be of the greatest importance to test whether the antibodies
that do return to the circulation in an accommodated recipient are able to
initiate hyperacute rejection.
2. The antigens expressed on the surface of endothelial cells of the graft may
change during the period when natural antibodies are absent. This possibil-
ity has already been raised (above) with respect to the A and B blood group
antigens. As discussed in Chap. 5, one of the terminal sugars on a glyco-
protein, that may well be an important target for natural antibodies in vivo,
is sialic acid. In Minneapolis. we have shown that removal of sialic acid sig-
nificantly increases the binding of natural antibodies to that structure. One
might envision that endothelial cells that are activated immediately after
transplantation express cell surface antigens from which sialic acid has
been lost, thus promoting access to natural antibodies that can bind and
thereby initiate hyperacute rejection. If the endothelial cells are given time
to "heal", the same molecule may now be present with terminal sialic acid
residues. thus blocking access to natural antibodies and decreasing the
probability of hyperacute rejection.
3. We envision the possibility that endothelial cells adapt in some manner dur-
ing the return of natural antibodies, as would occur when methods for natu-
ral antibody depletion are discontinued. Endothelial cells might well react
very differently to the natural antibodies as they are normally present as
opposed to the natural antibodies as they return after depletion.
As just one example, if an organ is engrafted in an unmanipulated xeno-

geneic recipient having the full repertoire of natural antibodies. then each en-
dothelial cell may have several thousand molecules of natural antibody attach-
ing to its surface, resulting in massive complement activation and very
extensive changes consequent to endothelial cell activation. Quite a different
response might be seen if one imagines that, as antibodies return to the circula-
tion. first one antibody molecule attaches to an endothelial cell, then two and,
slowly. an increasing number of natural antibodies become attached to cells.
Methods for the Inhibition of Complement 85
Perhaps under these circumstances, the endothelial cell would become "de-
sensitized" .
In some systems that have been studied, the delivery of what are "minimal
signals" results in "anergy" or nonresponse of the cells. In the case of T cells,
stimulation with the specific antigen, in the absence of other signals, can result
in development of anergy (or nonresponse to that antigen on a normal anti-
gen-presenting cell) at later times [27]. In a somewhat similar vein, providing
one signal to a macrophage, without other signals that are needed to activate
that macrophage, results in transcription of certain genes without their trans-
lation [28]. Under certain circumstances, those macrophages then become
nonresponsive to the later delivery of the other signals. Other examples could
be cited.
We hypothesize that accommodation could be achieved following the en-
graftment of a xenogeneic organ by the depletion of natural antibody or the in-
hibition of complement, or both, carried out for a finite period of time. Various
modalities potentially directed toward these ends are discussed below.
Methods for the Inhibition of Complement
The inhibition of complement in association with xenotransplantation has

been approached in two experimental conditions.
First, animals inherently deficient in the sixth component of complement
have been used as recipients, and significant graft prolongation has been ob-
served [29]. Although these rabbit recipients are unable to assemble the mem-
brane attack complex, they can generate C3a, C3b, and C5a, which could pro-
mote phagocyte activation, neutrophil adhesion to endothelial cells [30], and
endothelial cell activation [31].
An alternative approach has been the administration of cobra venom fac-
tor which depletes complement components by activating the alternative
pathway. Administration of cobra venom factor has led to very significant pro-
longation of vascularized xenografts in several models [9, 12,32]. Regrettably,
the period during which cobra venom factor is effective is limited by the devel-
opment of antibodies which block the action of the protein.
Dalmasso at the University of Minnesota has recently proposed an alterna-
tive strategy for the inhibition of complement [33]. There exist, in endothelial
cells, membrane-associated proteins which inhibit the activation of comple-
ment. For example, decay accelerating factor and CD46 impair the generation
of C3 convertases, and homologous restriction factor (CD59) impairs the as-
sembly of the membrane attack complex. The inhibitory properties of these
molecules are, to a certain extent, species restricted, which is to say the mem-
brane inhibitors in a porcine xenograft might not function effectively in the in-
hibition of primate complement.
We have proposed that appropriate complement inhibitors might be in-
serted in the endothelial cell membrane of a xenogeneic organ and as such
86 Accommodation
might confer significant protection against complement-mediated injury [20,

21, 33]. In fact, Dalmasso and colleagues have shown that human decay accel-
erating factor can be inserted into porcine endothelial cell membranes in suffi-
cient abundance to protect the endothelial cells from the cytotoxic effects of
human complement [33].
Alternatively, donor animals might be genetically engineered to abundant-
ly express human complement inhibitors on endothelial cell surfaces. In either
case it would be anticipated that the ability of the organ to withstand the injury
induced by complement activation would be significantly improved. A differ-
ent strategy is suggested by the very exciting work of Weisman and colleagues
[34] which demonstrates that the administration of CRl to experimental ani-
mals will inhibit complement-mediated tissue injury.
We are encouraged that new approaches directed at the inhibition of com-
plement may individually or in combination allow prolonged survival of vas-
cularized xenografts and, potentially, the achievement of accommodation.
The methods that have allowed the accommodation of vascularized
xenografts to date, however, have largely involved the depletion of natural an-
tibody. For that reason, we have provided below a discussion of those modali-
ties. It must be emphasized that some of the approaches for removal of natural
antibodies might well cause the temporary depletion of complement.
Methods of Removal of Preformed Natural Antibodies
This topic has been briefly reviewed by Van Breda Vriesman [35] and will be
expanded here. To date, five documented methods have been used for remov-
ing or neutralizing circulating preformed natural antibodies, and these are
outlined in Table 6.1.
Plasma Exchange
There is vast experience with plasma exchange, which has been used for a
large number of conditions unrelated to transplantation for many years. The
method has been clearly described and discussed in numerous publications
and basically consists of the removal of the subject's plasma with volume re-
placement, usually with fresh frozen plasma or albumin, though other fluids
such as hetastarch (Hespan) can be used. In human subjects usually not more
than one-and-a-half plasma volumes are exchanged at each treatment, and in
order to maintain a reduction in the antibody level, repeated exchanges, often
extending over weeks or months, may be necessary.
One disadvantage of the technique is that all circulating antibodies are re-
moved, not only those that are of pathogenic significance. As a result, the pa-
tient is depleted of many beneficial antibodies, and this can have serious se-
quelae, in particular leading to an increased risk of infection, for example,
from the reactivation of latent viruses.
Table 6.1. Selected clinical and experimental experience with various techniques to remove preformed natural antibodies
Method Clinical or experimental experience Authors [Reference]
1. Plasma exchange Clinical renal allografting in the presence of ABO incompatibility Alexandre et al. [16,36-39]
MacDonald et al. [40]
Clinical renal allografting in the presence of high levels of HLA antibodics Taube et al. [42,43]
Experimental renal xenografting (pig-to-baboon model) Alexandre et al. [25]
2. Non-spccific Clinical renal allografting in the presence of high levels ofHLA antibody Palmer et al. [17]
antibody sorbents (using protein A columns)
Experimental pig kidney hemoperfusion in man (using protein A columns) Welsh et al. (Chap. 32)
~
(1)
3. Specific Bone marrow transplantation in the presence of ABO-incompatibility Bensinger et al. [55] S-
antibody sorbents (using Biosynsorb columns) o
0..
CJ>
Clinical renal allografting in the presence of ABO-incompatibility o
,...,
i (using Biosynsorb columns) Bannett et al. [56,57]
i'::l
(1)
ii (using washed red blood cells) Slapak et al. [19,53,54]
Experimental cardiac xenografting (pig-to-baboon model) i3
o
<:
i (using pig organs) Cooper et al. [48] e:..
ii (using pig white blood cells) Edwards and Rose [52] o,...,
Experimental cardiac xenografting (pig-to-rhesus monkey model) Fischel et al. [49] "t:I
>;
(1)
(using pig organs) 0'
Experimental cardiac xenografting (pig-to-dog model)
i (using pig organs) Henry et al. [50,51]
3
(1)
0..
ii (using pig erythrocyte columns) Cooper et al. (unpublished data) Z
4. Injectable antigen Experimental renal xenografting (pig-to-dog model) '"2'
>;
i (using pig kidney homogenate) Perper and Najarian [58] e:..
ii (using pig erythrocyte stroma) Linn et al. [46] ;J>
g
5. Non-specific Dithiol-reducing agents Sanchez et al. [59] 0'
o
reducing agents 0..
(';.
CJ>
00
-....)
88 Accommodation
Another disadvantage, depending on the replacement fluid used, is the po-

tential depletion of plasma proteins such as antithrombin III and protein C.
These proteins, as just one example, would ordinarily oppose thrombus for-
mation in the graft.
Volume replacement may also lead to complications in that there is a risk of
passive transfer of viral infection in fresh frozen plasma or gamma globulin, in
particular hepatitis B and human immunodeficiency virus infection. There is
also a risk of anaphylactic reactions.
In the realm of organ transplantation, plasma exchange has been used (i) to
remove circulating anti-A or anti-B blood group antibodies and allow success-
ful bone marrow or organ transplantation across the ABO blood group bar-
rier, and (ii) in the removal of HLA antibodies in sensitized hosts thus allow-
ing successful kidney transplantation (Table 6.1).
Alexandre and his group in Belgium have been most active in using plasma
exchange to overcome the ABO blood group barrier and have written exten-
sively of their experiences in this field [16,36-39]. Their method is to carry out
repeated plasma exchange, thus reducing the titer of anti-A or anti-B antibod-
ies in the human subject, and to perform kidney transplantation when the anti-
body level is low. This group considers splenectomy to be an essential pre-
requisite for the success of this technique, though others have achieved
successful results without the need for splenectomy [40].
There is experimental evidence in rodents that splenectomy does not influ-
ence the rate of return of antibodies once they have been removed by plasma
exchange [41], but whether this is the case in man remains unclear. Even when
the anti-A or anti-B titers subsequently rise to levels higher than the original
natural level, rejection (vascular or cellular) is by no means inevitable. There
are now well-documented patients surviving many years following kidney
transplantation in whom the anti-A or anti-B antibody level is extremely high
and yet in whom a kidney expressing A or B surface antigens continues to
function satisfactorily. The possible mechanisms in play in such circumstances
have been discussed elsewhere in this chapter.
Alexandre's group has used the same approach in experimental xenograft-
ing in a pig-to-baboon model with function of one transplanted kidney for 22
days [25]. This experience is currently the most successful in the field of ex-
perimental discordant xenografting in any animal model.
The London (UK) group has used plasma exchange in a similar way to re-
move HLA antibodies in highly sensitized patients awaiting renal transplanta-
tion [42,43]. Essentially, their technique is similar to that used by Alexandre
and his colleagues, though they have not expressed the view that splenectomy
is an important requirement.
Repeated plasma exchanges are performed in the patient, who also re-
ceives cyclophosphamide therapy in an attempt to suppress B cell activity and
therefore reduce the production of antibody. When the antibody titer has
been low for some days, weeks or months, kidney transplantation is per-
formed. Whereas lymphocytotoxic cross-matching using stored (preplasma
exchange) serum will almost certainly be positive in such highly sensitized sub-
Methods of Removal of Preformed Natural Antibodies 89
jects, there is a high likelihood that the current (postplasma exchange) serum
will give a negative response. The lymphocytotoxic cross-match between
donor cells and current (postplasma exchange) recipient serum must clearly
be negative for transplantation to go ahead. The results have been encourag-
ing.
This method, although of proven success, is, however, a rather crude one,
and subsequent developments have been directed towards refining the tech-
nique so that only those antibodies that are pathogenic are removed from the
plasma; the remaining plasma, containing beneficial antibodies, is returned to
the subject.
Nonspecific Antibody Sorbents
The technique of nonspecific antibody adsorption differs from plasma ex-

change in that the plasma is separated from the cellular components of the
blood (as in plasma exchange) but is then passed through a column containing
a substance that removes (or adsorbs out) immunoglobulins from the plasma.
At the present time the commonly used sorbents are staphylococcal protein A
or protein G. These are especially efficient in removing IgG antibodies, by
binding through the Fc portion of the antibody, but also remove IgM antibod-
ies. Although the remaining plasma is returned to the subject, thus minimizing
the need for fluid replacement therapy, the sorbent is nevertheless nonspecific
and, therefore, removes all gamma globulins from the plasma, again resulting
in hypogammaglobulinemia, with its risks to the subject.
The advantage of this technique over plasma exchange is that the subject's
circulating volume is largely replaced by his or her own plasma. By reducing
the need for volume replacement with fresh frozen plasma or albumin, the
risks associated with the passive transfer of infectious agents are also reduced.
This method has been explored mainly by the London group who have
used staphylococcal protein A columns ("Citen 10" immunoadsorption sys-
tem, Excorin, Lund, Sweden) to absorb out IgG antibodies expressed against
HLA antigens [17). This is an extension of their work described above using
plasma exchange. They have found that where repeated plasma exchange
failed to reduce the antibody titer sufficiently, on occasions success has been
obtained using protein A columns.
In patients undergoing removal of HLA antibodies in this way, this group
has measured the titers of antipig antibodies (Chap. 31). The protein A
columns would appear to remove antipig antibodies successfully. On at least
one occasion prolongation of pig kidney function has been documented when
the organ has been perfused ex vivo with plasma from a patient depleted of an-
tipig antibodies. These studies encourage us to believe that it may be possible
to remove xenoantibodies using this or a similar approach.
90 Accommodation
Specific Antibody Sorbents
A further refinement of the sorbent technique is to attempt to remove only

those antibodies that are pathogenic, returning the remaining antibodies in
the plasma to the subject. The sorbent is directed only against the specific anti-
body whose removal from the plasma is desired, and must therefore consist of
either (i) the antigen itself (or a cloned or synthetic antigen), or (ii) a cross-re-
active antigen. A third possibility is that of developing an anti-idiotypic mono-
clonal antibody that resembles the antigenic binding site.
The plasma is separated from the cellular contents of the blood and is
passed through a column which contains, for example, the antigen. Passage of
the plasma across the column results in removal of the specific antibody which
is fixed to the antigen in the column. The plasma returning to the subject con-
tains all the remaining nonreactive antibodies. The subject is therefore neither
depleted significantly of volume nor of beneficial antibodies. The risk of reac-
tivation of latent viral or other infection is minimized, and, as replacement of
circulating volume with fresh frozen plasma or other fluid is also minimized,
the risk of introducing an infectious agent is also small. In the experimental an-
imal, however, volume replacement would still appear to be necessary, but can
be achieved satisfactorily with hetastarch or human albumin.
Van Breda Vriesman [35] has pointed out that this technique requires prior
accurate identification of the donor antigen to which the preformed natural
antibodies bind. It also requires the need for a standardized antibody assay
system so that the results of adsorption by such a column can be adequately
documented and compared. Neither an exact identification of the donor anti-
gen (or antigens) nor the development of a satisfactory assay method have yet
been achieved in the field of xenotransplantation, though recent work from
Minneapolis has shown progress in these areas [44,45].
As the exact nature of the donor antigen(s) remains uncertain, it has not
been possible to utilize a truly specific sorbent in such a system. Examples of at
least partially specific sorbents are several.
In Oklahoma, in an attempt to remove dog antipig xenoantibodies, we have
used columns containing pig erythrocyte stroma which successfully adsorb out
a significant percentage of the circulating dog antipig xenoantibodies. This is
an example of using the antigen itself as the sorbent, and is based on initial ob-
servations made by Linn and his colleagues in 1968 [46].
We are further attempting to develop an anti-idiotypic monoclonal anti-
body by eluting the dog antipig antibodies from the pig stroma, and injecting
them into mice to produce an anti-idiotypic antibody against the dog antipig
antibodies. This anti-idiotypic antibody will initially almost certainly be poly-
clonal, though subsequent refinements can be made to produce a monoclonal
antibody. The anti-idiotypic antibody produced could then be made up into a
column which would specifically remove the dog antipig (erythrocyte) anti-
body from the plasma which passes through it. Similar anti-idiotypic antibod-
ies could be made up after eluting antibody from columns made up of pig lym-
phocytes, endothelial cells, etc.
Work in 1983 by Cameron et al. [47] suggested that sheep serum adsorbed
out with dog red blood cells lost the ability to agglutinate dog white blood cells.
Similarly, sheep serum absorbed out with dog white blood cells lost its capaci-
ty to agglutinate dog red blood cells. In the Oklahoma experience, we remain
uncertain as to whether the pig stroma removes only hemagglutinating anti-
bodies or whether it also removes lymphocytotoxic antibodies. Whether these
two forms of antibody are one and the same in this model and whether the dog
has other antipig antibodies remains unclear.
Recent work in Minneapolis, however, indicates that, in pigs, vascular en-
dothelial cells and platelets express the same antigens [45]. Human antipig an-
tibodies can be adsorbed by these cells and also by pig erythrocytes and lym-
phocytes, which appear to express the same epitopes.
Van Breda Vriesman has also pointed out the need to prove the
pathogenicity of the preformed natural antibodies [35]. For example, the out-
come of the graft may differ depending on whether the antibodies are IgM or
IgG - there are examples where the presence of one leads to decreased graft
survival but the presence of the other leads to increased graft survival.
Much work was done in this area using the pig-to-dog and sheep-to-dog
models in the 1960s and early 1970s, and selected studies are outlined in Table
6.2. In brief, in these early experiments blood or plasma from the host dog was
perfused through a donor pig organ, such as the kidney or liver (used as the
"specific" sorbent) resulting in depletion of the dog antipig antibodies which
were adsorbed on to the vascular endothelium of the perfused organ. Though
this depletion of antibody was only temporary, if a second pig organ were im-
mediately transplanted into the dog (or perfused with dog blood) a significant
prolongation of function was achieved.
The use of a donor organ is a relatively crude method of removing the
species-directed antibody from the subject, and refinements to this technique
have subsequently been made. In essence, however, the same basic technique
has been used by several groups in recent years. Both the Cape Town [48] and
Minneapolis [26,49] groups have used a similar technique in removing antipig
antibodies from nonhuman primate species and have both shown prolonga-
tion of subsequent graft survival in these antibody-depleted hosts. Henry and
his colleagues in Ohio [50,51] have demonstrated that approximately 90% of
circulating antipig antibodies in the dog can be adsorbed from the plasma us-
ing a spleen or liver as the adsorbing organ. Survival of kidney xenografts
transplanted after such immunoadsorption using the spleen was increased 16
times and, using the liver, 30 times when compared with controls. Repeated
adsorption by this technique over several days using whole blood to perfuse
the organ may, however, induce sensitization [48].
Refinements are continuing to be explored using a column on which is
bound a "specific" antigen (or antigens) against which some or all of the anti-
bodies are directed. Reference has already been made to pig erythrocyte stro-
ma, and in Oklahoma we have also carried out pilot studies using columns of
pig lymphocytes, leukocytes (buffy coat), liver cells and spleen cells. The
Columbia-Presbyterian group in New York [52] has also experience using
Table 6.2. Selected experimental studies or kidney xenotransplantation in the pig-to-dog model utilizing plasmapheresis or antihody adsorption \0
N
Authors Year Therapy Period or organ survival Reference

;J;>
(')
(')
Perper and Najarian 1%6 Immunosuppression (azathioprine, prednisone, actinomycin) 1-5 min 15S] o
Antibody adsorption in vivo using pig kidney homogenate 1-5 min :3
:3
Linn et al. I%S Intravenous administration or pig stroma 152 min 146] 5.
Sequential pig kidney transplants 15x longer than control
;:;.
o·
:l
Bier et al. 1970 } Selective plasmapheresis, (glohulin depletion) >100 min 162,63]
Merkel cl al. 1971
Giles et al. 1970 Sequential pig kidney, spleen or liver transplantation 2x longer than control 164]
Moberg et al. 1971 Plasmapheresis 45-180 min 165]
Immunoadsorption using pig liver 60-240 min
Siapak et al. 1971 Immunoadsorption using pig liver 37 min 166]
Shonsetal. 1973 Plasma depletion, oxidation, electrophoresis 71-165 min 167]
Ghilchik and Morris a 1974 Sequential sheep kidney perfusion No increase over control (10-17 min) 16S]
Terman et al. 1979 Sequential perfusion through pig kidneys 180--480 min [69J
a Used the sheep-to-dog model.

porcine lymphocytes as a specific immunoadsorbent in baboons. As the donor
vascular endothelium is believed to be a prime target for the recipient anti-
body, it may prove necessary to make up a column of endothelial cells cultured
on beads. Whether such tissue culture might reduce or alter the antigenicity of
such cells remains uncertain.
In human subjects, this technique has been mainly explored by those inter-
ested in overcoming the ABO blood group barrier in allotransplantation.
Slapak and his colleagues used blood group A red blood cells as the adsorbing
agent [53] ; by reducing the titer of anti-A antibody they successfully reversed
humoral rejection of a group A kidney transplanted inadvertently into a group
o recipient. Two further cases of kidney allotransplantation across the ABO
barrier, where the patient's group 0 plasma was passed across group A
washed red cells, were reported by this group subsequently [54].
Work in patients undergoing bone marrow transplantation has utilized
commercially available columns made up of synthetic A or B human blood
group antigens in the form of trisaccharides (Biosynsorb, Chembiomed,
Edmonton, Canada) which adsorb out anti-A or anti-B antibody from the
plasma passed through the column [55]. This lead has been followed by the
renal transplant surgeons who have performed kidney transplantation from
living-related donors who were HLA identical or closely matched with the re-
cipient but ABO incompatible.
Bannett and his colleagues performed a small series of such transplants
with prior adsorption of the anti-A or anti-B antibody from the recipient us-
ing the same columns of trisaccharides [56,57]. Splenectomy was performed
in all recipients. In five of six cases the removal of such antibodies led to suc-
cessful kidney transplantation, though in the fifth case graft function was lost
from delayed hyperacute vascular rejection which could not be controlled by
further antibody adsorption or by increased immunosuppressive therapy
[57].
Injectable Antigen or Synthetic Antigen
The injectable antigen method is probably the least well-developed to date, al-
though experimentally it is not a new concept. The antigen, or a synthetic anti-
gen, is injected into the subject's blood and thus forms a complex with the cir-
culating antibody. The antibody is, therefore, no longer free to bind with the
antigen sites on an organ that might be grafted during this period. The antigen-
antibody (immune) complex, however, could have potentially serious detri-
mental effects, such as inducing a state of shock. The occlusion of small vessels
might render organs and tissues ischemic. Furthermore, the immune complex
so formed may be only temporary, thus freeing the antibody to bind to antigen
sites on the transplanted organ. Even if the complex thus formed were perma-
nent, new antibody is formed, thus necessitating repeated injections (or con-
stant infusion) of the antigen or synthetic antigen for a hitherto unknown peri-
odoftime.
94 Accommodation
Early work in this area was by Perper and Najarian [58] in 1966 who admin-
istered pig kidney homogenate intravenously to the potential recipient dog
15 min before the blood supply to a transplanted pig kidney was established.
The injection resulted in a shock-like state, requiring epinephrine therapy.
Although there was evidence of a reduction in the antipig antibody titer, no ef-
fect was observed on either the period of survival of the organ (Table 6.2) or on
the histopathological changes seen on microscopy.
In 1968, Linn and his colleagues injected antigen in the form of pig erythro-
cyte stroma intravenously into dogs, thus binding dog anti pig antibodies and
allowing prolongation of concomitant pig kidney graft survival in the dog [46]
(Table 6.2). The injected stroma was reported to have no adverse effects on
the dog.
The method may have a part to play in organ xenotransplantation if the
species antigen, or a synthetic antigen, can be injected intravenously or intra-
muscularly into the prospective recipient in a form that does not damage other
organs and in a dosage sufficient to prevent humoral rejection from occurring.
The length of time such an infusion needs to be maintained remains unknown,
nor is it known whether humoral rejection will occur once infusion of the anti-
gen is discontinued, although it would seem likely that accommodation might
occur.
Dithiol-Reducing Agents
Certain reducing agents, for example, 2-mercaptoethanol, dithiothreitol, and

penicillamine, when incubated with serum, are known to reduce antibody ac-
tivity. Only IgM activity is affected and there appear to be no adverse effects
on other measurable blood components at the concentrations required
(50 mM). The Columbia-Presbyterian group [59] has found that no detectable
precipitation occurs under such circumstances and the electrophoretic pattern
remains unaffected. Other immunoglobulin levels remain constant.
Such reducing agents are, however, toxic, and therefore the reduction of
antibody activity can only be carried out extracorporeally. Furthermore, a
method must be provided to remove the reducing agent before the serum is re-
turned to the human subject. The Columbia group is exploring the feasibility
of treating serum in this way via an extracorporeal circuit with subsequent dia-
lytic removal of the agent prior to reinfusion of the serum into the subject. If
preformed antibody activity against xenoantigens can be eliminated in this
way, hyperacute rejection in discordant xenografting would be postponed or
eliminated.
The method would appear to have the disadvantage that all IgM activity is
removed from the serum, and, therefore, certain beneficial antibodies will be
removed, possibly exposing the patient to risk of infection. The technique
therefore represents a nonspecific elimination of functional antibody, but pro-
vides a novel and distinct approach to this problem.
References 95
Comment
In the realm of xenotransplantation, the use of specific antigen sorbent

columns would appear to offer the most hope of successfully reducing the lev-
el of circulating preformed natural antibodies in the potential host. Whether
splenectomy is an essential or beneficial requisite to help inhibit antibody re-
bound remains uncertain, but, on the present evidence, seems unlikely.
Pharmacologic agents will undoubtedly be required not only to prevent cel-
lular rejection (as with allografts), but also to suppress B cell activity both be-
fore and after transplantation in an effort to reduce the production of the de-
pleted antibody. It is well known that depletion of an antibody may lead to
overproduction with a rebound effect in which the antibody level rises above
its original level [60]. This must clearly be guarded against during the period of
time (the "window") in which the graft must be transplanted if accommoda-
tion is to take place and it is to be successfully accepted by the host.
Which drug or combination of drugs will prove most successful in reducing
antibody production remains uncertain. Early studies by Terman et aI. sug-
gested that double or triple immunosuppressive drug therapy (rather than sin-
gle drug therapy) was more effective in attenuating or arresting antibody re-
bound [60]. More recent work by Thomas and his group suggests that
antilymphocyte globulin is a potent drug in this respect [61]. Some of the new-
er agents currently being extensively researched, such as FK-506, 15-de-
oxyspergualin, rapamycin, and RS 61443, clearly have some effect in suppress-
ing B cell activity. It is hoped that one or, more probably, a combination of
these agents may render the specific antibody-depleted recipient in a state
where a discordant organ graft might not be rejected.
Acknowledgements. The original work reported in this chapter and performed

at the University of Minnesota was supported by grants from the NIH
(DK13083), the March of Dimes, and the Minnesota Medical Foundation.
This is paper #566 from the Immunobiology Research Center at the
University of Minnesota.
References
1. Platt, 1.L., Fischel, Rl., Matas, A.l., Reif, S.A., Bolman, RM., Bach, F.H. Immuno-
pathology of hyperacute xenograft rejection in a swine to primate model.
Transplantation. In press.
2. Hamilton, K.K., Hattori, R, Esmon, c.T., Sims, P.l. Complement proteins C5b-9 induce
vesiculation of the endothelial plasma membrane and expose catalytic surface for assem-
bly of the prothrombinase enzyme complex. 1. BioI. Chern. 265,3809,1990.
3. Ryan, U.S. The endothelial surface and responses to injury. Fed. Proc. 45,101,1986.
4. Pober, 1.S. Cytokine-mediated activation of vascular endothelium. Am. 1. Pathol. 133,
426,1988.
5. Schleef. RR, Bevilacqua, M.P., Sawdey, M., Gimbrone, M.A. lr., Loskutoff. D.l.
Cytokine activation of vascular endothelium. Effects on tissue-type plasminogen activa-
tor and type I plasminogen activator inhibitor. 1. BioI. Chern. 263,4797,1988.
96 Accommodation
6. Brett. 1.. Gerlach. H .. Nawroth. P .. Stcinberg. S.. Godman. G .. Stern. D. Tumor necrosis
faetor/cachectin increases permeability of endothelial cell monolayers by a mechanism
involvingregulatoryG proteins.f. Exp. Med. 169.1977.1989.
7. Geng. 1.G .. Bevilacqua. M.P .. Moore. K.L.. Mcintyre. T.M .. Prescott. S.M .. Kim. 1.M.
Bliss. G.A .. Zimmerman. G.A .. McEvcr. R.P. Rapid neutrophil adhesion to activated
endothcIium mediated by GMP-140. Natllre. 343.757.1990.
8. Platt. 1.L.. Vercellotti. G.M .. Lindman. B.1 .. Oegema. T.R. Bach. F.H .. Dalmasso. A.P.
Release of heparan sulfate from endothelial cells: implications for pathogenesis of hy-
peracute rejection.f. Exp. Med. 171.1363.1990.
9. Kemp. E., SteinbrucheL D .. Starklint. H .. Larsen, S .. Henriksen. I., Dieperink. H. Renal
xenograft rejection: prolonging effect of captopriL ACE-inhibitors. prostacyclin and co-
bra venom factor. Transplant. Proc. 19.4471.1987.
10. Busch. G.l., Martins. ACP .. Hollenberg. N.K .. Moretz. RC. Wilson. RE.. Colman.
RW. Successful short-term modification of hyperacute renal allograft rcjection in thc
primate. Am. f. Pat/wI. 82.43.1976.
11. Colman. RB .. Habal. M .. Hollenberg. N.K., Birtch. AG., Busch. G.J. Hyperacute renal
allograft rejection in the primate. Am. f. Pat/wI. 82.25,1976.
12. Adachi, H .. Rosengard. B.R. Hutchins, G.M., Hall. T.S .. Baumgartner, W.A. Borken,
AM .. Reitz. B.A Effects of cyclosporine, aspirin, and cobra venom factor on discordant
cardiac xenograft survival in rats. Transplant. Proc. 19.1145,1987.
13. Makowka, L.. Chapman, F.A, Cramer. D.V .. Qian. S .. Sun. H .. Starzl, T.E. Platelet-acti-
vating factor and hyperacute rejection. Transplantation. 50.359. 1990.
14. Moberg, AW., Shons, A.R, Gewurz. H .. Mozes, M., Najarian. J .S. Prolongation of renal
xenografts by the simultaneous sequestration of preformed antibody. inhibition of com-
plement. coagulation and antibody synthesis. Transplant. Proc. 3,539,1971.
15. Chopek, M.W., Simmons. RL.. Platt, J.L. ABO incompatible renal transplantation: ini-
tial immunopathologic evaluation. Transplant. Proc. 19.4553.1987.
16. Alexandre. G.P.J .. Squifflet. J.P .. De Bruyere. M., Latinne, D., Reding, R, Gianello, P.,
Carlier, M., Pirson, Y. Present experiences in a series of 26 ABO-incompatible living
donor renal allografts. Transplant. Proc. 19,4538,1987.
17. Palmer, A, Welsh, K.. Gjorstrup. P., Taube, D., Bewick, M., Thick. M. Removal of anti-
Lancet. 1. 10. 1989.
18. Bannett, A.D., McAlack, RF., Raja. R, Baquero. A, Morris. M. Experiences with
known ABO-mismatched renal transplants. Transplant. Proc. 19.4543.1987.
19. Slapak. M., Digard, N., Ahmed, M .. Shell. T., Thompson, F. Renal transplantation across
the ABO barrier - a 9-year experience. Transplant. Proc. 22, 1425. 1990.
20. Platt. J.L., Vercellotti, G.M., Dalmasso, A.P., Bolman, RM., Matas, AJ., Najarian, J.S.,
Bach, F.H. Transplantation of discordant xenografts : a review of progress. Immunol.
Today. 11,450,1990.
21. Bach. F.H., Turman, M.A, Vercellotti, G.M., Platt, J.L., Dalmasso, A.P. Accommo-
dation: a working paradigm for progressing toward clinical discordant xenografting.
Transplant. Proc. 23.205, 1991.
22. Gleason, RE., Murray, 1.E. Report from kidney transplant registry: analysis of vari-
ables in the function of human kidney transplants. Transplantation. 5,343, 1967.
23. Paul, L.C, van Es, L.A, de la Reviere, G.B., Eernisse, G., de Graeff, J. Blood group B
antigen on renal endothelium as the target for rejection in an ABO-incompatible recipi-
ent. Transplantation. 26,268, 1978.
24. Clausen, H., Hakomori, S. ABH and related histo-blood group antigens; immunochem-
ical differences in carrier isotypes and their distribution. Vox. Sang. 56, 1, 1989.
25. Alexandre, G.P.1., Gianello, P., Latinne, D., Carlier, M., Dewaele, A, van Obbergh, L.,
Moriau, M., Marbaix, E., Lambotte, J.L., Lambotte, L., Squifflet, J.P. Plasmapheresis
and splenectomy in experimental renal xenotransplantation. In: Hardy, M.A (ed.)
Xenograft 25. New York, Elsevier Science Publishers, 1989, p. 259.
26. Fischel. Rl., Platt, J.L., Matas, A.J., Perry, E .. Dalmasso, A.P., Manivel, C, Najarian,
J.S .. Bach. F.H., Bolman. RM. Prolonged survival of a discordant cardiac xenograft in a
rhesus monkey. Transplant. Proc. 23,589,1991.
References 97
27. Mueller, D.L., Jenkins, M.K., Schwartz, RH. Clonal expansion versus functional clonal
inactivation: a costimulatory signalling pathway determines the outcome of T cell anti-
gen receptor occupancy. Ann. Rev. Immunol. 7,445, 1989.
28. Schindler, R, Lonnemann, G., Shaldon, S., Koch, K.M, Dinarello, c.A. Transcription,
not synthesis, of interleukin-I and tumor necrosis factor by complement. Kidney Int. 37,
85,1990.
29. Chartrand, c., O'Regan, S., Robitaille, P., Pinto-Blonde, M. Delayed rejection of car-
diac xenografts in C6-deficient rabbits. Immunology. 38,245, 1979.
30. Vercellotti, G.M., Platt, J.L., Bach, F.H., Dalmasso, A.P. Neutrophil adhesion to xeno-
geneic endothelium via C3bi.J. Immunol. 146,730,1991.
31. Friedl, H.P., Till, G.O., Ryan, U.S., Ward, P.A. Mediator-induced activation of xanthine
oxidase in endothelial cells. Faseb J. 3,2512,1989.
32. Gewurz, H., Clark, D.S., Cooper, M.D., Varco, RL., Good, R.A. Effect of cobra venom-
Transplantation. 5, 1296, 1967.
33. Dalmasso, A.P., Vercellotti, G.M., Platt, J .L., Bach, F.H. Inhibition of complement-me-
diated endothelial cell cytotoxicity by decay accelerating factor: potential for prevention
of xenograft hyperacute rejection. Submitted for publication.
34. Weisman, H.F., Bartow, T., Leppo, M.K., Marsh, H.C., Carson, G.R, Concino, M.F.,
Boyle, M.P., Roux, K.H., Weisfeldt, M.L., Fearon, D.T. Soluble human complement re-
ceptor type I: in vivo inhibitor of complement suppressing post-ischemic myocardial in-
flammation and necrosis. Science. 249, 146, 1990.
35. Van Breda Vriesman, P.J.c. The future of plasmapheresis in host manipulation. In:
M.A. Hardy (ed.) Xenograft 25. Elsevier Science Publishers, Amsterdam, New York,
Oxford, 1989, p. 267.
36. Alexandre, G.P.J., Squifflet, P.J., De Bruyere, M., Latinne, D., Moriau, M., Carlier, M.,
Lecomte, c., Pirson, Y. Kidney transplantation of human ABO-incompatible kidney
transplants in splenectomized recipients. In: Touraine, I.L. et al. (eds.) Transplantation
and Clinicallmmunology- Vo!' XVII. Amsterdam, Elsevier, 1985, p. 211.
37. Alexandre, G.P.J., Squifflet, J.P., De Bruyere, M., Latinne, D., Moriau, M., Idabu, N.,
Pirson, Y. Splenectomy as a prerequisite for successful human ABO-incompatible renal
transplantation. Transplant. Proc. 17, 138, 1985.
38. Reding, R., Squifflet, J.P., Latinne, D., De Bruyere, M., Pirson, Y., Alexandre, G.P.J.
Early postoperative monitoring of natural anti-A and anti-B isoantibodies in ABO-in-
compatible living donor renal allografts. Transplant. Proc. 19,1989,1987.
39. Alexandre, G.P.J., Squifflet, J.P. Significance of the ABO antigen system. In: G.I.
Cerilli (ed.) Organ Transplantation and Replacement. Lippincott, Philadelphia, 1988,
p.223.
40. MacDonald, A.S., Belitsky, P., Bitter-Suermann, H., Cohen, A., Gorelick, M., Gupta, R.
ABO-incompatible living related donor kidney transplantation: report of two cases.
41. Reding, R, White, D.I.G., Davies, H.S., Latinne, D., Delepaut, B., Lambotte, L., Caine,
R Effect of splenectomy on antibody rebound after plasma exchange. Transplantation.
48,145,1989.
42. Taube, D.H., Cameron, J.S., Ogg, c.S., Welsh, K.I., Williams, D.G., Bewick, M., Rudge,
c.l., Kennedy, L.A., Thick, M.G. Renal transplantation after removal and prevention of
resynthesis of HLA antibodies. Lancet. 1,824, 1984.
43. Taube, D.H., Welsh, K.I., Kennedy, L.A., et al. Successful removal and prevention of
resynthesis of anti-HLA antibody. Transplantation. 37,254, 1984.
44. Platt, J.L., Turman, M.A., Noreen, H.I., Fischel, RI., Bolman, RM. III, Bach, F.H. An
elisa assay for xenoreactive natural antibodies. Transplantation. 49, 1000, 1990.
45. Platt, 1.L., Lindman, B.I., Chen, H .. Spitalnik, S.L., Bach, F.H. Endothelial cell anti-
gens recognized by xenoreactive human natural antibodies. Transplantation. 50, 817,
1990.
46. Linn, B.S., Jensen, 1.A., Portal, P., Snyder, G.B. Renal xenograft prolongation by sup-
pression of natural antibody. J. Surg. Res. 8,211,1968.
98 Accommodation
47. Cameron. 0.1 .. Rajagopalan. P.R .. Fittis. CT.. Majeski. 1.A Characterization of
the preformed antibodies involved in the xenograft reaction. 1. Sllrg. Oncol. 22. 157.
1983.
48. Cooper. D.K.C. Lexer. G .. Rose. AG .• Keraan. M .. Rees. 1.. du Toit. E. Effects of
cyclosporine and antibody adsorption on pig cardiac xenograft survival in the baboon.
1. Heart Transplant. 7.238.1988.
49. Fischel. R.l .. Bolman. R.M. III. Platt. 1.L.. Najarian. 1.S .. Bach. F.H .. Matas. A.l.
Removal if IgM anti-endothelial antibodies results in prolonged cardiac xenograft sur-
vival. Tramplant Proc. 22. 1077. 1990.
50. Henry. M.L.. Han. L.K.. Davies. E.A, Orosz. CG .. Sedmak. D.D .• Ferguson. R.M. The
role of organ perfusion in xenotransplantation: influence of ex-vivo xenoantibody ad-
sorption on xenograft survival. In: M.A Hardy (ed.) Xenograft 25. Elsevier Science
Publishers, Amsterdam. New York. Oxford, 1989. p. 249.
51. Henry. M.L.. Han, L.K., Orosz, G.C. Sedmak, D.O., Ferguson, R.M. Modification of
xenograft hyperacute rejection via xenoantibody depletion. Transplant. Proc. 22. 1081.
1990.
52. Edwards. N.M .. Rose, E.A The use of non-human primates in xenotransplantation re-
search. In: M.A Hardy (ed.) Xenograft 25. Elsevier Science Publishers. Amsterdam,
New York, Oxford, 1989. p. 79.
53. Slapak, M., Naik, R.B .. Lee. H.A Renal transplant in a patient with major donor-recipi-
ent blood group incompatibility. Transplantation. 31,4, 1981.
54. Slapak, M., Evans, P., Trickett, L.. Harris, K.R., Gordon, P .. Matini. A, Mocelin, A.l.
Can ABO-incompatible donors be used in renal transplantation? Transplant. Proc. 16.
75,1984.
55. Bensinger. W.I.. Buckner, CD., Thomas, E.D .. Clift, R.A ABO-incompatible marrow
transplants. Transplantation. 33,427.1982.
56. Bannett. AD., McAlack, R.F., Raja, R., Baquero, A .. Morris, M. Experiences with
known ABO-mismatched renal transplants. Transplant. Proc. 19.4543,1987.
57. McAlack, R.F., Bannett, A.D., Raja, R., Kim, P., Romano, E .. Lauzon, G. Delayed hy-
peracute rejection in an ABO-incompatible renal transplant. Transplant. Proc. 19,4558.
1987.
58. Perper, R.l .. Najarian. 1.S. Experimental renal heterotransplantation. I. In widely diver-
gent species. Transplantation. 4.377,1966.
59. Sanchez, 1.. Berger, C. He. X., Reemtsma, K., Rose. E. Elimination of preformed anti-
body activity to xenoantigens utilizing dithiol-reducing agents. (Abstract) Transplant.
Proc. In press.
60. Terman. D.S., Garcia-Rinaldi, R., Dannemann, B., Moore. D., Crumb, C, Tavel, A,
Poser, R. Specific suppression of antibody rebound after extracorporeal immunoad-
sorption. 1. Comparison of single versus combination chemotherapeutic agents. Clin.
Exp. Immllnol. 34,32,1978.
61. Thomas, F.T., Demasi. R.l., Aranada, D., Marchman, W., Alqaisi, M., Larkin, E.W.,
Condie, R.M., Carobbi, A., Thomas, 1.M. Comparative efficacy of immunosuppressive
drugs in xenografting. Transplant. Proc. 22, 1083, 1990.
62. Bier, M., Beavers, D.C, Merriman, W.G., Merkel, F.K., Eiseman, B., Starzl, T.Z.
Selective plasmapheresis in dogs for delay of heterograft response. Trans. Amer. Soc.
Artif Int. Organs. 16,325,1970.
63. Merkel, F.K., Bier, M., Beavers, CD., Merriman, W.G., Wilson, C, Starz\' T.E.
Modification of xenograft response by selective plasmapheresis. Transplant. Proc. 3,
534,1971.
64. Giles, G.R., Boehmig, 1.H .. Lilley, 1., Amemiya, H., Takagi, H., Coburg, A.l.,
Hathaway, W.E., Wilson, CB., Dixon, F.l., Starz\, T.E. Mechanism and modification of
rejection ofheterografts between divergent species. Transplant. Proc. 2,522,1970.
65. Moberg, AW., Shons, A.R., Gewurz, H., Mozes, M., Najarian, 1.S. Prolongation of renal
xenografts by the simultaneous sequestration of preformed antibody, inhibition of com-
plement, coagulation and antibody synthesis. Transplant. Proc. 3,538,1971.
66. Slapak, M., Greenbaum, M., Bardawil, W" Saravis, C, Joison, 1., McDermott, W.v. lR.
References 99
Effect of heparin, arvin, liver perfusion, and heterologous antiplatelet serum on rejec-
tion of pig kidney to dog. Transplant. Proc. 3,558, 1971.
67. Shons, A.R, Beir, M., J etzer, J., Najarian, J .S. Techniques of in vivo plasma modification
for the treatment of hyperacute rejection. Surgery. 73,28, 1973.
68. Ghilchik, M.W., Morris, A.S. Platelet trapping by sheep kidney and heart xenografts in
the dog. 1. Surg. Res. 17,434,1974.
69. Terman, D.S., Garcia-Rinaldi, R, McCalmon, R, Crumb, c.c., Mattioli, c., Cook, G.,
Poser, R. Modification of hyperacute renal xenograft rejection after extracorporal im-
munoadsorption ofheterospecific antibody. Int. 1. Artif Organs. 2,35,1979.
Chapter 7
Mechanism of Cellular Xenograft Rejection

R.D. MOSES AND H. AUCHINCLOSS JR.
Introduction
While much is known about cell-mediated immune responses to allografts,

significantly less is known regarding the cellular response to xenografts [1].
Since xenoantigens are more foreign (relative to self) than alloantigens, one
might at first expect that cellular immune responses against the former would
be more vigorous, as appears to be the case for humoral immunity [2].
However, the strong T cell immunity directed against alloantigens probably
results in part from their similarities to self major histocompatibility complex
(MHC) molecules, since T cells are selected in the thymus for their affinity for
self antigens. Therefore, T cells might actually have weaker affinity for more
disparate xenoantigens. Furthermore, other molecular interactions involved
in allogeneic responses besides receptor-antigen binding, such as accessory
molecule and lymphokine interactions, might not operate efficiently when
there are species differences. With these considerations in mind, one wonders
whether xenogeneic cell-mediated immunity is stronger, weaker, or perhaps
different from allogeneic immunity.
The purpose of the present chapter is to examine the available information
regarding cellular immunity to xenoantigens and to compare this with allo-
geneic responses. The analysis will focus primarily on the results of in vitro
studies, from which most current know lege has been gained. Particular atten-
tion will be given to recent developments in the field since the last review of
this subject [1]. The material will be organized as follows: (a) general consider-
ations regarding xenogeneic cell-mediated immune responses, (b) cellular and
antigenic requirements of the response, (c) molecular requirements of the re-
sponse, (d) the question of direct versus associative recognition of xenoanti-
gens, (e) mechanisms and control of cell-mediated xenograft rejection, (f)
conclusions, and (g) some comments on possible avenues for future inquiry.
General Considerations
Early in vivo studies showed that cell-mediated immunity has a role in

xenograft as in allograft rejection [3,4]. A general question that arises is the
strength of the cellular immune response to xenoantigens. This has been ex-
amined in a number of in vitro systems.
102 Mechanism of Cellular Xenograft Rejection
For the helper phase of the response, most studies have examined the bulk
xenogeneic mixed leukocyte reaction (MLR) and have yielded conflicting re-
sults. Several studies found weak or no primary xenogeneic MLR for a range
of responder species. including the human [5.6], sheep [7,8]. goat [7]. guinea
pig [8,9], duck [8]. goose [8]. pigeon [8]. rat [7,8, 10-12], and mouse [13. 14]. In
contrast, others found the primary xenogeneic MLR to be equal in magnitude
to [2. 15-26] or even stronger than [27] the allogeneic MLR Only three groups
examined xenogeneic interleukin-2 (IL-2) production. again with contlicting
results [14. 16. 28J. Two groups reported intact primary xenogeneic IL-2 pro-
duction by human T cells in response to stimulation by mouse and pig cells [16.
28].ln contrast. we found weak or no IL-2 production by naive mouse T cells in
response to stimulation by monkey. pig. and human spleen cells [14] (Moses,
Winn, Auchincloss Jr. unpublished data). Quantitative studies ofxenogeneic
helper responses by limiting dilution analysis have not yet been reported.
The decreased helper xenogeneic responses found in some studies cannot
be attributed to an inhibitory effect of the xenogeneic cells in culture. since
brisk secondary in vitro responses have been a uniform finding. How then can
the discrepancy in results be explained? There are three likely (and not mutu-
ally exclusive) explanations. First, several of the reports of intact primary
xenogeneic helper responses involved phylogenetically close species combi-
nations, including human-chimpanzee [20]. sheep-goat [7, 19], duck-chicken
[8], and rat-mouse [21. 22. 24]. In these cases, the close relationship may make
the response more like an allogeneic reaction. Second. differences in xeno-
geneic responsiveness may exist among species. Many of the intact primary re-
sponses wcre reported for human xenogeneic responses [16-21.26]. Finally,
intact primary human antimouse [28] and mouse antihuman [15] responses
were found to be dcpendent on the presence of responder antigen-presenting
cells (APCs), suggesting that the xenoantigens were being recognized in asso-
ciation with responder MHC molecules rather than directly. Why there should
be a primary response to xenogeneic MHC peptides in association with self
MHC molecules and one to similarly presented MHC alloantigens [29]. but
not one to processed nominal antigens, is not known. One possibility is that the
intact responses actually reflect prior sensitization by cross-reactive environ-
mental antigens [10]. Whichever of thesc possibilities is most important in a
given xenogeneic situation. the bulk of evidence indicates that helper respon-
ses to xc no antigens are generally weaker than responses to alloantigens in un-
primed hosts. particularly as the phylogenetic disparity between responder
and stimulator increases.
For the effector (cytotoxic) phase of the response, in vitro xenogeneic cell-
mediated lympholysis (CML) assays using both bulk cultures and limiting di-
lution analysis have been performed. Results from bulk assays have again
been conflicting. Many studies reported diminished xenogeneic CML activity.
either as an isolated finding [30-33] or in direct comparison with allogeneic re-
sponses [34-37]. This was observed for human [32.34.36]. rat [30], and mouse
[31-35.37] responders. In contrast, a smaller number of studies reported hu-
man [16.28], guinea pig [7], rat [7.38.39], and mouse [7] xenogeneic CML re-
Cellular and Antigenic Requirements 103
sponses to be at the level of allogeneic responses. However, when examined
quantitatively by limiting dilution analysis (mostly for mouse responses), both
primary [40-42] and secondary [41,43-46] xenogeneic CML responses have
been uniformly quite low in comparison with the levels usually observed in al-
logeneic responses. Thus, although the range of responder species tested has
been relatively limited, the available evidence supports the notion that xeno-
geneic CML responses are diminished compared with allogeneic responses.
Cellular and Antigenic Requirements of the Xenogeneic

Cell-Mediated Immune Response
Early in vitro studies established that xenogeneic cellular immune responses,

like allogeneic responses, are mediated by T cells [47,48]. The availability of
monoclonal antibodies and a better understanding of the T cell subsets in-
volved in allogeneic responses have made it possible to study the cellular re-
quirements of the xenogeneic response in more detail.
The induction phase of the xenogeneic cellular response has been consis-
tently found to be dependent on Lyt-l+ or CD4+ helper T cells [13-16,28].
This has been the case for both human [16,28] and mouse [13-15] responses.
This differs from the finding that both CD4+ and CD8+ cells can generate
helper responses in MHC-disparate allogeneic combinations both in vitro [49]
and in vivo [50]. As in allogeneic responses, xenogeneic helper T cell respon-
ses require stimulation by APCs [13-15,26,28,51,52], a topic that will be dis-
cussed in detail in the section on direct versus associative recognition.
Xenogeneic class II MHC antigens have consistently been found to be the
principal target antigens recognized by xenospecific helper T cells [13, 15, 16,
51]. One report [51] also described weak helper T cell responses to xenogeneic
class I MHC and MIs antigens. Human and rat helper T cell responses to
mouse xenoantigens were found to be specific for the same polymorphic de-
terminants of the mouse MHC (H-2) as recognized by mouse allogeneic
helper T cells [5, 16,24,26,51, 52]. Other responses, including mouse antimon-
key [14], mouse antihuman [13, 15], and human antidog [18], were found to be
specific for monomorphic determinants of xenogeneic class II MHC
molecules. It is important to note, however, that the dominant role of class II
MHC molecules as the target antigens of xenoreactive helper T cells does not
imply direct recognition of those antigens in all cases. In fact, many of the re-
sponses appear to involve recognition of the MHC antigens after processing
and presentation in association with selfMHC molecules (to be discussed fur-
ther below). This raises the possibility that processed non-MHC xenoantigens
are also recognized as target structures by xenoreactive helper T cells in some
cases.
In summary, the cellular and antigenic requirements of xenogeneic helper
responses are characterized by a predominant role of CD4+ helper T cells rec-
ognizing monomorphic or polymorphic determinants of xenogeneic class II
MHC antigens presented by APCs, similar in many respects to the require-

ments for allogeneic responses. Primary differences from allogeneic responses
include the minimal role of the CD8+ helper subset and the recognition, in
some xenogeneic combinations, of monomorphic rather than polymorphic de-
terminants of class II MHC molecules and perhaps of peptides of non-MHC
molecules.
For the effector phase of the xenogeneic response, most studies of xeno-
geneic CML activity in vitro have shown a central role for T cells [47,48,53,
54J. The T cell-mediated cytotoxic activity has resided entirely in the CD8+
subset [16,28, 55-57J. This was observed not only for responses to xenogeneic
class 1 MHC antigens [16,56, 57J, but also to class II antigens [55-57J. The cyto-
toxic response against class 11 antigens was not blocked by anti-CD8 mono-
clonal antibody [55]. The exclusive role of the CD8+ subset in T cell-mediated
cytotoxicity differs somewhat from the allogeneic situation in the mouse,
where CD4+ cytotoxic T lymphocytes (CTL) can mediate responses to class II
MHC alloantigens [29J.
The possibility has been considered in a few studies of a non-Tcell-mediat-
ed effector mechanism in xenograft rejection, involving either antibody-de-
pendent cell-mediated cytotoxicity (ADCC) mechanisms, natural killer (NK)
cells, or macrophages [48, 58-62J. Recently, Doody and Winn (personal com-
munication) have characterized a popUlation of helper-dependent Thy-l +
CD4- CD8- NK1.1 + NK-like effector cells with potent xenogeneic cytotoxic
activity in bulk mouse antirat MLR cultures, These NK-like cells are not gen-
erated in allogeneic cultures and do not lyse allogeneic targets; thus, they may
constitute an important non-T cell cytotoxic effector population unique to
xenogeneic responses.
Several studies have examined the nature of the target antigens recognized
by xenospecific CTL. Responses have uniformly been found to be specific for
MHC antigens [16,30,32,53-57,63, 64J, similar to allogeneic responses. While
some groups have found CML activity to be directed solely against class I
MHC antigens [16, 30, 32, 53, 63, 64J, others have found the responses to be
distributed between class I and class II antigens [54, 56, 57J, and one group
even described activity directed exclusively against class II antigens [55J. An
unusually high incidence of MHC class II-specific CTL might be explained by
poor CD8 molecule function across species differences (see below), resulting
in a failure to channel CTL toward recognition of xenogeneic class I antigens.
Although a few studies have reported mouse CTL to be specific for monomor-
phic determinants of both class I and II MHC xenoantigens [32,35,41, 56J, the
majority of studies have found xenogeneic responses to be directed predomi-
nantly or exclusively against the polymorphic regions of those antigens [7, 16,
30,32,38,39,52,53,56,57, 63-66J. Recently, the fine specificity of several hu-
man antimouse CTL lines has been mapped to the identical polymorphic de-
terminants of mouse class I molecules as recognized by allospecific CTL [66J.
In summary, these results indicate that many of the cellular and antigenic
requirements of the effector phase of xenogeneic responses are similar to
those of allogeneic responses. Some differences include the exclusive use of
Molecular Requirements ofthe Xenogeneic Cell-Mediated Immune Response 105
the CD8+ subset for CTL-mediated xenogeneic responses, the tendency to-
ward CTL recognition of class II versus class I MHC xenoantigens, and the
recognition, in some cases, of monomorphic rather than polymorphic determi-
nants of MHC xenoantigens. Finally, a difference of potentially major impor-
tance is the possibility of a potent NK-like cytotoxic effector pathway for
xenoantigens.
Molecular Requirements of the Xenogeneic Cell-Mediated

Immune Response
The observation that primary xenogeneic cell-mediated immune responses in

vitro are weaker than allogeneic responses despite generally similar cellular
and antigenic requirements raises the question of the molecular mechanisms
underlying this difference. Any of several cell surface molecule interactions
used in all ore cognition could potentially be defective in xenogeneic responses
(Table 7.1). New techniques have provided an opportunity to examine this
question in some detail. In particular, transfection and transgenic methods
have permitted the construction of xenorecognition systems in which some of
the molecular interactions have been artificially "corrected". In this way, the
contributions of individual molecular interactions to xenorecognition can be
studied, even in the presence of simultaneous defects in multiple types of in-
teractions.
Considering the induction phase of the xenogeneic response, one of the
possible defects in molecular interactions between species might be the activi-
ty of lymphokines produced by one species on T cells of another. One study
showed that exogenous guinea pig but not mouse mitogen-stimulated super-
natant in the MLR culture reconstituted primary guinea pig antimouse CML
activity [7]. This suggested a xenogeneic lymphokine defect, presumably at the
Table 7.1 Cell surface molecule interactions that may be defective in T cell-xenogeneic cell
interactions
Type of interaction Responder cell Xenogeneic cell

surface molecule surface molecule
Antigen recognition T cell receptor MHC molecule al/a2 (cl I)

or all131 (cl II) domains
Accessory molecule CD4 Monomorphic class II MHC
molecule 132 domain
Accessory molecule CD8 Monomorphic class I MHC
molecule a3 domain
Accessory molecule LFA-l ICAM-lorICAM-2
Accessory molecule LFA-2 LFA-3
Lymphokine IL-l receptor IL-l
Lymphokine Other lymphokine receptor Other lymphokine
induction phase. Two studies measured the proliferation of highly purified,

unprimed human T cells in response to stimulation by mouse APCs [26, 52]
and found that, while the T cells did not proliferate in the absence of exoge-
nous factors, they did so after the addition of either autologous human APCs
[26. 52] or exogenous human IL-I [26, 52] or human IL-2 [26] to the culture.
These results suggested that human helper T cells could be stimulated directly
by mouse APCs in the presence of exogenous human lymphokines. However,
the possibility that mouse antigens were being recognized in association with
human MHC molecules on residual human APCs in these experiments was
not rigorously excluded, e.g., by attempts to block the responses with antihu-
man MHC class II antibodies. This could be an important control in view of the
finding in another study [28] that human helper T cells recognize mouse
xenoantigens primarily in association with human MHC molecules.
Furthermore. exogenous lymphokines did not reconstitute responder APC-
depleted human antimouse T cell responses in this latter study [28]. We have
also been unable to detect mouse T cell proliferation or IL-2 production in re-
sponse to stimulation by monkey or human cells even after the addition of ex-
ogenous recombinant mouse IL-l, IL-2, IL-4, or interferon (IFN)-gamma
(Moses. Winn, Auchincloss Jr, unpublished data). Thus, it appears likely that
ineffective lymphokine interaction across species differences is not the sole
defect in helper T cell xenorecognition.
Another possible defect in molecular interactions at the induction phase
might involve the function of T cell surface accessory molecules. One study
measured the ability of different transfected CD4 molecules to enhance adhe-
sion of CD4- human tumor cells to MHC class 11+ human B cells [67]. Native
human CD4 was found to be significantly more effective at enhancing adhe-
sion than a series of mutant human CD4 molecules carrying short stretches of
mouse CD4 sequences in nonconservative regions of the molecule. These re-
sults suggested a defect in the interaction of murine CD4 with xenogeneic class
II MHC molecules. On the other hand, another study [68] showed that trans-
fected murine and human CD4 equally enhanced IL-2 production by an anti-
gen-specific, CD4- mouse T cell hybridoma stimulated by murine cells ex-
pressing the appropriate murine class II MHC antigen. This indicated that
human CD4 could function across species differences. It should be noted that
the experimental system in the latter study was not quantitative and thus might
have failed to detect small differences between human and murine CD4
molecule interactions with murine class II MHC molecules.
Our laboratory has also investigated the molecular requirements of mouse
xenogeneic helper responses using partially "corrected" systems. We have
found (Moses, Winn, Auchincloss Jr, unpublished data) that primary mouse
xenogeneic helper T cell proliferation and IL-2 production were not reconsti-
tuted in response to any of the following stimuli: (i) to pig antigens. by T cells
that had matured in the presence of a transgenic pig class I MHC molecule
(PDI) in the thymus; (ii) to the pig class I MHC antigen PDI, when expressed
as a transgene on syngeneic mouse APCs; and (iii) to mouse class I and class II
MHC alloantigens, when expressed on a human APC (G3.B4) by a cell fusion
Molecular Requirements of the Xenogeneic Cell-Mediated Immune Response 107
technique, even in the presence of exogenous mouse lymphokines. These re-
sults suggest, respectively, that (i) defective thymic education is not solely re-
sponsible for decreased xenorecognition; (ii) there is a defect in T cell receptor
(TcR) and/or CDS interaction with xenogeneic class I MHC molecules; and
(iii) there is an additional defect in accessory molecule interaction across
species differences, possibly involving the lymphocyte function associated
antigen (LFA)-l or LFA-2 pathways.
In summary, these results indicate the presence of mUltiple molecular de-
fects in xenorecognition by helper T cells, most notably that of CD4 interac-
tion with xenogeneic MHC molecules. The data also support the existence of a
defect in the function of one or more of the accessory molecules (Table 7.1).
Indeed. completely normal function across species differences has not yet
been proven for any of the molecular interactions known to be involved in
all ore cognition (Table 7.1).
For the effector phase of the cell-mediated response. a greater body of
knowledge has been accumulated regarding the possible defects in xenorecog-
nition. A series of experiments has addressed the question of a defect in acces-
sory molecule interactions by examining the ability of human MHC (human
leukocyte antigen, HLA) class I-specific alloreactive CTL to lyse mouse tu-
mor cell targets transfected with the target HLA class I molecule. Several
studies using transfected mouse L cell and other mouse targets showed a fail-
ure of target lysis whether or not human b2m was supplied either exogenously
or by cotransfection [36,69-73]. These results were initially attributed to a de-
fect in accessory molecule interaction across species differences (such as LFA-
1 and intercellular adhesion molecule,ICAM-l). although the possibility was
later raised that the defect might be caused by a lack of expression of appropri-
ate human self peptides in association with the transfected human class I
molecules [73]. However, these experiments were complicated by the fact that
mouse L cells generally do not express even murine ICAM-l [74]. Indeed, it
was found that ICAM-l-expressing mouse PS15 tumor cells [32,75-77] and an
unusual ICAM-l-expressing mouse L cell variant [7S] were lysed well when
transfected with the appropriate xenogeneic class I MHC molecules. The lat-
ter results suggested that the LF A-I accessory molecule could indeed function
across species differences. This conclusion was strengthened by the observa-
tions that human anti-mouse [32,75], human antimonkey [36], and mouse an-
tihuman [55] CML responses were blocked by antiresponder LF A-I antibody.
While all of these studies support the notion that LFA-l functions across
species differences, it should be noted that the experiments were qualitative in
nature and did not address the possibility of partial dysfunction. On the other
hand. the second major accessory molecule pathway, i.e .. LF A-2 on the T cell
reacting with LFA-3 on the target cell, has been shown not to function in hu-
man antimouse responses [36,75, 7S, 79], although it does function in the hu-
man antimonkey combination [36].
Transfection and transgenic models have also been used to examine the
possibility of defects in TcR and/or CDS recognition of xenogeneic MHC
molecules. In one type of experiment, mouse CTL primed against a human or
pig class I MH C xenoantigen were tested in vitro for killing of mouse cells ex-
pressing the specific xenoantigen either by transfection or transgenic tech-
niques. Although one such study reported relatively normal killing [SO]. the
large majority of studies [33.40-42.45.46.81-83] reported weak CML activity
against the tranfected or transgenic xenoantigen-expressing targets. These in
vitro findings were confirmed in an in vivo study examining the rejection by
B 10 mice of syngeneic mouse skin grafts expressing a transgenic pig class I
MHC antigen (BlO.PD1) [84]. Graft survival was prolonged by anti-CD4 but
not anti-CD8 in vivo immunosuppression, suggesting an inability of CDS+ T
cells (helper and/or cytotoxic) to recognize the xenogeneic class I antigens di-
rectly. Thus, for CTL at least, it appears that a defect does exist in TcR and/or
CD8 interaction with xenogeneic class I MHC molecules.
In order to distinguish between these two possible defects, exon-shuffling
techniques have been used to create hybrid MHC molecules containing mouse
al and a2 domains and a human a3 domain, or human a1 and a2 domains and
a mouse a3 domain. In this way, the binding sites forTcR (a1 and a2 domains)
and CD8 (a3 domain) could be investigated separately. One study [43] found
that the precursor frequency of mouse CTL specific for a human class I MHC
xenoantigen transfected onto a mouse target cell was low whether the trans-
fected molecule expressed a human or a mouse a3 domain [43]. These results
were interpreted to indicate a defect in TcR recognition of the a1 and a2 do-
mains of xenogeneic MHC molecules rather than in CD8 interaction with the
xenogeneic a3 domain. On the other hand, another group of investigators
found diminished mouse CML responses to a transfected MHC alloantigen
when expressing a human rather than a mouse a3 domain [85J, although they
attributed this to an alteration in the configuration of the al and a2 domains
by the xenogeneic a3 domain rather than to a defect in CD8-xenogeneic a3
domain interaction.
Three other studies found that the primary defect resided in the CD8-xeno-
geneic a3 domain interaction [31, 86, 87]. One study [87] showed that allo-
primed human and mouse CML responses against targets transfected with the
appropriate allogeneic MHC molecules were diminished by the presence of a
xenogeneic a3 domain (mouse and human, respectively) in the transfected
molecules. Similarly, a second study [31] showed that xenoprimed mouse anti-
human CML responses against targets transfected with the appropriate xeno-
geneic MHC molecule were enhanced by the presence of a mouse a3 domain
in the transfected molecule. Finally, both the latter [31] and a third study [86]
showed that the induction of mouse allogeneic [86] and antihuman xenogene-
ic [31,86] CML responses depended on the presence of a mouse rather than a
xenogeneic (human) a3 domain in the transfected stimulating antigens. The
latter observations were made for both in vivo [86] and in vitro [31] stimula-
tions.It is difficult to reconcile these differences in results.
The weight of evidence favors the existence of a defect in CD8-xenogeneic
class I MHC molecule interaction. However, the possibility of an additional.
perhaps less severe defect in TcR-xenoantigen recognition cannot be exclud-
ed.
Direct Versus Associative Recognition 109
Assuming that a TcR-xenoantigen recognition defect does exist, the ques-
tion arises whether this deficient TcR repertoire for xenoantigens is encoded
in the TcR genes or is acquired during thymic selection because of a lack of
similarity between self and xenogeneic MHC molecules. In order to distin-
guish between these possibilities, the xenogeneic responses of transgenic mice
expressing a human class I MHC antigen in the thymus have been tested. If the
diminished TcR xenorepertoire were an acquired phenomenon, the presence
ofaxenogeneic MHC molecule during thymic education might correct this de-
fect. CTL from such mice were found to be able to recognize viral antigens in
association with the human MHC molecule in a restricted fashion [88],
demonstrating a role for recognition of the xenogeneic MHC molecule in pos-
itive thymic selection. Transgenic CTL did not lyse MHC-identical mouse
cells transfected with the human MHC molecule encoded by the transgene
[89], suggesting a role for recognition of the xenogeneic MHC molecule in
negative selection as well. However, the human MHC-restricted viral respon-
ses were found to be much weaker than those restricted by native (mouse)
MHC molecules [37]. Also, CML responses against human xenoantigens were
no greater in the transgenic mice than in nontransgenic mice [40,44,46]. Thus,
these experiments indicate that the presence of a xenogeneic MHC molecule
in the thymus only weakly influences the TcR repertoire for xenoantigens.
At face value, these results might be interpreted to indicate a genetic basis
of defective TcR xenorecognition. However, in view of the significant defect
in CD8-ligand interaction across xenogeneic barriers (see above) and the im-
portant role of the CD8 molecule in the thymic selection of class I-restricted
T cells [90-92], these results could just as well be explained by a defect in
CD8-xenogeneic MHC molecule interaction during thymic selection.
Distinguishing between these two possibilities will require the measurement
of xenogeneic responses in transgenic mice expressing exon-shuffled hybrid
MHC molecules similar to those described above. Thus, multiple molecular
defects are present in xenorecognition by cytotoxic T cells, similar to the situa-
tion for helper T cells. Specifically, LFA-2/LFA-3 and CD8/class I MHC
molecule interactions have been demonstrated to be defective for disparate
xenogeneic combinations. It is likely that the interaction of TcR with xeno-
geneic class I MHC molecules is also impaired, although it is unclear whether
this latter defect is genetically determined or acquired during thymic educa-
tion.
Direct Versus Associative Recognition
It is evident from the material reviewed thus far that the weak T cell xenogene-
ic responses observed in primary in vitro assays result from many defects in
molecular interactions between T cells and xenogeneic cells. Since direct T cell
recognition of xenoantigens is defective, the question arises: Do T cells prefer-
entially recognize xenoantigens as processed peptides in association with self
MHC molecules on host cells, in the manner of recognition of nominal anti-

gens? The answer to this question has important implications regarding mech-
anisms of cell-mediated xenograft rejection.
Utilizing responder and/or stimulator APC-depleted cultures, we found
that mouse antimonkey helper T cell responses were strictly dependent on the
presence of responder (mouse) APCs and were blocked by antimouse MHC
class II antibody [14]. Similar results have been found by others for mouse an-
tihuman [13, 15] and human antimouse [28] helper responses, both indirectly
by blocking with antihost MHC class II antibodies [13,15] and directly by APC
depletion [28]. These findings have been demonstrated for both primary [15,
28] and secondary [13,14] in vitro helper responses, and for experimental pro-
tocols describing either intact [15,28] or absent [13, 14] primary responses. In
the human antimouse helper T cell responses [28], chloroquine treatment of
responder APCs, which interferes with the processing of exogenous antigens,
and paraformaldehyde treatment of stimulator cells, which prevents shedding
of xenoantigens, each abrogated the generation ofxenospecific CTL, presum-
ably by blocking the response at the induction phase. These common observa-
tions demonstrate that mouse and human helper T cells recognize disparate
xenoantigens after processing and presentation of those antigens in associa-
tion with self class II MHC molecules on host APCs, i.e" associatively rather
than directly.
Contrasting with these results, the only study thus far [28] to examine the
APC requirements of human helper T cell responses to a species less disparate
than the mouse found that human antipig proliferation could occur in the ab-
sence of human APCs. Furthermore, the generation of human CTL specific
for the xenoantigens of a range of species, including the rhesus, pig, rabbit, and
even rat, was found to be dependent on the presence of stimulator and not re-
sponder APCs during the bulk MLR culture [28]. These results suggest that
human helper T cells can recognize xenoantigens less disparate than the
mouse directly on xenogeneic APCs. Further studies are needed to confirm
this potentially important finding and to quantify the degree to which human
helper T cells can interact directly with xenogeneic as compared with allo-
geneic APCs.
Virtually every study of the effector phase of the response has shown some
ability of cytotoxic T cells to lyse xenogeneic targets and therefore to recog-
nize xenoantigens directly. However. as described earlier, features of this in-
teraction are unusual, including the exclusive use of the CD8+ CTL subset
even for recognition of class II MHC xenoantigens, the lack of utilization of
the CD8 accessory molecule, and the relatively even distribution of responses
directed against class I and class II MHC xenoantigens. These observations
suggest that the xenoreactive T cells measured in these assays might represent
relatively rare CTL clones that do not require CD8 molecule function because
of very high TcR affinity for xenoantigens. This raises the possibility that
xenogeneic CTL may more frequently recognize xenoantigens in association
with self MHC molecules. In order to study this question, a series of experi-
ments were performed in which CML responses of mouse T cells primed
Implications for Mechanisms of Xenograft Rejection 111
against human or pig xenoantigens were compared using xenogeneic targets
versus H-2-identical transfected or transgenic mouse targets expressing the
appropriate xenogeneic class I antigen [33,40-42,44,45,81,82,93]. The latter
targets would present xenoantigens not only in native form but also as pep-
tides in association with host (mouse) MHC molecules. Although two studies
[33,42] appeared to favor direct over associative recognition of xenoantigens,
the large majority of such studies [40,41,44,45,81,82,93] demonstrated pre-
dominantly or exclusively associative recognition. The evidence for predomi-
nant associative recognition included significantly greater lysis of transfected
mouse targets than xenogeneic targets [40,41,81,82,93], blocking ofthe lysis
oftransfected targets by antihost H-2 antibodies [40,41,44,81,93], and lysis of
transfected mouse targets co expressing self but not allogeneic H-2 molecules
[41,44,45, 81]. The identification of the specific oligopeptides of the xeno-
geneic class I MHC antigens recognized in association with self MHC
molecules by the xenoreactive CTL [45, 94] provided further confirmation of
the existence of an associative recognition pathway. Thus, the bulk of evi-
dence from these studies supports the notion that CTL predominantly recog-
nize disparate xenoantigens associatively rather than directly, similar to the
case for helper T cells.
Implications for Mechanisms of Xenograft Rejection
The observation that T cells recognize disparate xenoantigens primarily as

peptides in association with self MHC molecules on host cells rather than di-
rectly on xenogeneic cells has important implications for mechanisms of cell-
mediated xenograft rejection. These implications are based in part on the so-
called three-cell model of cytotoxic T cell activation [95]. According to the
three-cell model, the activation of CTL by helper T cells requires intimate
physical contact between the two types of T cells bound to the same APe. A
dependence on associative recognition of xenoantigens by helper T cells
would lead to the activation of CTL recognizing xenoantigens in a form not
expressed on the somatic cells of the xenograft, as shown in Fig. 7.1. The re-
sulting absence of xenograft-specific CTL would imply either an atypical
CTL-mediated mechanism of rejection or, more likely, a CTL-independent
mechanism (Fig. 7.1). The helper-dependent, NK-like, xenoreactive cytotox-
ic cells characterized by Doody and Winn (see above) could be the mediators
of such a CTL-independent rejection mechanism. Whether or not the three-
cell model of cytotoxic T cell activation is valid, overwhelmingly associative
recognition of xenoantigens by cytotoxic T cells themselves would have simi-
lar implications regarding nonclassical mechanisms of xenograft rejection
(Fig. 7.1).
112 Mechanism of Ccllular Xenograft Rejection
T Cell Subset with

Obligatory Associative
Xeno·Recoqnition
A) None IL2(
"-------_.....
C) IL2(
,
G)~
IL2 C"[
Xeno peptide
IL2( h
Fig. 7.1. Implications of obligatory associative recognition of xenoantigens on mechanisms

of xenograft rejection in the context of thc threc-cell model of cytotoxic T cell activation
[95]. If both helper (Th) and cytotoxic (Te) T cells can recognize xenoantigens directly on
xenogeneic cells (A). then Th will activate Te specific for the antigens expressed on the so-
matic cells of the xenograft. and the graft will be rejected by a classical Tc-mediated mecha-
nism. If Th (B) and/or Tc (C) are unablc to recognize xenoantigens directly on xenogeneic
cells. then Tc specific for the antigens expressed on the somatic cells of the xenograft will not
be activated. Instead. the Tc that are activated (D) will bc specific for xenoantigens in a form
not expresscd on the somatic cclls of the xenograft. i.e., xenogeneic peptides in association
with host MHC molecules. Thus. the graft will be rejectcd by a Tc-independent mechanism
(D), perhaps involving a population (Xc) of Th-dependent. antigen-nonspecific cytotoxic
effector cells
Implications for the Control of Xenograft Rejection
As noted earlier, in vitro studies have demonstrated that the induction phase
of the xenogeneic response is mediated overwhelmingly by the CD4+ subset
of helper T cells. This observation. together with the central role of helper
T cells in the initiation and control of immune responses. raiscs the possibility
that xenograft rejection might be controlled more easily and with more specif-
Conclusions 113
ic immunosuppression than allograft rejection. Indeed, it has been shown that
CD4-specific (but not CD8-specific) immunosuppression in vivo significantly
prolongs xenogeneic but not whole MHC-disparate allogeneic skin graft sur-
vival in the mouse [96]. This and similar observations in murine models of pan-
creatic islet [97-101] and heart [102] xenografting are well explained by the in
vitro findings. The skin and pancreatic islet grafts used in most of these studies
are thought to be resistant to antibody-mediated rejection. The demonstra-
tion that rejection was relatively easily controlled in the absence of humoral
rejection raises the possibility that a similar result might be obtained in clinical
xenografting if effective control of the humoral xenogeneic response could be
achieved.
Conclusions
Current knowledge of the cell-mediated immune response to xenografts can

be summarized as follows:
1. Helper and cytotoxic T cell responses to xenoantigens are generally weaker

than to alloantigens. This results from diminished frequencies of xenoreac-
tive helper and cytotoxic T cell precursors in unprimed hosts and is mani-
fested by decreased or undetectable responses in primary in vitro bulk cul-
tures (MLR, IL-2 production, and CML). Although somewhat dependent
on the particular responder species, xenogeneic responses generally be-
come weaker as the phylogenetic disparity increases.
2. The cell populations mediating xenogeneic responses and the target anti-
gens recognized by xenoreactive T cells are largely the same as those found
in allogeneic responses. Some unique features of xenogeneic responses in-
clude the recognition of both monomorphic and polymorphic MHC deter-
minants, the comparable frequencies of MHC class I- and class II-specific
CTL, and the probable existence of a potent CTL-independent, NK-medi-
ated effector pathway.
3. The weakness of xenogeneic T cell responses results from defects in molec-
ular interactions at the cell surface between T cells and xenogeneic cells.
Although the results of studies in this area have been somewhat conflicting,
the accumulated evidence suggests the existence of defects in several of the
types of molecular interactions outlined in Table 7.1. The number and
severity of defective interactions probably become more significant as the
phylogenetic disparity increases. Although the available information is in-
complete, the evidence is clearest for the existence of defects in CD4/CD8
and LFA-2 interactions across species differences. There are probably addi-
tional, albeit less pronounced, defects in the TcR repertoire for xenoanti-
gens and in certain incompletely defined lymphokine interactions. There is
probably the least evidence for a major defect in LFA-l function across
species differences.
4. A consequence of the defective xenogeneic molecular interactions is that

T cells more easily recognize disparate xenoantigens in association with
host MHC molecules on host cells, in the manner used for recognition of
nominal antigens, rather than directly on xenogeneic cells. in the manner
used for recognition of MHC alloantigens. This phenomenon applies to
both helper and cytotoxic T cells. The preference for associative recognition
of xenoantigens increases as the phylogenetic disparity increases. As a con-
sequence of the requirement for associative recognition of xenoantigens,
the only pathway used for the induction phase of the response consists of
CD4+ helper T cells recognizing xenoantigens in association with self (host)
class II MHC molecules.
5. The requirement for associative recognition of disparate xenoantigens has
important implications for the mechanisms of xenograft rejection.
Assuming that the postulates of the three-cell model of cytotoxic T cell acti-
vation are correct, a requirement for associative recognition of xenoanti-
gens by helper T cells would result in the activation of cytotoxic T cells spe-
cific for antigens not present on the somatic cells of the xenograft. This
would imply an atypical CTL-mediated or a CTL-independent mechanism
of rejection. A requirement for associative recognition by cytotoxic T cells
themselves would have similar implications, whether or not the three-cell
model is correct. Thus. these observations raise the possibility of a CTL-in-
dependent pathway of cell-mediated xenograft rejection.
6. The dependence of the induction phase of the xenogeneic response on the
CD4+ subset raises the possibility that cellular xenograft rejection might be
more easily controlled than allograft rejection. Indeed, this has been con-
firmed by the efficacy of in vivo CD4-specific immunosuppression in pro-
longing the survival of skin, pancreatic islet, and heart xenografts but not
whole MHC-disparate allografts in murine models. Whether or not this par-
ticular approach to immunosuppression will be effective in clinical
xenografting, current knowledge suggests that cell-mediated immunity to
xenoantigens is no stronger and if anything is weaker than allogeneic immu-
nity. This implies that the development of a means to control the humoral
immune response to xenografts might enable easier transplantation of
xenografts than allografts.
Future Directions
Future studies in several areas would be helpful in improving our understand-

ing of cell-mediated immune responses to xenografts:
1. While helper T cell responses to xenoantigens have been found to be dimin-

ished in bulk in vitro assays, not a single study has addressed the matter
quantitatively by limiting dilution analysis. Future studies should quantify
xenogeneic helper T cell responses for a range of species combinations.
Future Directions 115
2. Many questions remain unanswered concerning the function of cell surface
molecules across species differences (Table 7.1). The interpretation of re-
sults in existing models is complicated by the likelihood of redundancy in
the function of some of the molecular pathways. For instance, if the TcR-
xenoantigen interaction is of particularly high affinity in a given experimen-
tal system, then concomitant defects in one or more of the accessory
molecule pathways might be masked. This mechanism probably underlies
many of the conflicting results in current studies. Future studies would be
most helpful if they focused on a single molecular interaction at a time by ar-
tifically "correcting" potential defects in all other pathways. Successful
identification of specific defects in molecular interactions would improve
understanding of T cell biology and might suggest immunosuppressive
methods for controlling xenograft rejection.
3. A critical question is the relative role of associative versus direct recognition
of xenoantigens. This has been studied rather extensively for the human-
mouse combination. However, only one group has investigated the question
for the more clinically relevant case of human T cell responses to large ani-
mal species [28]. Their observation that human helper T cells can recognize
xenoantigens other than those of the mouse in an allo-like direct manner is
an important finding that should be confirmed by other laboratories and in-
vestigated in a quantitative fashion. Furthermore, human T cell responses in
vitro should be screened for a range of stimulator species in order to identify
those species stimulating the weakest responses. Such results might suggest
a particular species for use in clinical xenografting.
4. Xenogeneic systems in which T cells recognize xenoantigens strictly in an
associative manner could serve as useful models for studying cell interac-
tions during T cell activation and mechanisms of graft rejection. For exam-
ple, this kind of model would provide an opportunity to study whether
helper T cells can activate cytotoxic T cells without the need for intimate
physical contact on the same APC, as current understanding would suggest.
Systems in which cytotoxic T cells themselves are strictly dependent on as-
sociative recognition of xenoantigens would provide an opportunity for
studying nonclassical mechanisms of graft rejection.
5. Finally, from a practical perspective, the in vitro observation that xenogene-
ic cell-mediated responses are no stronger (and possibly weaker) than allo-
geneic responses raises the possibility of clinical xenografting in situations
where humoral rejection is not a significant problem. Those situations
might at present include the use of grafts inherently resistant to humoral re-
jection, such as cultured pancreatic islets or the liver.
Acknowledgements. This work was supported in part by Usphs grants

HL36372 and HL18646 and by grant 11 9C1 from the Cystic Fibrosis
Foundation.
References
1. Auchincloss H Jr. Xenogeneic transplantation. A review. Transplantation 1988: 46: 1.

2. Shons AR, Najarian JS. Xenograft rejection in human beings. Rev. Surg. 1975; 32: 70.
3. Lance EM, Medawar PB. Survival of skin heterografts under treatment with antilym-
phocytic serum. Lancet 1968; 1: 1174.
4. Manning D D, Reed ND, Shaffer CF. Maintenance of skin xenografts of widely divergent
phylogenetic origin on congenitally athymic (nude) mice. J. Exp. Med. 1973; 138: 488.
5. Asantila T, Vahala J, Toivanen P. Response of human fetal lymphocytes in xenogeneic
mixed leukocyte culture: phylogenetic and ontogenetic aspects. Immunogenetics 1974:
1: 272.
6. Hersh EM. Blastogenic responses of human lymphocytes to xenogeneic cells in vitro.
Transplantation 1971; 12: 287.
7. Woolnough JA, Misko IS, Lafferty KJ, Cytotoxic and proliferative lymphocyte re-
sponses to allogeneic and xenogeneic antigens in vitro. Aust. J, Exp. BioI. Med. Sci.
1979; 57: 467.
8, Lafferty KJ, Jones MAS. Reactions ofthe graft-versus-host (OVH) type. Aust. J. Exp.
BioI. Med. Sci. 1969;47: 17.
9. Oreineder DK, Rosenthal AS. Macrophage activation of allogeneic lymphocyte prolif-
eration in the guinea pig mixcd lcukocytc culturc. J. Immunol.1975; 114: 1541.
10. Wilson DB, Fox DH, Quantitative studies on the mixed lymphocyte interaction in rats:
VI. Reactivity of lymphocytes from conventional and germfree rats to allogeneic and
xenogeneic cell surface antigens. J. Exp. Med. 1971; 134: 857.
11. Wilson DB, Nowell Pc. Quantitative studies on the mixed lymphocyte interaction in
rats: IV. Immunological potentiality of the responding cells. J. Exp. Med, 1970; 131: 391.
12. Nielsen HE. Reactivity of lymphocytes from germfree rats in mixed leukocyte culture
and in graft-versus-host reaction. J. Exp. Med.1972; 136: 417.
13. Viguier M, Lotteau V, Charron D, Debre P. Xenogeneic recognition of soluble and cell
surface HLA class II antigens by proliferative murine T cells. Eur. J. Immunol. 1987; 17:
1540.
14. Moses RD, Pierson RN, Winn HJ, Auchincloss H Jr. Xenogeneic proliferation and
IL-2 production are dependent on CD4+ helper T cells and self antigen-presenting cells
in the mouse. J. Exp. Med. 1990; 172: 567.
15. Y oshizawa K, Yano A. Mouse T lymphocyte proliferative responses specific for human
MHC products in mouse anti-human xenogeneic MLR. J. Immunol. 1984; 132: 2820.
16. Swain SL, Dutton RW, Schwab R, Yamamoto J. Xenogeneic human anti-mouse Tcell
responses are due to the activity ofthe same functional T cell subsets responsible for al-
lospecific and major histocompatibility complex-restricted responses. J. Exp. Med.
1983; 157: 720.
17. Peck AB, Alter BJ, Lindahl KF. Specificity in T cell mediated lympholysis: identical ge-
netic control of the proliferative and effector phases of allogeneic and xenogeneic reac-
tions. Transplant. Rev. 1976; 29: 189.
18. Hirshberg H, Rankin B, Thorsby E. Specificity of human lymphocytes proliferating in
response to allogeneic and xenogeneic cells in vitro. Transplantation 1974; 17: 593.
19. Shons AR, Kromrey C, Najarian JS. Xenogeneic mixed leucocyte response. Cell.
Immunol. 1973; 6: 420.
20. Bach FH, Engstrom MA, Bach ML, Sell KW. Histocompatibility matching. VII. Mixed
leukocyte cultures between chimpanzee and man. Transplant. Proc. 1972; 4: 97.
21. Widmer MB, Bach FH. Allogeneic and xenogeneic response in mixed leukocyte cul-
tures.J. Exp. Med. 1972; 135: 1204.
22. Knight SC, Thorbecke OJ. Ontogeny of cellular immunity: development in rat thymo-
cytes of mixed lymphocyte reactivity to allogeneic and xenogeneic cells. Cell. Immunol.
1971;2: 91.
23. Rudolph RH, Hered B, Epstein RB, Thomas ED. Canine mixed leukocyte reactivity
and transplantation antigens. Transplantation 1969; 8: 141.
References 117
24. Hollander N, Ginsburg H, Feldman M. In vitro generation of memory lymphocytes re-
active to transplantation antigens. J. Exp. Med.1974; 140: 1057.
25. Toivanen P, Asantila T. Ontogeny of allogeneic and xenogeneic cell recognition. II.
Studies on fetal lamb [Abstr]. Scand. J. Immuno!.1974; 3: 891.
26. Alter BJ, Bach FH. Cellular basis of the proliferative response of human T cells to
mouse xenoantigens. J. Exp. Med. 1990; 171: 333.
27. Widmer MB, Sondel PM, Bach FH. Responsiveness of germfree mice in mixed leuko-
cyte culture: re-examining the nature of the responding unit. J. Exp. Med. 1972; 136:
398.
28. Lucas PJ, Shearer GM, NeudorfS, Gress RE. The human anti-murine xenogeneic cyto-
toxic response: I. Dependence on responder antigen presenting cells. J. Immuno!.
1990; 144: 4548.
29. Golding H, Mizuochi T, McCarthy SA, Cleveland CA, Singer A. Relationship among
function, phenotype, and specificity in primary allospecifie T cell populations: identifi-
cation of phenotypically identical but functionally distinct primary T cell subsets that
differ in their recognition of MHC class I and class II allodeterminants. J. Immuno!.
1987; 138: 10.
30. Burakoff SJ, Dorf ME, Benacerraf B. Specificity of rat xenogeneic cell-mediated cyto-
lysis for the products of the K and D loci of the mouse H-2 complex. J. Immuno!. 1977;
118: 1795.
31. Irwin MJ, Heath WR, Sherman LA. Species-restricted interactions between CD8 and
the alpha3 domain of class I influence the magnitude of the xenogeneic response.
J. Exp. Med. 1989; 170: 1091.
32. Achour A, Begue B, Gomard E, Paul P, Sayagh B, van Pel A, Levy l. Specific lysis of
murine cells expressing HLA molecules by allospecific human and murine H-2-restrict-
ed anti-HLA T killer lymphocytes. Eur. l. Immuno!.1986; 16: 597.
33. Bluestone lA, Pescovitz MD, Frels WI, Singer DS, Hodes RJ. Cytotoxic T lymphocyte
recognition of a xenogeneic major histocompatibility complex antigen expressed in
transgenic mice. Eur. J. Immuno!.1987; 17: 1035.
34. Stejskal V, Holm G, Perlmann P. Differential cytotoxicity of activated lymphocytes on
allogeneic and xenogeneic target cells. I. Activation by tuberculin and by staphylococ-
cus filtrate. Cel!. Immuno!. 1973; 8: 71.
35. Burakoff SJ, Ratnofsky SE, Benacerraf B. Mouse cytolytic T lymphocytes induced by
xenogeneic rat stimulator cells exhibit specificity for H-2 complex alloantigens. Proc.
Nat!. Acad. Sci. USA 1977; 74: 4572.
36. Mentzer SJ, Barbosa lA, Strominger JL, Biro PA, Burakoff SJ. Species-restricted
recognition of transfected HLA-A2 and HLA-B7 by human CTL clones. J. Immuno!.
1986; 137: 408.
37. Dill 0, Kievits F, Koch S, Ivanyi P, Hammerling GJ. Immunological function ofHLA-C
antigens in HLA-Cw3 transgenic mice. Proc. Nat!. Acad. Sci. USA 1988; 85: 5664.
38. Berke G, Ax W, Ginsburg H, Feldman M. Graft reaction in tissue culture: II.
Quantification of the lytic action on mouse fibroblasts by rat lymphocytes sensitized on
mouse embryo monolayers. Immunology 1969; 16: 643.
39. Ginsburg H. Graft versus host reaction in tissue culture: I. Lysis of monolayers of em-
bryo mouse cells from strains differing in the H-2 histocompatibility locus by rat lym-
phocytes sensitized in vitro. Immunology 1968; 14: 621.
40. Barra C, Perarnau B, Gerlinger P, Lemeur M, Gillet A, Gibier P, Lemonnier FA.
Analysis of the HLA-Cw3-specific cytotoxic T lymphocyte response of HLA-B7 x hu-
man beta2m double transgenic mice. J. Immuno!.1989; 143: 3117.
41. Holterman MJ, Engelhard VH. HLA antigens expressed on murine cells are preferen-
tially recognized by murine cytotoxic T cells in the context of the H-2 major histocom-
patibility complex. Proc. Nat!. Acad. Sci. USA 1986; 83: 9699.
42. Kievits F. Wijffels J, Lokhorst W, Ivanyi P. Recognition of xeno-(HLA. SLA) major
histocompatibility complex antigens by mouse cytotoxic Tcells is not H-2 restricted: A
study with transgenic mice. Proc. Nat!. Acad. Sci. USA 1989; 86: 617.
43. Engelhard VH, Le AT, Holterman MJ. Species-specific structural differences in the al-
phal and alpha2 domains determine the frequency of murine cytotoxic T cell precur-
sors stimulated by human and murine class I molecules. 1. Immunol. 1988: 141: 1835.
44. Hammerling Gl. Hunt T. Dill O. Moreno 1. Cytotoxic T lymphocyte recognition of
HLA-A2 antigens in normal and HLA-Cw3-transgenic mice. Eur. 1. Immunol. 1989:
19: 599.
45. Pala P, Corradin G, Strachan T, Sodoyer R, 10rdan BR, Cerottini 1, Maryanski lL.
Mapping of HLA epitopes recognized by H -2-restricted cytotoxic T lymphocytes specif-
ic for HLA using recombinant genes and syntheticpeptides. 1. Immunol. 1988: 140: 871.
46. Le AT, Bernhard E1, Holterman M1, Strub S, Parham p, Lacy E, Engelhard VH.
Cytotoxic T cell responses in HLA-A2.l transgenic mice. Recognition of HLA al-
loantigens and utilization of HLA-A2.1 as a restriction element. 1. Immunol. 1989: 142:
1366.
47. Newlands ES, Roitt 1M. Evidence for two cell populations involved in the rejection of a
xenograft in mice. Transplantation 1975: 20: 13.
48. Dennert G. Effector mechanisms of cell-mediated immunity to xenogeneic cell anti-
gens. 1. Immunol. 1974; 113: 201.
49. Mizuochi T, Ono S, Malek TR, Singer A. Characterization of two distinct primary Tcell
populations that secrete interleukin 2 upon recognition of class I or class II major histo-
compatibility antigens. 1. Exp. Med. 1986: 163: 603.
50. Sprent 1, Webb SR, Function and specificity of T cell subsets in the mouse. Adv.
Immunol. 1987; 41: 39.
51. Lindahl KF, Bach FH. Genetic and cellular aspects of xenogeneic mixed leukocyte cul-
ture reaction. 1. Exp. Med. 1976; 144: 305.
52. Brunswick M, Lake P. Functional interactions of human and murine lymphoid cells. I.
Analysis of primary and secondary responses. Cell. Immunol. 1986: 103: 441.
53. Carnaud C, Fadai-Ghotbi M, Lesavre P, Bach IF. Education of human lymphocytes
against mouse cells: specific recognition of H-2 antigens. Eur. 1. Immunol. 1977: 7: 81.
54. Burakoff Sl, Engelhard VH, Kaufmann 1, Strominger lL. Induction of secondary cyto-
toxicTlymphocytes by Iiposomescontaining HLA-DR antigens. Nature 1980: 283: 495.
55. Greenstein 1L, Foran 1A, Gorga 1C, Burakoff S1. The role ofT cell accessory molecules
in the generation of class II-specific xenogeneic cytolytic T cells. 1. Immunol. 1986; 136:
2358.
56. Engelhard VH. Benjamin C, Isolation and characterization of monoclonal mouse cyto-
toxic T lymphocytes with specificity for HLA-A, -B, or -DR alloantigens. 1. Immunol.
1982; 129: 2621.
57. Engelhard VH, Kaufman 1F, Strominger 1L, Burakoff S1. Specificity of mouse cytotox-
ic T lymphocytes stimulated with either HLA-A and -B or HLA-DR antigens reconsti-
tuted into phospholipid vesicles. 1. Exp. Med. 1980: 152: 54S.
58. Escobar MR, Sigel MM. Effects of lymphoid cells and serum on target cells in xeno-
geneic cell-culture systems. Proc. Soc. Exp. BioI. Med. 1970; 134: 744.
59. Grani CK, Avis Pl, Hali 1G, Alexander P. Evidence for the role of antibody in the cyto-
toxic action of lymph cells on xenogeneic target cells. Transplantation 1971; 11: 356.
60. Harding B, MacLennan ICM. The distribution of antibody and antibody-producing
cells after immunization with xenogeneic cells. Immunology 1972; 23: 35.
61. Moller G, Svehag SE. Specificity of lymphocyte-mediated cytotoxicity induced by in
vitro antibody coated target cells. Cell. Tmmunol. 1972; 4: I.
62. Chaussy C, Hammer C, Pongratz H, von Scheel 1, Pfeifer K1, Land W, Sollinger HW,
Pielsticker K. Prolongation of graft survival in rats and dogs by a specific anti-
macrophage serum (AMS). Transplant. Proc. 1975: 7: 779.
63. Lindahl KF, Bach FH. Human lymphocytes recognise mouse alloantigens. Nature
1975; 254: 607.
64. Engelhard VH, Benjamin C. Xenogeneic cytotoxic T-cell clones recognize alloanti-
genic determinants on HLA-A2. Immnnogenetics 1983; 18: 461.
65. Berke G, Clark WR, Feldman M. In vitro induction of a heterograft reaction: immuno-
logical parametcrs of the sensitization of rat lymphocytes against mouse cells in vitro.
Transplantation 1971; 12: 237.
References 119
66. Gress RE, Nathenson SG, Lucas PJ. Fine specificity of xenogeneic antigen recognition
by human T cells. Transplantation 1989; 48: 93.
67. Clayton LK, Sieh M, Pious DA, Reinherz EL. Identification of human CD4 residues af-
fectingclass II MHC versus HIV-l gpl20binding. Nature 1989; 339: 548.
68. von Hoegen P, Miceli MC, Tourvieille B, Schilham M, Parnes JR. Equivalence of hu-
man and mouse CD4 in enhancing antigen responses by a mouse class II-restricted T
cell hybridoma. J. Exp. Med.1989; 170: 1879.
69. Barbosa JA, Mentzer SJ, Minowada G, Strominger JL, Burakoff SJ, Biro PA.
Recognition of HLA-A2 and -B7 antigens by cloned cytotoxic T lymphocytes after
gene transfer into human and monkey, but not mouse, cells. Proc. Nat!. Acad. Sci. USA
1984; 81: 7549.
70. Bernabeu C, Finlay D, van de Rijn M, Maziarz RT, Biro PA, Spits H, de Vries J,
Terhorst CPo Expression of the major histocompatibility antigens HLA-A2 and HLA-
B7 by DNA-mediated gene transfer. J. Immuno!. 1983; 131: 2032.
71. van de Rijn M, Bernabeu C, Royer-Pokora B, Weiss J, Seidman JG, de Vries J, Spits H,
Terhorst C. Recognition of HLA-A2 by cytotoxic T lymphocytes after DNA transfer
into human and murine cells. Science 1985; 226: 1083.
72. Bernabeu C, Maziarz R, Spits H, de Vries J, Burakoff SJ, Terhorst C. Co expression of
the human HLA-A2 or HLA-B7 heavy chain gene and human beta2-microglobulin
gene in L cells. J. Immuno!. 1984; 133: 3188.
73. Bernhard EJ, Le AT, Y annelliJR, Holterman MJ, Hogan KT, Parham P, Engelhard VH.
The ability of cytotoxic T cells to recognize HLA-A2.1 or HLA-B7 antigens expressed
on murine cells correlates with theirepitope specificity.J. Immuno!.1987; 139: 3614.
74. Golde WT, Kappler JW, Greenstein J, Malissen B, Hood L, Marrack P. Major histo-
compatibility complex-restricted antigen receptor on T cells. Viii. Role of the LFA-l
molecule. J. Exp. Med.1985; 161: 635.
75. Koller TD, Clayberger C, Maryanski JL, Krensky AM. Human allospecific cytolytic T
lymphocyte lysis of a murine cell transfected with HLA-A2.J. Immuno!. 1987; 138: 2044.
76. Maryanski JL, Moretta A, Jordan B, de Plaen E, van Pel A, Boon T, Cerottini J. Human
T cell recognition of cloned HLA class I gene products expressed on DNA transfec-
tants of mouse mastocytoma P815. Eur. J. Immuno!. 1985; 15: 1111.
77. Gomard E, Begue B, Sodoyer S, Maryanski JL, Jordan BR, Levy JP. Murine cells ex-
pressing an HLA molecule are specifically lysed by HLA-restricted antiviral human T
cells. Nature 1986; 319: 153.
78. Cowan EP, Coligan JE, Biddison WE. Human cytotoxic T-lymphocyte recognition of
an HLA-A3 gene product expressed on murine L cells: the only human gene product
required on the target cells for lysis is the class I heavy chain. Proc. Nat!. Acad. Sci. USA
1985; 82: 4490.
79. Barbosa JA, Mentzer SJ, Kamarck ME, Hart J, Biro PA, Strominger JL, Burakoff SJ.
Gene mapping and somatic cell hybrid analysis of the role of human lymphocyte func-
tion-associated antigen-3 (LFA-3) in CTL-target cell interactions. J. Immuno!. 1986;
136:3085.
80. Epstein H, Hardy R, May JS, Johnson MH, Holmes N. Expression and function of
HLA-A2.l in transgenic mice. Eur. J. Immuno!. 1989; 19: 1575.
81. Maryanski JL, Accolla RS, Jordan B. H-2 restricted recognition of cloned HLA class I
gene products expressed in mouse cells. J. Immuno!. 1986; 136: 4340.
82. Chamberlain JW, Nolan JA, Gromkowski SH, Kelley KA, Eisenstadt JM, Herrup K,
Janeway CA, Weissman SM. Cell surface expression and alloantigenic function of a hu-
man class I MHC heavy chain gene (HLA-B7) in transgenic mice. J. Immuno!. 1988;
140: 1285.
83. Taurog JD, Lowen L, Forman J, Hammer RE. HLA-B27 in inbred and non-inbred
transgenic mice. Cell surface expression and recognition as an alloantigen in the ab-
sence of human beta2-microglobulin. J. Immuno!.1988; 141: 4020.
84. Auchincloss H Jr, Moses RD, Conti D, Sundt T, Smith C, Sachs DH, Winn HJ.
Rejection of transgenic skin expressing a xeno-class I antigen is CD4-dependent and
CD8-independent. Transplant. Proc.1990; 22: 1059.
85. Maziarz RT, Burakoff SJ. Bluestone JA. The alpha-3 domain of class I MHC proteins
influences cytotoxic T lymphocyte recognition of antigenic determinants located with-
in the alpha-l and alpha-2 domains. J. Immuno!. 1988; 140: 4372.
86. Samberg NL Scarlett EC, Stauss HJ. The alpha3 domain of major histocompatibility
complex class I molecules plays a critical role in cytotoxic T lymphocyte stimulation.
Eur. J. Immuno!. 1989; 19: 2349.
87. Maziarz R, Allen H, Strominger JL. Flavell R. Biro P A. Burakoff SJ. Recognition of in-
terspecies hybrid class I histocompatibility antigens by antigen-specific cytolytic T lym-
phocytes. Proc. Nat!. Acad. Sci. USA 1985; 82: 6276.
88. Kievits F. Ivanyi P, Krimpenfort P, Berns A, Ploegh HL HLA-restricted recognition of
viral antigens in HLA transgenic mice. Nature 1987; 329: 447.
89. Bernhard EJ, Le AT. Barbosa lA, Lacy E, Engelhard VH. Cytotoxic T lymphocytes
from HLA-A2 transgenic mice specific for HLA-A2 expressed on human cells. J. Exp.
Med. 1988; 168: 1157.
90. Smith L. CD4+ murine Tcells develop from CD8+ precursors in vivo. Nature 1987; 326:
798.
91. Kisielow P. Bluthmann H. Staerz UD, Steinmetz M, von Boehmer H. Tolerance in T-
cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes.
Nature 1988; 333: 742.
92. Shaw WC, Nelson CA, Newberry RD, Kranz DM. Russell JH, Loh DY. Selective ex-
pression of an antigen receptor on CD8-bearing T lymphocytes in transgenic mice.
Nature 1988; 335: 271.
93. Engelhard VH, Powers GA. Moore LC, Holterman MJ. Correa-Freire Me. Cytotoxic
T lymphocyte recognition of HLA-A/B antigens introduced into EL-4 cells by cell-
liposome fusion. J. Immuno!. 1984; 132: 76.
94. Maryanski JL, Pal a p, Corradin G, Jordan BR. Cerottini J. H-2-restricted cytolytic
Tcells specific for HLA can recognize a synthetic HLA peptide. Nature 1986; 324: 578.
95. Mitchison NA, O'Malley e. Three-cell-type clusters of T cells with antigen-presenting
cells best explain the epitope linkage and noncognate requiremcnts of the in vivo cy-
tolytic response. Eur. J. Immunol.1987; 17: 1579.
96. Pierson RN, Winn HJ, Russell PS, Auchincloss H Jr. Xenogeneic skin graft rejection is
especially dependent on CD4+ T cells. l. Exp. Med. 1989; 170: 991.
97. Ricordi C, Lacy PE, Sterbenz K, Davie JM. Low-temperature culture of human islets or
in vivo treatment with L3T4 antibody produces a marked prolongation of islet human-
to-mouse xenograft surviva!' Proc. Natl. Acad. Sci. USA 1987; 84: 8080.
98. Kaufman DS, Kong CS, Shizuru JA, Gregory AK, Fathman CG. Use of anti-L3T4 and
anti-Ia treatments for prolongation of xenogeneic islet transplants. Transplantation
1988; 46: 210.
99. Wilson JD, Simeonovic CJ, Ting JHL, Ceredig R. Role of CD4+ T-Iymphocytes in re-
jection by mice of fetal pig proislet xenografts. Diabetes 1989; 38 (Suppll): 217.
100. Lacy PE, Ricordi C, Finke EH. Effect of transplantation site and anti-L3T4 treatment
on survival ofrat, hamster. and rabbit islet xenografts in mice. Transplantation 1989; 47:
761.
101. Kawai M, Gotoh M, Monden M, Yamamoto H, Ichikawa T, Valdivia LA, Mori T,
Uenaka A, Nakayama E. Effect of L3T4 and Lyt-2 monoclonal antibodies on islet
xenograft (rat to mouse) rejection. Transplant. Proc. 1989; 21: 2709.
102. Sakakibara N, Click RE, Condie RM, Jamieson SW. Rejection/acceptance of
xenografts. Transplant. Proc. 1989; 21: 524.
Chapter 8
Xenotolerance Through Bone Marrow

Transplantation
M. SYKES, I. AKSENTIJEVICH, Y. SHARABI, AND D.H. SACHS
Introduction
The use of xenogeneic bone marrow transplantation (BMT) may provide an

approach to induction of specific hyporeactivity toward a xenogeneic organ
donor, while preserving otherwise normal immune function. Numerous stud-
ies in allogeneic animal models have demonstrated that specific transplanta-
tion tolerance can be produced by transplanting hematopoietic cells across full
major histocompatibility (MHC) barriers [1-7]. This approach obviates the
need for chronic immunosuppressive therapy with its attendant risks, and the
likelihood that donor-type grafts will be rejected is minimized while
chimerism is maintained [8-10].
Unfortunately, however, the use of this approach to the induction of trans-
plantation tolerance in man is currently infeasible, due to the unacceptable
risks associated with myeloablative conditioning regimens necessary to
achieve bone marrow engraftment in adults, and the difficulty in achieving
bone marrow engraftment across major histocompatibility barriers. The latter
difficulty has represented a major limitation to the use of clinical bone marrow
allotransplantation [11, 12], and, as illustrated below, resistance to bone mar-
row engraftment may be even greater when BMT is attempted across xeno-
geneic barriers.
While the use of lethal whole body irradiation (WBI) and BMT has been
shown to permit induction of transplantation tolerance across xenogeneic bar-
riers in animal models [2, 13-16], the clinical applicability of this approach is
limited by the toxicity of preparative regimens which involve lethal WBI
[17-19], by graft-versus-host disease (GVHD) [20-22], and by difficulty in
achieving engraftment [12,23,24]. Therefore, if BMT is to be a useful method
of inducing transplantation tolerance in the clinical setting, it will be essential
to develop conditioning regimens which specifically target those host ele-
ments which resist marrow engraftment, rather than to use regimens which
nonspecifically destroy proliferating cells such as hematopoietic stem cells and
gut epithelium. Furthermore, it will be important to develop means of achiev-
ing xenoengraftment without GVHD. Elimination of GVHD-producing T
cells in bone marrow has been associated with an increased incidence of fail-
ure of engraftment of allogeneic marrow in both clinical [23,25,26] and exper-
imental [27,28] models. To develop a nontoxic conditioning regimen which
permits engraftment ofT cell-depleted (TCD) xenogeneic bone marrow with-
122 Xenotolerance Through Bone Marrow Transplantation
out GYHD, therefore, animal models will be needed to determine which host
elements should be targeted for elimination.
An additional obstacle must be addressed when considering BMT across
complete histocompatibility barriers (allogeneic or xenogeneic). Since MHC
restriction specificity is determined by host-type antigen-presenting cells
(APC) present in the recipient's thymus during the time when T cells are re-
generating post-BMT [29], a source of APC bearing the same MHC pheno-
type is required to present antigen effectively in the periphery. Since such pe-
ripheral APC are bone marrow-derived. they will eventually consist only of
donor-type cells in fully allogeneic or fully xenogeneic chimeras. Thus. a state
of immunoincompetence would exist, since the restricting elements required
to present antigen to host-restricted T cells would be absent from the periph-
ery [30].
One way of circumventing this difficulty would be to provide a source of
host-type bone marrow cells (BMC) and thus produce a mixed chimera. so
that a continuous source of host-type APC is provided. Indeed, superior im-
munocompetence has heen demonstrated in mixed allogeneic chimeras com-
pared with fully allogeneic chimeras prepared across complete MHC barriers
[29-33]. Thus. preparation of mixed xenogeneic chimeras might be an optimal
approach to the use of BMT for the induction of transplantation tolerance
across species barriers.
A Nonmyeloablative Conditioning Regimen for Induction

of Mixed Xenogeneic Chimerism
Recent studies have demonstrated that mixed chimerism and transplantation

tolerance can be induced across complete allogeneic MHC barriers in mice us-
ing a relatively nontoxic. nonmyeloablative preparative regimen (Fig. 8.1).
This regimen involves elimination of host T lymphocytes using monoclonal
antibodies (mAbs) specific for CD4-positive and CD8-positive T cells. fol-
lowed by a low dose (3 Gy) ofWBI and a higher dose (7 Gy) of local irradiation
to the thymus [34] (Fig. 8.1).
More recently. we have attempted to extend this model to a xenogeneic rat-
to-mouse species combination [35]. Tolerance and chimerism were not
achieved after administration of TCD F344 strain rat BMC using the same
preparative regimen which was successful in allogeneic combinations. When
mAbs mAbs Fig. 8.1. Time course of

I I treatment with nonmyeloab-
-6 -1 0 7 14 21
~I______~------~I--------~I-------+1--- lative conditioning regimen
for induction of mixed allo-
300 RAD WBI CHIMERISM geneic (B I O.D2 donors) or
700 RAD TI DETERMINED BY FCM
60-S0x 106 TCD RAT BM ANALYSIS OF PBl
mixed xenogeneic (F344 rat
OR 15x 106 TCD Bl0.D2 BM donors) chimerism
Induction of Donor-Specific Transplantation Tolerance 123
Table 8.1. Engraftment ofTCD rat BMC in mice conditioned by a non-mycloablative regi-
men
Group mAb Treatment" Fraction of chimeric anima ISh

(% Donor cells)
2-3 Weeks 5-6 Weeks
I Anti-CD 4+Anti-CD 8 1/4 (3) 0/4

2 Anti-CD4+Anti-CD8+Anti-NK 1.1 2/6(3-14) 1/6 (4)
3 Anti-CD4+Anti-CD8+Anti-Thy 1.2 6/6 (6-10) 5/6(1-9)
4 Anti-CD 4+Anti-CD8+Anti-Thy 1.2+Anti-NK 1.1 10/10 9/9
(9-24,66') (1-9,77<)
a B 10 recipients were treated with 3 Gy W8I, 7 Gy thymic irradiation. and 6x10 7 F 344 rat
BMC on day O. Rat BMC were TCD using anti-CDS mAb RI-3B3 and complement.
Indicated mAbs were administered i. p. on days -6 and -1.
h Xenochimerism was detected by now cytomctric analysis.
C Values obtained from a single outlier animal.
antibodies against host Thyl-positive cells and natural killer (NK) cells were
added to the preparative regimen, however, chimerism and specific transplan-
tation tolerance were successfully induced.
As shown in Table 8.1 (group 4), administration ofmAbs against recipient
CD4+, CD8+, Thy1+, and NKl.1+ cells led to optimal development of
chimerism in the early post-BMT period. Nevertheless, chimerism was also in-
duced in animals which received mAbs against CD4+, CD8+, and Thy1 + cells,
without anti-NKl.l (Table 8.1, group 3). An example of the flow cytometry
profile produced by peripheral blood leukocytes (PBL) from an animal in
group 4 at two different time points post-BMT is shown in Fig. 8.2. These ani-
mals did not develop GVHD, although clinically obvious GVHD was pro-
duced in similarly treated recipients of rat marrow which was not TCD (data
not shown). Thus, our regimen allows the engraftment of TCD rat marrow
without GVHD.
MAbs against NKl.l alone (not shown), or against CD4 and CD8 with or
without anti-NKl.l (Table 8.1, groups 2 and 1, respectively), were not permis-
sive for the engraftment ofTCD rat BMC.
Induction of Donor-Specific Transplantation Tolerance
Figure 8.3 shows that the acceptance of donor-type skin grafts correlates with
mixed xenogeneic chimerism. Animals preconditioned with all four mAbs
(i.e., group 4 in Table 1) demonstrated markedly prolonged acceptance of
donor-type skin grafts; nondonor-type rat (Wistar-Furth) skin grafts were
rapidly rejected, indicating that the chimeras were immunocompetent, and
were specifically tolerant to the bone marrow donor.
124 Xenololerance Through Bone Marrow Transplantation
810 RAT RAT --+ 810

DAY 16 DAY 50
.-
UJ u.. a
U L!)
Z
UJ .D
U ~
(f) ~
UJ t-
a: Z
0 <{
::> ANTI-RAT LCA (OX - 1)
-I
u..
0 C"! d
UJ
a: >
0 ..c
t- I
61.6
t!J
t-
~: I~}J I
0
-I Z
<{ rom"
ANTI -CD5 (OX - 19)
•
LOG1O GREEN FLUORESCENCE
Fig. 8.2. Development of xenochimerism in B I0 mice pre treated with anti-CD4, anti-COX.
anti-Thy 1.2, and anti-NK 1.1 mAbs followed by the regim e n shown in Fig. X.l and infusion
on day Oof6xl0 7 TCD F344 ra t BMC. Two color immunofluoresce nce profiles ofPBL from
B I 0 control (a and b), F344 rat control (e and d), and a typical chimera (e- h). Top lin£' shows
contour plots afte r staining with fluoresceinated anti rat leukocyte common antigen (OX-I)
mAb, which stains all rat le ukocytes (green fluorescence, horizontal a xis) and biotinylated
anti-Kb (SF1) mAb plus Texas Red Streptavidin (TRA), staining all cells of B I 0 origin (red
fluorescence , vertical axis). Sol/om line shows contour plots a fter staining with fluores-
ceinated antirat CDS (OX-19), which stains rat T cells (gree n fluorescence , horizontal axis),
and biotinylated anti-Thyl.2 plus TRA, staining murine T cells (red fluoresce nce , vertical
axis). Percentages of cells in each rectangle were determined by subtraction of background
stainin g obtained by staining with Leu4-ncga tive control mAb. (From the Jo urnal of
Experimental Medicine. 1990, 172: 19S-202, [3S])
Animals preconditioned with three of the four mAbs without anti-NK1.1

(group 3, Table 8.1) also demonstrated donor-specific skin graft prolongation
in most instances. However, in this group, tolerance was only observed in the
three of five animals in which donor rat T cells could be detected among PBL
by 5 weeks following BMT, as shown in Table 8.2. In contrast, all six animals
preconditioned with all four mAbs produced detectable rat T cells by this time
(Table 8.2). Notably, neither rat nor murine T cells were detectable in the cir-
culation 2-3 weeks following BMT (e.g., Fig. 8.2).
This observation is consistent with the interpretation that rat T cells devel-
oped de novo in the recipients ' thymuses along with murine T cells, and were not
progeny of mature T cells contaminating TCD BMC inocula. Consistent with
this hypothesis, rat cells were also detected in the bone marrow, spleen, and thy-
mus of a mouse sacrificed 45 days following BMT [35] . Thus, stem cell engraft-
ment and de novo T cell maturation could be demonstrated in this model.
Gradual Disappearance of Chimerism in Tolerant Mice 125
1001-.....-----,1 F344
90 r- I
I
80 ... ~---,,
,,
70 ... II ,
60 ~--------------------,,
I ,
Fig. 8.3. Survival of donar- 50 ~
type (top, F344) and non- 40 L, '.,
il: ,
donar-type (bottom, WF) 30 - III:
,,,
rat skin grafts placed 123 ill 1_-.,
days after BMT on recipi- 20 - ill
;i
,
,,
ents of7 Gy thymic irradi-
ation,3 Gy WBI, and
> 10
::; 0 !.'I:. :: ,
6xl0 7 TCD F344 BMC, a:
~ 100 --,
following pretreatment W.F.
with anti-CD4 plus anti- '# 90
CDR mAbs (long dashes); 80 -
anti-CD4, anti-CDR, plus
anti-NKl.1 mAbs (dotted 70
line); anti-CD4, anti-CDR, 60
plus anti-Thy1.2 mAbs
(short dashes); anti-CD4,
50 r-
anti-CDS, anti-Thy 1.2, 40 ~
plus anti-NKl.1 mAbs
'i
30 ~
(continuous line). Skin
graft survival on untreated 20 ~
B I 0 controls is indicated 10 ~
by the dashed and dotted
O~~~~~~~~~~~~---~~
line. (From the Journal of
Experimental Medicine,
o 10 20 30 40 50 60 70 80 90 100110
1990,172:195-202. [35]) TIME (DAYS)
Gradual Disappearance of Chimerism in Tolerant Mice
Despite the initial mixed chimerism in these animals, however, PBL

chimerism was gradually lost, and was no longer detectable by 6 months fol-
lowing BMT [35]. The time course of loss of chimerism is illustrated in Fig. 8.4.
Additional time points not shown in the figure were also evaluated.
Importantly, the animals had completely or almost completely lost their
chimerism by day 120, the time when rat skin was grafted on to these mice,
leading to the graft survival curves shown in Fig. 8.3. Thus, donor-specific skin
graft acceptance was still observed during the period when chimerism was dis-
appearing in these mice. These results suggested that either loss of chimerism
was due to a non-T cell-mediated immune mechanism, such as that due to
B cells or NK cells, or was not an immune-mediated phenomenon.
126 Xenotoleranee Through Bone Marrow Transplantation
Table 8.2. Rat T cell repopulation in PBL of mice conditioned with a non-mycloablative
regiment
MAb pretreatment Animal % RatTcellsin PBL" Skin graft

(weeks)b survival
(days)"
2- 3 5 7 26
Anti-CD4+Anti-CD R+ 1 0.4 5.8 4.7 1.2 74

Anti-Thy 1.2 2 0 () () 0 18
3 0 1.3 0.1 0 82
4 0.1 0 0 0 8
5 0 2.0 3.4 0.7 74
Anti-CD 4+Anti-CD 8+ I 0.2 3.3 4.1 1.3 >110
Anti-Thy 1.2+Anti-NK 1.1 2 0.3 3.8 5.8 0.8 28d
3 0.8 3.9 5.8 0.2 >74C
4 0.2 3.0 3.0 0.1 >110
5 0.3 1.7 3.8 0.1 >110
6 0 1.8 0.3 0 >110
" Determined by flow cytometry using anti-CDS mAb OX 19.

b Weeks following BMT.
C Days following skin grafting. Grafting with F344 rat skin was performed approximately
18 weeks fo llowing BMT.

d Although entire graft became firm and dry on day 28, contraction and scarring did not oc-
cur until > 150 days following grafting.
C Animal died 74 days after skin grafting with intact graft.
t From reference [351
25 r---------------------------.-----------------------------~
7 Gy TI/ 3 Gy WBI/ RAT BMC 7 Gy TI/ 3 Gy WBI/ RAT BMC
• mAb AGAINST CD4/ C08/ Thy 1.2 • mAb AGAINST C04/ CDB/ Thy 1.21NK 1. 1
20
1/1
j 15
~
~
.q;
a: 10
*-
15 50 90 183 o 15 50 90 183
TIME (DAYS)
Fig. 8.4. Rat PBL chimerism at various times following BMT, as determined by flow cytome-
try. Each type of bar represents a sin gle animal a t the different time points shown after BMT
(horizontal axis). Lefi-hand chart shows OX I-positive cells among PBL of BlO mice pre-
treated with anti -CD4, anti-CD8, plus anti -Thy1.2 mAbs; right-hand chart shows OX1-posi-
tive cells among PBL of BID mice pretreated with anti-CD4, a nti-CDS. anti-Thyl.1, plus
anti-NK 1.1 mAbs. All animals received 7 Gy TI plus 3 Gy WBI prior to infusion of 6xl 0 7
TCD rat BMC. (From the Journal of Experimental Medicine. 1990. 172: 195-202. [35])
Absence of Antidonor Lymphocytotoxic Antibody 127
Absence of Antidonor Lymphocytotoxic Antibody
While donor-type skin graft survival was markedly prolonged in mixed xeno-
geneic chimeras, these grafts eventually underwent chronic rejection (Table
8.3). Furthermore, repeat donor-type skin grafts were rejected more rapidly
than the first grafts, although their survival was still significantly prolonged in
several instances (Table 8.3). These results were consistent with the possibility
that donor-reactive T cells eventually developed in the thymuses of such ani-
mals after chimerism was lost. Alternatively, skin graft rejection may have
been due to reactivity to skin-specific minor histocompatibility antigens [36,
37], to which animals would not be tolerized by BMC. If immunity towards
antigens expressed on BMC-derived cells developed in these animals, such
Table 8.3. Cytotoxic antibody following second donor skin graft
MAb Pretreatmenta First graft Second graft Maximum percent

survival (days)b survival (days)" specific cytotoxicity
VS.F344 VS.F344
splenocytesd BMce
Normal B 10 (ungrafted) 21.7 65.4

Normal B 10 (grafted) 8 7 84.1 78.8
8 7 ND ND
uCD4+uCD8 8 8 96.8 85.5
+uNK1.1 ND 11 98.7 88.1
uCD4+uCD8 124 43 3.9 35.9
+uThy 1.2+uNK 1.1 >322 f >59 15.5 34.5
173 13 ND ND
174 ND ND ND
ND,notdone
a Animals which received mAbs were pretreated with the irradiation protocol shown in
Fig. 8.1.
b Donor-type F344 skin grafted 123 days following BMT. Survival time indicates number of
days following skin grafting.
C Donor-type F 344 skin grafted 406 days following BMT, 283 days following first skin graft.
Survival time indicates number of days following placement of second skin graft.
d Antibody-dependent complement-mediated cytotoxicity assay using sera obtained
47 days after placement of second F 344 skin graft. Targets were 51Cr-labeled F 344 spleen
cells. Maximum percent specific cytotoxicity was calculated from percent specific lysis
(PSL) values using the formula:
Maximum PSL of experimental serum+C'-PSL of C' alonexlQO%
Maximum PSL of positive control immune serum+C'-PSL of C' alone
e Antibody-dependent complement-mediated cytotoxicity assay using sera obtained
47 days after placement of second F 344 skin graft. Targets were 51Cr-labeled F 344 BMC.
Percent specific cytotoxicity was determined as in (d) above.
f Although the graft was not completely rejected by day 322, evidence for chronic rejection
was apparent by day 150.
Table 8.4. Lymphocytotoxic antibody following BMT and skin grafting
MAb Pretrcatment" Maximum percent specific cytotoxicity"
Following BMTC Following skin grafting"
Mean SEM 11 Mean SEM 11
None (no BMT)C 3.6 2.0 5 84.0 1

aCD4/CD8 7.6 1 71.5 14.4 4
aCD4/CD8/NK1.1 19.7 4.4 12 60.8 7.5 5
aNKl.l 71.0 3.2 6 97.4 1.8 5'
aCD 4/CD 8/Thy 1.2 2.1 0.7 17 0.0 0 2
aCD 4/CD 8/NK 1.1!Thy 1.2 4.6 1.3 7 4.6 4.6 4
a Animals were pretreated with indicated mAbs on days -6 and -1, and on day 0 received
3 Gy WBI, 7 Gy thymic irradiation, and 6xl 0 7 TCD F344 rat BMC.
b Determined as shown in legend to Table 8.3.
C Serum samples were obtained 15-120 days following BMT.
" Serum samples were obtained 60 days following grafting with F344 and WF rat skin,
183 days following BMT.
C These animals did not receive conditioning or BMT.
I Skin grafting was not performed on this group of mice.
immunity might also be reflected in the development of donor-specific cyto-

toxic antibody. It was, therefore, of interest to evaluate the sera of these ani-
mals for the presence of antibody.
In groups of animals pretreated with mAb combinations which did not in-
clude anti-Thy1.2 and in which skin graft tolerance was not observed, donor-
reactive cytotoxic antibody appeared in the serum within 15-30 days following
BMT (Table 8.4) [38]. The highest antibody levels were observed in the group
pretreated only with anti-NKl.1 mAb prior to BMT (Table 8.4) [38,39], sug-
gesting that elimination of T cells prior to BMT, as in the group pretreated
with anti-CD4 and anti-CD8 in addition to anti-NK1.1, led to a reduction in
the amount of cytotoxic antibody produced. Sera from normal, untreated BlO
mice which were age-matched to the BMT recipients did not contain cytotoxic
antibody against F344 spleen cells (Table 8.4) [38]. Thus, administration of rat
BMC led to immunization and development of a humoral response, even after
pretreatment of recipients with depleting doses of anti-CD4, anti-CD8, and/or
anti-NK1.1 mAbs, regardless of whether or not chimerism developed initially.
In contrast, sera from animals in the group of mice pretreated with anti-
CD4, -CD8, and -Thy1.2 mAbs did not contain significant levels of cytotoxic
antibody against donor spleen cells at any time point examined between 15
and 120 days following BMT (Table 8.4) [38]. Thus, antibody was not detected
even at late time points when chimerism had completely or almost completely
disappeared from peripheral blood [35]. Similar results were obtained for sera
from animals pretreated with all four mAbs (i.e., aCD4, aCD8, aThy1.2, and
aNKl.l), which were evaluated only at later time points [38] (Table 8.4). Thus,
cytotoxic antidonor antibody did not appear in the serum during the period of
Absence of Bone Marrow-Specific Cytotoxic Antibody 129
gradual decline of chimerism in animals preconditioned with a mAb regimen
which permitted induction of initial chimerism and prolonged donor-type skin
graft survival. These results indicate that humoral tolerance also existed in
these animals during the period of gradual loss of chimerism.
The levels of cytotoxic antidonor antibody in sera taken 183 days following
BMT and 60 days after skin grafting, are also shown in Table 8.4. Among nor-
mal BlO controls and animals preconditioned with mAb regimens not permit-
ting induction of skin graft tolerance, high levels of cytotoxic antibody were
detectable in the serum 60 days after grafting. In contrast, sera from the ani-
mals in the two groups demonstrating skin graft prolongation did not contain
cytotoxic antibody 60 days after skin grafting (day 183 following BMT) (Table
8.4).
In order to boost the degree of immunity induced by antigens on donor-
type skin grafts, the previously tolerant animals were regrafted with a second
donor-type skin graft. As shown in Table 8.3, these second grafts were rejected
more rapidly than the first grafts on the same animals, but, in two of three in-
stances, survival was significantly prolonged compared with that of second
grafts in groups preconditioned with mAb combinations not permitting toler-
ance induction. Despite rejection of second skin grafts, significant antibody-
dependent cytotoxicity against donor-type spleen cells did not appear in sera
from these mice (compared with normal, unimmunized control mouse, Table
8.3). In contrast, sera from animals preconditioned with a mAb combination
not permitting tolerance induction contained markedly increased levels of
anti-F344 cytotoxic antibody (Table 8.3).
Absence of Bone Marrow-Specific Cytotoxic Antibody
We have recently demonstrated that, under certain conditions, normal, un-

treated mice develop natural antibody (nAb) which is cytotoxic for rat BMC
but not for rat spleen cells (see below). We, therefore, considered the possibil-
ity that such "BMC-specific" antibody developed in chimeric animals, causing
gradual loss of chimerism. The results shown in Fig. 8.5 mitigate against this
possibility, as donor bone marrow-reactive cytotoxic antibody was not detect-
ed in sera from animals in groups demonstrating skin graft tolerance during
the period between days 30 and 180 when chimerism declined and gradually
disappeared. It therefore seems unlikely that a humoral immune mechanism
accounted for the loss of chimerism in these animals.
In contrast to the results in tolerant mice, significant lysis of F344 BMC was
produced by sera from animals in groups preconditioned with regimens which
did not permit development of skin graft tolerance. Similar to results using
splenocyte targets, the highest levels of bone marrow cytotoxic antibody were
observed in sera from animals preconditioned with anti-NK1.1 only (Fig. 8.5).
In summary, these results demonstrate a lack of humoral immunity against
antigens expressed on donor BMC or lymphocytes during the period when
100 0
•••
.
0
0
>< 80
a o 0
{:, •
I-
a
~
0
60 l-
0
w
a...
CI)
•
'if!. 40
• •
~
~ • • ...
20
• • •
~f)
•• • {:,
0
• •• 00(.\ '" 00
0 011 "'.0 .... iiii ... fU ...
---.
.... 6.........
••• :.1:... ", ......
DAY 15 D.30 D.90 D.1201 D.183

S.G.
Fig. 8.S. Cytotoxic antibody against donor-type F344 rat BMC in sera of mice after condi-
tioning with nonmyeloablative regimen and F344 BMT. Horizontal axis, number of days fol-
lowing BMT (BMT was performed on day 0); vertical axis, maximum percent specific cyto-
toxicity, calculated as described in legend to Tablc S.3 (SG, skin graft). Donor-type F344 and
third party Wistar-Furth rat skin were grafted on day 123 (white triangle, normal B10
mouse). BMT recipients pretreated with mAbs: black circle, aCD4/CDS; hlack square.
aCD4/CDS/NKl.l; white square, aNK1.l; white circle. aCD4/CDS/Thy1.2; black trial/gle,
aCD4/CDS/NK1.l/Thy1.2 (I. Aksentijevich ct al.. manuscript submitted)
chimerism is lost or following skin grafting in mice preconditioned with the

complete tolerizing mAb regimen. Thus, it is unlikely that antibody mediates
either bone marrow graft failure or skin graft rejection in these mice. In gener-
al, our results suggest a persistence of specific tolerance in these mice to anti-
gens expressed on bone marrow-derived cells, despite the gradual loss of
chimerism. While further studies are needed to clarify this issue, skin graft re-
jection appears to be most likely due to recognition of skin-specific antigens.
Possible Mechanisms Accounting for Loss of Chimerism
Results of the above studies therefore failed to explain the gradual loss of rat
chimerism in mixed xenogeneic chimeras prepared following conditioning
with the nonmyeloablative regimen. One possible explanation is that a non-T
cell-, non-B cell-mediated immune mechanism, such as that mediated by NK
cells, effects gradual rejection of nt BMC. Indeed, our studies indicated that
NK cells playa greater role in resisting engraftment of xenogeneic rat BMC
than of allogeneic BMC in mice, since chimerism and tolerance could be read-
The Natural Antibody Problem 131
ily induced in allogeneic combinations using anti-CD4 and -CDS mAbs and
without a requirement for anti-NK1.1 or anti-Thy1.2 antibodies [34].
It seems unlikely that the requirement, in the xenogeneic combination, for
anti-Thyl.2 mAb reflects a requirement for more exhaustive depletion of con-
ventional T cells, since, in some strain combinations, mouse antirat xenograft
responses appear weaker than alloresponses [40,41], perhaps due to ineffi-
cient MHClaccessory molecule interactions across species [42-45]. Instead,
the need for this mAb may reflect a contribution to xenoresistance of a distinct
Thyl +, CD4-, CDS- cell subset. These might be NK cells, some of which ex-
press Thy1 [46]. It is possible that recipient NK cells may not be "tolerized" by
induction of mixed chimerism, and may recover after initial depletion from
mAb to cause gradual destruction of donor stem cells.
An additional explanation for the gradual loss of chimerism in mixed xeno-
geneic chimeras should also be considered. Administration of mixed TCD syn-
geneicplus rat BMC inocula to lethally irradiated mice [2, 13,47], or of rat BMC
to nonmyeloablated recipients [35] does not lead to permanent chimerism, de-
spite markedly prolonged donor-specific skin graft survival. The skin graft ac-
ceptance observed in these models [2, 13, 35, 47] argues against an immune
mechanism as being responsible for xenogeneic marrow graft failure.
In marked contrast to these results, studies reported in the literature show
that prolonged donor-type chimerism can be induced in lethally irradiated
mice reconstituted with rat BMC alone [16, 22, 4S). Thus, rat hematopoietic
stem cells are clearly capable of survival and long-term reconstitution in
murine recipients. It appears, therefore, that whenever a source of murine
BMC is available, mouse cells eventually repopulate the lymphohematopoiet-
icsystem.
These observations suggest that, when placed in a murine stromal environ-
ment, rat stem cells may be at a competitive disadvantage compared to murine
hematopoietic stem cells. This disadvantage may be due to a need for species-
specific growth factors [49] or to cell-cell interactions requiring species-specif-
ic receptors [50]. Studies are in progress in our laboratory to evaluate these
possibilities.
The Natural Antibody Problem
When primarily vascularized organs are grafted between discordant species

[51), an additional component of the immune system that plays a critical role in
preventing engraftment consists of nAb [52, 53). Such antibody, which ap-
pears to be directed against antigens expressed on the surface of vascular en-
dothelial cells, causes rejection within minutes of vascularization, due to acti-
vation of the complement and coagulation cascades [52, 53], which leads to
immediate thrombosis and infarction.
Natural antibody, by mechanisms to be discussed below, may also present
an impediment to the engraftment of xenogeneic marrow. Even if nAb does
132 Xcnotolerancc Through Bone Marrow Transplantation
not impair xenogeneic marrow engraftment. it will be critical to determine

whether or not continued nAb formation is inhibited by the successful induc-
tion of lymphohematopoietic chimerism across species barriers. While induc-
tion of chimerism is generally associated with induction of both T and B cell
tolerance [1-7.31. 38]. little is known about the determinants recognized by
nAb against cells of other species.
Evidence suggests that these antibodies recognize antigens expressed on
endothelial cells; however. to our knowledge. no systematic evaluations have
been performed to determine whether or not these antigens are also expressed
on BMC. If not, then it is possible that induction of chimerism would not sup-
press the development of endothelial cell-specific nAb. In this case. successful
induction of xenogeneic chimerism might not be sufficient to prevent hyper-
acute rejection of vascularized organ grafts from the same xenogeneic donor.
Resolution of these questions will be essential in order to determine the best
possible approach to inducing specific tolerance across species barriers.
Natural Antibody Against Rat BMC in Sera of Norrnal Mice
The rat-mouse species combination is considered to be concordant [51], as hy-

peracute rejection does not occur when vascularized organs are transplanted
between these species [40.53.54 J. Since hyperacute rejection is believed to re-
flect the presence of preexisting antibodies (usually of the immunoglobulin M.
IgM class), and lymphocytotoxic antibodies against rat cells have not been de-
tected in normal mouse sera [13. 53J, it has previously been concluded that
mouse antirat nAb does not exist. However, studies have not been reported in
which rat BMC rather than lymphocytes were used as targets for evaluation of
normal mouse sera.
Since much greater numbers ofTCD rat BMC than of murine BMC are re-
quired for induction of chimerism using similar conditioning regimens [2. 13.
34,35]. we considered the possibility that nAb might exist in sera of normal
mice with specific reactivity against rat BMC, but at a level low enough to be
absorbed by the excess rat BMC. We utilized flow cytometric analyses and an-
tibody-dependent complement-mediated cytotoxicity assays to address this
question.
The results of these studies are summarized in Table 8.5, which shows that
normal murine serum indeed contained antibody with greater binding activity
to rat BMC than to rat splenocytes. This antibody was ofthe IgM and IgG, sub-
classes. with the latter subclass binding exclusively to BMC and not to spleno-
cytes. In addition. IgG 2b in normal mouse serum bound weakly to a small sub-
population of both splenocytes and BMC. Furthermore. normal mouse serum
mediated cytotoxicity against rat BMC and not against spleen cells, consistent
with the greater binding to BMC than to splenocytes of natural IgM antibody.
The development of cytotoxic nAb was dependent upon the environment in
which animals were housed; sera from animals raised in a specific pathogen-free
Bone Marrow Transplantation and Natural Antibody 133
Table 8.5. Natural antibody against rat BMC in serum of normal mice
Ig Subclass' Binding to rat cells
Splenocytes BMC
IgA
IgG,
IgG 2 •
IgG 2b + +
IgG 3 ++
IgM + +++
Cytotoxicityb +++
a Binding of nAb of each subclass was determined by incubating rat cells with 10 JlI of nor-
mal mouse serum or negative control mAb of the appropriate subclass, followed by incu-
bation with a FITC-labeled rat-anti-mouse Ig subclass-specific secondary reagent. Flow
cytometric analyses were performed using a FACSCAN. The subclass specificity of each
secondary reagent was confirmed by incubating positive and negative control H-2-specific
mAbs of each subclass on appropriate cell populations from various mouse strains.
b Determined by antibody-dependent complement-mediated cytotoxicity assays. s'Cr-
labeled F344 rat spleen cells or BMC were incubated with normal mouse serum in
microtiter plates, washed, then incubated with rabbit complement, and s'Cr release was
measured.
(SPF) microisolator environment contained very little cytotoxic antibody com-

pared to age-matched mice raised in a non-SPF conventional mouse colony.
The development of such nAb was somewhat age-dependent, with increasing
levels of antibody developing with increasing age in animals raised in a non-SPF
colony. In older mice raised in such an environment, low levels of cytotoxicity
against rat spleen cells, similar to that shown for a normal BlO mouse in Table
8.3, have also been observed. However, the level of cytotoxicity against rat
BMCwas always greater than that against spleen cells (e.g., Table 8.3) [63].
Bone Marrow Transplantation and Natural Antibody
The most widely studied nAbs in man, those which are directed against ABO
blood group determinants, can lead to significantly delayed erythropoietic re-
covery following bone marrow transplantation [55-57]. Importantly, such an-
tibodies eventually disappear from the circulation after a stable allograft is
achieved [57]. The prolonged presence of these antibodies after BMT suggests
that not all B cells are eliminated by pre-BMT conditioning regimens [57], and
the eventual disappearance of natural nAb therefore suggests that BMT may
induce tolerance among nAb-forming B cells. If nAb production against the
donor species can indeed be suppressed by the induction of chimerism, then
BMT might be used to eliminate all of the major impediments to grafting of
vascularized organs across species barriers.
The observation that rat BMC-specific nAbs exist in sera of normal mice
provides a unique opportunity to evaluate the relationship between
chimerism, tolerance, and nAb, and to address specifically the question of
whether or not induction of chimerism and transplantation tolerance leads to
suppression of nAb formation. A systematic analysis of the relationship be-
tween chimerism and nAb has not been performed in mixed xenogeneic
chimeras prepared according to the nonmyeloablative conditioning regimen.
The low level of antidonor BMC cytotoxicity (which was less than that ob-
served for a normal, ungrafted BlO mouse), shown in Table 8.3, for mixed
xenogeneic chimeras which had long since lost their chimerism suggests that
nAb may develop in such mice after the disappearance of chimerism. The pat-
tern of cytotoxicity in these sera, which show greater cytotoxicity against
donor BMC than against spleen cells, was typical of the nAb observed in sera
of normal mice, and differs from the pattern observed for sensitized mice (e.g.,
Table 8.3, skin grafted BI0 mice and mice conditioned with nontolerizing
mAb regimen of aCD4, aCD8, aNK1.1), in which greater cytotoxicity was
produced against spleen cells than against BMC.
While these results suggest that nAb appears in sera of tolerant mice after
the loss of chimerism, a comparison to age-matched controls beginning soon
after the induction of chimerism will be required to determine whether or not
the production of such antibody is suppressed during the period when
chimerism is still detectable. Additional studies will be needed to address this
question in discordant species combinations.
Since initial chimerism and transplantation tolerance can be induced in the
rat-to-mouse species combination using BMT, the type of nAb we have de-
tected clearly does not prevent marrow engraftment. However, this does not
imply that nAbs do not reduce the level of engraftment of rat BMC, and, in-
deed, their presence might explain the large numbers of rat BMC which are re-
quired in order to achieve engraftment in mice [2, 13,34,35]. Antibody could,
in theory, influence bone marrow engraftment by several possible mecha-
nisms, including NK cell-mediated ADCC, complement-mediated cytotoxici-
ty, and opsonization followed by uptake by cells of the reticuloendothelial sys-
tem.
While ADCC has been implicated as a mechanism of allogeneic bone mar-
row rejection [58], the murine IgG 2b nAb we have detected binds only a small
subpopulation of rat BMC. Definition of the cell subpopulations recognized
by this nAb subclass will be required to determine whether or not it signifi-
cantly hinders rat BMC engraftment. On the other hand, IgM nAb strongly
binds a large proportion of rat BMC, and is probably responsible for the bone
marrow-specific complement-mediated cytotoxicity we observed. IgM nAb
might reduce engraftment of rat BMC in vivo through complement activation,
which may lead to opsonization and uptake by cells of the reticuloendothelial
system.
The other major subclass of nAb we detected, IgG 3, showed the greatest
specificity for BMC. The high level of binding of IgG 3 to rat BMC is especially
striking in light of the relatively minor contribution made by this subclass to to-
References 135
tal serum Ig content [59]. IgG 3 fixes complement poorly [59], but does bind a
macrophage Fc receptor [60, 61]. A chimeric form of CD4-depleting mAb
GK1.5 bearing a murine IgG 3 Fc was ineffective at mediating cellular clear-
ance in vivo [62], suggesting that this subclass of nAb might not lead to elimi-
nation of BMC in vivo.
Studies are in progress in our laboratory to evaluate the effects of nAb on
rat BMC engraftment in T and B cell-deficient severe combined immunodefi-
ciency (SCID) mice receiving nAb passively transferred in normal mouse
serum. Results of preliminary studies suggest that nAb is indeed capable of re-
ducing the level of rat BMC engraftment in mice.
Comment
BMT is a promising approach to the induction of both humoral and cellular

tolerance across species barriers, and might potentially provide a solution to
the critical organ shortage which currently exists. While studies to date have
indicated that this approach can be used with success in concordant animal
species combinations, our understanding of the relationship between nAb,
chimerism, and tolerance is in its infancy. Greater understanding ofthese rela-
tionships and extension of these studies to discordant species combinations
should help to establish the priorities necessary for the development of clini-
cally applicable methods of using BMT to induce donor-specific tolerance
across species barriers in man.
Acknowledgements. We thank Dr. Henry J. Winn for helpful review of the

manuscript.
References
1. Billingham, RE., L. Brent. and P.B. Medawar. 1953. "Actively acquired tolerance" of
foreign cells. Nature 172: 603-606.
2. Ildstad, S.T., and D.H. Sachs. 1984. Reconstitution with syngeneic plus allogeneic or
xenogeneic bone marrow leads to specific acccptance of allografts or xenografts. Nature
307(5947):168-170.
3. Sykes, M., M. Sheard, and D.H. Sachs. 1988. Effects ofT cell depletion in radiation bone
marrow chimeras. I Evidence for a donor cell population which increases allogeneic
chimerism but which lacks the potential to produce GVHD. l.Immunol.141: 2282-2288.
4. Marrack, P., D. Lo, R Brinster. R Palmiter. L. Burkly. R.H. Flavell, and J. Kappler.
1988. The effect of thymus environment on T cell development and tolerance. Cell 53:
627-634.
5. Slavin. S. 1987. Total lymphoid irradiation. Immunol.Today 3: 88-92.
6. Pierce, G.E. 1990. Allogeneic versus semiallogeneic Fl bone marrow transplantation
into sublethally irradiated MHC-disparate hosts. Effects on mixed lymphoid chimerism,
skin graft tolerance, host survival, and alloreactivity. Transplantation 49: 138-144.
7. Mayumi, H., and RA. Good. 1989. Long-lasting skin allograft tolerance in adult mice in-
duced across fully allogeneic (multimajor H-2 plus multi minor histocompatibility) anti-
gen barriers by a tolerance-inducing method using cyclophosphamide. J Exp.Med. 169:
213-238.
8. Lubaroff. D.M., and W.K. Silvers. 1973. The importance of chimerism in maintaining
tolerance of skin allografts in mice. J .Immunol. 111: 65-71.
9. Abramowicz. D., C Bruyns. and M. Goldman. 1987. Chimerism and cytotoxic T lym-
phocyte unresponsiveness after neonatal injection of spleen cells in mice. Trans-
plantation 44: 696-701.
10. Roser. B.J. 1989. Cellular mechanisms in neonatal and adult tolerance. Immunologic
Rev 107: 179-202.
11. O'Reilly, R.J .. N.H. Collins, N. Kernan, and et al. 1985. Transplantation of marrow de-
pleted of T cells by soybean lectin agglutination and E-rosette depletion: major histo-
compatibility complex-related graft resistance in leukemic transplant recipients.
Transplant.Proe. 17: 455.
12. Anasetti. C. D. Amos, P.G. Beatty, F.R Appelbaum, W. Bensinger, C.D. Buckner, R
Clift, C. Doney. P.J. Martin, E. Mickelson, B. Nisperos, J. O'Quigley, R Ramberg, J.E
Sanders, P. Stewart, R Storb. K.M. Sullivan, RP. Witherspoon, ED. Thomas, and J.A.
Hansen. 1989. Effect of HLA compatibility on engraftment of bone marrow transplants
in patients with leukemia or lymphoma. New EngU.Med. 320: 197-204.
13. Ildstad, S.T., S.M. Wren, S.O. Sharrow, D. Stephany, and D.H. Sachs. 1984. In vivo and
in vitro characterization of specific hyporeactivity to skin xenografts in mixed xenogene-
ically reconstituted mice (B 10 + F344 rat - BlO). J.Exp.Med. 160: 1820-1835.
14. Santos, G.W., and L.J. Cole. 1958. Effects of donor and host lymphoid and myeloid tissue
injections in lethally X-irradiated mice treated with rat bone marrow. J .Natl.Cancer Inst.
21: 279-293.
15. Bau, J., and S. Thierfelder. 1973. Antilymphocytic antibodies and marrow transplanta-
tion. Transplantation 15: 564-567.
16. Muller-Rucholtz, W., H.K. Muller-Hermelink, and H.U. Wottge. 1979. Induction of last-
ing hematopoietic chimerism in a xenogeneic (rat-to-mouse) model. Transplant.Proc.
11: 517-521.
17. Deeg, H.J., R Storb, and ED. Thomas. 1984. Bone marrow transplantation: a review of
delayed complications. Br.J.Haematol. 57: 185.
18. Travis, E.L.. L.J. Peters. J. McNeill, ED. Thames, and C. Karolis. 1985. Effect of dose-
rate on total body irradiation: lethality and pathologic findings. Radiother.Oncol. 4:
341-351.
19. Freirich, E.J., E.A. Gehan, D.P. Rail, L.H. Schmidt, and H.E. Skipper. 1966. Quantita-
tive comparison of toxicity of anti-cancer agents in mouse, rat, hamster, dog, monkey,
and man. Cancer Chemotherapy Rep. 50: 219.
20. Sullivan, K.M., RP. Witherspoon, R Storb, C. Anasetti, F.R Appelbaum, C Bigelow, J.
Clark, K. Doney, R Hill, T. Loughran, D.O. Matthews, J. Nims, F. Petersen, J. Sanders,
F. Schuning, A. Shields, S. Strom, and E.D. Thomas. 1989. Chronic graft-versus-host dis-
ease: recent advances in diagnosis and treatment. In Bone Marrow Transplantation:
Current Controversies. RP. Gale and R Champlin, eds. Alan R Liss, Inc., New York,
P.51!'
21. Clift, RA., and R Storb. 1987. Histoincompatible bone marrow transplants in humans.
Ann.Rev.Immunol. 5: 43-64.
22. Makinodan. T. 1956. Circulating rat cells in lethally irradiated mice protected with rat
bone marrow. P.S.E.B.M. 92: 174-179.
23. Gale, RP., and Y. Reisner. 1986. Graft rejection and graft-versus-host disease: mirror
images. Lancet i: 1468-1470.
24. Poynton, C.H. 1988. T cell depletion in bone marrow transplantation. Bone Marrow
Transplant. 3: 265-279.
25. Martin, P.J., J.A. Hansen, C.D. Buckner, J.E. Sanders, H.J. Deeg, P. Stewart, F.R
Appelbaum, R Clift, A. Fefer, R.P. Witherspoon, M.S. Kennedy, K.M. Sullivan, N.
Flournoy, R Storb, and E.D. Thomas. 1985. Effects of in vitro depletion of T cells in
HLA-identical allogeneic marrow grafts. Blood 66: 664-672.
References 137
26. Kernan, N.A., N. Flomenberg, B. Dupont, and R.J. O'Reilly. 1987. Graft rejection in
recipients of T-cell-depleted HLA-nonidentical marrow transplants for leukemia.
Transplantation 43: 842-847.
27. Soderling, CCB., CW. Song, B.R. Blazar, and D.A. Vallera. 1985. A correlation be-
tween conditioning and engraftment in recipients of MHC-mismatched T cell-depleted
murine bone marrow transplants. J.lmmunol. 35: 941.
28. Ferrara, J.L.M.,J. Lipton, S. Hellman, S. Burakoff, and P. Mauch. 1987. Engraftment fol-
lowing T-cell-depleted marrow transplantation. Transplantation 43: 461-467.
29. Zinkernagel, R.M., G.N. Callahan, A. Althage, S. Cooper, P.A. Klein, and J. Klein. 1978.
On the thymus in the differentiation of "H-2 self-recognition" by T cells: evidence for
dual recognition?J.Exp.Med.147: 882-896.
30. Singer, A., K.S. Hathcock, and R.J. Hodes. 1981. Self recognition in allogeneic radiation
chimeras. A radiation host element dictates the self specificity and immune response
gene phenotype ofT-helper cells. J.Exp.Med. 153: 1286-1301.
31. Ildstad, S.T., S.M. Wren, J.A. Bluestone, S.A. Barbieri, and D.H. Sachs. 1985.
Characterization of mixed allogeneic chimeras. Immunocompetence, in vitro reactivity,
and genetic specificity of tolerance. J.Exp.Med. 162: 231-244.
32. Zinkernagel, R.M., A. Althage, G. Callahan, and R.M. Welsh Jr.. 1980. On the immuno-
competence of H-2 incompatible irradiation bone marrow chimeras. J.Immunol. 124:
2356-2365.
33. Ruedi, E., M. Sykes, S.T. Ildstad, CH. Chester, A. Althage, H. Hengartner, D.H. Sachs,
and R.M. Zinkernagel. 1989. Antiviral T cell competence and restriction specificty of
mixed allogeneic (PI +P2-> PI) irradiation chimeras. CelLImmuol. 121: 185-195.
34. Sharabi, Y., and D.H. Sachs. 1989. Mixed chimerism and permanent specific transplan-
tation tolerance induced by a nonlethal preparative regimen. J.Exp.Med.169: 493-502.
35. Sharabi, Y., 1. Aksentijevich, T.M. Sundt III, D.H. Sachs, and M. Sykes. 1990. Specific tol-
erance induction across a xenogeneic barrier: production of mixed rat/mouse Iymphohe-
matopoieticchimeras using a nonlethal preparative regimen. J.Exp.Med. 172: 195-202.
36. Wachtel, S.S., H.T. Thaler, and E.A. Boyse. 1977. A second system of alloantigens ex-
pressed selectively on epidermal cells of the mouse. Immunogenetics 5: 17-23.
37. Steinmuller, D., and S.S. Wachtel. 1980. Transplantation biology and immunogenetics of
murine skin-specific antigens. Transplant.Proc. 12, Suppl.1: 100-106.
38. Aksentijevich, 1., D.H. Sachs, and M. Sykes. 1990. Humoral tolerance in xenogeneic
BMT recipients conditioned with a nonmyeloablative regimen. Manuscript submitted.
39. Aksentijevich, 1., D.H. Sachs, Y. Sharabi, T.M.III Sundt, and M. Sykes. 1991. Humoral
tolerance in mixed xenogeneic chimeras prepared by a nonmyeloablative conditioning
regimen. Transplant.Proc. 23: 880-882.
40. Burdick, J.F., S.V. Jooste, and H.J. Winn. 1979. Variations in the responses of mouse
strains to rat xenografts. J .Immunol. 123: 954-955.
41. J ooste, S.V., and H.J. Winn. 1977. Strain variations in the responses of mice to xenografts
of skin. Transplant.Proc. 9: 375-376.
42. Irwin, M.J., W.R. Heath, and L.A. Sherman. 1989. Species-restricted interactions be-
tween CD8 and the a.3 domain of class I influence the magnitude of the xenogeneic re-
sponse. J.Exp.Med.170: 1091-1101.
43. Pierson, R.N., H.J. Winn, P.S. Russell, and H. Auchincloss Jr. 1989. Xenogeneic skin
graft rejection is especially dependent on CD4+ T cells. J .Exp.Med. 170: 991-996.
44. Lucas, P.J., G.M. Shearer, S. Neudorf, and R.E. Gress. 1990. The human antimurine
xenogeneic cytotoxic response. 1. dependence on responder antigen-presenting cells.
J.lmmunoI.144: 4548-4554.
45. Moses, R.D., R.N. Pierson III, H.J. Winn, and H.Jr. Auchincloss.1990. Xenogeneic pro-
liferation and Iymphokine production are dependent on CD4+ helper T cells and self
antigen-presenting cells in the mouse. J.Exp.Med. 172: 567-575.
46. Trinchieri, G. 1989. Biology of natural killer cells. Adv.lmmunol. 47: 187-376.
47. Ildstad, S.T., S.M. Wren, D. Stephany, and D.H. Sachs. 1986. Effect of selective T cell de-
pletions in mixed xenogeneic reconstitution on specific hyporeactivity to transplanta-
tion across a species barrier. Transplantation. 41: 372-376.
48. Zaalberg, O.B., O. Vos, and D.W. Van Bekkum. 1957. Surviving rat skin grafts in mice.
Nature 180:238-239.
49. Morris, c.F., I.G. Young. and A.1. Hapel. 1990. Molecular and cellular biology of intcr-
Icukin-3. In Colony-stimulating factors. T.M. Dexter. J.M. Garland and N.G. Testa. eds.
Marcel Dekker. Inc .. New York. P.l77.
50. Allen. T.D .. and T.M. Dexter. 1990. Marrow biology and stem cells. In Colony-stimulat-
ing factors. T.M. Dexter. J.M. Garland and N.G. Testa. cds. Marcel Dekker, Inc .. New
York. P.1.
51. CaIne, RY. 1970. Organ transplantation between widely disparate species.
TransplanLProc. 2: 550.
52. Hammer. c., C. Chaussy, and W. Brendel. 1973. Preformed natural antibodics in ani-
mals and man; outlook on xenotransplantation. Eur.Surg.Res. 5: 162.
53. Hardy, M.A., G. Todd, and K. Reemtsma. 1984. Xeno-transplantation. In Bone marrow
and organ transplantation. S. Slavin. ed. Elsevier, B. V .. P.515.
54. Corry, R.1 .. and S.E. Kelley. 1974. Immunological enhancement of primarily vascular-
ized rat heart xenotransplants in mice. Transplantation 18: 503.
55. Gale, R.P., S. Feig. W. Ho, P. Falk. C. Rippee. and R. Sparkes. 1977. ABO blood group
system and bone marrow transplantation. Blood 2: 185-194.
56. Bensinger, W.I., C. Dean Buckner, E. Donnall Thomas, and R.A. Clift. 1982. ABO-in-
compatible marrow transplants. Transplantation 33: 427-429.
57. Barge. A.J., G. Johnson, R. Witherspoon, and B. Torok-Storb. 1989. Antibody-mediat-
ed marrow failure after allogeneic bone marrow transplantation. Blood 74: 1477-1480.
58. Dennert, G .. and J.F. Warner. 1985. Bone marrow rejection as a function of antibody-
directed natural killer cclls. J .Exp.Med. 161: 563-576.
59. Grey, H.M., l.W. Hirst, and M. Cohn. 1971. A new mouse immunoglobulin: IgG3.
J.Exp.Med. 133: 289-304.
60. Ralph, P., I. Nakointz, B. Diamond, and D. Yelton. 1980. All classes of murine IgG anti-
body mediate macrophage phagocytosis and lysis of erythrocytes. J.Immunol. 125:
1885-1888.
61. Unkeless. J.c., E. Scigliano, and V.H. Freedman. 1988. Structure and function of human
and murine receptors for IgG. Ann.Rev.Immunol. 6: 251-281.
62. Alters. S.E., K. Sakai, L. Steinman, and V.T. Oi. 1990. Mechanisms of anti-CD4-mediat-
ed depletion and immunotherapy: A study using a set of chimeric anti-CD4 antibodies.
J.lmmunol. 144: 4587-4592.
63. Aksentijevich, I., D.H. Sachs, and M. Sykes. 1991. Natural antibodies against bone mar-
row cells of a concordant xenogeneic species. J. Immunol. 147: 79-85.
Chapter 9
Immunobiology of Xenografting in Rodents

F.T. THOMAS, W. MARCHMAN, A. CAROBBI, R. DEMASI, D. ARANEDA,
T. PATSELAS, E. LARKIN, K. PITTMAN, M. ALQAISI, C. HAISCH, ANDJ. M. THOMAS
Introduction
Rodents have always been favored species for laboratory research. The ani-
mals are inexpensive, their lodging and care are simple and cost-effective, and
they can be bred in a well-controlled environment to develop pure strains with
known histocompatibility. Their use in organ transplantation research has
previously been limited by their small size. Recently, the development of mi-
crosurgery has permitted the performance of a large number of whole-organ
grafts in rodents, including kidney, pancreas, spleen, liver, lung, and heart
grafts. The utility of rodents in skin grafting is legendary. One of the most use-
ful papers on skin grafting technique was published by Billingham over 40
years ago using rodents and remains a recommended up-to-date reference on
experimental skin grafting [1]. Perhaps the most important factor in the utility
of these animals is the ability to perform large numbers of solid-organ grafts
in a cost-effective manner consistent with modern concerns for animal wel-
fare.
Over the past 3 years, our group has worked extensively with over 2000 ro-
dents in xenograft studies [2-16]. This chapter summarizes our experience us-
ing rodent models to develop techniques of effective immunosuppression for
xenografting. It quickly became apparent that conventional immunosuppres-
sive agents, including cyclosporine, were ineffective in xenografts. Emphasis
was therefore shifted to newer immunosuppressive modalities, including
FK-506, 15-deoxyspergualin (DSG), quality-controlled rabbit antithymocyte
globulin (RATG), and total lymphoid irradiation (TU).
Studies of infiltrating cells in rejecting xenografts were utilized in an at-
tempt to explain the basic processes of humoral and cell-mediated immunity
in xenografting. Antibody studies were utilized to determine the effect of
xenograft antidonor antibody, as well as to study blocking of antibody forma-
tion by immunosuppressive drugs.
Finally, important insights were gained into the basic nature of xenograft-
ing by studies in genetically immunodeficient rodent strains. The immunodefi-
cient models have included the X-linked immunodeficiency (xid) mouse, nude
(Nu) mouse, and mice having various combinations of these genetic muta-
tions, as well as studies in the nude rat.
Most recently, we have utilized the model of a primarily vascularized heart
graft in these immunodeficient mice to study mechanisms of xenograft rejec-
140 Immunobiology of Xenografting in Rodents
tion. In addition, studies done in collaboration with investigators at the

University of Vermont established that the endothelial cell was a major stimu-
lus to development of in vitro xenogeneic reactivity in rodents. T cell immuni-
ty as well as antibody-dependent cellular cytotoxicity (ADCq, and leukocyte
adhesion deficiency (LAD) killer cells were developed against the endothelial
cell by xenogeneic cells [15].
These studies provide the basis for a number of postulates regarding
xenograft immunobiology and potential avenues to effective immunosuppres-
sion and will be reviewed here in their overall context.
An Overview of Xenograft Models
A wide variety of animal species have been used in xenograft studies. The ar-
ray of experimental models is nearly as kaleidoscopic as the animal kingdom
itself. It includes primates, large domesticated animals such as pigs, goats, and
dogs, a host of rodent xenograft models involving primarily the rat, mouse,
hamster, and guinea pig, and even studies of lower amphibians and other non-
mammalian species. Our experience, to be reviewed here, involved primarily
three rodent species: the mouse, the rat, and the hamster.
Skin Grafting in the Rodent
Early in our experience, it became clear that the rodent skin graft was an excel-
lent model for studying xenograft immunity and immunosuppressive drug po-
tency and toxicity. To begin with, the elegant genetic controls possible in these
animals provided an immunological milieu with a uniform pattern and tempo
of rejection. The in vivo studies demonstrated a vigorous, well-defined, and
rapid progression of mouse, rat, and hamster skin rejection, often complete
within 24-48 h of the first indication of impending rejection.
Equally consistent was the inability of immunosuppressive agents currently
used to effect delay in rejection of this model. Every immunosuppressive drug
studied, when used as monotherapy, failed to significantly modify graft survival
of these xenografts, with the exception of RATG, which slightly prolonged the
skin grafts in monotherapy studies. Therapy with two or three drug combina-
tions, however, produced some significant improvements in skin graft survival,
with delay of rejection beyond 30-40 days post-transplantation in some cases.
Most significantly, the skin xenograft models showed patterns of rejection
and prolongation which closely mimicked other xenograft models (such as the
heterotopic cardiac xenograft and pancreas islet xenografts). Figure 9.1 shows
the survival ofxenografts of skin and heart in the hamster-to-Lewis rat and the
linear correlation of prolongation by different drug combinations, which has
been found to be very much the rule in this model. There was a similar tempo
and vigor of rejection between the two organ grafts.
An Overview of Xenograft Models 141
Days survival
30
25
20
Fig. 9.1. Linear correlation
of xenografting results using 15
heart (H) or skin (S) in 10
Lewis rats. CYA, cyclo-
sporine; RATG, rabbit anti- 5
thymocyte globulin; DOS?, O~~"~--~---
15-deoxyspergualin Control CyA RATG/DOSP
Days survival
35
30
25
Fig. 9.2. Skin xenograft sur-
vival with different drug 20
combinations. C, control; 15
TLl, total lymphoid irradia-
tion; CYA, cyclosporine; 10
RATG rabbit antithymo- 5
cyte globulin; DOS?, 15- O~~----~--~ __ ~ __ ~ ____- L__- L____L -
deoxyspergualin C TLi/CyA RATG/FK506 RATG/DOSP
Cyclosporine, even at toxic doses of 25-50 mg/kg per day, did not produce
significant prolongation of graft survival or modification of the basic histologi-
cal pattern of xenograft rejection in either model. Other studies, not shown
here, demonstrated the inability of the conventional agents, prednisone and
azathioprine, even at high doses, to modify rejection of either xenografted or-
gan when used as monotherapy or together. As shown in Fig. 9.1, however,
combinations of RATG with DSG produced significant graft prolongation in
both the heart and skin graft.
Thus, there is a high concordance of graft survival and prolongation in
these two models despite the use of different donor organs. We view the skin
test as a sort of "screening assay" to permit rapid inexpensive testing of a wide
variety of suppressive agents. Agents shown to be suppressive in screening as-
says then receive more definitive confirmation of their potency in heart or
pancreas islet xenografting.
Figure 9.2 shows survival in the rat-to-mouse skin xenograft model, using
combined therapy. Control grafts (shown on the left) survived a mean of
5.2±1.6 days. TLI and cyclosporine were able to prolong graft survival to only
8.6±3.2 days. The ineffectiveness of TLI and cyclosporine has been a consis-
tent finding in our studies and in studies of other groups using the heart and
other organ xenograft models [2,5,6,13,14,17-21].
Excellent results in prolongation of skin xenograft survival were seen with
a combination of RATG and FK-506 [2,4] and RATG plus DSG [5] at 2.5
mg/kg (shown in Fig. 9.2). This prolongation was achieved with a low rate of
toxicity, and demonstrates the generally low toxicity seen with use of RATG
combined with FK-506 and DSG. This relatively simple model permitted the
testing of large numbers of drug combinations at different doses.
Perhaps most importantly, the results seen in skin xenografting using a
wide variety of suppressive modalities were highly consistent with results from
whole primarily vascularized organs, as previously discussed. Thus, the utility
of the skin graft as an overall test model for xenograft immunosuppression was
established by these studies. Despite the simplicity of the skin xenograft, this
model must be recognized as one of high potential utility and validity for fu-
ture xenograft studies, especially with the current dependency on empirical
data and need for widespread screening of new suppressive drugs.
The skin xenograft has another advantage - the ability to access drug toxic-
ity in a relatively pure form, unfettered by vagaries of microsurgery and anes-
thesia involved in other more stressful xenograft models. In contrast to
xenografts of solid primarily vascularized organs such as heart, kidney, pan-
creas, and liver, the anesthesia and transplant morbidity and mortality for skin
grafts approaches 0%. With only a few months experience, most researchers
can perform literally hundreds of skin xenografts without technical morbidity
or mortality. In this situation it is possible to accurately ascribe morbidity, and
especially mortality, to the effects of immunosuppressive drugs.
For example, in one experiment, in over 40 control skin grafts with no im-
munosuppression and 30 grafts given RATG only, there was no mortality. In
the next ten animals grafted by the same investigator in the same manner, but
given DSG at 5 mg/kg, mortality was 70%, despite the use of the same ship-
ment of animals, same source of animal supply, similar anesthesia, operating
techniques, postoperative care, and housing and caging of the rodents. We
viewed such findings as incontrovertible evidence of drug toxicity.
Anesthesia for Primarily Vascularized Organ Grafts
Despite limitations, primarily vascularized organs, especially the heart, can be

established as models in a skilful manner to the point where mortality from the
procedure is minimal. Anesthetic mortality is a serious and important consid-
eration, and xenograft studies yielding high validity are not possible until the
test laboratory has perfected a safe and standardized manner of animal anes-
thesia. Concerns of humane animal care during surgery necessitate that ani-
mals be effectively anesthetized. In these small animals, there is a fine line be-
tween effective and toxic anesthesia.
Our group has utilized a number of anesthesia protocols to effectively anes-
thetize rodents for xenografting. The most satisfactory of all has been the ke-
tamine-xylazine anesthesia using 0.5 mg/kg ketamine and 0.2 mg/kg xylazine. In
difficult animal models, especially in some of the genetically immunodeficient
rodents which are less hardy than normal rodents, it may be necessary to utilize
reduced levels of systemic ketamine-xylazine anesthesia and supplement this
anesthesia with local injections ofxylocaine into the site of the xenograft.
An Overview of Xenograft Models 143
Drug Toxicity
Studies from our laboratory and others have established that virtually all
agents with the ability to prolong xenograft survival are associated with signif-
icant morbidity and mortality in rodents. It, therefore, behoves the xenograft
investigator to accurately and carefully look for morbidity and mortality asso-
ciated with these toxic immunosuppressive agents. A number of publications
in the literature imply, for example, that doses of 5 and 10 mg/kg DSG can be
given with no significant toxicity. Mortalities of up to 80% can be seen, but are
often not attributed to toxicity associated with these agents. Deaths after skin
xenografting have been described as due to "technical complications". Our
studies suggest that this is not a plausible explanation, and that the deaths in
animals following skin grafting done by skilled personnel are almost certainly
attributable to the immunosuppressive agent if anesthetic toxicity is excluded.
Another example of this principle is seen in papers in the literature, using
cyclosporine at doses of 30-50 mg/kg for prolonged periods in xenografts. In
our experience, the administration of this dose of cyclosporine is essentially
100% toxic if given to rodents for any extended period of time. We have found
15 mg/kg per day to be a maximum tolerated dose if given our several weeks,
and this should be the dose used for xenograft testing.
Immunosuppressive drug toxicity is currently one of the major Achilles
heels of xenografting, and its occurrence must be carefully monitored. There
can be little meaningful discussion of immunosuppression for xenografting at
present without careful attention to the potential or actual toxicity of potent
new immunosuppressive agents, since toxicity is so frequently present at the
doses necessary to prolong xenografts.
Effect of Vascularization of Skin Grafts
Rodent xenograft models have exhibited analogies to allotransplantation and

also illustrate novel immunobiologic principles. Jooste, Winn and colleagues
[22] have done elegant studies on rodent skin xenografts and their acute rejec-
tion from antibody injections. They have shown that the mouse skin xenograft
is resistant to antibody-mediated attack for the first 5-7 days of its residence in
the host. For a period of about 7-16 days, the graft then becomes sensitive to
an accelerated rejection induced by the injection of antidonor antisera.
Following 16 days, the graft again becomes resistant. This group has estab-
lished that this novel immunological finding, unique to xenografting, is appar-
ently related to the vascular supply of the xenograft.
For the first 7 days the xenograft does not possess a direct vascular supply.
Presumably, the donor endothelium is isolated and protected from vascular
rejection induced by an antidonor antibody during this period. After 5-7 days,
the xenograft develops its own blood supply and is connected to the host blood
capillaries, thus making the xenograft vascular tree susceptible to antibody-
mediated rejection. After 14-16 days, the donor endothelium is gradually re-
placed by host endothelium and the graft again becomes resistant to antidonor
antibody rejection.
In contrast. discordant xenografts with primary and direct vascularization
such as heart and kidney xenografts are frequently rejected in a hyperacute or
accelerated manner within minutes. Messmer's group [23] have used elegant
techniques to study xenograft microvasculature and have shown it to be invul-
nerable to immune attack for the first 6 days after xenografting.
The Vascular Endothelial Cell
In studies of the xenogeneic response to the vascular endothelial cell (VEC),

Haisch et al. [7, 15] showed that mouse VECs isolated in vitro induce strong
xenogeneic T cell responses as well as ADCC and complement-dependent cy-
tolysis. Haisch also demonstrated that the mouse VECs induce a much
stronger xenogeneic reaction than splenocytes in vitro. These findings with
the VEC are provocative, since they correlate well with the in vivo xenograft
rejection reaction, which is much stronger than alloreactivity. In contrast, dis-
parate xenograft combinations show low T cell reactivity with lymphocyte tar-
gets, and this is clearly an invalid in vitro correlate. The findings also indicate
the importance of the VEC in both the afferent and efferent arms of the
xenograft immune response.
Pancreatic Islet Transplantation
The pancreatic islet deserves special mention as a rodent xenograft model.

The islet, like the skin graft. undergoes a type of secondary neovascularization
and survives for the first few days in the host without primary vascular connec-
tions with the host circulation. A number of investigators have suggested that
the islet is susceptible to an almost immediate 24- to 48-h hyperacute-type re-
jection [41]. Certainly, the islet xenograft and its counterpart, the islet allo-
graft, are unusually susceptible to rejection. Often, the rejection mimics pri-
mary nonfunction in that the grafts fail to lower blood sugar at any time in the
post-transplant period. Though primary failure of the xenograft may be due to
early severe accelerated rejection, a number of authors have attributed early
islet failure to deficiencies of isolation and culture techniques which impair
islet viability.
Studies from our laboratory have recently provided evidence that so-called
primary nonfunction of the islet xenografts in rodents is related to an immuno-
logical attack upon the islets shortly after grafting, since primary islet
xenograft nonfunction can be reduced to 5%-10% by the use of some of the
more effective new immunosuppressive agents in combination with splenec-
tomy. This is yet another area where xenografting in rodents should prove to
be fruitful for gathering of empirical data and development of plausible hy-
potheses ofxenograft immune reactivity.
Xenograft Studies in Immunodeficient Rodents 145
Xenograft Studies in Immunodeficient Rodents

A number of elegant studies focused on genetic mutations leading to specific
immunological dysfunctions have been carried out in rodent species, most
particularly the mouse and rat [24-26]. In addition, the mouse histocompati-
bility complex has been well mapped out, and a number of genes responsible
for hereditary dysfunction of the immune system have been identified. Thus,
selective genetically immunodeficient mutants have recently become avail-
able. Their high rate of inbreeding, coupled with the frequent occurrence of
spontaneous mutations, permits the selective development of mutant species
which demonstrate phenotypically a critical genotypic immune dysfunction.
A number of rodent models with specific immune deficiencies, including
the nude mouse (T cell-deficient and athymic), the beige mouse (killer,K, and
natural killer, NK, cell-deficient, specifically), the xid mouse (excellent im-
munodeficiency showing B cell and antibody deficiencies), and SCID mouse
(severe combined immunodeficiency mouse, with both T and B cell deficien-
cies and normal K and NK cells), and genetic combinations of these four ani-
mals have recently become available [24]. Animals with specific immune defi-
ciencies, including complement deficiencies and other dysfunctions of the
immune system are also available, but will not be discussed in detail here.
This section will focus on the best studied mutations in mice, the scid muta-
tion, the nude (Nu) mutation, the beige (Be) mutation, and the xid animals,
and combinations of these mutants, as well as the nude rat. Over 30 single-
gene mutations leading to immunodeficiency have been characterized and in-
bred. In addition, ten mice strains with multiple mutations of immunodefi-
ciency have been described, as well as rat, guinea pig, and hamster mutants
[37,38].
The Nude Rodent
The nude (Nu) genetic aberration has been described in both the mouse and
the rat [24,26,27,29]. The nude mouse was one of the earliest animals studied
for immunodeficiency, and the nude rat has been the most popular rat strain
for immunodeficiency studies. The nude rat developed as a mutation in a
group of animals in Scotland, and separately in New Zealand.
Using the basic Rowlett nude rat and backcrossing it with outbred strains,
including Lewis, Buffalo and Wistar Furth, National Institutes of Health
(NIH) staff have developed a nude rat which is herein referred to as the NIH
nude rat. The animal in the homozygous state is hairless, athymic, and known
to have marked T cell deficiencies. Animals used should be younger than 8-10
weeks old, since older animals show development ofT cell expansion and dif-
ferentiation [27]. These animals show severe depletion of lymphocytes in the
thymus-dependent pericortical area of lymph nodes, as well as in the thymus-
dependent splenic periarteriolar sheaths. Peyer's patches are small and lym-
phocyte counts (presumably B cells) are high. No identifiable defect in poly-
morphonuclear cells (PMNs) is present. Serum immunoglobulins vary from

normal to slightly elevated. Antigen retention by the follicular dendritic cells
is essentially normal. NK activity is normal to increased in nude rats, as it is in
nude mice.
In the thoracic duct, B cells are normal or increased, but T cells are marked-
ly reduced «5%). T cells in the peripheral lymph node tissues are less than 2%
of total lymphocytes in young animals, up to age 4-8 weeks. After this age, the
number ofT cells in the pericortical areas increases and some T cell function is
seen as measured by alloreactivity in mixed lymphocyte cultures (MLC), de-
velopment of cytotoxic cells, development of helper cells producing inter-
leukin-2 (IL-2), and responses to phytohemagglutinin (PHA) and con-
canavalin A (Con A).
In short, the nude rat is an elegant model of an athymic pure T cell defect
[30]. Other systems appear to work well in immune reactivity if they do not de-
pend upon T cell help. This is a limitation in evaluating these animals for so-
called lymphokine activated killer (LAK) cells, since these cells depend upon
T helper cell-produced IL-2 for activation. When combined with other geneti-
cally controlled immune deficiencies, these models may serve as elegant tools
for study of functions of the lymphocyte subpopulation function in immune re-
activity.
The Beige Mouse
The beige (Be) mutant is a light -colored variant of the C57BlI6J mouse. These
animals show hypopigmentation, and the genetic mutant has arisen in a num-
ber of different laboratories [31]. The phenotypic manifestations resemble the
Chediak-Higashi syndrome in humans. The animals have a basically intact
T cell system. Deficiencies in macrophage pinocytosis have been reported.
There are abnormally large lysosomal granules in most of the leukocytes in
bone marrow and peripheral blood and many other cells of the immune sys-
tem, which presumably are antigen-presenting cells. The defective granules do
not function well, and there is a general motility defect in these animals'
PMNs.
The animals have a selective impairment of NK cell function. NK cells do
not mount an antibody-dependent or antibody-independent cytolysis of tu-
mor cells in vivo. However, the NK cells can be activated to cytolysis in vitro.
The defect of macrophage pinocytosis is apparently not related to antibody
defects, since immunoglobulin levels in these animals are comparable to nor-
mal controls. The animals have a high susceptibility to pyogenic infections and
spontaneous pneumonitis. The main defect here is a suppression of K and NK
activity, which appears to be an isolated defect, although reports of defects in
cytotoxic T cells can be found in the literature. The animals apparently have
LAK cell precursors and presumably normal LAK cell activity. In short, this is
an elegant model for studying isolated K and NK cell activity as it might relate
to graft rejection and/or tumor growth.
Xenograft Studies in Immunodeficient Rodents 147
Results ofXenografting in Immunodeficient Rodents
Our group has now studied xenograft rejection in a wide variety of genetically
immunodeficient strains. Results are summarized in Fig. 9.3. In these studies,
allografts and xenografts of skin were placed on selectively immunodeficient
animals and their rejection times recorded. In addition, biopsies of the grafts
were examined for the nature and degree of infiltrating cells in these grafts.
Control skin grafts survived 5.4±3.6 days. A striking and unexpected obser-
vation was that the nude rat rejects non-neoplastic tissue xenografts in a nor-
mal manner and tempo of rejection. As noted, the graft survival times of the
skin xenografts (also heterotopic cardiac allografts, now shown here) were
normal and, if anything, slightly decreased. These findings contradict a num-
ber of observations in the literature. However, most of the studies of
xenografts in nude animals have used tumor xenografts. It is possible that the
choice of donor xenograft tissue could explain these apparent discrepancies.
This matter clearly needs further clarification.
Another striking finding was the delay of rejection of both allografts and
xenografts in the beige animals which have defective NK and K cell function.
Since Band T cell function appear to be normal in these animals, it would ap-
pear that NK and K cell activity is important in the early acute rejection pro-
cess. To our knowledge, there is nothing in the literature which contradicts
these findings. Further experimentation will be necessary to determine pre-
cisely the role ofNK and K cells in effector mechanisms of allo- or xenorejec-
tion.
The xid mutation is a marked deficiency in T cell-independent B cell immu-
nity, although there is also some degree of suppression ofT cell-dependent B
cell immunity [25]. As seen in Fig. 9.3, the xid animals rejected their xenografts
in a normal manner. This finding suggests a need to examine more precisely
the role of antibody in xenograft rejection, since humoral immunity is thought
to have a major role in this response. The SCID animals and the Be/Nu animals
Days survival
35
* * *
* Indefinite
30
25
20
15
10
5 T T T
o l ~
J I ~ I l ~ I
C (Lewis) Xid mouse Nude mouse SCID mouse Nu/Be Nu rat
Fig. 9.3. Survival ofxenografts in immunodeficient rodents. C, control; Nu, nude; Be, beige
all maintained their skin grafts for an indefinite period and showed a marked
blunting of the xenogeneic response in histopathologic sections. These overall
results are difficult to explain at present. but they do provide an important ba-
sis for conceptualizing the basic mechanisms of xenograft rejection.
The findings in xenografting of the immunodeficient nude rats raise a num-
ber of possibilities as to the xenograft rejection mechanism. This recently re-
ported finding that nude animals reject skin and heart grafts (seen on the right-
hand side of Fig. 9.3) suggests that T cell deficiency does not lead to
prolongation of xenografts in many cases. It is possible that these results are
due to a "leakiness" of T cells in athymic animals. The late differentiation of
T cells into mature immune effector cells has been described in a number of
species of immunodeficient rodents. Our use of young animals. however. sug-
gests that this explanation is not adequate. Furthermore. the nude rats used in
this study showed no rejection of allografts, suggesting that they are indeed
T cell deficient. In addition. studies of peripheral T cells with monoclonal
markers, as well as mitogen responses, indicated that the T cell system was di-
minished as well as poorly responsive to known T cell mitogens. These results
indicate that there is a need to re-evaluate the role of the T cell system in
xenograft rejection.
The SCID animals. showing both T and B cell deficiency [32]. maintain
xenografts and allografts indefinitely (as shown in the middle of Fig. 9.3). The
beige/nude/xid animals maintained skin grafts indefinitely. Although knowl-
edge is incomplete on the beige/xid animals. the best evidence available is that
this species has no significant deficit in NK activity. Taken together. the results
suggest that K and NK activity alone. while contributing to xenograft rejec-
tion. is not by itself sufficient to reject xenografts without T or B cell help.
Studies of the Immunobiology ofXenografts
In early studies. our group looked at some basic immunobiologic principles of

xenograft rejection in rodents. These studies were designed as one part of a
comprehensive overview of xenografting in an attempt to develop more ratio-
nal perspectives leading to a more plausible and rational understanding of
xenograft biology.
Strain Specificity
One of the first areas we explored was the question of strain specificity in
xenografts. Other authors have maintained that xenograft rejection can be
highly dependent upon the strain of the species utilized in xenograft experi-
ments in mice [33]. One experiment we designed as a stringent test of this con-
cept was to compare the survival of cardiac allografts in the weak allograft re-
sponder ACI rat recipient to survival of cardiac xenografts in the high
Studies of the Immunobiology of Xenografts 149
responder Lewis strain. The ACI strain is known for its low responsiveness
and will accept a number of fully allogeneic cardiac allografts with only minor
immunological manipulation maintained for short periods of time.
Despite these differences, the hamster-to-rat cardiac xenograft survived
for equal periods (3.8±1.2 versus 4.l±O.6 days, respectively) in Lewis versus
ACI recipients. Similarly, skin grafts done between a hamster and the ACI
versus the Lewis rats showed no difference in graft survival or in histopatho-
logical infiltrate of rejected grafts. Interestingly, this occurred despite marked
differences in natural titers of antihamster antibodies; the ACI rat has signifi-
cantly lower levels of antihamster antibody than the Lewis rat. In general, we
found an association of anti donor antibody in many of the xenograft models of
rejection, but the antibody levels in no way explained many of the subtle varia-
tions in xenograft rejection.
Our conclusion from this study is that the rat strain differences exert minor,
if any, effects on xenograft survival, although others report strain differences
of xenograft rejection in the mouse. This matter needs to be studied in more
detail, since different xenograft reactivity between strains suggests that either
specific histocompatibility differences (perhaps in minor histocompatibility
antigens) or different VEC responses might produce a difference in strain
responsiveness or that the difference could be related to the overall im-
munoresponsiveness of a given strain. Either of these concepts could have
practical implications in future clinical xenografting.
Antidonor Antibodies
An area of great interest is the role of antibodies in xenograft rejection. The

failure of skin grafts to hyperacutely reject is a curious phenomenon, since, in
many of the donor-recipient xenograft models studied, there was a high level
of anti donor cytotoxic antibody in the recipient. Furthermore, investigators
have demonstrated a relative ease of induction of the skin analog of hyper-
acute rejection in allografts, the so-called "white graft". However, the hamster
heart placed in the Lewis rat, and also the hamster liver, do not hyperacutely
reject, despite high levels of antihamster antibody in Lewis sera. The cardiac
xenograft findings also stand in stark contrast to the relative ease of induction
of allogeneic hyperacute rejection in cardiac allografts in the rat, as shown by
Forbes, Guttman, and others [34].
A common explanation for the failure of rats to develop hyperacute rejec-
tion is the low level of complement seen in these animals. It was striking for us,
therefore, to observe in some other experiments a hyperacute rejection pat-
tern in the Lewis rat in 15-20 min, in a typical hyperacute rejection pattern
with massive swelling, epicardial ecchymoses, a dilated and cyanotic heart and
cessation of heart beat within the first 15 min post-transplant.
Using a plasmapheresis technique we developed, with mUltiple centrifuga-
tion of the plasmapheresed aliquots, we were able to show that plasmapheresis
of 10 ml blood daily for 2 consecutive days pretransplant was able to markedly
prolong the guinea pig heart graft to 2-3 h. At the end of this time, the heart
looked completely normal, without any signs of hyperacute rejection, but the
experiment had to be terminated because of blood leakage at the suture line,
presumably due to defects in clotting factors removed during plasmapheresis.
The guinea pig studies were important in showing that the Lewis rat can ex-
hibit normal complement-dependent cytotoxicity and a vigorous hyperacute
rejection ofaxenografted heart without deliberate induction of preformed an-
tidonor antibody. Interestingly enough, there was no significant difference be-
tween antiguinea pig antibody levels in the Lewis rat and the antihamster anti-
body levels, irrespective of the failure of the Lewis rat to generate an effective
hyperacute rejection of the hamster, but not the guinea pig heart. While there
is much reason to believe that antibody in the primary effector in this model,
there are a large number of observations leading to the conclusion that levels
of antibodies do not show a direct positive correlation with rejection in many
situations. Thus, it seems highly possible that factors other than complement-
dependent antibody (NK cell, antibody-dependent cellular cytotoxicity, LAK
cells, platelets, etc.) may interact with humoral antibody to produce effector
mechanisms generating hyperacute rejection.
Again, the xenograft models can be very useful in dissecting out the basic
elements of hyperacute rejection seen in discordant xenografts. Previous stud-
ies of hyperacute rejection in xenografts (especially pig-to-dog) have outlined
roles for platelets, PMNs, leukocytes, hemagglutinating red blood cells, com-
plement, and antidonor antibody in hyperacute rejection. Our studies in ro-
dents, as well as those of others, also suggest that multiple mechanisms may be
involved, since humoral antibody and/or complement alone do not seem suffi-
cient to explain the disparate results seen.
Of great importance is the demonstration in our studies that early block
of antixenodonor antibody block rejection in disparate xenografts.
Furthermore, these animals often sustained a reappearance of high levels of
xenodonor antibodies similar to pre transplant levels, but not associated with
graft rejection. In other words, a "humoral adaptation" occurred similar to the
"immunological adaptation" first described by Woodruff in which early, vig-
orous rejection activity wanes in the later post-transplant course, despite a re-
duction of immunosuppression. The validity of the concert of "humoral adap-
tation" (or "accommodation", see Chap. 6) has been shown in situations
where natural antibodies or induced antibodies are associated with hyper-
acute rejection which can be clocked by acute removal of these antibodies.
Later, the antibodies can come back without recurrence of an acute rejection
of a xenograft (Chap. 6).
Killer Cells
The major defect in knowledge of xenograft rejection lies in a lack of under-

standing of the sensitizing and effector arms of the response, which is undoubt-
edly mediated by a combination of humoral antibody and cellular mecha-
Studies of the Immunobiology ofXenografts 151
nisms. Interplay of these mechanisms occurs with ADCC and it is also quite
likely that natural killer cells, or the more recently described LAK cells are in-
volved in xenograft rejection. The mechanisms and the precise effector cells
involved in the ADCC, NK, and LAK cell effector mechanisms are largely un-
resolved in tumor and allograft biology, and the role ofthese cells in xenograft
rejection is virtually unknown [35,36].
The major differences of the NK and LAK cells are that their killing, unlike
that of the CTL of T cell lineage is a non-major histocompatibility complex
(MHC) restricted and is often directed at viral, tumor, or autologous antigens.
The NK cells, and especially the LAK cells, can be activated in response to a
variety of lymphokines, especially IL-2 [37]. The killer cells are predominantly
of two lineages: (i) the CD3-TCE-cell with a morphology of large granular
lymphocytes, and (ii) a CD3+ CD8+ cell of unknown lineage, but presumably
derived from T cell precursors. Unfortunately, much of the knowledge of
these cells derives from in vitro cultures in which a recombinant IL-2 is added
and, thus, the precise in vivo significance of these cells is not well delineated.
It has been suggested that the term "NK" should be applied to killer cells
with non-MHC restricted cytotoxicity, which arise without an evident acute
immune stimulus. The problem with the use of the more common term "NK
cell" is that cells of a heterogenous origin, such as neutrophils, macrophages
and bone-marrow-derived lymphocytes all execute non-MHC restricted cyto-
toxicity, yet are not usually considered classical NK killers. Indeed, the prob-
lem is even more complex in that these cells can be shown to execute im-
munoregulatory function of a noncytotoxic nature, such as down-regulation of
the immune response, presumably by suppressor mechanisms and cytokine
production. The whole matter is also confused by a lack of knowledge of the
precise mechanisms of cytotoxicity produced by conventional cytotocix T lym-
phocytes of T cell lineage and these cells which exhibit different patterns of
cytotoxicity.
Non-T Cell Cytotoxicity
Despite this confusion, some interesting early in vitro/in vivo correlates in

xenograft rejection can be suggested from known data concerning non-T cell
cytotoxicity, especially LAK killing in rodents. Studies in our laboratory and
those of others have shown low levels of xenogeneic-induced classical T cell
cytotoxicity. Indeed, an afferent stimulation as shown by MLC reactivity is
low with xenogeneic stimulation in our experience and that of others. T cell re-
activity to endothelial antigens of xenogeneic origin is, however, quite brisk.
Endothelial studies by Haish et al. (Fig. 9.4) show the rather extraordinary lev-
els of both afferent stimulation and development of classical antibody-depen-
dent cellular-mediated cellular cytotoxicity and complement-dependent cyto-
toxicity against endothelial antigens, but not against nonendothelial
xenoantigens. Thus, it seems quite possible that the xenograft response will be
shown to be highly involved with non-T cell cytotoxicity.
152 Immunohiology of Xenografting in Rodents
60 VEC - induced response compared to

similar lymphocyte stimulation
o VEC • LY
40
20
0-'---'---
H3 Thymidine uptake x10 3 % Cytotoxicity
Fig. 9.4. Evidence of induction of stimulatory and effector immune responses in vascular
endothelial cells (VEe; Ly, lymphocyte)
Early studies in immunodeficient rodents support this concept. since ani-

mals with marked deficiencies in T cells (such as nude rats) can reject
xenografts in a rapid, vigorous manner with apparently normal patterns of cel-
lular reactivity. In addition, xenograft rejection in the hamster-to-Lewis rat
heart model is apparent at 24-48 h and peaks by 72 h. This accelerated destruc-
tion of grafts occurs far before development of significant T cell cytotoxicity at
7-10 days. LAK killer cell activity is easily inducible in 48-72 h with sufficient
levels of IL-2 in culture. Thus, the early in vivo cytotoxicity seen in these ro-
dent xenograft models is probably a non-T cell type cytotoxicity, most likely
LAK andlor ADCC killing.
Immunodeficient models which show a marked delay in xenograft rejec-
tion (or even indefinite survival of xenografts) such as the SCID model, the
xidl Nul Be model, and the Nul Be model all have defects in LAK killing. In con-
trast, animals with LAK killing intact or relatively normaL such as the xid
model, all reject xenografts normally, although NK defects will lead to a slight
prolongation of xenografts models from 7014 days, as shown by our group in
the Be mouse. These findings are highly reproducible and suggest that LAK
cell killing is a possible primary mechanism of xenorejection.
Another fascinating aspect of these findings is that they would go far to ex-
plain the reason why agents with strong anti-T cell activity, especially against
the lymphokine-producing helper cells, such as RATG and anti-L3T4 mono-
clonal antibodies, are potent blockers of xenogeneic responses, as reported by
Pierson and Auchincloss [38]. This concept is further supported by results in
our lab with RA TG, which most effectively blocks T helper cells and also is a
relatively good blocker of xenograft rejection. A similar explanation for the
effectiveness of TLI in blocking xenograft survival seems appropriate and ra-
tional. Identification of afferent and efferent effector arms of xenogeneic re-
activity would seem to be critical in developing a basic understanding of this
immune response.
Studies of the so-called xid deficiency may provide insight into the role of B
cell activity in xenograft rejection. since these animals will reject a xenograft
normally. These animals have normal T cell, NK activity, and normal LAK ac-
Role of the Spleen in Xenograft Rejection 153
tivity. Furthermore, mutants known to be defective in LAD cell activity, such
as the beige/nude/xid and the SCID animals, all have defective xenograft re-
jection. While the results of these studies need to be confirmed and expanded
by immunological reconstitution experiments, the early findings are provoca-
tive. Hopefully, these findings in rodents will permit a better understanding of
the mechanism of xenograft rejection, which may permit the development of
better techniques for immunomodulation of the xenograft response.
A recent finding of major interest is the discovery that nude rats with
marked T cell deficiency reject cardiac xenografts in a completely normal tem-
po and fashion [9, 10]. In addition to this striking finding, further studies
demonstrated that splenectomy partially ablated this response and prolonged
graft survival to a mean of 18 days. These results demonstrate that T cell-defi-
cient animals can have normal xenogeneic rejection responses which are par-
tially dependent upon the spleen. Finally, the splenic component of the xeno-
geneic response may not be related to antibody, since titers of antidonor
antibody are not changed by splenectomy.
Role ofthe Spleen in Xenograft Rejection
Because antibody responses are felt to be important in xenografting, it seemed

logical to us that the spleen, important in the antibody response to intra-
venously injected antigens, might well be important in xenoantibody respon-
ses. Therefore, we performed splenectomy in a number of animals, both pre-
transplant and at the time of transplantation, and measured xenograft survival
[11].
In the hamster-to-Lewis rat heart recipient, splenectomy produced a de-
crease in the humoral antibody response, but no significant prolongation of
graft survival. Splenectomy plus 15 mg/kg cyclosporine, however, produced a
rather marked mean xenograft prolongation to nearly 22 days in this model.
Of even greater interest, however, was the ability of FK-506, when used in
combination with splenectomy, to prolong graft survival to a mean of 33-40
days. Splenectomy and DSG given at 2.5 mg/kg also prolonged graft survival
to 27 days, and this too was highly significant. These graft survivals were some
of the longest survivals seen to date in over 1000 hamster-to-Lewis rat cardiac
xenografts.
All prolongations were associated with decreased antibody titers in the re-
sponding animals. The most effective decrease in antibody titers was pro-
duced by splenectomy plus DSG. These results clearly demonstrate that
splenectomy can act with suppressive drugs in a synergistic manner and sug-
gests that splenectomy may be important in xenografting, despite its poor abil-
ity to prolong either primary or secondary allografts. Our studies suggest that
the spleen may have a primary role in xenograft rejection.
The most recent studies in our laboratory indicate that the effects of
splenectomy on xenograft rejection may be independent of blockade of anti-
body production by the spleen. There was a poor correlation of antibody block
by splenectomy and xenograft prolongation [11]. These findings suggest that
splenectomy may act to prolong xenograft survival by mechanisms other than
a block of antibody formation. Some current studies, too detailed to describe
in full here, suggest that splenectomy removes an effect coterie of cells in-
volved in xenograft sensitization of the host. Another highly provocative find-
ing recently reported by our group is the ability of splenectomy to block the
xenograft rejection in the nude rat [10], an animal which paradoxically rejects
xenografts at a normal tempo and intensity of rejection. We have no satisfacto-
ry explanation for these results at present, but the results suggest an important
role for the spleen in xenograft immunobiology.
Histopathology of Xenograft Rejection
One of the most poorly understood areas of xenograft rejection is the

histopathology of xenograft rejection. There have been only a few reports on
this subject, and a large number of these were done prior to the modern era of
immunobiologic sophistication including monoclonal antibody markers of in-
filtrating cells. Our studies began with standard histopathology obtained with
hematoxylin and eosin stains in hamster hearts removed after rejection by
Lewis rat recipients [12, 13].
The early lesions of the accelerated rejection model with preformed anti-
bodies were a vasculitis with endothelial disruption, pyknosis, swelling, and
overall disruption of endothelial integrity. Associated with this, there was of-
ten a severe hemorrhagic infiltrate, high numbers of PMNs, but little myocyte
necrosis. Most of the myocyte necrosis seemed to be secondary to infarcts,
which appeared in stage II xenograft rejection in this model. In this stage,
there were multiple micro infarcts and mUltiple areas of obstruction of the ves-
sels and plugging by platelet thrombin-fibrin complexes with intermeshed
cells. In a milder stage of xenograft rejection seen in immunosuppressed ani-
mals at a later time, there was a more marked infiltrate of cells into the intersti-
tium and myocardial swelling and myocyte necrosis which was more uniform,
suggesting a direct and local attack upon the cells and not an ischemic necrosis
as suggested by the earlier phase of vasculitis and thrombosis within the blood
vessels.
Some studies were performed with monoclonal antibody stains and
showed dense infiltrates ofT cells, a high percentage of macrophages in the in-
filtrate, as well as cells resembling large granular lymphocytes (presumably, K,
NK or LAK cells). We performed fluorescence-activated cell sorting (FACS)
analyses of infiltrating cells using monoclonal antibodies. In most early hu-
moral rejection (up to 10 days), no significant cellular infiltrate was seen. In
the later rejections, in animals treated with DSG and/or FK-506, a significant
cellular infiltrate was observed. This infiltrate, by F ACS analysis, was com-
posed of 10%-40% T cells with T helper cells slightly predominating over
Study of Immunosuppressive Modalities in Rodent Xenograft 155
T cytotoxic/suppressor cells. We were unable to confirm the findings of Click
et aI., who reported that rodent xenograft rejection was characterized by a ho-
mogenous infiltrate of Thy 1-IgG- cell [39]. In none of these sections did we
observe a homogenous Thyl- Ig~ell infiltrate to be a hallmark of rejection in a
rat-to-mouse xenograft.
In addition to vasculitis, early xenograft rejection is characterized by small
vessel thrombosis. Microinfarcts are often seen in the adjacent tissues. More
commonly, the interstitium shows a hemorrhagic pattern with edematous,
separated intermyocyte spaces. The major hallmark of discordant xenografts
is thus a severe vasculitis with clotting infarction and disruption of endothelial
integrity. This process, undoubtedly mediated by humoral antibody, can be
blocked by some suppressive agents, especially DSG.
Study ofImmunosuppressive Modalities in Rodent Xenografts
There is reasonable cause to believe that the single major impediment to appli-
cation of clinical xenografting in the near future is the problem of short- and
long-term rejection of the xenogeneic graft accentuated because of the great
disparity of xenoantigens between the donor and host. Three years ago, our
group began a comprehensive study of immunosuppression in xenografting us-
ing rodents. Rodents were selected deliberately because we felt from prelimi-
nary studies that a number of new drugs given in varying doses would have to be
screened in order to obtain effective suppression of the xenograft response.
Furthermore, we knew that these drugs were quite toxic in almost every in-
stance, both because of the need for such a vigorous blocking of the immune sys-
tem an also because so many of the drugs were effective only at high doses.
Monotherapy
Preliminary studies showed, for example, such drugs as methotrexate,

Adriamycin, azathioprine, and other antimetabolites known to be immuno-
suppressive in allografts simply did not at all block the xenograft response,
which was far more vigorous than allograft responses. As our work continued,
more sobering data appeared, showing, for example, that even toxic doses of
cyclosporine did not at all block xenograft response in the models tested.
The first glimmer of hope for suppressing the xenograft response came with
the discovery that a quality-controlled RATG produced a mild prolongation
of skin xenografts in the mouse-to-rat and the rat-to-mouse combinations.
Although the prolongation of graft survival was minimal, it was clearly repro-
ducible and could also be seen with other models utilizing hamster-to-mouse
skin, etc.
FK-506 was kindly made available to us by the Fujisawa Company, but we
found this drug to be ineffective in monotherapy, as did Ochiai in later pub-
lS6 Immunobiology of Xenografting in Rodents
lished studies [36]. DSG, a new immunosuppressive agent, was made available
first from the Nippon Kyoto Corporation and later by Bristol-Myers for study.
This drug, too, was ineffective in prolonging xenografts in monotherapy.
FK-S06 and cyclosporine were quite toxic, with animal mortality rates of
30%-80%, even when no effective immunosuppression was achieved. These
results clearly indicated that monotherapy with currently available immuno-
suppressive agents (and, in fact, even newer experimental ones) would not be
sufficient to control xenograft rejection.
Combination Drug Therapy
Our first breakthrough in controlling the vigorous immune response in skin

and heterotopic cardiac xenografts occurred with the use of a combination
therapy with RATG and FK-S06, as well as with RATG and DSG. Control
grafts of both heart and skin survived only about 3-S days. A 3-day course of
RATG, plus 2.S mg/kgDSG or 1.S mg/kg FK-S06 prolonged both skin and car-
diac xenograft survival to a range of 20-30 days in almost all experiments. In
addition, studies of pancreas islet xenografts showed similar prolongation and,
perhaps more importantly, the abolition of primary graft nonfunction by com-
bination RATG/FK-S06 and RATGIDSG immunosuppression. These results
suggested that primary nonfunction of xenograft islets is clearly related to im-
munological rejection. These early results were encouraging and represented
some of the first definitive prolongations of xenografts seen using these diffi-
cult models.
Combination Therapy Including Total Lymphoid Irradiation
A previous report from another laboratory showed a graft survival of lOS days
using the difficult hamster-to-Lewis rat heterotopic cardiac allograft, and sup-
pression with total lymphoid irradiation and cyclosporine [40]. Over SO ani-
mals were tested with a variety of TLI protocols, generally using lS00 gamma
plus S-lS mg/kg cyclosporine in an attempt to reproduce these results. We
have been unable to do this to date. Instead, our results with TLI and cy-
closporine were discouraging. We attempted to modify the TLI regimen as
recommended by others, but were still unable to obtain these long survivals
[6].
Later experiments, however, showed some far superior survivals using TLI
and DSG [12]. The feeble prolongation of the hamster-to-Lewis rat cardiac
xenograft of about 10 days with cyclosporine and TLI is inferior compared
with the 20-to 2S-day mean graft survival seen with TLI and DSG. Some of the
TLIIDSG animals have prolongation of their grafts beyond 30 days, which was
the first prolongation greater than 1 month that we saw with this model.
Histopathological studies demonstrated a clear abolition of the early hu-
moral immunity and prolongation of the graft and the subsequent develop-
Comment 157
ment of cellular infiltrates clearly indicating the shift in immune reactivity
away from humoral-type vascular reactivity to cell-mediated immunity. These
observations gave rise to the hypothesis that a type of humoral immunity
adaptation could occur similar to that reported earlier for cell-mediated im-
munity by Woodruff and others.
These results were all obtained with a 14-day injection schedule of DSG.
When the injection schedule was lengthened to 21 and 28 days, the prolonga-
tion of graft survival beyond 30 days was seen again with a marked abolition of
humoral vasculitis and the clear development of late cellular immune infil-
trates. A small group of five animals given DSG and RATG with repeated in-
jections had a mean heterotopic cardiac allograft survival of over 35 days with
all animals surviving over 30 days.
Following the unsuccessful combination ofTLI with cyclosporine in a vari-
ety of drug protocols detailed by DeMasi et aI., we combined TLI with im-
munosuppression using DSG and FK-506. Prolonged graft survival was seen
in combinations of these agents. Marchman et aI. first reported the marked
prolongation of survival of the Syrian hamster-to-Lewis rat heterotopic car-
diac allograft to near 30 days using the combination of TLI and DSG. These
authors also found a low toxicity rate seen with doses of2.5 mg/kg, which were
effective in combination with 1500 rads TLI. Similarly, recent experiments
have shown prolongations in the range of 30 days using a combination of TLI
and FK-506. These experiments and those of Marchman also demonstrated a
marked block of development of antidonor humoral antibody, especially with
the TLI/DSG therapy. This inhibition of formation of antidonor xenoantibod-
ies was striking. Clearly, this block is related to prolongation of graft survival
and showed the characteristic lack of development of the severe vascular le-
sions with endocytolysis, intravascular and perivascular cell infiltration, and
destruction.
These combinations of agents were able to markedly delay the rejection re-
action seen in the difficult xenograft model of the hamster heart to the Lewis
rat. Thus, RATG, TLI, splenectomy, FK-506 and DSG appear to be the best
available agent combinations for suppression of rodent xenorejection as
gauged by prolonged survival and histopathology. We conclude that these im-
munosuppressive modalities should be targeted for further study, especially
the use of combinations of these suppressives, for xenografting.
Comment
Extensive studies of xenografting using over 1000 rodents have demonstrated

an important utility for rodents in testing of immunosuppressive agents, espe-
cially in the use of skin grafts as screening for suppressive agents followed by
more definitive testing of these agents on the heterotopic cardiac xenografts
from hamsters to Lewis rats. These studies indicate that conventional suppres-
sive agents are poorly effective in prolonging xenografts, but some agents
(TLI, RA TG, FK-506, and DSG) prolong well, especially when used in combi-
nations.
Recent studies of genetically controlled inbred rodents with selective im-
munodeficiency have shed light on some possible mechanisms of xenograft re-
jection. Splenectomy has been found to exert a strongly beneficial role in
blocking xenograft rejection. The early primary nonfunction and acute graft
rejection seen with isolated pancreatic islet xenografts could be blocked effec-
tively by splenectomy, RATG, and DSG. These studies also suggest some
striking paradoxes and indicate the need to carefully re-evaluate the role of
both the T cell and B cell immune systems in xenograft rejection.
Acknowledgements. This study was supported by NIH grant #ROl Al22293

and Diabetes and Educational Fund grant #B39-II89.
References
1. Billingham, RE., Medawar, P.B. Free skin grafting in mammals. 1. Exper. BioI. 28.385.
1951.
2. Thomas, F.T., DeMasi, RJ., Daniel, H., Araneda, D., Hsu, S., Moon, D .. Thomas, J.M.
Comparative immunosuppression for xenografting. In: Xenograft 25. Hardy, Mark A.
(ed.), Elsevier Science Publishers, Amsterdam, 1989, p. 55.
3. Thomas, F.T., Thomas, J.M., Ganghoff, 0., Gross, U. Mechanisms of cell-mediated re-
jection. In: Kidney Transplant Rejection. Williams, G.M., Burdick, J.F. and Solez, K.
(eds.) Marcell Dekker, Inc., New York and Basel, In press.
4. Hsu, S., Daniel, H., Thomas, J.M., Thomas, F.T. Synergism ofFK-506 and rabbit antithy-
mocyte globulin (RA TG) in prolongation ofxenografts (XG). Surg. Forum. 39,374,1988.
5. Thomas, F.T., DeMasi, R., Araneda, D., Marchman, W., Alqaisi, M., Larkin, E.,
Dabney, L., Condie, R, Thomas, J.M. Comparative efficacy of immunosuppressive
drugs in xenografting. Transplant. Proc. 22,1083,1990.
6. DeMasi, R, Alqaisi, M., Araneda, D., Nifong, W., Thomas, J.M., Gross, u., Thomas,
F.T. Re-evaluation of total lymphoid irradiation (TLI) and cyc1osporine therapy in
the Syrian hamster to Lewis rat cardiac xenograft model. Transplantation. 49, 639,
1990.
7. Haisch, c., Lodge, A., Huber, S., Thomas, F.T. In vitro human versus murine xenogene-
ic reactions. Transplantation. 50,528, 1990.
8. Marchman, W., Araneda, D., DeMasi, R, Taylor, D .. Larkin, E., Alqaisi, M., Thomas,
F.T. Combination therapy with 15-deoxyspergualin and total lymphoid irradiation pro-
longs xenograft survival in the hamster-to-rat cardiac xenograft model. Transplantation.
1991, In press.
9. Marchman, W., Thomas, F.T., Araneda, D., Carobbi, A., Thomas, J.M. Xenograft rejec-
tion in immunodeficient mice. Surg. Forum. 41,376, 1990.
10. Thomas, F.T., Marchman, W., Carobbi, A., Araneda, D., Pryor, W., Thomas, J.M.
Immunobiology of the xenograft response: xenograft rejection in immunodeficient ani-
mals. Transplant. Proc. 23,308,1991.
11. Carobbi, A., Araneda, D., Patselas, T., Thomas, J .M., Mosca, F. Effect of splenectomy in
combination with FK-506 and 15-deoxyspergualin on cardiac xenograft survival.
12. Marchman, W., Araneda, D., DeMasi, R., Taylor, D., Carobbi, A., Larkin, E., Alqaisi,
M .. Thomas, F.T. Therapy of total lymphoid irradiation and 15-deoxyspergualin blocks
survival in the hamster-to-rat xenograft model. Transplant. Proc. 23,201,1991.
References 159
13. DeMasi, R., Araneda, D., Gross, u., Daniel, H., Larkin, E., Thomas, J.M., Swanson, M.,
Nifong, W., Thomas, F.T. Improved xenograft survival with continued infusion de-
oxyspergualin and RATG.f. Invest. Surg. 1991, In press.
14. DeMasi, R, Araneda, D., Gross, U., Daniel, H., Thomas, F.T. 15-Deoxyspergualin
(DOSP) is a potent immunosuppressive agent in xenografting. Eur. Surg. Res. 21,35,
1989.
15. Haisch, C, Lodge, A, Huber, S., Thomas, F.T. The vascular endothelial cell is central to
xenogeneic immune reactivity. Surgery. 108,306,1990.
16. Patselas, T., Thomas, F.T., Araneda, D., Thomas, J.M. Vascularized heart transplanta-
tion in immunodeficient mice. Curro Surg. 1990, In press.
17. Steinbruchel, D.A, Madsen, H., Neilson, B., Larsen, S. Total lymphoid irradiation, cy-
c\osporine-A, and a monoclonal anti-Tcell antibody in hamster-to-rat heart transplanta-
tion. Transplant. Int. 3,36,1990.
18. Steinbruchel, D.A, Madsen, H.H., Nielsen, B., Kemp, E., Larsen. S., Koch, C Graft sur-
vival in a hamster-to-rat heart transplantation model after treatment with total lymphoid
irradiation. cyclosporine A, and a monoclonal anti-T-cell antibody. Transplant. Proc. 22,
1088,1990.
19. Cooper, D.K.C, Human, P.A, Reichart, B. Prolongation of cardiac xenograft (vervet
monkey-to-baboon) function by a combination of total lymphoid irradiation and im-
munosuppressive drug therapy. Transplant. Proc. 19,4441,1987.
20. Bowman, E., de Bruin, RW.F., Marquet, R., leekel, J. Prolongation of graft survival in
hamster to rat xenografting. Transplant. Proc. 21,540,1989.
21. Downing, T.P., Sadeghi, A.M., Bieber, CP., Aziz, S., Reitz, B.A, Shumway, N.E.
Preoperative total lymphoid irradiation, splenectomy, and postoperative cyc\osporine
in the rat cardiac heterograft mode\. Transplantation. 39,81,1985.
22. Jooste, S.V., Colvin, RB., Winn, H.J. The vascular bed as the primary target in the de-
struction of skin grafts by antiserum. II. Loss of sensitivity to antiserum in long-term
xenografts ofskin.f. Exp. Med. 154,1332,1981.
23. Menger. M.D., Jager, S., Walter, P., Hammersen, F., Messmer, K. A novel technique for
studies on the microvasculature of transplanted islets of Langerhans in vivo. Int. f.
Microcirc. Clin. Exp. 9,103,1990.
24. de Jong, W.H., Steerenberg, P.A., Ursem, P.S., Osterhaus, A.D.M.E., Vos, J.G.,
Ruitenberg, E.J. The athymic nude rat. III. Natural cell-mediated cytotoxicity. Clin.
Immunol.lmmunopathol. 17,163,1980.
25. Sprent, J., Bruce, J. Physiology of B cells in mice with X-linked immunodeficiency (xid)
III. Disappearance ofxid B cells in double bone marrow chimeras.f. Exp. Med. 160,711,
1984.
26. Roene, RAP. Rejection of skin grafts in the nudc mouse. Nature. 251,67,1974.
27. Festing, M.F.W., May, D., Connors, T.A An athymic mutation in the rat. Nature. 274,
365,1978.
28. Gershwin, M.E., Merchant, B. (eds.) Immunologic Defects in Laboratory Animals. New
York, Plenum, Vol. 1, 1981.
29. Rygaard, J., Paulsen, C Xenogeneic tumors grow in nude mice. Acta. Patho!. Microbiol.
Immunol. Scand. (A). 77,758,1969.
30. Mjaaland, S., Fossum, S. Ag processing normal in nude rats. Scand. 1. Immunol. 26, 141.
1987.
31. Roder, J., Duwe, A The beige mutation in the mouse selectively impairs natural killer
cell function. Nature. (London). 278, 451,1979.
32. Dorshkind, K., Pollack, S., Bosma, M., Phillips, R NK cells are present in SCID mice.
f. Immunol. 134,3798,1985.
33. Jooste, V., Winn, H. Strain variations in the responses of mice to xenografts of skin.
34. Forbes, R.D.C, Kuramochi, T., Guttmann, RD. A controlled sequential morphologic
study of hyperacute cardiac allograft rejection in the rat. Lab. Invest. 33,280,1975.
35. Auchincloss, H. Xenografting: a review. Transplant. Reviews. 4, 14, 1990.
37. EisenthaL A.. Rosenberg. S. Systematic induction of cells mediating antibody depen-
dent cellular cytotoxicity following administration of interleukin 2. Cancer Res. 49.6953.
19i\9.
38. Pierson. N .. Winn. H.J .. Russell. P.S .. Auchincloss. H. Xenogeneic skin graft rejection is
especially dependent on CD4 + T cells. 1. Exp. Med. 170.991. 1989.
39. Sakakibara. N .. Click. R .. Sakakibara. K .. Aziz. S. T cell subsets involved in rejection of
xenografts. Transplantation. 1991. In press.
40. Knechtle. S.. Halperin. E.c.. Bollinger. R. Cardiac xenograft survival using total lym-
phoid irradiation and cyclosporine. Heart Tramplant. 4.605. 1985.
41. Kaufman. D.B .. Rabe. F.L.. Dunn. D.L.. Sutherland. D.E.R. Effect of host immunomod-
ulation on the prevention of islet allograft primary nonfunetion in a murine model.
Transplant. Proc. 22.857.1990.
Chapter 10
Immunosuppression in Xenotranspiantation
J.B. V AN DEN BOGAERDE AND D.1.G. WHITE
"The immediate reason for this revival of interest in xenotransplantation must

at least partially stem from a belief, whether true or false, that some of the
newer immunosuppressive agents offer better promise of controlling the
xenograft reaction than the conventional agents used prior to 1980" (Frank
Thomas in [1 D.
Introduction
The remarkable successes of clinical allotransplantation in the last two

decades have not been parallelled by similar advances in the field of xeno-
transplantation, either experimentally or clinically. Indeed, if the early work
of Reemtsma is considered [2-4], where 9-month survival of chimpanzee kid-
neys transplanted into a patient was achieved using a now obsolete immuno-
suppressive regimen, it could be said that the field ofxenotransplantation has
regressed in the last 25 years. A great deal of knowledge has been acquired re-
garding the mechanisms of xenogeneic rejection (Chaps. 4-7), but long-term
suppression of xenogeneic rejection remains clinically unattainable, and can
be achieved in most animal models only with considerable difficulty.
The first section of this chapter will discuss immunosuppression in concor-
dant species combinations, while the second section will deal with discordant
combinations. The concordant and discordant terminology, as first described
by CaIne [5], divides species combinations according to whether acute (first-
set) or hyperacute (second-set) rejection occurs when tissue is transplanted
between the species.
The nature of the transplanted tissue affects the mechanism of rejection,
and, therefore, the effectiveness of immunosuppression. A hierarchy exists in
allograft rejection whereby skin grafts are most easily rejected [6], whereas liv-
er grafts are rejected least easily and can sometimes survive in untreated major
histocompatibility complex (MHC)-disparate recipients [7].
This hierarchy of organ graft survival has not been extensively studied in
xenografts, but it is indisputable that certain tissues such as skin or pancreatic
islet cells are seldom rejected hyperacutely when transplanted into discordant
hosts [8]. Prolonged survival of human islet cells in mouse recipients has been
achieved by anti-CD4 monoclonal antibody therapy [9], and discordant skin
162 Immunosuppression in XenotranspIantation
grafts survived significantly longcr than allografts when mice were treated
with anti-CD4 therapy [10, 11].
In stark contrast to this, no regimen which suppresses cellular immunity
prolongs survival of primarily vascularized discordant xenografts. It, there-
fore, seems likely that the humoral mechanisms which appear to destroy dis-
cordant vascular xenografts in minutes or hours (whether by antibody [12] or
the alternative complement pathway [13]) do not destroy single cell, or skin
xenografts [8].
Skin grafts might evade the host antibody since they are not immediately
connected to the recipient vasculature. We can offer no logical explanation as
to why discordant xenogeneic pancreatic islet cells evade the host
antibody/complement immune system. It is attractive to speculate that such
cells lack critical antigens or epitopes. At present there are no data to support
or refute such a hypothesis.
Rejection of a xenograft which is directly connected to the recipient blood
supply would be an all or nothing event, since even partial damage or activa-
tion of the endothelium would result in thrombosis and infarction of the graft.
The immunological insult would not have to be directed at the endothelial cell
itself, since indirect damage to endothelial cells caused by destruction of other
xenograft components could damage endothelium sufficiently to cause
thrombosis.
We will not indulge in further speculation about immune mechanisms of
different graft destruction, beyond concluding that single cell and skin
xenografts are amenable to conventional immunosuppression, similar to that
which successfully prolongs allografts. We will, therefore, limit ourselves to
discussing the suppression of rejection of vascular xenografts, which are not
only unique as regards immunosuppression, but also of more clinical rele-
vance.
Immunosuppression for Concordant Xenografts
Clinical Experience
Several immunosuppressive regimens have been used in different experimen-

tal models and in clinical xenotransplantation to prolong concordant graft sur-
vival. Reemtsma et al. [2-4] achieved prolongation of survival of chimpanzee
kidneys in patients using a combination of azathioprine, actinomycin C
steroids, and irradiation of the transplanted organ (Chaps. 2, 33). Two patients
survived with functioning kidneys for 63 days and 9 months, respectively.
Using a similar regimen, Starzl achieved more modest survival of baboon kid-
neys in humans, with 10-60 days graft function [14] (Chap. 33). Three out of
these six transplants were performed in donor/host combinations in which an
ABO blood group incompatibility existed, without apparently detrimental
effects.
Immunosuppression for Concordant Xenografts 163
Barnard et al. [15] and Bailey et al. [16], working in separate centers
attempted baboon-to-human cardiac transplantation without success
(Chap. 34). A baboon heart transplanted into Baby Fae, using cyclosporine
(CsA) in the immunosuppressive regimen, survived for 3 weeks. Histological
examination of this single rejected xenograft demonstrated cellular infiltra-
tion in less than 10% of the graft and deposition of antibodies in the graft. The
alleged reason for the rejection of this xenograft was an ABO mismatch be-
tween human and baboon, although the role of newly produced antibodies
was not excluded.
Nonhnman Primate Models
Nonhuman primates have been extensively used to study both discordant and
concordant xenografting (Chaps. 22, 24). Nonhuman primates are closely re-
lated to man, and primate models of concordant xenografting have an advan-
tage over other experimental animal models since the results obtained are
more applicable to clinical practice. In addition, concordant xenografting in
man will involve the transplantation of primate organs into human patients, so
research using primate models directly examines the survival of primate
xenografts across concordant species barriers.
The survival of cynomolgus-to-baboon xenografts has been reported to be
similar to allograft survival when CsA/steroid immunosuppression is used [17,
18]. In contrast to this, extension of xenograft survival after the heterotopic
transplantation of vervet (African green) monkey hearts into Chacma ba-
boons has proved to be far more difficult [19-21]. In spite of various combina-
tion therapies with immunosuppressive drugs (CsA, methylprednisolone, aza-
thioprine' antithymocyte globulin, 15-deoxyspergualin), and total lymphoid
irradiation, no regimen produced long-term graft survival. Histology of reject-
ed grafts generally showed a combination of cellular and humoral rejection.
The data from these and other studies do not provide sufficient information to
allow speCUlation about the mechanisms of concordant xenograft rejection in
primates, beyond the observation that in certain species combinations
xenograft rejection is more difficult to suppress than allograft rejection.
Canine Models
Valuable data have been generated using interspecies transplantation with

organs from dingos (Canis dingo), foxes (Vulpes vulpes), and wolves (Canis
lupus) being transplanted into dogs [22]. A surprising observation of these
studies was that survival of wolf kidneys was significantly longer in untreated
dog recipients than was that of allografts; cellular mechanisms appeared to
have caused rejection of these xenografts [23]. In contrast, fox kidneys or
hearts, transplanted into dog recipients, were rejected more rapidly than allo-
grafts [22,24] and long-term survival in this xenogeneic combination was not
164 Immunosuppression in Xenotransplantation
achieved by CsA/methylprednisolone therapy [25]. Histological examination

of rejected fox organs demonstrated a combination of cell-mediated and
humoral rejection mechanisms.
Hare-to-Rabbit Model
CsA therapy has been shown to extend survival of hare kidneys in rabbits [26].
and rejection in this interspecies combination appears to be analogous to allo-
graft rejection.
Comment
This brief review has illustrated that in certain closely related species combi-
nations (cynomolgus-to-baboon. wolf-to-dog, hare-to-rabbit), rejection of
xenografts is easily controlled using methods similar to those which successful-
ly suppress allograft rejection. Other concordant xenografts (vervet monkey-
to-baboon, fox-to-dog) are rejected despite treatment which would ensure al-
lograft survival. Concordant species combinations can thus be divided into
"easy" and "difficult". Easy combinations are those in which prolonged
xenograft survival can be achieved with conventional immunosuppressive
therapies. which also prolong allograft survival. These conventional immuno-
suppressive regimens do not achieve prolonged xenograft survival between
the more challenging difficult concordant combinations.
It would appear that the chimpanzee-to-human species difference is an
easy (allograft-like) species combination since long-term survival of chim-
panzee kidneys in man has been reported [2--4]. It is difficult to assess, from the
published literature, whether baboon-to-human transplants represent an easy
or a difficult concordant xenograft combination. No long-term survival of ba-
boon xenografts in man has been reported, although Starzl et al. havc
achieved 60 day survival of a baboon kidney in a human recipient [14]. The re-
jected baboon grafts. however. appeared histologically different from chim-
panzee grafts, and some evidence suggested that humoral mechanisms werc of
importance in the rejection process. Histological cxamination of a recent ba-
boon-to-human heart transplant showed cellular infiltration in less than 10%
of the graft. and antibody and complement deposition was seen in necrotic
myocardial tissue [16]. These data suggest that baboon-to-human transplanta-
tion approximates the difficult type of concordant xenograft rejection (vervet
monkey-to-baboon, fox-to-dog), both according to similarities in histological
appearances and in unsuccessful application of conventional immunosuppres-
sion.
This is a very important distinction to make, since it seems unlikely that
chimpanzees could ever be used clinically as organ donors on a large scale due
to logistic and ethical problems [27]. Baboons are far easier to breed in captiv-
ity and are not an endangered species.
The Syrian Hamster-to-Rat Model 165
With these considerations in mind, it therefore seemed appropriate to us to

study the mechanisms of concordant rejection in an animal model which was
both cheap and represented a difficult species combination. An additional ad-
vantage of studying rejection between difficult species is that humoral mecha-
nisms could be contributing to rejection, and elucidation of this process might
provide some insight into the rejection of discordant organs.
The Syrian Hamster-to-Rat Model in the Study

of Concordant Xenotransplantation
Transplants between Syrian hamster (M esocricetus auratus) and rat (Rattus

norwegicus) represent a concordant species difference, since hamster heart
grafts are rejected by recipient rats in approximately 3 days [28]. The advan-
tages of using hamster donors and rat recipients are that vascular organ trans-
plantation is technically feasible and both species are easy to maintain in con-
ventional animal facilities, which makes this research affordable.
This animal model has been used extensively to study concordant
xenograft rejection [28-38]. Knechtle et a1. [28] demonstrated that combina-
tion therapy with CsA and total lymphoid irradiation produced long-term
hamster heart xenograft survival in rat recipients. These results have proved
difficult to reproduce [34,35], nor has the mechanism of rejection been clari-
fied. The general approach followed in these previous reports has been to use
ever-increasing doses of immunosuppressive drugs, or immunoablative com-
bination therapies, which have obscured the underlying immune processes.
Table 10.1. Histological appearances of transplanted hamster organs in unmodified rat re-
cipients
Reference Histopathology
Homan et al. 1981 [29] Dense infiltration with histiocytes. neutrophils; few lympho-
cytes; large area of focal infarction; interstitial hemorrhage.
Knechtle et a1. 1987 [28] Hemorrhage. edema. necrosis. and neutrophil mononuclear
cell infiltrate.
Rosengard et al. 1986 [37J Cellular infiltration and infarction.
Steinbruchel et al. 1990 [34J Total acute infarction. vascular rejection. and subendocar-
dial inflammation.
Valdivia et al. 1990 [38J Infarction. with mononuclear cell infiltrate lining some
blood vessels. and evidence of vascular occlusion.
Nakajima et al. 1989 [73] Hemorrhage. with neutrophil and macrophage infiltrate: no
T cell staining in rejected hearts.
Other workers have found that combinations of immunosuppressive regi-

mens (including CsA, antilymphocyte globulin, cyclophosphamide, 15-de-
oxyspergualin, FK-506) splenectomy, total lymphoid irradiation, antirat T cell
monoclonal antibodies, and selective lymphoid irradiation with palladium-
109 hematoporphyrin failed to produce long-term survival. Where any signifi-
cant extension of survival was obtained, this was achieved only when the ani-
mal was very severely immunosuppressed, with a resulting high incidence of
toxic and infectious complications. The new immunosuppressive drug, FK-
506, which has a mode of action similar to CsA but is far more powerful, did
not improve concordant xenograft survival [33]. The striking feature of all of
this work is that regimens which would easily induce long-term survival in allo-
geneic combinations had little or no effect on concordant xenograft survival.
The histology of certain of these grafts showed that rejection was caused by
humoral as well as cellular mechanisms (Table 10.1).
One problem confronting investigators in this field is that the combinations
of immunotherapy used had such multiple effects upon the immune system
that the mechanism of concordant xenogeneic rejection could not be elucidat-
ed from the data.
T Cell Depletion
An extended series of experiments was undertaken in our laboratory using

monoclonal antibodies to deplete rat recipients of (i) CD4 T cells, (ii) CD4
plus CD8 T cells, and of (iii) cells bearing the T cell receptor. It became clear
that no significant improvement in survival time was found since rats with no
functional T cells could still reject hamster hearts [39]. CsA therapy was also
used in conjunction with monoclonal antibody therapies, but this also failed to
improve survival time. This failure of T cell immunosuppression to prolong
hamster hearts in rats was confirmed by Lim et al. [40], who showed that nude
(athymic) rats rejected hamster hearts in the same period of time as untreated
normal rats. Histology of these hearts demonstrated features of humoral re-
jection (Fig. 10.1). Because of this spectacular failure of T cell immunosup-
pressive therapies, the next series of experiments, the next series of experi-
ments was undertaken using cobra venom factor (CVF) to deplete the C3
component of complement.
Cobra Venom Factor
Presensitization of allogeneic recipients has been shown to result in antibody-

mediated hyperacute rejection both experimentally [41], and in clinical prac-
tice [42-45]. Since the mechanism of hyperacute allogeneic rejection appears
similar to hyperacute rejection of discordant xenotransplants [8], it seemed
possible that antibody/complement-mediated mechanisms could be responsi-
ble for rejection of concordant xenografts.
Fig. 10.1. Light micrograph of hamster heart graft rejected by an untreated rat on day 3,
showing total myocardial necrosis in the absence of cellular infiltration (H & E, x400)
100
80
co> 60
CVF
"E
::J
til
~ 40
' '·'·'1
No RX ~
20
~~
"""""'' ' )
0
0 2 3 4 5 6 7 8 9 10 11 12
Days post transplant
Fig. 10.2. Survival of hamster hearts grafted into rats (n=6) which have been deeomplement-
ed by treatment with cobra venom factor (CVF). Survival in untreated control rats (n=6; no
RX) is also shown
This hypothesis was tested by using CVF to deplete the C3 component of

the complement cascade [46, 47] (Chaps. 4, 5). C3 represents the common
component of both the alternative and the classical complement pathways,
and depletion of C3 thus disarms both pathways. CVF has been used in vivo to
delay the hyperacute rejection response during transplantation between dis-
cordant species combinations [48-52] but has not so far been tested for its abil-
ity to produce long-term graft survival of concordant xenografts.
1 0 0 4 - - -......
80
'i'1I1I111II1111
~
> 60
~
:J
<II
40
Jl'CSA
oS!.
NO RX~~
20 ;""11111",111
0
0 2 3 5 8 9 10 11 12
Fig.tO.3. Survival of hamster hearts grafted into rats (n=6) treated with cyclosporine «('sA)
(20 mg/kg on alternate days). The survival of untreated control rats (N() RX)is also shown
(n=6)
100
80
I1
~
> 60
~
:J
VI
oS!. 40
k"'NO RX
20
0
0 20 40 60 80 100
Fig. 10.4. Survival of hamster hearts grafted into rats (n=IO) treated with both cyclosporine
(20 mg/kg on alternate days) and cobra venom factor (CVF + CsA). Survival in untreated
control rats (N() RX) is also shown (n=6)
In our laboratory, CYF injected on its own increased hamster heart graft
survival in rats from 3 to 5 days (Fig. 10.2). This improvement of graft survival
was significant, in contrast to all therapies which depleted or inhibited T cells.
When combined with 20 mg/kg of CsA on alternate days, which on its own
leads to no improvement of survival time (Fig. 10.3), long-term survival was
achieved (Fig. lOA). In all these experiments, levels of antihamster lytic anti-
body rose to titers of 1/16000-1/32000 (Fig. 10.5) in rats which, despite the
presence of these antibodies, still retained beating hamster hearts.
Histological examination of these hearts showed normal myocardium, and im-
2.0
1.8
1.6
Untreated
E
c 1.4
III
r-
'<t"
1.2
ro
Q) 1.0
I/)
0)
Q)
0.8
~
.D 0.6
::t:
0.4
0.2
0.0
~ '<t" 00 W ~ '<t" 00 W ~ '<t" 00 W ~ '<t" 00 W
~ W ~ III ~ '<t" m m 00 W ~
~ III 0 0 0 ~ ~ III
~ '<t" 00 W ~ III
~ W
T i te r
Fig. 10.5. Titers of hemolytic anti hamster antibodies 5 days after transplantation of hamster
hearts into (i) untreated rats (controls), (ii) rats treated with an anti-CD4 monoclonal anti-
body (anti-CD4) , or (iii) athymic nude rats (Nudes) that are Tcell deficient
munohistochemical analysis demonstrated deposition of rat immunoglobulins

(Ig) in the absence of any C3 binding. Depletion ofT helper cells did not signif-
icantly inhibit the increase in antibody titer. The major lytic and, therefore,
complement-binding component of this antihamster antibody is in the IgM
fraction (Fig. 10.6). Infusion of IgM, purified from the serum of an animal
7 days post transplant, caused hyperacute rejection of a beating hamster heart
in an untreated rat within approximately 10 min.
The knowledge gained from this body of experimental work, including in-
terpretation of data from clinical transplantation and primate and rodent stud-
ies, allows some insight into the mechanisms of concordant xenogeneic rejec-
tion, and therefore the methods of immunosuppression most likely to succeed.
Firstly, allogeneic rejection is very different from difficult concordant rejec-
tion. This sounds like a fatuous repetition of the obvious, but workers in the
field have tended to approach the problem of xenografting as a similar, but
more aggressive, form of allogeneic rejection. The second point is that al-
though T cells do playa part in rejection of xenografts, it would appear that - at
least in the hamster-to-rat model - antibody/complement mechanisms are
more important. There is no reason to believe that other difficult concordant
models which involve accelerated graft rejection are any different. A third
point is that abrogation of T cell help does not inhibit the formation of anti-
bodies against antigens on the surface of cells from other species.
c 300
0
.~
~
'0
.fe- 200
>
"D
m
0
:3- 100
283031323334353637383940414245475052545560 64 68 74 8090 95
No. of fraction
Fig. 10.6. Histogram of the relative hemolytic titers of antihamster antibodies in G200 frac-
tionated rat serum removed 7 days after hamster heart xenografting. The majority of the lyt-
ic antibodies are in the IgM fraction
This T cell-independent nature ofxenogeneic antibody response calls for a

new approach aimed at inhibiting the effect and/or the formation of anti-
xenograft antibodies. It may be possible that the B cell population can be erad-
icated or disarmed in such a way that antibody formation can be stopped. The
elimination of both functional T and B cells would seem to be too hazardous
for clinical practice. Perhaps a compound similar in action to CVF needs to be
found which depletes complement and prevents the cascade of events leading
to thrombosis and graft destruction.
Although it seems unlikely that patients who are immunosuppressed and
complement deficient could live normal lives [53,54], preliminary results from
our laboratory suggest that recipients of concordant xenografts need not be
permanently complement depleted. Hamster heart grafts which survive in
CVF-treated animals for approximately 28 days are not subsequently rejected
after complement levels have been allowed to return to normal in the presence
of high lytic antihamster antibody titers.
Immunosuppression for Discordant Grafts
The hyperacute rejection of discordant tissue by the recipient has made the
prevention of this response a priority before specific recruited graft rejection
mechanisms can be addressed. The thrust of research in discordant xenogene-
ic transplantation has been concerned with modifying this humorally mediat-
ed hyperacute rejection. Two approaches have been followed.
The first has been the attempted elimination of preformed antibody in the
host prior to transplantation by (i) plasmapheresis or plasma exchange
Immunosuppression for Discordant Grafts 171
[55-57], (ii) adsorption of antibody by circulation of the host blood through a
discordant organ before transplanting an organ of the same species into the
specific antibody-depleted donor ([58] and reviewed by Henry et al. in [59]),
or (iii) removal of antibody by passing donor blood through protein A
columns which bind to the Fc portion of IgG and IgM antibodies.
The second approach has been the inhibition of the cascade of events initi-
ated after antibody/complement binding to xenografts by (i) inhibition of
platelet aggregation or thrombosis [60-62], (ii) inhibition of inflammatory me-
diators produced by the complement cascade, or (iii) depleting complement
components [48, 63]. A brief discussion of these methods follows.
Plasmapheresis
Plasmapheresis or plasma exchange is an accepted form of treatment in some

autoimmune conditions, and is clinically well tolerated. Several workers have
shown that antibodies to xenoantigens can be reduced in titer by plasma ex-
change; this topic has been reviewed in Chap. 6. There is, however, arebound of
antibody. and within 1-5 days the antibody titers can equal or exceed the prede-
pletion level [55]. The addition of immunosuppressive drugs or splenectomy
has been reported to produce inhibition ofthis rebound antibody formation.
Alexandre et al. [57] achieved a remarkable 22 day survival of a discordant
xenograft (minipig-to-baboon) by plasmapheresing a splenectomized animal,
and administering a combination of CsA, azathioprine and methylpred-
nisolone. It must be noted, however, that the plasmapheresis did not com-
pletely eliminate the antixenograft antibody; it just decreased the titer. In all
cases the antibody titer increased 5 days after transplantation, although the
levels did not reach pretransplantation levels.
Similar results have been achieved by other workers using plasma ex-
change techniques. One point which the authors of these papers fail to empha-
size is that. in addition to removing antibodies, plasmapheresis influences a
spectrum of species-specific plasma proteins, including cytokines and comple-
ment components. It is very difficult to determine what role. if any. is played by
these proteins in the initiation of the hyperacute rejection response, how their
depletion affects this response, and what is the effect of their resynthesis and
entry into the circulation.
Antibody Depletion
Several groups have attempted depletion of preformed antibody by perfusing

an organ of the donor species with the blood or plasma of the recipient, and
then transplanting another organ of the donor species into the recipient. In
theory, the specific antixenograft antibody binds to determinants in the graft,
thereby removing the specific antibodies which cause hyperacute rejection.
This can also be achieved by the serial transplant of organs, which also pro-
duces reduction of preformed antibodies [58]. Unfortunately. antibody deple-

tion is once again not complete. For example. when the spleen is used for ex
vivo depletion. antibody levels are reduced to approximately 10% of pre-
adsorption levels [59]. The liver is probably the best depleting organ. possibly
due to its size. its large reticuloendothelial cell population. or the cross-sec-
tional surface area of its endothelium.
Experiments using the technique of antibody adsorption have clarified the
role of specific antibodies in xenograft rejection. and have a theoretical advan-
tage over nonspecific antibody depletion regimens. since nonspecific plasma
proteins are not removed. In principle. however. the deposition of antibodies
on the endothelium of an ex vivo organ would initiate the complement cas-
cade. with depletion of both serum components and cellular elements that par-
ticipate in this process. The depletion of antibody is. therefore. once again ac-
companied by various other effects, which could contribute to the improved
survival of subsequent xenografts.
One other feature of this technique is that total depletion of antixenograft
antibodies is not achieved. Ex vivo passage through one organ (such as the liv-
er) might not deplete antibodies which bind to another organ (such as the
heart). Alternatively. the antibody binding sites on the depleting organ may
become saturated. Another possible explanation is that the antibodies which
do not bind to the depicting organ are of low affinity. and do not cause rejec-
tion of the transplanted xenograft. Experiments depleting antibody by passing
plasma over protein A or other 19 affinity columns have produced increased
survival times of organ transplants between discordant species combinations.
but no long-term discordant graft survival has been achieved.
Even if total depletion of antibody could be achieved. it is possible that oth-
er mechanisms contribute to hyperacute rejection of discordant xenografts. A
causative role has been ascribed to the alternative complement pathway in hy-
peracute rejection of guinea pig hearts in rat recipients [64]. However. these
studies did not conclusively prove that preformed antibodies were not present
at the time of hyperacute rejection.
Recent work in our laboratory has demonstrated that hyperacute rejection
of rabbit hearts occurs in pre suckled pig recipients which have no im-
munoglobulin. Stringent antibody assays demonstrated that there was no dif-
ference in rejection times between animals that were totally deficient of anti-
body and animals possessing significant titers of antigraft antibodies [13].
These data suggest that antibody depletion on its own would not be sufficient
to produce discordant xenograft survivaL and needs to be combined with ther-
apies which deplete complement.
Platelet Inhibitors
Experiments have been performed by several groups showing that inhibition

of coagulation can delay the hyperacute rejection response. Inhibitors of
platelet aggregation extend discordant xenograft survival slightly [65,66], and
Immunosuppression for Discordant Grafts 173
some success has been achieved with heparin in the transplantation of guinea
pig or rabbit hearts into rats. It seems obvious that the coagulation cascade
plays a vital role in the hyperacute rejection response, but it must be kept in
mind that major transplantation surgery is hazardous in the absence of a good
coagulation profile.
Inhibition ofinflammatory Mediators
The hyperacute response is associated with antibody/complement deposition

and nonspecific cellular activation, which release many inflammatory media-
tors. Platelet-activating factor (PAF) has been implicated as central to the re-
jection response, and the use of specific pharmacologic agents which inhibit
PAF resulted in a slight increase in the survival time of discordant grafts [67).
Unfortunately a very large number of mediators are released, including
leukotrienes, thromboxane A 2 , prostaglandins, and organic peroxidases. To
inhibit all these mediators of inflammation individually by pharmacologic
means seems an overwhelming endeavor.
Inhibition of the Complement Cascade
The complement cascade appears to be of central importance in the hyper-

acute rejection response. In one report, guinea pig hearts survived for 3 days in
rats treated with CVF and CsA [52]. Long-term discordant xenograft survival,
however, has not been achieved by regimens which depleted complement and
inhibited cellular immunity [8,48-50). There are three likely explanations for
these somewhat disconcerting findings.
The first is that depletion of complement was not 100%, and the residual
complement was sufficient to destroy the graft. This is improbable, since rat
recipients in our laboratory, treated with CsA and CVF and shown to be total-
ly complement depleted, still rejected guinea pig hearts within 72 h.
The second explanation is that T cells are recruited and destroy the discor-
dant xenograft in complement-depleted recipients. This seems unlikely since
various immunosuppressive regimens which are known to inhibit T cell func-
tion have been used in combination with complement depletion.
The third, and most likely, explanation is that discordant xenograft de-
struction results from factors other than T cells and antibody/complement
mechanisms. Thus, graft destruction could be mediated by natural killer cells,
neutrophils, antibody-dependent cytotoxicity (ADCC), macrophages, or acti-
vation of inflammatory proteins by as yet unidentified mechanisms. Indirect
proof for this comes from antibody depletion experiments in which organs re-
jected by animals depleted of antidonor antibody showed increased infiltra-
tion of polymorphonuclear cells [59). The donor antigens which stimulate
these putative "nonspecific" cellular responses have not been identified.
It is also possible that discordant xenografts are destroyed, not by immuno-
logical, but by "physiological" mechanisms, such as platelet aggregation and
coagulation [62, 68]. The presence of such physiological barriers to discordant

xenograft survival, which would be extremely difficult to surmount, have been
predicted [5].
The Future ofimmunosuppression of Discordant Vascular Grafts
The rejection of discordant vascular xenogeneic grafts must be seen as a series

of mechanisms which individually are sufficient, but not essential, to cause
graft rejection. Undoubtedly, hyperacute rejection can be caused by pre-
formed antibodies, but in the absence of antibody other mechanisms become
important.
The inescapable conclusion is that the probability of achieving long-term
acceptance of discordant xenografts in patients, using currently available
methods of immunosuppression, is slight. T cells, B cells, and probably non-
specific immune cells, such as natural killer (NK) cells and macrophages,
would have to be inhibited or eliminated. In short, the minimum requirement
would be total immunosuppression, and perhaps even this would not be suffi-
cient. The hazards of such immunosuppression to the recipient are probably
too great to be contemplated.
The most promising approach to achieving long-term discordant xenograft
survival would probably entail a shift in emphasis away from changing the im-
mune environment of the recipient, to the modification of donor tissue. This
concept was suggested by CaIne in 1988: "Custom-tailored MHC-engineered
transgenic animals might provide donor organs which would be less aggres-
sively rejected as xenografts in both concordant and discordant models" [69].
Target antigens, which are recognized by preformed antidonor antibodies,
could be modified or removed from donor tissues by techniques of genetic en-
gineering. The polyclonal nature ofthe antibody response induced by a discor-
dant xenograft in situ, however, would probably make this approach unre-
warding.
A more promising avenue of research is the production of transgenic ani-
mals expressing human complement-inhibiting proteins. Decay accelerating
factor (DAF) and membrane cofactor protein (MCF) are membrane in-
hibitors of complement which are present on a wide variety of cell types [70].
These inhibitors block the activity of autologous complement, but not of xeno-
geneic complement from a distantly related species. For example, rabbit ery-
throcytes are unable to block the activity of human complement and are con-
sequently lysed in human serum [70]. Lysis of human cells by human
complement occurs when these regulatory proteins are deficient, as has been
demonstrated for paroxysmal nocturnal hemoglobinuria [71,72]. Preliminary
data have shown that the expression of human complement-inhibiting
molecules on rodent cells protects them from human antibody/complement-
mediated lysis (White et al. in preparation). The production of transgenic
donor animals expressing human complement-inhibiting molecules is an ex-
citing avenue of future research.
References 175
In conclusion, the rewards of overcoming this, perhaps final, great chal-

lenge of transplantation biology are large enough to warrant the tremendous
resources that are now being brought to bear on the problem of discordant
xenograft survival.
References
1. Thomas, F.T., DeMasi. RJ .. Hugh, D., Areneda. D .. Hsu. S.. Moon. D., Thomas. J.
Comparative immunosuppression for xenografting. In: Xenograft 25. Hardy, M.A. (ed.)
Elsevier, Amsterdam. New York, Oxford. 1989, p. 55.
2. Reemtsma. K .. McCracken. B.H .. Schlegel. J.Y .. Pearl. M.A.. Pearce. CW .. DeWitt.
CW., Smith, P.E .. Hewitt, RL., Flinner, RL., Creech, O. Jr. Renal heterotransplanta-
tion in man. Ann. Surg. 160.384.1964.
3. Rcemtsma. K.. McCracken, B.H., Schlegel. J.Y., Pearl, M.A .. DeWitt. CW., Creech, O.
Jr. Reversal of early graft rejection after renal heterotransplantation in man. lama. 187.
691,1964.
4. Reemtsma. K.. McCracken. B.H .. Schlegel, J.Y., Pearl, M.A. Heterotransplantation of
the kidney: two clinical experiences. Science. 143,70,1964.
2.550.1970.
6. Barker. C.F.. Billingham, RE. Comparison of the fates of Ag-B locus compatible homo-
grafts of skin and heart in inbred rats. Nature. 225,851.1971.
7. Caine, RY., Sells, RA., Pena, J.R .. Davis, D.R, Millard. P.R, Herbertson, B.M., Binns,
R.M., Davies, D.A.L. Induction of immunological tolerance by porcine liver allografts.
Nature. 223.472,1969.
8. Auchincloss. H. Jr. Xenogeneic transplantation. A review. Transplantation. 46,1,1988.
9. Ricodi, C, Lacy, P.E.. Sterbenz, K., Davie, J.M. Low temperature culture of human
islets or in vivo treatment with L3T4 antibody produces a marked prolongation of islet
human to mouse xenograft survival. Proc. Natl. Acad. Sci. USA. 84,8080,1987.
10. Auchincloss, H. Jr., Moses, R, Conti, D., Sundt, T., Smith, C, Sachs, D.H., Winn, H.J.
Rejection of transgenic skin expressing a xeno-class I antigen is CD4-dependent and
CD8-independent. Transplant. Proc. 22, 1059, 1990.
11. Pierson, RN. III, Winn, G .J., Russell, P.S., Auchincloss. H. Jr. Xenogeneic skin graft re-
jection is especially dependent on CD4+ T cells. l. Exp. Med. 170,991,1989.
12. Perper, RJ., Najarian, J .S. Experimental renal heterotransplantation. I. In widely diver-
13. Johnston, P.S., Wang, M.W., Lim, S.M., Wright, L., White, D.J.G. Hyperacute xenograft
rejection takes place in the complete absence of antibodies but requires the presence of
functional complement. Transplant. Proc. 23,877.1991.
14. Starzl, T.E., Marchioro, T.L., Pcters, G.N., Kirkpatrick, CH., Wilson, W.E.C. Porter,
K.A., Rifkind, D., Ogden, D.A., Hitchcock, CR. Waddell, W.R Renal heterotrans-
plantation from baboon to man: experience with six cases. Transplantation. 2,752,1964.
15. Barnard, CN., Wolpowitz, A., Losman. J.G. Heterotopic cardiac transplantation with a
xenograft for assistance of the left heart in cardiogenic shock after cardiopulmonary by-
pass. S. Afr. Med. l. 52, 1035.1978.
16. Bailey. L.L., Nehlsen-Cannarella, S.L.. Concepcion. W .. Jolley, W.B. Baboon-to-human
cardiac xcnotransplantation in a neonate. lama. 245.3321,1985.
17. Kurlansky, P.A., Sadeghi, A.M., Michler. RE.. Smith, CR. Marboe, CC. Thomas.
W.G .. Colpey. L.J .. Reemtsma, K.. Rose. E.A. Comparable survival of intraspecies and
cross species primate cardiac transplants. Clirr. Slirg. 43,413,1986.
18. Sadeghi, A.M .. Robbins. RC, Smith. CR. Kurlansky. P.A., Michler, RE.. Reemtsma.
K., Rose, E.A. Cardiac xenograft survival in baboons treated with cyclosporin in combi-
nation with conventional immunosuppression. Transplant. Proc. 19, 1149,1987b.
19. Cooper, D.K.C, Human, P.A., Rose, AG., Rees, J., Keraan, M., Reichart, B., du Toit,
E., Oriol, R. The role of ABO blood group compatibility in heart transplantation be-
tween closely related animal species. An experimental study using the vervet monkey to
baboon cardiac xenograft model. 1. Thorae. Cardiovasc. Sllrg. 97,447,1989.
20. Cooper. D.K.C, Human, P.A .. Reichart, B. Prolongation of cardiac xenograft (vervet
monkey to baboon) function by a combination of total lymphoid irradiation and im-
munosuppressive drug therapy. Tramplant. Proc. 19,1987,1987.
21. Cooper, D.K.C, Rose, AG. Experience with experimental xenografting in primates. In:
Xenograft 25. M.A Hardy (ed.) Elsevier; Amsterdam, New York, Oxford, 1989, p. 95.
22. Hammer, C, Saumweber, D., Krombach, F. Xenotransplantation in canincs. In:
Xenograft 25. M.A Hardy (ed.) Elsevier; Amsterdam, New York, Oxford, 1989, p. 67.
23. Hammer, C, Chaussy, C, von Scheel, J., Pongratz, H., Roscher, E., Brendel, W. Survival
times of heart and kidney grafts within different canine species in relation to their genet-
ic markers. Transplantation. 7,439,1975.
24. Hagl, S., Geck, G., BrendeL W., Land, W., Mayr, N., Menler, N., Pielsticker, K.,
Sebening, F. Xenogene Herztransplantation im nah stammesverwandten Spezies-
System Fuchs-Hund. D. Med. Wsclzr. 296,217,1971.
25. Bohm, D., Kromback, F., Hammer, C, Gebhard, F., BrendeL W. Fine needle aspiration
cytology in cyclosporin-treated xenogeneic kidney rejection. Transplant. Proc. 17,2128,
1985.
26. Kemp, E., Starklint, H., Larscn, S., Dieperink, H. Cyclosporin in concordant renal hare-
to-rabbit xenotransplantation: prolongation and modification of rejection and adverse
effects. Transplant. Proe. 17,1351, 1988.
27. Goodall, J. Ethical concerns in the use of animals as donors. In: Xenograft 25. M.A
Hardy (ed.) Elsevier; Amsterdam, New York, Oxford, 1989, p. 335.
28. Kneehtle, S.J., Halperin, E.C, Bollinger, R.R Xenograft survival in two species combi-
nations using total lymphoid irradiation and eyclosporin. Transplantation. 43, 173, 1987.
29. Homan, W.P., Williams, K.A, Fabre, J.W., Millard, P.J., Morris, P. Prolongation of car-
diac xenograft survival in rats receiving cyclosporin A Transplantation. 31, 164, 1981.
30. Hardy, M.A, Oluwole, S., Fawwaz, R, Kazunari, S., Novygrod, R, Reemtsma, K.
Selective lymphoid irradiation. III. Prolongation of cardiac xenografts and allografts in
presensitized rats. Transplantation. 33,327,1982.
31. Monden, M., Valdivia, L.A, Gotoh, M., Hasuike, Y., Kubota, N., Kanai, T., Okamura,
J., Mori, T. Hamster-to-rat orthotopic liver transplantation. Transplantation. 43, 745,
1987.
32. Kemp, E., Dieperink, H., Jensenius, J.C, Koch, S., Larsen, S., Madsen, H.H.T., Nielsen,
8., Starklint, S., Steinbruchel, D.A Hope for successful xenografting by immunosup-
pression with monoclonal antibody against CD4, total lymphoid irradiation and cy-
closporin. Scand. 1. Urol. Nephrol. 24,79, 1989.
33. Gudas, V.M., Carmichael, P.G., Morris, R.E. Comparison of the immunosuppressive
and toxic effects ofFK 506 and cyclosporin in xenograft recipients. Transplant. Proc. 21,
1072,1989.
34. Steinbruchel, D.A, Madsen, H.H.T., Nielsen, B., Larsen, S., Koch, C, Jensenius, J.C,
Hougescn, C, Kemp, E. Treatment with total lymphoid irradiation, cyclosporin A and a
monoclonal anti-T cell antibody in a hamster to rat heart transplantation model: graft
survival and morphologic analysis. Transplant. Int. 3,36, 1990.
35. DeMasi, R, Alqausi, M., Araneda, D., Nifong, W., Thomas, J., Gross, U., Swanson, M.,
Thomas, F. Re-evaluation of total lymphoid irradiation and cyclosporin therapy in the
Syrian hamster to Lewis rat cardiac xenograft model. Transplantation. 49,3,1990.
36. Van den Bogaerde, J.B., White, D., Roser. B .. Kempinga, J., Aspinall, R. In vitro and in
vivo effects of monoclonal antibodies against T-cell subsets in allogeneic and xenogene-
ic responses in the rat. Transplantation. 50,918,1990.
37. Rosengard, B.R, Adachi, H., Ueda, K., Hall, T.S., Hutchins, TG.M., Herskovitz, A.,
Borkon, AM., Baumgartner, W.A, Reitz, B.A. Differences in the pathogcnesis of first-
set allograft rejection and acute xenograft rejection as determined by sequential mor-
phologic analysis. 1. Heart Transplant. 5,263, 1986.
References 177
38. Valdivia, L.A., Monden, M., Gotoh, M., Nakano, Y., Tono, T., Mori, T. Evidence that
deoxyspergualin prevents sensitization and first-set cardiac xenograft rejection in rats by
suppression of antibody formation. Transplantation. 50, 132, 1990.
39. Van den Bogaerde, 1.B., Aspinall, R, Wright, L., Wang, M.W., Carey, N., White, D.l.G.
The induction oflong-term survival of hamster heart xenografts in rats. Transplantation.
In press.
40. Lim, S.M.L., Wee, A., Chong, M., White, DJ.G. Analysis ofxenograft rejection in the
nude rat model. Transplant. Proc. 22,2095,1990.
41. Guttman, RD. Genetics of acute rejection ofrat cardiac allografts and a model of hyper-
acute rejection. Transplantation. 17,383,1974.
42. Kissmeyer-Nielsen, F., Olsen, S., Petersen, V.P., Fjeldborg, O. Hyperacute rejection of
Lancet. 1,662, 1966.
43. Paul, L.C, Baldwin, W.M .. Van Es, L.A. Vascular endothelial alloantigens in renal
transplantation. Transplantation. 40, 117, 1985.
44. Baldwin, E.M., Paul, L.C, Claas, F.H.l., Daha, M.R Antibody mediated events in trans-
plantation: challenges to past dogma and hopes for the future. In: Progress In
Transplantation, Vol. 3 Morris. P.l. and Tilney, N.L. (eds.) Churchill Livingstone:
Edinburgh. London, Melbourne, New York. 1986, p. 85.
45. Trento, A., Hardesty, RL., Griffith, P., Zerbe, T., Kormos, RL., Bahnson, H.T. Role of
antibody to vascular endothelial cells in hyperacute rejection in patients undergoing car-
diac transplantation.J. Thorae. Carriiovasc. Surg. 95,37,1988.
46. Cochrane, CG., Muller, H.l., Eberhard, B., Aikin, S. Depletion of plasma complement
in vivo by a protein of cobra venom: its effect on various immunologic reactions.
J. lmmllnol. 105,55,1970.
47. Vogel, CW., Smith, CA .. Muller, H.l., Eberhard, B. Cobra venom factor: structural
homology with the third component of human complement. J. lmmllnol. 133, 3235,
1984.
48. Gewurz, H., Clark, D.S., Cooper, M.D., Varco, RL., Good, RA. Effect of cobra venom
Transplantation. 5, 1296,1967.
49. Mejia Lagun, 1.E., Martinez Paloma, A., Lopez Soriano, F., Garcia Cornejo, M.
Morphologic study of the participation of the complement system in hyperacute rejec-
tion of renal xenografts. Am. J. Pathol. 69,71,1972.
50. Kemp, E., Kemp, G., Starklint, H., Larsen, S. Immunosuppression with cobra venom
factor, anti-platelet aggregator, and cyclosporin A in renal xenotransplantation.
51. Rosengard, B.R, Hall, T.S., Imagawa, D.K., Ueda, K., Baumgartner, W.A., Borkon, M.,
Smith, RRL., Hutchins, G.W., Reitz, B.A. Hyperacute rejection of cardiac xenografts:
two new models showing differences in pathogenesis. Surg. Forum. 36,361,1985.
52. Adachi, H., Rosengard, B.R, Hutchins, G.M. Effects of cyclosporin, aspirin, and cobra
venom factor on discordant cardiac xenograft survival in rats. Transplant. Proc. 19,1145,
1987.
53. Alper, CA., Colten, F.S., Rabion, A.R, Macnab, G.M., Gear, 1.S.S. Homozygous defi-
ciency of C3 in a patient with repeated infections. Lancet. 2, 1179, 1972.
54. Botto, M., Fong, K.Y., So, A.K., Rudge, A., Walport, M.l. Molecular basis of hereditary
C3 deficiency. J. Ciin. Invest. 86, 1158, 1990.
55. Reding, R, White, D.l.G., Davies, H.fLS., Wright, L.l., Marbaix, E., CaIne, R.Y.
Beneficial effect of plasma exchange on guinea pig-to-rat xenograft survival. Ellr. Surg.
Res. 20, 172, 1988.
56. Van de Stadt, 1., Vendeville, B., Wiell, B., Crougneau, S., Michel, A., Filliponi, F.. Icard,
P., Renoux, M .. Louvel, A., Houssin, D. Discordant heart xenografts in the rat.
Additional effects of plasma exchange and cyclosporin, cyclophosphamide or splenecto-
my in delaying hyperacute rejection. Transplantation. 45,514,1988.
57. Alexandre, G.P.l., Gianello, P., Latinne, D., Carlier, M., Dewaele, A., Van Obbergh, M.,
Moriau, E., Marbaix, E., Lambotte, 1.L., Lambotte, L., Squifflet, 1.P. Plasmapheresis
and splenectomy in experimental xenotransplantation. In: Xenografl 25. M.A. Hardy

(cd.) Elsevier: Amsterdam. New York. Oxford 1989. p. 259.
51l. Linn. B.S .. Jensen. J.A .. Portal. P .. Snyder. G.B. Renal xenograft prolongation by sup-
pression of natural antibody. 1. Slirg. Res. 8.211. 1968.
59. Henry. M.L.. Han. L.K .. Davies. E.A .. Orosz. CG .. Sedmak. 0.0 .. Ferguson. RM. The
role of organ perfusion in xenotransplantation: influence of ex vivo zenoantibody ab-
sorption on xenograft survival. In: Xenografl 25. M.A. Hardy (ed.) Elsevier:
Amsterdam. New York. Oxford. 19119. p. 249.
60. Rosenberg. J.C. Broersma. R1.. Bullemer. G .. Mannen. E.F.. Lenaghan. R.
Rosenberg. B.F. Relationship of platelets blood coagulation and fibrinolysis to hypcr-
acute rejection of renal xenografts. Transplantatioll. 8. 152. 1969.
61. Rosenberg. J.C. Hawkins. E .. Rector. F. Mechanisms of immunological injury during
antibody mediated hyperacute rejection of renal heterografts. Transplanlalion. II. lSI.
1971.
62. Jefferson. K .. Tyerman. K.. McLeish. M .. Collier. ST. 1.. Thiru. S. Donor pretreatment
prolongs survival of discordant xenografts. Transplant. Proc. In press.
63. Good. A. Effect of cobra venom induced inhibition of complement activity on allograft
and xenograft rejection reactions. TrallSpla11lalion. 5. 1296. 1967.
64. Miyagawa. S.. Hirose. H .. Shirakura. R. Naka. Y., Nakata. S .. Kawashima. Y .. Seya. T..
Matsumoto. M .. Uenaka. A .. Kitamura. H. The mechanisms of discordant xenograft re-
jection. TrallSplalltatioll. 46, 825, 1981).
65. Forbes. RD.C. Guttmann. RD. Histopathology and mechanisms of rejection in xeno-
transplantation. In: Xenografr 25. M.A. Hardy (ed.) Elsevier: Amsterdam. New York,
Oxford. 1989.p. 133.
66. Jorgensen, K.A.. Kemp. E.. Barfort. P .. Starklint. H .. Larsen. S.. Munk-Anderson. G.
Platelet aggregation is not essential for xenograft rejection. Thromb. Res. 43.1l7. 1986.
67. Makowka, L., Miller. C. Chapchap. P .. Podesta. L.. Pan C. Pressley. 0 .. et a!.
Prolongation of pig to dog renal xenograft survival by modification of the inflammatory
mediator response. A nil. Sllrg. 206,412,1987.
68. Platt, J.L., Vercelotti, G.M .. Lindman. B.L Oegema. T.R. Bach. F.H .. Dalmasso. A.G.
Release of hepar an sulfate from endothelial cells. Implication for pathogenesis of hyper-
acute rejection..!. Exp. Med. 171. 1363. 1990.
69. CaIne. R.Y. Xenograft: functional definition. In: Xenograft 25. M.A. Hardy (ed.)
Elsevier: Amsterdam. New York. Oxford. 1989, p. 3.
70. Atkinson, J.P .. Farries. T. Separation of self from non-self in the complement system.
lmmllnol. Toda\'. 8.212. 1987.
71. Nicholson-Weller, A .. March, J.P., Rosenfeld. S.l. Austen. K.F. Affected erythrocytes of
patients with paroxysmal nocturnal hematuria are deficient in the complement regulato-
ry protein, decay accelerating factor. Proc. NaIl. Acad. Sci. USA. 80,5066. 1983.
72. Pangburn. M.K .. Schreiber, RD .. UllIer-Eberhard. H.J. Deficiency of an erythrocyte
membrane protein with complement regulatory activity in paroxysmal nocturnal
hemoglobinuria. Proc. Natl. Acad. Sci. USA. 1l0, 5430, 1983.
73. Nakajima, K., Sakamoto, K.. Ochiai. T.. Asano. T.. 150no. K. Effects of 15-deoxysper-
gualin and FK 506 on the histology and survival of hamster- to-rat cardiac xenotransplan-
tation. Transplantatioll. 21.546. 1989.
Section III
Histopathology of
Xenograft Rejection
Chapter 11
Histopathology of Kidney Xenograft Rejection

S. LARSEN AND H. STARKLINT
Introduction
Human renal allotransplantation has in recent years become a frequent proce-

dure in the treatment of terminal renal failure, and the pathology of trans-
planted grafts constitutes a complicated part of renal pathology.
The morphological and immunological changes that develop in the renal
graft, as evaluated qualitatively as well as quantitatively, depend on the genet-
ic relationship between donor and recipient. Based on the genetic differences,
five transplantation systems can be defined:
1. Autogeneic - tissue transplanted from one place to another in the same indi-
vidual
2. Isogeneic - tissue transplanted from one individual to another with an iden-
tical genetic constitution (monovular twins)
3. Allogeneic - tissue transplanted from one individual to another of the same
species
4. Xenogeneic (concordant) - tissue transplanted to an individual of a closely
related species (e.g., chimpanzee to man)
5. Xenogeneic (discordant) - tissue transplanted to an individual of a distantly
related species (e.g., pig to man)
Based on knowledge of the morphological changes that occur in human kid-

ney graft biopsies, mainly obtained in situations with impaired renal graft
function [1], and from studies on experimental xenografting, the lesions at-
tributed to transplantation may be divided into two basic groups: (i) lesions
caused by histoincompatibility specific for the transplant situation, and (ii) le-
sions due to noxious influences (physical, infectious, or side effects of medical
treatment including immunosuppressants).
The histoincompatibility-mediated lesions are traditionally divided into
three types of rejection: (i) hyperacute (vascular, humoral), (ii) acute (cellu-
lar), and (iii) chronic. However, there are sometimes no sharp limits between
these conditions; a clear-cut distinction is, morphologically as well as immuno-
logically, difficult because two (or more) types of rejection may be present si-
multaneously. Furthermore, changes secondary to noxious influences may
modify any of the histoincompatibility-related lesions both quantitatively and
qualitatively.
182 Histopathology of Kidney Xenograft Rejection
The morphology of xenografts varies with the animals used and with the
discrepancy between the species. The mechanisms behind these variations are
largely unknown. However, in some xenografted organs the morphological
manifestations are very much like those found in human allografts, implicating
the possibility of similar pathogenetic mechanisms, and that is why we use the
same descriptive terminology as in clinical transplantation.
The purpose of this chapter is solely to illustrate the morphological pat-
terns that create a spectrum of lesions as seen in concordant and discordant re-
nal xenografts, and furthermore to illustrate the changes found in kidneys that
have been perfused in vitro with xenogeneic blood. Our personal experience is
based on studies on transplantation of kidneys from different species (about
500 animals), and on isolated kidneys after perfusion with human blood
(whole blood, platelet-poor, or leukocyte-poor) and plasma, and also with
blood from other animals. Details of the results of these experiments have
been published previously [2-17]. Genetic and immunological considerations
in xenograft transplantation have been discussed in a review by Auchincloss
[18].
From our material (obtained mainly from untreated animals) some pat-
terns have become clear, and these will be illustrated by typical examples. In
the legends to each figure, comment will be made of other situations where
similar appearances have been documented. The lesions seen in various ex-
perimental models are summarized in Tables 11.1-11.3, which demonstrate
the light microscopic "patterns" found in different xenotransplant situations.
Methods
The macroscopic changes that occurred in the donor kidneys were described
by the colleagues who performed the transplantations and hemoperfusions.
The results were recorded but were not made available to the two pathologists
who received biopsies. One of us (S.L.) received match-like pieces the size of
needle biopsies, whereas the other (H.S.) received median frontal slices of the
whole kidney.
For light microscopy, the tissue was fixed in buffered formalin. Paraffin sec-
tions were stained with hematoxylin and eosin (H&E) supplemented with
methenamine-silver for basement membranes and with periodic acid-Schiff
(PAS) and Frazer-Lendrum's stains for platelets and fibrin respectively.
For electron microscopy, cortical tissue was "teased" into small fragments
and fixed in 2.5% glutaraldehyde in a modified Tyrode buffer. Following post-
fixation in osmium tetroxide, specimens were treated conventionally, and ul-
trathin sections were stained with uranyl acetate/lead citrate.
Tissue for immunofluorescence microscopy was frozen using dry ice and
blocked in Tissue-Teck(R)-gelatine (Ames Laboratory) before 1-2 11m sec-
tions were cut at-24°C. The sections were mounted on clean slides and treated
by a direct fluorescent technique [16].
Table 11.1. Experimental renal xenografting in a concordant model
Group Donorlrecipient n Treatment Longest graft Histopathologya Reference

survival
Harelrabbit 10 Control 6 days Thrombocyte aggregation, vascular endothelial [4]

hyperplasia, interstitial mononuclear cell infiltration,
vascular wall nccrosis, thrombosis
2 Hare/rabbit 5 Steroids, cytostatic 6 days Thrombocyte aggrcgation, vascular wall necrosis, [4]
agcnts thrombosis
3 Hare/rabbit 10 Cyclosporinc 9il days Mesangial cell hyperplasia, glomerular endocapillary [4]
hyperplasia, interstitial mononuclear cell infiltration,
tubular necrosis
4 Harelrabbit 3 Cyclosporine 10 days Thrombocyte aggregation [1l]
(10 mg/kg per day)
5 Harelrabbit 7 Cyclosporine 91 days Mesangial cell hyperplasia, thromhocyte aggregation [1l]
(15 mg/kgperday)
$:
(0
6 Harelrabbit il Presensitized 60 min (killed) Mesangial cell hyperplasia, glomerular endocapillary [!6]
with blood or hyperplasia, thromhocyte aggregation, S-
o
0-
skin grafts glomerular thromhosis V>
a No immune deposits were detected on the rejected kidney except in group 6 where goat anti-rahhit polyglohulin and goat anti-rabbit C3 were f-'
00
present. V-l
Table 11.2. Experimental renal xenografting in discordant models >-'
'Y)
~
Group Donor/recipient n Treatment Longest graft Histopathology" Reference

survival ::r::
Vi'
Rabbit/cat 16 Steroids, cytostatic agents, 72h Vascular endothelial hyperplasia, [12] 0
"0
co
cobra venom factor tubular nccrosis ;.
co
2 Rabbit/cat 40 Steroids, cytostatic agents 10 min Thrombocyte aggregation, thrombosis [12] 0"
CfQ
,<
3 Rabbit/cal 5 Ticlopidine h , eyclosporine, 4 days Tubular necrosis [10] co-.
cobra venom factor 0.
0-
4 Pigirabbit 5 Dazoxiben 40 min Thrombocyte aggrcgation l8] ::l
(')
'<
5 Pig/rabbit 5 Control 60min Thrombocyte aggregation l8] ><
n
::l
6 Rabbit/pig 5 Control 45 min Thrombocyte aggregation [15] co
CfQ
...,
7 Rabbit/piglet 5 Control 60 min (killed) Thrombocyte aggregation [ 15] co
;::;>
8 Rabbit/cat 13 Cobra venom factor 7 days Tubular necrosis 114] :N
"'-'.
(')
9 Rabbit/cat 50 Various agents 60 min Thrombocyte aggregation [14] ;2.

(no cobra venom factor) o·
::l
a Immune deposits on the rejected kidney were not looked for in group 3 and were undeteclable in the olher groups.
h An antiplatelet agent.
Description and Definition of Lesions 185
Table 11.3. Experimental hemoperfusion of rabbit kidneys using human blood (discordant
model)
Group n Treatment Longest graft Histopathology" Reference

survival
5 Control 30 min Thrombocyte aggregation, [6J

vascular wall inflammation,
tubular necrosis (on electron
microscopy the endothelium
was seen to be loosened)
2 5 Control 24 min Thrombocyte aggregation [9J
3 5 Platelet-poor 60 min Thrombocytc aggregation [9J
human blood
4 5 Prostacyclin 60 min Thrombocyte aggregation [9J
a Rabbit antihuman IgM and IgG were detected on the kidney in all groups and rabbit anti-
human C3 was detected in groups 2,3, and 4.
Description and Definition of Lesions (Tables 11.1-11.3)
1. Mesangial cell hyperplasia (Fig. 11.1). Increased number of cells localized to

the mesangium (compared with controls).
2. Glomerular endocapillary hyperplasia (Fig. 11.2). Increased number of in-
tralumenal cells, which may be of endothelial origin or inflammatory
mononuclear cells. Often a combination of mesangial cell and endocapillary
cell hyperplasia is seen in the same glomerulus (endomesangial hyperpla-
sia).
3. Thrombocyte aggregation (Fig. 11.3). Presence of thrombocytes localized to
the lumena, focally or disseminated, as small clots or occluding aggregates
in the arterial vessels, juxtaglomerular arterioles, and capillary loops of the
glomeruli.
4. Vascular endothelial hyperplasia (Fig. 11.4). Increased number of endothe-
lial cells resulting in a reduction or occlusion of vascular lumena.
5. Interstitial mononuclear cell infiltration (Fig. 11.5). The cells in interstitial
infiltrates are mainly mononuclear cells, i.e., small and large lymphocytes,
macrophages and, to a lesser degree, plasma cells.
6. Vascular wall inflammation (Fig. 11.6). The lesions are most severe in medi-
um-sized and small arteries where the endothelial cells are slightly hyper-
plastic with hydropic degeneration and often piled up in two layers. The inti-
ma is thickened due to edema, and accumulation of mononuclear cells is
often present. Fibrin may cover parts of the lumenal surface or is seen as
droplets in the thickened intima. The media is often infiltrated by mononu-
clear cells.
7. Vascular wall necrosis (Fig. 11.7). The lesion is often fibrinous in its staining
quality. Initially, the inner part of the media is involved. Larger areas with
Fig. 11.1. Diffuse and global mesangial widening in a hare kidney transplanted into a rahhit:
bar, 40 f.lm, methenamine-silver
. ....-,-
-
",'
Fig. 11.2. Focal proliferation of mesangial and endothelial cells with accentuated mesangium
at the vascular pole in a hare kidney transplanted into a rabbit: bar, lOO f.lm, H&E_
Glomerulonephritis-like appearances were especially found in the hare -to-rabhit modeL
but were also seen in (i) rabbit kidneys autotransplantcd following perfusion with human
blood. (ii) hare kidneys transplanted into splenectomized rabhits, and (iii) rabbit kidneys
flushed with human whole blood or plasma
Fig. 11.3. Tightly packed platele ts in the glomerular capillary tuft, the efferent a rterioles and
an interstitial arteriole. The section is from a rabbit kidney transplanted into a eat without
treatment; bar, 100 11m, H&E -PAS
Fig. 11.4. Acute rejection of kidney transplanted from hare to rabbit. The interlobular artery
shows an intense endothelial proliferation, and the surrounding mononuclear infiltrate is
pleomorphic with blasts and lymphocytes; bar, 50 11m, H&E - PAS. These findings were
seen almost exclusively in xenotransplants between hare and rabbit, but on a few occasions
were also observed in autotransplanted rabbit kidneys after they had been pe rfused with
human blood
Fig. U.S a, b. Acute rejection of kidney transplanted from hare to rabbit. a The kidney tis-
sue is fairly well prese rved. A mantle of mononuclear cells is see n around the vessels: bar.
200 ~m. H&E, x63. b A severe endothelial proliferation exists segmentally which obscures
the different components of the vessel wall. The mitotic figure is in an endothelial cell (ar-
row) ; bar, 50 ~m. H&E, x63 )
Fig. 11.6. a Interlobar artery with a nearly empty lumen showing a loosened endothelium
surrounded by granulocytes (including eosinophils) and mononuclear cells which are pre-
sent in the intima and media; bar, 100 ~m , H&E. b Immunofluorescence microscopy illus-
trating an interlobar artery with granular deposits of rabbit antihuman immunoglobulin
(Ig)M localized to the endothelium, which in small areas is loosened. An equal pattern and
distribution were shown using antihuman fibrin/fibrinogen and C3 . Inflammatory reactions
related to the endothelial lining (intima and/or media) showed a wide spectrum of morpho-
logical alterations in different experiments but also in different kidneys in the same experi-
mental model. [t was found in perfusion and flush experiments using rats, rabbits and pigs as
donors and humans (blood and plasma) , rabbits and cats as recipients. [t could also be seen
when kidneys from hares or pigs were transplanted into presensitized rabbits
190
..••-.. ,." -.. . ... . .
Histopathology of Kidn ey Xe nograft Rejection
....- . .- . .... .. ... \ "' .", !.

; • 'J ,
• ... " . •• •
,
••-.,.• "-.! .
..... ..
, ..•. ~
,~, .
..
1
.. •
I
.... •
~ '. I -
.. .......... .
~
...
,;
-: ,
~;
"'=.,
\
•
..:i
• •1IIII1iIIiI •• .•
' .,"
,... . •
".:'
~,~.
, ......"~ .
. '.
.
;.•.
Iof"
, --A •
4. - • • • , ...
-" ;. ','
..
' ,;,
.t
.1 .,. • ;:O'I,"~ ..
.. •
I
11
- .' •
~
~
• • • • it..I"~"
'I
'..
Fig. 11.7. Hyperacute rej ection of kidney transplanted from hare to rabbit. Tu the le]i, an in ·
terlobul a r artery shows necrosis and deposition of fibrin in its wall. To the right , a typical
glomerulus conta ins fibrin that tapers and occludes the capill ary lumen. The tubules are
seve rely dama ged by ischemia with heavy interstitial edem a and few inflammatory cells:
bar. 40 ~m. Frazer-Lendrum. This pattern was nearly exclusively found in the hare·to-rabbit
model. A few examples were seen wh e n rabbit kidn eys were autotransplanted followin g
perfusion with human blood
destruction of the internal elastica and intima may be seen in later stages. If
arteria l necrosis is accompanied by le ukocyte infiltration in the media, the
use of the term "arteritis" may be justified.
8. Tubular necrosis (Fig. 11.8). Partial tubular necrosis, most often subcapsu-
lar, is seen in a few tubular profiles or as sma ll areas surrounded by groups of
preserved tubules. Necrosis may affect single tubular cells, small groups or
the total tubular epithelium. The nuclei show pyknosis; later, all cytologic
detail may disappear. Calcification of necrotic proximal tubular cells may
be seen.
9. Thrombosis (Fig. 11.9). Arterial vesscls or glomerular capillary loops where
lumena are partially or totally occluded with fibrin matcrial containing red
blood cells and/or thrombocytes and/or granulocytes.
Results 191
Fig. 11.S. Subcapsular area in a rabbit kidney transplanted into a cat, which survived for
1 week while receiving cobra venom factor once every day. Totally and partially necrotic
tubules, some calcified, are seen. A few mitotic figures (arrow) suggest some capacity for re-
generation; bar, 40 Ilm, H&E. Tubular necrosis was found in many xenografted models, but
was most frequent and extensive in cats treated with cobra venom factor (Table 11.2). This
suggests that this compound is tubulotoxic, as are certain other snake poisons. However, in
other experimental groups small areas demonstrated necrosis of what appeared to be proxi-
mal tubules. Such changes were observed in hare-to-rabbit transplants and following perfu-
sion of goat kidneys with blood from rabbits, cats, and man. Many of the transplanted ani-
mals received cyclosporine, usually in dosages of approximately 15 mg/kg per day, which , in
our experience, is not toxic to the tubules to the extent seen in the experiments [19]
Results
From a technical point of view, most of the investigated tissues were satisfacto-
rily preserved. Some of the experimental situations, however, led to heavy tis-
sue damage, making detailed interpretation difficult or even impossible.
Methenamine-silver stain for basement membranes, which in the human kid-
ney is of great importance for diagnosis, was often difficult to interpretate in
the experimental animals. The "staining-window" between good visualization
of peripheral membranes and overstaining, especially of the mesangial area, is
very narrow. In doubtful cases we, therefore, recommend the use of a series of
impregnation times in order to ensure a clear picture of the different struc-
tures. The main purpose of the electron microscopic studies was to verify the
192 Histopathol ogy of Kidn ey Xenograft Rejection
. ..·G
":" - ..".....".
;...
• •
Fig. 11.9 a, b. In some cases of hare~to~ra bbit co ncord ant xenotransplantation a mixed pic~
ture of hyperacute and acute rejection was seen. In a, th e glomerulus contains fibrin along
the capillary walls, but this was not seen in all glomeruli. Apart from the presence of edema
and interstitial bleeding, the tissues arc rather well preserved; bar, 40 J.1m, Frazer~Lendrum.
In b, a great number of parenchymal vessels showed mantles of mononuclear cells; bar,
40 J.1m , H& E. This mixed picture was solely found in hare~to~rabbit kidn ey transplants
Results 193
Fig. 11.10 a, b. For legend, sec over

Fig. 11.10 a-c. Chronic glomerular rejection was diagnosed in a few long-term surviving
nephrectomized rabbits transplanted with a hare kidney. a Some interstitial fibrosis and
mononuclear cell infiltration can be seen; bar. 200 ~m, H&E. b A slight glomerular hyper-
cellularity and thickened peripheral basement membranes; bar, 40 ~m , H&E. In c these
findings are duplicated in some loops (arrow); bar. 40 ~m . methenamine-silver. These were
rare findings, seen only in rabbits transplanted with hare kidneys and who had received
eyclosporine (15 mg/kg per day i.m.) for 30 days. the animals surviving from 9 to 13 weeks
presence of platelets, and to cvaluate both their granularity and relation to

endothelial cells.
The morphological features seen can be summarized under the following
diagnostic headings.
Morphological Conditions Seen Mainly in Concordant Xenograft Rejection

(Table 11.1)
1. Hyperacute rejection. A widespread glomerular microthrombosis was

found, i.e., not confined to a single area of the kidney and therefore presum-
ably not related to infarction of the transplanted organ (Fig. 11.7).
2. Acute rejection. Perivascular and interstitial infiltrates of lymphoid cells ac-
companied by varying degrees of endothelial proliferation were found (Figs.
11.4,11.5). The compositionofthe infiltrates varied with the maturity and dif-
ferentiation of lymphocytes, some exhibiting a great number of plasma cells.
Rcs ults 195
Fig. n .ll a, b. For legc nd. see ove r

Fig. n.11 a-c. Electron micrographs from glomerular capillary loops in a rabbit kidney
transplanted into a cat. a The endothelial cell in the center is edematous but preserved, as is
the fenestrated endothelial cytoplasm peripheral to the erythrocytes (arrows); har. 5 flm,
uranyl -lead. b The framed area in a, showing the contact between a platelet (asterisk) and
an endothelial cell (bar. 2 flm). c Basement membranes from two neighboring capillary
walls. To the /eli. endothelial cell cytoplasm with edema and proximity to red blood cells. To
the right, fenestrated and preserved endothelial cytoplasm in apposition to platelets; the
foot-processes in between (asterisk) are flattened (bar. I flm)
3. Mixed hyperacllte and (lClIte rejection (Fig. 11.9). This was seen in some cases
and related to accelerated rejection.
4. Chronic rejection ofglofl1erular type (Fig. 11.10). Glomerular cellular pro-
liferations and membranous-like patterns were found in long-term sur-
vivors accompanied by immune complex deposition diagnosed by fluores-
cence.
5. Glomerliionephritis-like alterations. This was observed in cases with either
focal segmental or diffuse global mesangial cell hyperplasia and/or endo-
capillary hyperplasia (Figs. 11.1.11.2). Not all of these were accompanied by
immune deposits. Differentiation from the chronic rejection of glomerular
type (as described in point 4) was certainly problematic and mainly related
to graft survival time. Glomerulonephritis-like alterations were found morc
frequently than chronic rejection of glomerular type .
Results 197
Fig. 11.12. Parenchymal artery from rabbit kidney transplanted into a nephrectomized cat
without treatment. The endothelial cells (E. C.) are intact in spite of close apposition to part-
ly degranulated platelets (P.) (E, clastic lamina; S M, smooth muscle cell): bar, 4 J.lm, uranyl-
lcad. This appearance, with severe aggregation of platelets in vessels without morphological
damage to the endothelium and with no inflammatory response apart from edema, was seen
in (i) rabbit kidneys transplanted into cats, (ii) rabbit kidneys transplanted into baby pigs
that were presumed to be antibody-free, and (iii) following perfusion of rabbit, rat and goat
kidneys with human blood
Morphological Conditions Seen Mainly in Discordant Xenograft Rejection

(Tables 11.2-11.3).
6. Tubular necrosis. Necrotic and desquamated epithelium were seen mainly

in proximal tubules, and not attributed to infarction. The basement mem-
branes of the necrotic tubules were intact. No inflammatory reaction was
found. A possible sequel, in the form of calcified tubular epithelium, was a
frequent finding in all animals, especially rabbits (Fig. 11.8).
7. Platelet aggregation. Diagnosed by light microscopy or, preferably, ultra-
structurally (Figs. 11.3, 11.11). Platelets were found in glomerular capillar-
ies and sometimes in parenchymal arteries. Aggregation could be accompa-
nied by fibrin deposits and the presence of either neutrophils or, especially
in rabbits, eosinophilic granulocytes. The number of platelets varied with
the experimental design and the species used. The significance of these find-
ings remains unclear.
198 Histopathology of Kidncy Xenograft Rcjection
Fig. 11.13. a Glomerular loop with a few platelcts sticking to thc dcnudcd bascmcnt mcm-
branc of a rabbit kidncy aftcr perfusion with human plasma: /Jar. 10 !-1m. uranyl-lead. Thc
frallled area in b shows nearly completcly degranulatcd platelets. The structurcs of the b<lsc-
mcnt membranc and thc ovcrlying podocytcs arc normal: hilI'. 2 !-1m . uranyl-Icad
Fig. I1.J4a, b. Two capillary loops from the same glomerulus in a rabbit kidney perfused
with human plasma. a Denuded basement membrane with possible residuals of endothelial
cytoplasm (arrows). b Preserved endothelium with granulated platelets and sparse fibrin
(asterisk) in the lumen; bar, 4 f.lm, uranyl-lead. Accumulation of platelets with endothelial
damage, but without significant inflammatory response (with initially granulocytes but also
fibrin) was found mainly in perfusion experiments simulating discordant xenografting. It
was also seen following transplantation of hare kidneys into rabbits presensitized with re-
peated skin grafts or transfusions
Fig. 11.ts a-c Rabbit kidney perfused with human blood. a Two glomeruli containin g
platelets (slraigill arrow) anu a few granulocytcs (Cllrv ea arrows) in th e capillary lumcna;
har, 40 ~m, H&E. b Immunofluorescence microscopy showing a glomerulus with dcposits
of rabbit antihuman IgG in a gra nular pattern along the basement membranes in the capil -
lary loops and to a lesser dcgrc e in th e mcsangium. c A glomerulus (from the samc cxperi-
ment as b) sho ws granular pattcrn and distrihution of ra bhit antihuman C , very similar to
that of rahhit antihum a n IgG
Results 201
Fig. 11.16. Areuate artery from rabbit kidney perfused with human blood . To the left, in the
partially obliquely cut lumen, are red blood cells; in the middle, platelets (arrow); 10 the right.
fibrin; bar. 40 J.lm , H&E
202 Histopathology of Kidney Xe nog raft Rejection
Fig.n.1 7 a, b. Baby pig kidney flush e d with hum a n blood. a The partl y immature glol11eru -
Ius contains a moderat e number of platelets. In the interlobular artery. the lumen is slUfred
with neutrophil s. platelets. and spa rse fibrin: har. 40 11m . H& E . b Iml11unofluorescenee
microscopy illustra ting rat antihuman IgG locali ze d along th e endothelium or the arterioles
and the capillary loops in the glomeruli
Results 203
Fig. 11.18. a Straight part of nephrons in the medullary ray; har, 50 Ilm , H&E. b Extensive
pyknosis and loosening of the epithelial cells; bar, 40 Ilm , H&E. This phenomenon has only
been met after perfusion of rabbit kidneys with human blood. It is not clear whether it is the
result of a technical problem or a feature of ischemia secondary to platelet aggregation
Platelets could demonstrate preserved granules or be degranulated; the sig-

nificance of this difference remains uncertain. Pseudopodia could be found
in relation to the surfaces of endothelial cells, without apparent reaction in
the latter (Figs. 11.12-11.14).
Platelet aggregation was sometimes accompanied by damage and/or re-
moval of endothelial cells, and, in such cases, the platelets were frequently
found together with granulocytes. This could occur in glomerular capillaries
and/or parenchymal arteries (Figs. 11.6,11.15,11.16).
8. Glomerular micro thrombosis. This consisted mainly of fibrin but was often
accompanied by granulocytes and, in the parenchymal arteries, by throm-
bosis and granulocytic adhesion to endothelial cells, the latter often show-
ing degeneration and/or necrosis (Fig. 11.17).
9. Distinct tubular pyknosis. This was presumably located in the straight part
of the proximal tubules or the thick ascending part of the distal tubules, and
was found in the medullary ray with well-preserved collecting tubules
(Fig. 11.18). This condition was seen exclusively in the hemoperfusion ex-
periments (Table 11.3).
Comment
Light, electron, and immunofluorescence microscopy alone have demonstrat-

ed significant morphological results. However, these methods are restricted to
structural alterations and are of limited value in giving us a detailed under-
standing of graft rejection. The multifactorial influences on the graft and the
complexity of the mechanisms involved in the immune response are major fac-
tors making the histological interpretation difficult.
The application of methods that detect changes at the macromolecular level
have proved valuable in further enlightening problems in human allografting.
DNA technological methods (e.g., Northern blotting, Southern blotting, in
situ hybridization, peR, etc.) and immunochemical techniques (immunohis-
tochemistry, enzyme-linked immunosorbent assay, etc.) have provided valu-
able information concerning gene expression, mRNA, protein, and glycan
synthesis of the cellular components constituting the immune response.
Further research employing these methods in vitro and in vivo with special
regard to the cellular constituents (phenotypes, receptors, signal hormones
(cytokines), major histocompatibility complex (MHC) system (Ia antigens),
adhesion molecules, etc.) involved in the activation and effectuation of the im-
mune response is needed. These methods, together with histological examina-
tion, will increase our understanding of rejection. Appropriate treatments for
the various pathogenetic types of rejection may then be achieved.
References 205
References
I. Brun. C, Olsen, S.lItlas of Renal Biopsy. Munksgaard, Copenhagen, 1981.
2. Dieperink, H., Steinbruchcl, D., Stark lint, H., Larsen, S., Kemp, E. Improvement in
hare-to-rabbit kidney transplant survival. Transplant. Pmc. 19,1140,1987.
3. Green, CJ., Kemp, E., Kemp, G., Larsen, S., Starklint, H., Simkin, S. Prolongation of
concordant renal xenografts in rabbit recipients by a short course of cyclosporine A
treatment. In Cyclosporin A. DJ.G. White (ed.) Elsevier, Amsterdam, New York,
Oxford, 1982, p. 155.
4. Jorgensen, K.A .. Kemp, E., Olsen, T.K., Barfort, P., Starklint, H., Petersen, P.H.,
Larsen, S., Munk-Andersen, G. Activation of fibrinolysis during xenoperfusion.
Thrombosis Res. 46.473. 19P>7.
5. Jorgensen, K.A., Kemp. E.. Barfort. P., Starklint, H., Larsen, S .. Abildgaard-Jacobsen,
I.. Dieperink, H., Frifelt, J.J., Munk-Andersen, G. Xeno- and auto-perfusion of rabbit
kidney. Machine perfusion with blood at 37"C Acta. Path. Microbial. Immunol. Scand.
(Sect. A), 93, 305. 19P>5.
6. Jorgensen, K.A., Kemp, E., Barfort, P., Starklint. H., Larsen, S., Munk-Andersen. G. On
the role of platelets and leukocytes in renal xenoperfusion. Acta. Path. Microbiol.
IfIlunol. Scand. (Sect. A), 94, 223, 19P>6.
7. Jorgensen, K.A., Kemp, E., Barfort, P., Starklint, H., Larsen. S., Petersen. P.H.,
Knudsen, J.B. The survival of pig to rabbit renal xenografts during inhibition of throm-
boxane synthesis. Thrombosis Res. 32, 5P>5, 19P>3.
8. Jorgensen, K.A., Kemp. E., Barfort, P., Stark lint, H., Larsen, S .. Munk-Andersen. G.
Platelet aggregation is not essential for xenograft rejection. Thrombosis Res. 43, P>7, 19P>6.
9. Kemp, E., Kemp. G., Starklint, H., Larsen, S. Immunosuppression with cobra-venom
factor, anti-platelet-aggregator and cyclosporin A in renal xenotransplantation.
Transplant. Proc. 14,116.1982.
10. Kemp, E., Kemp. G., Larsen, S., Starklint, H., Green, CJ. Xeno-banking. In Organ
Preservation, Present and Future. D.E. Pegg, LA. Jacobsen, N.A. Halasz (cds.). MTP
Press Ltd., London, 1982, p. 291-301.
II. Kemp, E., Kemp, G .. Larsen, S. Survival of discordant renal xenografts up to 3 days: as-
sessment of function, light and immunol1uorescent microscopy. SCi/nd. f. Urol. Nephrol.
(Suppl.) 54, 150, 19P>0.
12. Kemp. E .. Starklint, H., Larsen, S.. Dieperink. H. Cyclosporine in concordant renal
hare-to-rabbit xenotransplantation: prolongation and modification of rejection. and ad-
verse effects. Transplant. Pmc. 17. 1351. 1985.
13. Kemp. E., Steinbruchel, D., Starklint, H .. Larsen, S.. Henriksen. I., Dieperink. H. Renal
xenograft rejection: prolonging effect of captopril, ACE-inhibitors. prostacyclin. and
cobra venom factor. Transplant. Proc. 19. 4471. 19P>7.
14. Kemp. E., White, D .. Dieperink, H .. Larsen. S .• Starklint. H .. Steinbruchcl. D. Delayed
rejection ofrabbit kidneys transplanted into baby pigs. Transplant. Proc. 19. 1143-1144.
19P>7.
15. Larsen. S .. Starklint. H., Dieperink, H., Kemp. E. Immunofluorescence microscopy in
experimental rcnal al1o- and xenografts. Transplant. Proc. 22. 1061. 1990.
16. Larsen, S. Immunofluorescent microscopy findings in minimal or no change-disease and
slight generalized mesangioproliferative glomerulonephritis. Acta. Path. Microbiol.
Scaml. (Sect. A). P>6. 531. 197P>.
17. Starklint. H .. Larsen. S .. Jorgensen. K.A .. Dieperink, H., Kemp, E. Flush perfusion of
rabbit kidneys with auto- al1o- and xenogeneic blood. Scand. f. Urol. Nephrol. 25. 45.
1991.
1P>. Auchincloss. H. JR. Xenogeneic transplantation. Transplantation. 46. I. I 98P>.
19. Dieperink. H. Cyclosporin A ncphrotoxicity. Dan. Med. Blill. 36.235.1989.
Chapter 12
Histopathological, Immunofluorescent,
and Electron-Microscopic Features of Hyperacute
Rejection in Discordant Renal Xenotransplantation
I.R. MARINO, S. CELLI, G. FERLA, A.C. STIEBER, N. Maggiano, and P. Musiani
Introduction
Limited availability of human organs for transplantation is the most immedi-

ate problem for the coming decade, now that organ transplantation has be-
come a well-established treatment modality for end-stage disease of a number
of solid organs [1,3]. However, even if the survival rate has improved signifi-
cantly [4J, thanks to both improved techniques [5-8] and immunosuppressive
therapy [9, 10], a shortage of donor organs persists [11].
This fact is particularly critical in pediatric transplantation, because chil-
dren with organ failure need prompt organ replacement with a size-compati-
ble graft if they are to achieve normal physical and psychological develop-
ment. At present, many pediatric patients, especially in Europe, can only
receive long-term artificial organ support (e.g., hemodialysis in the case of re-
nal failure) [12], which hinders their growth, or, if awaiting a liver or heart. may
die while awaiting a vital donor organ [13].
Furthermore, a wider availability of organs for transplantation would allow
an expansion of the indications for transplantation [14] and at the same time a
relaxation of the patient selection criteria [15, 16]. All these facts clearly justify
a renewed interest in xenotransplantation [17].
Historical Background
The original concept of xenografting, meaning the transplantation of cells, tis-

sues, or organs between different species with the resulting survival and nor-
mal function of a chimeric organism, is so ancient that it is easily recognizable
in Greek and Roman mythology. In fact, the centaur Chiron [18] (the teacher
ofEsculapius) and the Chimera [19] are legendary examples of discordant [20]
xenogeneic creatures. However, only during this century have scientists start-
ed to work on experimental [21-25] and clinical xenografts [26].
The first reports of successful clinical xenografting appeared in the literature
as recently as the 1960s [27-30]. Although several studies in the last 20 years
have described many aspects of the rejection mechanism in xenogeneic trans-
plantation [17]. the immunological processes involved in discordant xenograft
rejection [20,31-33] and their sequences [34-37] still remain largely unclear.
208 HistopathologicaL Immunofluorescent. and Electron-Microscopic Features
Practical Considerations
It is well known that only 25-50 chimpanzees per year are available in the
United States for all biologic and medical research purposes (including the an-
imal model for acquired immunodeficiency syndrome, AIDS) [30], and only a
total of 70 chimpanzees is theoretically available worldwide for organ trans-
plantation [17]. Moreover, the emotional and ethical implications [38,39] of
breeding apes for organ transplantation preclude any serious push in this di-
rection. Consequently, xenograft research today should be more appropriate-
ly directed to discordant rather than concordant models.
The aim of this chapter is to review the histopathological, immunofluores-
cent, and electron-microscopic features of hyperacute rejection as observed in
a renal discordant xenograft model (pig-to-rabbit). This model was developed
in 1987 at the University of Pittsburgh. Further research was performed by
members of that team at the Catholic University in Rome.
Selection of Experimental Model
Our aim in designing a model to study hyperacute rejection in a discordant

xenograft was to obtain a reproducible, low cost, small animal model. Because
of well-known immunological behavior, the rabbit [40J seemed to be the ideal
recipient. Previous classical studies suggested that the greater the donor-re-
cipient species differences, the faster would xenograft rejection occur, due to
pre-existing antibodies [1,2,20,41 J. Consequently, the Landrace pig appeared
to us as a perfect discordant kidney donor in order to achieve hyperacute re-
jection in the New Zealand rabbit, which was selected as the recipient.
Early studies suggested that the rabbit was a mediocre model for kidney
transplantation [42-47] due to technical problems related to anesthesia man-
agement [42-45J and large-vessel clamping for the performance of the vascu-
lar anastomoses [42,45-47]. Thus our initial effort was aimed at solving these
technical difficulties and obtaining a reliable and easily replicable model [39,
48]. A simple and effective anesthesia protocol was thus designed, while some
technical refinements described by other researchers in the interim [42,
45-47] were introduced successfully, such as the use of a donor venous cuff
prepared on the bench. Technical details have been reported elsewhere [32,
48].
Discordant organ hyperacute rejection was then studied by two different
methods. An initial series of experiments involved the study of transplanted
kidneys in vivo [32, 33J. A second series of experiments was directed at the
study in vitro of frozen sections of nontransplanted Landrace pig kidney
treated with normal New Zealand rabbit serum [33, 49, 50J. In the in vivo
study, xenograft biopsies for histological, immunohistochemical, and ultra-
structural studies were taken at 4, 10, 15,20,30,45 and 120 min after kidney
reperfusion.
Features of Discordant Hyperacute Rejection 209
Preparation of Tissue
Light and Electron Microscopy
Renal fragments were processed for light and electron microscopy. One part
of the tissue was fixed in 10% buffered formaldehyde and embedded in paraf-
fin. Sections cut every 4 flm were stained with hematoxylin and eosin (H & E)
and periodic acid-Schiff (PAS) reagent; another part of the tissue was fixed in
Karnowsky solution, postfixed in 1% osmium tetroxide and embedded in
Epon 812. Thin sections were stained with uranyl acetate and lead citrate and
examined under a Philips EM 400 electron microscope.
Immunohistochemical Studies
Tissue samples for immunohistochemical studies were embedded in the opti-

mum cutting temperature medium (Miles Scientific, Naperville, IL), snap
frozen in liquid nitrogen, and maintained at-80°e.
Sections 5 flm thick, prepared from frozen tissue were thaw-mounted on
poly-L-lysine hydrobromide (Polysciences Inc., Warrington, PA); the coated
slides were allowed to air dry for 3 h and were then fixed for 10 min in 100%
acetone. The sections were incubated with FITC-conjugated goat anti rabbit
immunoglobulin M (IgM), IgA, IgG, and C3 antibodies (Cappel, Organon
Teknika Corporation, Veedijk, Belgium) at room temperature for 30 min.
Negative controls were performed by using phosphate-buffered saline!
bovine serum albumin and normal goat Ig instead of the above mentioned
FITC-conjugated antibodies.
For the in vitro phase of the study, tissue samples of nontransplanted
Landrace pig kidney were snap frozen in liquid nitrogen and then sectioned.
After 30 min of incubation with normal New Zealand rabbit serum, and after
being washed in buffer solution, the kidney samples were incubated with
FITC-conjugated goat anti rabbit IgG, IgA, IgM, and C3 for 30 min.
Features of Discordant Hyperacute Rejection
In Vivo Studies
Light Microscopy and Immunofluorescence

Specimens obtained 15 min after kidney reperfusion showed some degree of
interstitial edema around the capillaries which branched between the tubules
as the intertubular plexus. Immunofluorescence techniques demonstrated the
presence of linear deposits of preformed IgG and IgA rabbit antibodies in the
wall of peritubular capillaries (Fig. 12.1). These antibodies most probably pre-
210 Histopathological. Immunofluoresccnt, allll Electron -Microscopic Features
Fig. 12.1. Immunofluoresccnce ofaxcnografted kidney (pig-to -rabbit) tissue sam pIc ob-
tained 15 min aftcr reperfusion. Deposits of IgG in the wall s of peritubular capillaries are
clearly visible. Deposits of Ig A have a similar di stribution. x 25()
Fig. 12.2. Immunofluorescence ofaxenografted kidney tissu e sample obtained 15min after
reperfusion . Linear deposition of C3 along peritubul a r capillary walls can be observed. x250
cipitate on the capillary endothelium, and were associated with C3 deposition
along peri tubular capillary walls (Fig. 12.2). Our data do not show whether the
C3 deposition precedes, follows, or occurs at the same time as the antibody de-
position. In theory, from an immunological point of view, C3 deposition fol-
lows antibody deposition, but at present this remains speculation.
In fact, White et al. [51] have provided evidence that complement does not
need an antigen-antibody reaction in order to be activated in a xenografted or-
gan, whereas others take exactly the opposite position [52]. White's hypothe-
sis is supported (i) by studies showing that the complement system recognizes
and reacts with foreign materials without the mediation of antibodies [53], and
(ii) by perfusion studies in rat kidneys with heat-inactivated and absorbed an-
tibody-free dog serum [54]. In this latter experiment, the addition of rat com-
plement alone to the system resulted in a "typical" rapid xenograft rejection.
Thus the authors suggest that xenograft rejection is possible in the presence of
an intact complement system alone, and that it does not necessarily require the
presence of preformed antibodies.
Other authors have reported some in vivo and in vitro experiments indicat-
ing that rejection in a guinea pig-to-rat heart model is caused by primary acti-
vation of complement via the alternative pathway [55].
IgM does not playa significant role in the early phases of hyperacute
xenograft rejection in the pig-to-rabbit model. Immunofluorescence of kidney
Fig. 12.3. Immunofluorescence of a xenografted kidney tissue sample obtained 30 min after
reperfusion. Linear and granular deposits of JgA are present in the peri tubular capillary
walls. A weak positivity can be observed also in the interstitium. xlOO
212 Histopathological. Immunofluorescent, and Electron-Microscopic Fcatures
Fig. 12.4. Immunofluorescence ofaxenograftcd kidney tissue sample obtained ()U min after
reperfusion. Deposits of IgA in the peritubular capillary walls and in the interstitium are
seen, whilc the tubules are filled in a patchy manner. Deposits of IgA and C3 prcsent with a
similar distribution. IgM fluorescence was almost negative; only a weak linear positivity
could be observcd along thc cndothelium of isolatcd arteries. x I 00
xenograft tissue obtained 45 min after organ reperfusion does not demon-
strate any significant deposits of IgM in the arterial endothelium, in the peri-
tubular capillary walls, or in the interstitium [32]. Instead, in the specimens ob-
tained 30 min after reperfusion, the immunofluorescence studies show
conspicuous linear and granular deposits of IgG and IgA along the peri tubular
capillary walls, while a mild reaction is present in the neighboring interstitium
(Fig. 12.3). This is probably related to antibody endothelial deposition with
consequent endothelial cell damage and basement membrane tears. In this
way, antibody passage and deposition in the interstitium is facilitated. The
confusing literature on the relation of antibodies, their classes, and comple-
ment activation has been summarized by Platt et al. [52].
In the specimens obtained 60 min after xenograft reperfusion, IgG, IgA,
and C3 are diffusely present in the arterial endothelium, in the peritubular
capillary wall, and in the interstitium. Renal tubules are filled with antibodies
in a patchy manner which demonstrates that they have already caused damage
to the tubular basement membrane (Fig. 12.4).lgM does not seem to play an
important role in this process (Fig. 12.4). The glomerular reactivity is very
mild, with only a weak and irregular positivity along the capillary tufts.
During the second hour of xenograft reperfusion, antibody and C3 deposits
are evident in all the renal tissues. IgG is widely distributed in the peri tubular
Fig. 12.5. Immunofluorescence ofaxenografted kidney tissue sample obtained 120 min after
re perfusion. IgO is widely distributed in the peritubular capillary walls and in the intersti-
tium. A mild positivity can be obse rved in the glomerular capillary loops. Large amounts of
IgO can be seen in the interstitium surrounding Bowman 's capsule. xlOO
capillary wall and in the interstitium (Fig. 12.5). Tubules are severely dam-
aged; IgG, IgA and C3 are present in large amounts in the lumena of the prox-
imal convoluted tubules [32]. A mild positivity can be observed in the
glomerular capillary loops. Large amounts of IgG are seen in the interstitium
surrounding Bowman's capsule (Fig. 12.5). This supports our theory [33] that
the glomerulus withstands antibody activity longer than do the peri tubular
capillaries, and that glomerular lesions most probably start from the outside,
namely the interstitium surrounding the epithelial coating of Bowman's cap-
sule.
Ultrastructure
Electron microscopy confirms at the ultrastructural level the sequence of
pathological events that is suggested by immunofluorescence studies.
At 15 min after xenograft reperfusion, electron microscopy demonstrates
aggregates of nondegranulated platelets close to the endothelium of peritubu-
lar capillaries, in which scattered foci of polymorphonuclear leukocytes and
erythrocytes are frequently found (Fig. 12.6). These microthrombi are most
probably triggered by endothelial lesions associated with the antibody deposi-
tion observed by immunofluorescence.
214 Histopathological, Immunofluorescent, and Electron-Microscopic Fcatures
Fig. 12.6. Electron photomicrograph of a xc no grafted kidney tissue sample ohtained 15 min
after reperfusion. Tn the perituhular capillary, a monocyte , erythrocytes, and platelets
(showing some adhcrence to th e endothelium) can he observed. The interstitium is edema-
tous. x4600
These specimens already show a wide separation of tubules, mainly those

of smaller diameter, revealing the presence of interstitial edema (Fig. 12.6).
The edema is secondary to immunological damage of the endothelium and
basement membrane of peritubular capillaries.
At 20 min after xenograft reperfusion the interstitium is even more edema-
tous, and some peritubular capillaries are obstructed by aggregates consisting
of intact or degranulated platelets, fibrin, polymorphonuclear leukocytes, and
Fig. 12.7. Electron photomicrograph ofaxenografted kidney tissue sample obtained 20 min
after reperfusion. The peritubular capillary is obstructed by platelet aggregates and erythro-
cytes. The epithelial cells of the convoluted tubules are not seriously damaged. x3600
erythrocytes (Figs. 12.7, 12.8). The epithelial cells ofthe convoluted tubules do
not seem seriously damaged at this point (Fig. 12.7).
At 30 min the endothelial cells are severely damaged, and the cell plasma
membranes are frequently dissociated and disrupted (Fig. 12.9). Erythrocytes
start to leak from the vessels and some micro hemorrhages become evident in
the interstitium (Figs. 12.9, 12.10). Tubular epithelial cells are still edematous,
but otherwise intact (Fig. 12.9).
216 Histopathological, Immunofluorescent, and Electron-Microscopic Fea tures
Fig. 12,8, Electron photomicrograph ofaxe nografted kidney tissuc sample obta incd 20 min
after repcrfusion. In the capillary lumen is a microthrombus, constituted by polymorpho-
nuclear leukocytes. Apparently intact or degranulated platelcts, fibrin, and erythrocytes
are present. Interstitial edema is evident. x3600
In the specimens obtained at 45 min, the damage to the tubular epithelium

becomes evident. The tubular epithelial cells show cytoplasmic vacuolization,
swollen mitochondria, and simplification of basal infoldings of the tubular cell
membrane (Fig. 12.11). The intertubular capillary lumena are frequently
blocked by microthrombi, and red blood cells and platelets are found outside
of the blood vessels in the interstitium (Fig. 12.12).
As shown by the immunofluorescence studies, glomerular damage does
not appear early, and electron microscopy confirms this finding. In fact, even
Fig. 12.9. Electron photomicrograph of a xenografted kidney tissue sample obtained 30 min
after reperfusion. The endothelial cells are severely damaged, and the cell plasma mem-
branes are frequently dissociated and disrupted. An erythrocyte is visible outside the vessel.
There are no obvious lesions of the tubular epithelial eells. x3600
45 min after reperfusion the glomerular capillary loops are not obstructed and
only the epithelial cells of Bowman's capsule show some ultrastructural dam-
age, characterized by intracytoplasmic vacuoles and plasma membrane dis-
ruption (Fig. 12.12). This is consistent with our hypothesis that the glomerular
lesions start in the interstitium - in other words, from the external epithelial
coating of Bowman's capsule,
At 90 min after the onset of xenograft reperfusion, the kidney appears very
heavily damaged. The epithelial cells of the proximal convoluted tubules are
218 Histopathological, Immunofiuorcscent, and Elcctron-Microscopic Features
Fig. 12.10. Electron photomicrograph ofaxenografted kidney tissue sample obtaincd

45 min after repcrfusion. The capillary lumen is obliterated by a microthrombus. Sevcral
erythrocytes are visible outside the vessel in the edematous interstitium. x6000
severely damaged, with swollen mitochondria and intracytoplasmic vacuoles

(Fig. 12.13).
At 120 min the interstitium is frankly edematous, and there are diffuse focal
hemorrhages and collections of fibrin. Microthrombi are evident in the small
venules and glomerular capillaries and sometimes in the arterioles. The
tubules are severely damaged. The epithelial cells of Bowman's capsule are
dramatically damaged, and cell and platelet debris occupy the urinary space of
Bowman's capsule (Figs. 12.14, 12.15). Interstitial edema and hemorrhages
Fig. 12.11. Electron photomicrograph ofaxenografted kidney tissue sample obtained

45 min after reperfusion . The tubular e pithelial cells show some ultrastructural damage
(cytoplasmic vacuolization, swollen mitochondria, and simplification of basal infoldings
of the tubular cell membrane). x2800
are very prominent, causing a wider than normal intertubular space. This find-
ing is frequently diffuse, but in some cases it is significantly increased particu-
larly at the corticomedullary junction.
The tubular changes consist of focal tears of the tubular basement mem-
brane, cytoplasmic vacuolization, and simplification of basal infoldings of the
tubular cell membrane. At this time several tubular epithelial cells with severe
degenerative changes have been shed by the tubular basement membrane
[32].
220 Histopathological, Immunofluorescent, and Electron-Microscopic Features

45 min after reperfusion. Capillary microthrombi are present; outside of the blood vessels,
red blood cells and platelets can be observed in the interstitium. The glomerular capillary
loops are not obstructed; only the epithelial cells of th e Bowman's capsule show some ultra-
structural damage (intracytoplasmic vacuoles and plasma membrane disruption) . x2~OO
In Vitro Studies
Immunofluorescent and electron-microscopic features of hyperacute rejec-

tion in our renal discordant xenograft model indicated that the underlying
pathogenesis can be traced back to a humoral mechanism. Our data suggest
that rejection is elicited by the action of preformed recipient humoral antibod-
ies to donor antigens. This is similar to the mechanism of hyperacute allograft

90 min after reperfusion. The epithelial cells of the proximal convoluted tubules are se-
verely damaged with swollen mitochondria and intracytoplasmic vacuoles. x3600
rejection where the humoral antibody reaction is triggered by donor major

histocompatibility complex (MHC) Class I antigens [56].
However, it follows that, if these antibodies are indeed preformed, they
should be present in the normal serum ofthe New Zealand rabbit. This was the
rationale behind the in vitro phase of our studies. We believed that an identical
humoral reaction should be elicited not only by a xenotransplanted Landrace
pig kidney, but also by a tissue sample of nontransplanted Landrace pig kidney
222 Histopathological, Immunotluorescent, and Electron-Microscopic Features
Fig. 12.14. E lectron photomicrograph of a glom erular capilla ry loop from a xenografted kid-
ney tissue sample obtained 120 min after reperfusion. The capillary contains a polymor-
phonuclear leukocyte and aggregated pl atelets. Cell and platelet debris are present in the
Bowman 's space. x3600
incubated with normal New Zealand rabbit serum. In fact , if the antibodies
contained in the normal New Zealand rabbit serum had reacted with struc-
tures of the non transplanted Landrace pig kidney, it would have been possible
to identify their distribution by FITC-conjugated antirabbit antibodies and
C3. Such a model would also have offered the possibility of identifying the na-
ture of the preformed natural antibodies and the structures eliciting their de-
position in the renal tissue [33,49,50] .

120 min after reperfusion. The urinary space of the glomerulus is completely occupied by
cell debris, and the epithelial cells of the Bowman's capsule are dramatically damaged.
x4600
Our in vitro results clearly demonstrate that preformed natural antibodies

are present in the normal serum of the New Zealand rabbit (Fig. 12.16).
Specifically, rabbit IgG and IgA react with molecular structures of the en-
dothelium in the peritubular and glomerular capillaries (Fig. 12.17). In con-
tradistinction, natural preformed IgM does not react with endothelial cells but
with cytoplasmic components of epithelial tubular cells, and only on the endo-
lumenal side. We postulate that IgM binds to molecules of the brush border of
the renal tubules [33] (Fig. 12.18).
Fig. 12.16. Immunohistochemical analysis of a Landrace pig kidney specimen tested with
normal New Zealand rabbit serum. The fluorescence staining (yellow) was perfo rmed with
goat antirabbit antibodies (IgG, IgA , IgM). Counterstaining was carried out using EV<lns
blue (red). Deposits of immunoglobulins are widely distributed in the peritubular capillary
walls and in th e glomerular capillary loops. A clear positivity in the cytoplasm of the tubular
epithelial cells can bc observed. x I 00
Fig. 12.17. Immunohistoch em ical analysis of a Landrace pig kidncy specimen tcsted with
normal New Zealand rabbit serum. Thc fluorescence staining was performed with goat an-
tirabbit IgG. IgG deposits arc present in th e glomerular capillary loops and in the peritubu-
lar capillary walls. Deposits or IgA were present with a similar distribution. x200
Comment 225
Fig. 12.18. Immunohistochemical analysis of a Landrace pig kidney specimen tested with
normal New Zealand rabbit serum. The fluorescence staining was performed with goat an-
tirabbit IgM. A clear IgM positivity can be observed in the cytoplasm of the tubular epithe-
lial cells, while the glomeruli and peri tubular capillaries are almost completely devoid of it.
x200
Comment
All these experimental findings support the theory that discordant hyperacute
xenograft rejection is triggered by natural preformed xenoantibody (IgG and
IgA) deposition on antigen determinants expressed by xenograft peritubular
capillary endothelium. These xenoantibody deposits activate complement ac-
tion that is then followed by a characteristic event cascade (recruitment of
polymorphonuclear leukocytes, platelet aggregation, and intravascular coag-
ulation) [57-61].
Endothelial cell antigens constitute biologically relevant targets of natural
antibodies in other xenograft models between discordant species [17, 31].
Recently, it was reported that in normal human serum, natural antibodies re-
acting with antigens on porcine endothelial cells are present [62]. These IgM
antibodies predominantly recognize carbohydrate determinants located on
glycoproteins associated with the endothelial cell membranes. The binding of
natural antibodies triggers complement activation that causes an endothelial
cell response. These events convert endothelium from its normal antithrom-
botic to a prothrombotic status. This is probably related to a dramatic loss of
heparan sulfate proteoglycan associated with endothelial cells in normal
blood vessels, leading to a loss of endothelial integrity [63].
226 Histopathological, Immunofluorescent, and Electron-Microscopic Features
While IgG and IgA promptly react with endothelial cell antigens, mainly in
the peritubular capillaries, IgM does not seem involved in this process in our in
vivo model [32,33,49,50]. Our in vitro study confirms and clarifies these data,
demonstrating no IgM deposits in the endothelial cells. However, at high mag-
nification, the immunohistochemical in vitro search for IgM shows fluores-
cence in the convoluted tubule cells (mainly on the endolumenal side), indicat-
ing IgM binding to some molecules of the brush border. Consequently, in our
discordant xenograft model IgM does not seem to playa significant role, at
least in the initial and most important phase of hyperacute rejection. The rea-
son for this would appear to be that in order to react in vivo with specific anti-
gen determinants on the brush border of convoluted tubules, IgM must cross
several structures (capillary endothelium, interstitium, and cell membrane of
tubular epithelium).
In our model, the glomeruli appear to be minimally affected. This is not
surprising, since, in discordant xenograft hyperacute rejection, the severity of
the glomerular lesions is related to the animal species utilized. It has been re-
ported that in some species the glomeruli are precociously and profoundly
damaged, while in other species (e.g., cat-to-rabbit model) a low incidence of
glomerular thrombosis is found [64]. We observed that in vivo IgG reacts
with the glomerular capillary loop structures 120 min after reperfusion,
whereas in vitro IgG reacts promptly with the glomerular endothelial net-
work. This confirms the hypothesis that the endothelial cells of glomerular
and peritubular capillaries express similar antigen determinants. It is possible
that still unclear mechanical and hemodynamic factors (e.g., blood pressure
gradient and flow) facilitate antibody deposition in peritubular capillary en-
dothelium, while preventing its deposition in the glomerular endothelium in
vivo.
The pathophysiology of acute inflammatory reactions promoted by xeno-
transplantation was first described almost 25 years ago [1, 2, 65, 66]. Although
the processes leading to hyperacute failure of the transplanted graft were dis-
cussed in the 1970s [58,67], and recently analyzed further [17, 37, 61] (and new
inflammatory mediators such as acetyl glycerol ether phosphorylcholine have
been discovered [36,68]), knowledge of the pathophysiological events leading
to hyperacute rejection and eventually to the ischemic necrosis of the
xenograft remains incomplete.
Procedures which may modulate the acute inflammatory response trig-
gered by xenotransplantation represent one of the most important goals in
present xenograft research [69-70]. We personally believe that these studies
should be aimed at achieving control of the rejection mechanism between
species that are not phylogenetically close, as it will not be possible to use pri-
mates as a source of organs for humans.
The immunofluorescent and electron-microscopic features seen in our dis-
cordant xenograft studies suggest that hyperacute rejection is initiated by pre-
formed antibodies (IgG, IgA, and later IgM) that do not react with all the
donor graft molecules, but are characterized by a specific restricted activity.
Consequently, identification of the restricted activity of these antibodies and
References 227
of the possible role of hemodynamic elements leads us to hope that their reac-
tivity can be modulated successfully to allow xenograft survival.
Acknowledgements. We would like to express our gratitude to Thomas E.

Starzl, M.D., Ph.D., Oscar L. Bronsther, M.D., and Howard R. Doyle, M.D.,
for reviewing the chapter and for their suggestions. We also wish to thank Judy
Belechak and Donna Ross for their valuable assistance in preparing the
manuscript. This study was partially supported by C.N.R. Target Project -
Biotechnology and Bioinstrumentation."
References
1. Starzl, T.E. Experience in Renal Transplantation. W.B. Saunders, Philadelphia, 1964.

2. Starzl, T.E. Experience in Hepatic Transplantation. S.B. Saunders, Philadelphia, 1969.
3. Starzl, T.E., Iwatsuki, S., Van Thiel, D.H., Gartner, J.C, Zitelli, B.J., Malatack, J.,
Schade, RR, Shavu, B.W., Hakala, T.R, Rosenthal, J.J., Porter, K.A. Evolution of liver
transplantation. Hepatology. 2,614,1982.
4. Terasaki, P.I. (ed.) Clinical Transplants. Los Angeles; UCLA Tissue Typing Laboratory,
1990.
5. Starzl, T.E., Iwatsuki, S., Esquivel, CO., Todo, S., Kam, I., Lynch, S., Gordon, RD.,
Shaw, B.W. Jr. Refinements in the surgical technique ofliver transplantation. Seminars
in Liver Disease. 5,348,1985.
6. Jamieson. N.V., Sundberg. R, Lindell, S., Ciaesson, K., Moen, J., Vreugdenhil, P.K.,
Wight, D.G.D., Southard, J.H., Belzer, F.O. Preservation of the canine liver for 24-48
hours using simple cold storage with UW solution. Transplantation. 46,517,1988.
7. Todo, S., Nery, J., Yanaga, K., Podesta, L., Gordon, R., Starzl, T.E. Extended preserva-
tion of human live grafts with UW solution. lAMA. 261,711,1989.
8. Bismuth, H., Houssin, D. Reduced-sized orthotopic liver graft in hepatic transplantation
in children. Surgery. 95,367,1984.
9. Starzl, T.E., Todo, S., Fung, J.J., Groth, C (eds.) FK-506. A potential breakthrough in
immunosuppression. Clinical implications. Transplant. Proc. 22,5,1990.
10. Starzl, T.E., Abu-Elmagd, K., Tzakis, A., Fung, J.J., Porter, K.A., Todo, S. Selected top-
ics on FK506: with special references to rescue of extrahepatic whole organ grafts, trans-
plantation of "forbidden organs", side effects, mechanisms, and practical pharmaco-
kinetics. Transplant. Proc. 23,914, 1991.
11. Evans, RW. Xenotransplantation: a panel discussion of some non-clinical issues. In:
Xenograft 25. Hardy, M.A. (ed.) Elsevier; Amsterdam, New York, Oxford, 1989, p. 359.
12. Marino, I.R, Celli, S., De Francisci, G., Perelli, L., Crucitti, F. Vascular access for
hemodialysis in small children by conversion of A-V shunt to A-V fistula. (Abstract) VII
Congress of the International Pediatric Nephrology Association. Toronto, Canada,
1989.
13. Shaw. B.W. Jr., Wood. RP., Kaufman, S.S., Williams, L., Antonson. D.L., Vanderhoof,
J. Liver transplantation therapy for children: Part 1. l. Ped. Gastr. Nutr. 7.797,1988.
14. Land. W., Hammer, C, Brynger, H. (eds.) Indication for organ transplantation.
Transplant. Proc. 18, (Supp\. 3),1,1989.
15. Merriken, K.J., Overcast, T.D. Patient selection for heart transplantation: when is a dis-
criminatory choice discrimination? l. Health Polito Policy Law. 10,7, 1985.
16. Starzl, T.E., Todo, S., Gordon, R, Makowka, L., Iwatsuki, S., Marsh, W., Esquivel, CO.,
Van Thiel, D. Liver transplantation in older patients. N. Eng/. l. Med. 165,343,1987.
17. Auchincloss, H. Jr. Xenogenic transplantation. Transplantation. 46, 1, 1988.
18. Homer. Iliad, Book XI: 832, VIII Century B.C
228 HistopathologicaL Immunofluorescent. and Electron-Microscopic Features
19. Homer. Iliad. Book VI: 175, VIII Century B.C

20. CaIne. R Y. Organ transplantation between widely disparate species. Transplant. Pmc.
2.550. 1970.
21. Ullmann. E. Experimentelle Nierentransplantation. vorhiufige Mitteilung. Wim. Klin.
Wochenschr.15.2K1.1902.
22. Princeteau, M. Grcffe renale.l. Mell. Bordeaux. 26.549.1905.
23. Unger. E. Nierentransp1antationen. Berlin Klin. Wehnsehr. 47,573. 1910.
24. Neuhof. H. The TrallSplantation of Tissues. Appleton and Co., New York, 1923.
25. Avramovici. A. Les transplantation de rein. Lyon Chir. 21.734. 1924.
26. Jaboulay. M. De reins au pli du coude par soutures arterielles et veineuses. Lyon Med.
107.575.1906.
27. Reemtsma, K., McCracken, B.H .. SchlegeL J.V .. PearL M. Heterotransplantation of the
kidney: two clinical experiences. Science. 143.700.1964.
28. Hitchcock. CR. Kiser. J.C. Tclander. RL.. Seljeskoh, E.L. Baboon renal grafts.
lAMA. lK9. 934.1964.
29. Starzl, T.E .. Marchioro. T.L.. Peters. G.N .. Kirkpatrick. CH .. Wilson. W.E.C, Porter.
K.A., Rifkind, D., Ogden, D.A .. Hitchcock. CR. WaddelL W.R. Renal heterotrans-
plantation from baboon to man: experience with six cases. Transplantation. 2.752.1964.
30. Starzl, T.E. Baboon renal and chimpanzee liver heterotransplantation. In: Xenograft 25.
Hardy. M.A. (ed.) Elsevier: Amsterdam. New York. Oxford. 1989. p. 17.
31. Forhes, R.D.C Guttman, R.D. Histopathology and mechanisms of rejection in xeno-
transplantation. In: Xenograft 25. Hardy. M.A. (ed.) Elsevier: Amsterdam. New York.
Oxford, 19K9. p. 133.
32. Marino, I.R, Feria, G., Celli, S., Stieber, A., Muttillo, 1.. Maggiano. N .. Mazzaferro. V ..
Perrelli, L., Musiani, P. Hyperacute rejection of renal discordant xenograft (pig-to-rab-
bit): model assessment and rejection mechanisms. Transplant Proc. 22. 1071,1990.
33. Marino, I.R, Ferla. G., Celli. S .. Stieher. A .. Muttillo. I.. Maggiano. N .. Perrelli, L..
Musiani, P. In vivo and in vitro study of hyperacute rejection mechanism of renal discor-
dant xenografts. Transplant Pmc. 23.620.1991.
34. Forbes, RD.S., Guttmann, RD. Pathogenetic studies of cardiac allograft rejection using
inbred rat models. Immunol. Rev. 77,5,1984.
35. Jorgensen, K.A., Kemp, E .. Barfort. P.. Starklint. H .. Larsen. S.. Munk-Anderson, G.
Platelet aggregation is not essential for xenograft rejection. Throll1h. Res. 43. K7. 1986.
36. Ito, S., Camussi, G., Tetta. C, Milgrom. F., Andres. G. Hyperacute renal allograft rejec-
tion in the rahbit. The role of platelet-activating factor and of cationic proteins derived
from polymorphonuclear leukocytes and from platelets. Lab. Invest. 51. 14K. 19K4.
37. Makowka, L.. Miller, C, Chapehap, P., Podesta, L., Pan, C, Pressley. D .. Mazzaferro, V ..
Esquivel, CO., Todo, S .. Banner, B., Jaffe, R, Saunders. R, StarzL T.E. Prolongation of
pig-to-dog renal xenograft survival by modification of the inl1ammatory mediator re-
sponse. Ann. Surg. 206.482,1987.
38. Goodall, J. Ethical concerns in the use of animals as donors. In: Xenograft 25. Hardy.
M.A. (ed.) Elsevier: Amsterdam, New York, Oxford. 1989. p. 335.
39. Sanfilippo, F. The use of non-human primates in xenotransplantation. In: Xenograft 25.
Hardy. M.A. (ed.) Elsevier; Amsterdam, New York, Oxford, 1989, p. 351.
40. Sittisombut, N., Knight, K.L. Rahhit major histocompatibility complex. 1. lmnlunol. 136.
1871,1986.
41. Kissmeyer-Nielson. F., Olsen, S., Pctcrson, V.P., Fjeldborg, O. Hypcracute rejection of
'W.·idney allografts. associatcd with pre-existing humoral antibodies against donor cclls.
Lancet. 2,662, 1966.
42. Bayuk, J.M .. Schmidlapp. CJ. Kidney transplantation in the rabbit. A method for the
study of transplantation immunology. Invest. Uro/. 4,378.1967.
43. Holter, A.R, McKearn, T.J., Neu, M.R., Fitch, F.W., Stuart. F.P. Renal transplantation
in the rabbit. 1. Development of a model for the study of hyperacute rejection and im-
munological enhancement. Transplantation. 13.244,1972.
44. Dunn. D.C Orthotopic renal transplantation in the rabbit. Transplantation. 22. 427.
1976.
References 229
45. Jacobsen, LA. Renal transplantation in the rabbit: a model for preservation studies. Lab.
Anim.12,63,1978.
46. Jorgensen, K.A, Kempe, E., Barfort, P., Starklint, H., Larsen, S., Petersen, P.H.,
Knudsen, J.B. The survival of pig-to-rabbit renal xenografts during inhibition of throm-
boxane synthesis. Thromb. Res. 32,585,1983.
47. Heron. I. Kidney transplantation in the rabbit. A new method. Acta. Path. Microbiol.
Scand. (Sect. A) 78, 90,1970.
48. Marino.I.R, Feria. G .. Celli. S.. Stieber. A .. Muttillo.l.. Perrelli, L. Studio sperimentale
di un modello originale di xenotrapianto renale. Archivio e atti della Societa Italiana de
Chirurgia, Volume Comllnicazioni, 1679, 1989.
49. Marino, I.R, Feria, G., Celli, S., Stieber, A, Muttillo, I.. Magiano, N., Perrelli, L.,
Musiani, P. Hyperacutc rejection in pig-to-rabbit kidney xenograft is due to preformed
antibodies with restricted reactivity. 1. Invest. Sllrg. In press.
50. Marino, I.R, Feria, G .. Celli, S., Stieber, A, Maggiano, N .. Castellino. F., Perrelli, L..
Musiani. P. Immunofluorescence and electron microscopy aspects of hyperacute rejec-
tion in pig-to-rabbit renal xenograft. Min. Chi,.. In press.
51. Johnston, P., Lim, S., Wright, L.. White, D. Hyperacutc xenograft rejection takes place
in the complete absence of antibodies but requires the presence of functional comple-
ment. Transplant. Proc. In press.
52. Platt, J.L., Vercellotti, G.M., Dalmasso, A.P., Matas, A.J., Bolman, RM., Najarian, J.S.,
Bach, F.H. Transplantation of discordant xenografts: a review of progress. Immunol.
Today. 11. 450.1990.
53. Okada. H .. Tanaka. H. Species-specific inhibition by glycophorin of complement activa-
tion via the alternative pathway. Mol. Immunol. 20, 1233, 1983.
54. Schilling, A., Land, W .. Pratschke, E., Piclsticker, K., Brendel, W. Dominant role of
complement in the hyperacute xenograft rejection reaction. Surg. Obstet. Gynecol. 142,
29,1976.
55. Miyagawa, S., Hirose. H .. Shirakura, R. Nakata, S., Naka, Y., Kitagawa, S., Nakano, S.,
Matsumoto. M .. Seya. T., Kitamura. H. The mechanism of discordant xenograft rejec-
tion. Transplant. Proc. 21,520,1989.
56. Guttmann, R.D., Forbes, RD.e, Cramer, D.V., Gill, T.G. III Cardiac allograft rejection
and enhancement in natural recombinant rat strains. Transplantation. 30,216,1980.
57. Williams, G.M., Lee, H.M., Weymouth, RF., Harlan, W.R Jr., Holden, K.R, Stanley,
eM., Millington, G.A., Hume, D.M. Studies in hyperacute and chronic renal homograft
rejection in man. Surgery. 62,204,1967.
58. Giles, G.R, Boehmig, H.1 .. Lilly. J .. Amemiya. H .. Takagi. H .. Coburg. A.1., Starzl. T.E.
Mechanism and modification of rejection of heterografts between divergent species.
Transplant. Proc. 2,522, 1970.
59. Boehmig, H.J., Giles. G.R, Amemiya. H. Wilson, CB .. Coburg, A.J., Genton. To,
Bunch, D.L., Dixon, F.J., StarzL T.E. Hyperacute rejection of renal homografts: with
particular references to coagulation changes, humoral antibodies and formed blood ele-
ments. Transplant. Proc. 3, 1105, 1971.
60. Myburgh, J.A. Cohen. 1.. Gecetter. L.. Meyers. AM .. Abrahams. C. Furman. K.l ..
Goldberg. B., Van Blerk. P.I.P. Hyperacute rejection in human kidney allografts:
Schwartzman or Arthus reaction? N. Engl. 1. Med. 281. 131. 1969.
61. Makowka, L., Chapman, F .. Cramer. D .. Sher. L.. Podesta. L.. Howard. T.. StarzL T.E.
The role of inflammatory reactions in xenotransplantation. In: Xenograft 25. Hardy.
M.A. (ed.) Elsevier; Amsterdam, New York, Oxford, 1989. p. 159.
62. Platt. J .L., Lindman. B.L Chen, H .. Spitalnik, S.L., Bach. F.H. Endothelial cell antigens
recognized by xenoreactive human natural antibodies. Transplantation. 50.817.1990.
63. Platt, J.L., Vercellotti. G.M., Lindman, B.J., Oegema, T.R, Bach. F.H .. Dalmasso. A.P.
Release of heparan sulfate from endothelial cells. Implications for pathogenesis of hy-
peracute rejection. 1. Exp. Med. 171.1363.1990.
64. Kemp, E., Kemp, G., Jacobsen, Ib.A., Lundborg, eJ. Prolongation of survival ofrenal
xenografts by infusion of donor blood. Acta. Path. Microbia!. Scand. 85,267,1977.
65. Starzl, T.E .. Lerner. R.A., Dixon, F.K., Froth, CG .. Brettschneider, L.. Terasaki. P.1.
230 Histopathological. Immunofluorescent. and Electron-Microscopic Features
Schwartzman reaction after human renal transplantation. N. Engl. 1. Med. ns. 642.
1965.
66. Williams, G.M., Hume, D.M., Hudson, RP. Jr., Morris, P.L Kano. K .. Milgrom. F.
··Hyperacute·· renal-homograft rejection in man. N. Eng/. 1. Med. 279,611, 1965.
67. Mejia-Laguna, J.E., Martinez-Palomo. A .. Lopez-Soriano. F., Garcia-Cornejo, M .. Biro.
C.E. Prolonged survival of kidney xenografts in leukopenic rabbits. Imlllilnulogr. 21.
S73. 1971.
6S. Pinckard, RN. The ··new·· chemical mediators of int1ammation. In: Currell! Topics in
Inflamlllation and Infection. Majno, G., Cotran, R.S., Kaufman, N. (eds.) Monogr.
Patho/. 23,38. 19S2.
69. Council on Scientific Affairs. Xenografts. Review of the literature and current status .
.lAMA. 254.3353, 19S5.
70. Shapiro. R, Tzakis, A.G., Scantlebury, Y., Makowka, L., Watt. R, Oks, A., Yanaga, K.,
Podesta, L.. Casavilla, A., Wos, S., Murray, J. Opal. A., D'Andrea. P., Banner, B., StarzL
T.E. Immunodepletion in xenotransplantation . .I. Invest. Surg. 3,39, 1990.
Chapter 13
Histopathology of Cardiac Xenograft Rejection

A.G.ROSE
Introduction
Widespread acceptance of cardiac transplantation as the treatment for end-

stage heart failure has placed ever increasing demands upon human organ
donation. Despite increased public acceptance of organ donation, human
sources of transplanted hearts continue to be insufficient to meet the demand.
Whilst formidable problems, particularly that of hyperacute rejection, stand in
the way of cross-species organ transplantation, the pressing paucity of human
donor hearts has led to renewed interest in xenotransplantation and, conse-
quently, in finding a means of preventing hyperacute rejection.
Although the pathology of both acute and chronic cardiac allograft rejec-
tion has been well documented [1, 2], the morphology of hyperacute (anti-
body-mediated, vascular, humoral) rejection is less well known, since cross-
species transplants have seldom been used clinically. Cardiac xenografts have
been implanted in isolated instances in humans in attempts to save the life of a
patient for whom no human donor heart was immediately available [3-5]
(Chap. 34). Many experiments have been performed to try and further our un-
derstanding of the etiology and pathogenesis of humoral rejection [6-36]. Our
present knowledge of the pathology of humoral rejection is thus mainly based
upon the findings in experimental animals.
Acute rejection results from the development of cellular immunity which is
mediated via T lymphocytes; acute rejection is therefore rarely encountered in
the first week after cardiac transplantation. Macrophages and natural killer
(NK) cells may also playa role in mediating acute cardiac rejection [37]. In
contradistinction, hyperacute (and possibly chronic) cardiac rejection appears
to be antibody-mediated.
Hyperacute rejection is believed to be initiated by the presence of pre-
formed antibodies which may have resulted from prior antigenic stimulation
by blood transfusions, pregnancy, or previous tissue grafts or be associated
with ABO blood group incompatibility or cross-species differences. The latter
is presently not a clinical problem (since cardiac xenografts are not routinely
used to treat patients), but pathologists may encounter hyperacute rejection
where cross-species experimental transplants have been performed. Whether
there are significant morphological differences between the hyperacute rejec-
tion that occurs in allografts and that which occurs in discordant xenografts re-
mains as yet unclear.
232 Histopathology of Cardiac Xenograft Rejection
Varying levels of antibody may be present in one species against a closely

related species. As a general working principle. the greater the phylogenetic
disparity of the two species. the greater will be the titer of species-specific anti-
body. Antibody-mediated rejection will therefore generally take place rapidly
(within a few minutes or hours) when an organ is transplanted between widely
disparate species. but may take several days to develop in more closely related
species. There is increasing evidence. however. that even when transplanta-
tion is between closely related species the presence or development of anti-
bodies plays a significant role in the development of rejection.
In our own studies. we not infrequently observed typical histopathological
features of antibody-mediated rejection several days after transplantation of
the organ. Such rejection. though clearly antibody-mediated and identical to
that seen in early hyperacute rejection. is best termed vascular or humoral re-
jection to differentiate it from the hyperacute rejection that occurs within a
few hours of transplantation.
In order to gain an understanding of the pathogenesis of hyperacute rejec-
tion. animal experimentation has focused upon various components that may
be important in the humoral rejection process. Whilst neutrophils have been
regarded as an important component of hyperacute rejection analogous to
their role in the Arthus phenomenon, hyperacute cardiac allograft rejection in
the rat model shows the usual pattern of vascular and myocardial damage in
the absence of polymorphonuclear leukocytes. In our own hyperacutely re-
jected xenografts. in which extensive interstitial hemorrhage was the predom-
inant feature, a significant number of neutrophils accompanied the interstitial
hemorrhage. The ratio of white to red blood cells was the same as that encoun-
tered in the peripheral blood. Forbes et al. [17] do not exclude the possibility
that neutrophils may playa role in the production of myocardial injury in the
later stages of hyperacute rejection.
Others have focused upon the role that various components of complement
may play in the pathogenesis of hyperacute rejection [23]. Experiments in C6-
deficient rabbits showed that activity of the sixth component of complement is
needed for hyperacute rejection to occur. Likewise, in studies on recipient
hemolytic C3 activity it has been shown that complement activation by graft-
bound antibody is an important effector mechanism for humoral rejection in
an inbred rat model [35]. Rats with a hereditary platelet function defect are
able to effect hyperacute rejection [17]. The role of antibody to the vascular
endothelial cells in hyperacute rejection in patients undergoing cardiac trans-
plantation is receiving attention [38, 39].
Macroscopic Appearances ofthe Donor Heart During

Early Hyperacute Rejection
Heterotopically transplanted hearts, whether they are from closely related or
widely disparate donor species, usually begin beating satisfactorily in sinus
rhythm, either spontaneously or after electrical defibrillation. The hyper-
Histopathology 233
acutely rejecting heart initially becomes swollen and distended with blood
over a period of only a few minutes. In some cases a thrombus forms within the
pulmonary artery, and blood ceases to flow through the organ. The normal
pink color of the myocardium is replaced by a dusky hue.
Histopathology of Xenograft Rejection
Some concordant xenografted hearts, when heavily immunosuppressed with

pharmacologic agents (Table 13.1), undergo typical cellular rejection. In our
experience, however, unmodified cellular rejection is not common in nonim-
munosuppressed cardiac xenograft models.
Hyperacute (Antibody-Mediated, Vascular, Humoral) Rejection
The preformed antibodies which generate vascular rejection come into imme-
diate contact with the donor heart's vascular endothelium where, in associa-
tion with complement deposition, they may cause direct injury to or destroy
the endothelial lining cells. The classical concept is that, if the antibody titer is
high enough, such damage to the vascular endothelium may occur within min-
utes or hours following transplantation. Recent experimental work suggests
that the humoral rejection response may be suppressed in some closely related
animal models for days or weeks by a combination of certain drugs, some of
which are not yet available for clinical use [32,36].
Loss of the endothelial lining will lead to the deposition of platelet and/or
fibrin thrombi on the exposed basement membrane in keeping with Virchow's
triad of factors predisposing to intravascular thrombosis. Thrombosis may
also be triggered by the formation of immune complexes within the
xenograft's microcirculation. Surviving endothelial cells may show prominent
mitotic activity in an attempt to re-endothelialize the vessel; such attempts are
usually inadequate.
The changes in the small blood vessels, particularly in the capillaries, lead
to increased permeability resulting in prominent interstitial edema in the graft
(Figs. 13.1, 13.2). Since the integrity ofthe capillaries is very dependent upon
the intactness of the endothelial lining cells, hyperacute rejection in
xenografts is characterized by widespread dissolution of capillaries with resul-
tant extensive interstitial hemorrhage within the graft (Figs. 13.1, 13.3, 13.4).
When it occurs, hyperacute rejection is usually severe and the combination
of microcirculatory obstruction by thrombi plus capillary destruction rapidly
produces severe functional disturbance of the graft with cessation of heart
beat. Sometimes the graft becomes totally necrotic, making histological as-
sessment difficult. Our experience in xenografting performed between donor
monkeys or pigs and recipient baboons is that microvascular thrombi are
rarely observed by light microscopy, whereas massive capillary destruction
Table 13.1. Histopathology of rejection and survival periods of selected heterotopic cardiac allografts and xenografts in the baboon N
w
....
Group n Type of rejection a Graft survival
:::r::
(;;.
Vascular Mixed Cellular None Mean (days) SO
v
0"
~
Allografts (baboon-to-baboon) ;.
1. No IS 10 0 0 10 0 11.0 5.6 0
0"
(JQ
2. ABO incompatible, no IS 9 2 2 5 0 12.4 11.7
'<
3. CSA,MP 10 0 0 1 9 (I) >30.0 S,
4. ABO incompatible, CSA MP 8 2 1 4 (1) I 22.5 12.0 n
~
...,
Concordant Xenografts (vcrvet monkey-to-baboon) n.
p;;'
5. No IS 9 5 4 0 0 10.3 5.2 (')
6. ABO incompatible, no IS 9 3 2 4 0 7.3 5.6 ><

(!)
::l
7. CSA,AZA,MP 6 1 0 5 (2) 0 13.0 8.2 0
(JQ
8. ABO incompatible, CSA. AZA MP 5 I 1 3 (I) 0 11.4 11.2 ...,
~
9. CSA, AZA MP i.v.MP 5 I 0 3 I (I) 19.0 21.8 ;:::>
10. RATG, CSA, AZA, MP therapy for 6 I 0 2 3 (3) 43.3 18.5 ;;0
..£l:.
11. IS-OS, CSA, AZA, MP rejection 7 I 0 2 4 (4) 20.1 11.5 (!)
(')
12. 15-DS, CSA, MP episodes 5 0 3 I I (I) 35.6 14.2 -o·
13. TLI,CSA,AZA,MP 5 0 1 (I) 3 (3) I (I) 16.2 9.8 ::l
14. ABO incompatible, TLI, CSA AZA, MP 5 2 (1) 0 I (I) 2(1) 17.8 10.5
~iscordant Xenografts (pig-to-baboon) Individual Experiments
IS. No IS 4 4 0 0 0 40,60, 180.480 min
16. Splenectomy 3 3 0 0 () 30.360.480 min
17. CSA AZA, MP 5 5 0 () () 15,15,40,75 min+5 days
18. Antibody adsorption 7 7 0 0 0 36(),48() min+0.5, 4, 4, 4, 5 days
19. Antibody adsorption, CSA, AZA MP 4 (1) 4 () 0 0 480 min+0.5, L 4 days
IS, Immunosuppressive therapy: CSA cyclosporine: AZA azathioprine: MP, methylprednisolone: IS-OS, 15-deoxyspergualin: TLI. pretransplant
total lymphoid irradiation: RATG, rabbit antithymocyte globulin.
a Figures in parentheses denote number of recipients that died.
Histopathology 235
Fig. 13.1. Massive interstitial edema and moderate hemorrhage in a hyperacutely rejected
cardiac xenograft (vervet monkey-to-baboon), H & E, x150
Fig. 13.2. Hyperacute cardiac rejection showing fibrin thrombi occluding capillaries, severe
interstitial edema with some hemorrhage. The myocytes show contraction band necrosis
(vcrvet monkey-to-baboon), H & E, x150
236 Histopathology of Cardiac Xenograft Rej ection
Fig. 13.3. Severe interstiti al hemorrhage with mild edema due to hyperacute rejection fol-
lowing clinical cardiac xenotransplantation (chimpanzee-to-man), H & E, x l50
Fig. 13.4. Seve re hyperacute rejection with hemorrhage a nd loss of or dam age to myocyt es
(vervet monkey-to-baboon) , H & E, x150
Histopathology 237
Fig. 13.S. Mixed cellular and vascular rejection is characterized by massive interstitial edema
and hemorrhage leading to wide separation of bundles of myocytes plus focal collections of
lymphocytes in relation to small blood vessels (vervet monkey-to-baboon), H & E, x80
Fig. 13.6. High-powe r view of mixed ce llular and vascular rejection with focal (centrally situ-
ated) groups of lymphocytes surrounded by severely edematous, hemorrhagic myocardium
(vervet monkey-to-baboon), H & E, x150
with severe interstitial hemorrhage is the usual pathology characterizing hy-

peracute rejection (Figs. 13.3, 13.4). Secondary degenerative changes are evi-
dent in the myocytes (Fig. 13.4) and contraction band necrosis may be evident.
Mixed Cellular (Acute) and Vascular (Antibody-Mediated, Humoral,

Hyperacute) Rejection
Mixed cellular and vascular rejection [34] (Figs. 13.S, 13.6) is characterized by
prominent focal, perivascular collections of lymphocytes, which extend in a
limited fashion between adjacent myocytes. Elsewhere, the myocardium
shows typical features of humoral rejection, as detailed earlier. The modest
number of lymphocytes present does not appear to be sufficient to explain the
very extensive microvascular damage or the myocyte necrosis on the basis of
either moderate or severe acute rejection. Lymphocytic infiltration has not
been a characteristic feature of hyperacute rejection in our experimental non-
immunosuppressed cardiac xenografts (nor was it seen in two clinical
xenografts - Chap. 34).
Modification of Xenograft Rejection by Pharmacological

Immunosuppression
In Concordant Xenografts (Vervet Monkey-to-Baboon)
Survival of vervet monkey hearts transplanted into ABO-compatible nonim-

munosuppressed baboons (Table 13.1, Group S) did not differ significantly
from survival of allografts. Vascular or mixed cellular and vascular rejection
was seen, however, in all cases, and rejection within 24 h occurred in one ani-
mal. When xenografting was performed between ABO-incompatible pairs
(Group 6), mean survival time was reduced mainly through the occurrence of
hyperacute rejection occurring within 1 h in 30% of the hearts.
Post-transplant immunosuppression with cyclosporine (CSA), azathio-
prine (AZA), and methylprednisolone (MP) did not significantly prolong car-
diac xenograft survival in either ABO-compatible (Group 7) or ABO-incom-
patible (Group 8) pairs, and there was still an incidence of vascular rejection,
particularly in the ABO-incompatible group. When rejection episodes were
treated by bolus MP therapy (Group 9), graft survival was significantly ex-
tended.
The addition of rabbit antithymocyte globulin therapy to the maintenance
immunosuppressive regimen and antirejection therapy in the form of bolus
MP extended graft survival significantly (Group 10), but there was a SO% re-
cipient mortality from infection, diarrhea and other side effects. When main-
tenance CSA, AZA, and MP therapy was combined with IS-deoxyspergualin,
and rejection episodes again treated with bolus MP (Group 11), graft survival
Comment 239
was prolonged but four of the seven recipients (57% ) died of treatment -relat-
ed complications such as infection or diarrhea. When 15-deoxyspergualin was
combined with maintenance CSA and MP only, and MP used for acute rejec-
tion episodes (Group 12), there was a reduction in the treatment-related mor-
tality to 20%, and subsequently a prolongation ofxenograft survival.
In the myocardial biopsies taken from the grafted hearts in these four
groups (Groups 9-12), acute (cellular) or mixed rejection predominated,
though pure vascular rejection was still seen in Groups 9-11. The mean num-
ber of rejection episodes seen in these four groups was as follows: Group 9,1.5
episodes; Group 10,2.5 episodes; Group 11,0.5 episodes; and Group 12, 0.8
episodes per animal.
In Discordant Xenografts (Pig-to-Baboon)
Discordant xenografting between pig and baboon resulted in histopathologi-

cal features of hyperacute rejection in every case. Neither splenectomy (Table
13.1, Group 16) nor pharmacologic immunosuppression (Group 17) increased
mean survival time of the grafts, but pretransplant hemoperfusion of donor
kidneys by the recipient baboon (to adsorb out the preformed antibodies on
the kidney before exposing the heart) increased graft survival significantly in
four of seven donor hearts (Group 18). Nevertheless, all 4 hearts showed typi-
cal features of antibody-mediated rejection at the time that function ceased,
even though cessation of function took 4 or 5 days to occur. The addition ofim-
munosuppression to pretransplant hemoperfusion (Group 19) did not lead to
any further prolongation of graft survival.
Comment
The significant progress that has been made in the relatively short time since
clinical heart transplantation was first performed, leads one to feel optimistic
that the problems militating against the use of animal hearts for human cardiac
transplantation will be ultimately overcome.
From the point of view of the histopathologist, concordant xenografts
would appear to hold a more promising potential than discordant xenografts
for clinical application. The presently available pharmacologic immunosup-
pressive agents showed some potential in overcoming rejection between con-
cordant xenografts, but showed no success with discordant xenografts.
Humoral factors, which might lead to vascular rejection, appear to playa less
important role in concordant xenografting, and the drugs presently available
appear to have some effect in suppressing these factors.
It seems unlikely, however, that nonhuman primates will prove to be suit-
able donors for man. In general, the baboon does not grow to a size large
enough to make it a suitable heart donor for adult humans, though there may
240 Histopathology of Cardiac Xcnograft Rejcction
be a role for this animal as a donor for children. Other higher primates are in
short supply themselves, and the numbers will be insufficient for transplanta-
tion purposes. There will, in addition, almost certainly be ethical and moral
objections to the use of such animals.
Attention must therefore be directed to xenotransplantation between ani-
mals of widely different species, though transplantation will be greatly compli-
cated by the development of hyperacute rejection, which the present study has
shown to be virtually uniform when transplantation is performed across this
wide species barrier. Transplantation between discordant species clearly
awaits the development of a satisfactory method of removal of preformed an-
tispecies antibodies before success is likely to be achieved.
References
1. Rose. A.G .. Uys. C.1. Pathology of acute rejection. In: D.K.C. Cooper and D. Novitzky
(cds.) The Transplantation and Replacement of Thoracic Organs. Kluwcr: Dordrecht.
Boston. London. 1990. p. 115.
2. Rose. A.G .. Uys. c.J. Pathology of graft atherosclerosis (chronic rejection). In: D.K.C.
Coopcr and D. Novitzky (eds.) The Transplantation and Replacement of Thoracic
Organs. Kluwer: Dordrecht. Boston. London, 1990, p. 161.
3. Hardy, J.o .. Chavez, C.M., Kurrus, F.E., Neely, W.A., Webb, W.R .. Eraslan, S .. Turner.
M.D., Fabian, L.W., Labecki, J.D. Heart transplantation in man: development studies
and report of a casc..!. ArneI'. Med. Assoc. 188,1132,1964.
4. Barnard, C.N., Wolpowitz, A., Losman, J.G. Heterotopic cardiac transplantation with a
pass. S. Atr. Med. 1.52,1035,1977.
5. Bailey, L.L., Nelsen-Cannarella, S.L., Concepcion, W .. Jolley. W.B. Baboon-to-human
cardiac xenotransplantation in a neonate. 1. Amer. Mell. Assoc. 254,3321. 1985.
6. Forbes. R.D.C., Guttmann, R.D .. Kuramochi. T., Klassen. 1., Knaack, 1. Non-essential
role of neutrophils as mediators of hyperacute cardiac allograft rejection in the rat. Lab.
invest. 34.229.1976.
7. Forbes, R.D.C.. Kuramochi, T., Guttmann, R.D., Klassen, 1., Knaack, 1.A. A controlled
sequential morphologic study of hyperacute rejection in the rat. Lab. Invest. 33, 280,
1975.
8. Forbes, R.D.C.. Guttmann, R.D. Mechanisms of humoral mediated cardiac allograft re-
jcction in vivo: controlled comparative morphological studies utilizing inbred rat mod-
els.1. Heart Transplant. 1,196,1982.
9. Sharma, H.M., Rosenweig, 1., Chatterjee, S., Moore. S., De Champlain. M.L. Platelets in
hyperacutc rejection of heterotopic cardiac allografts in presensitizcd dogs. Am. 1.
Pathol. 70, 155, 1973.
10. Cavcs, P.K., Dong, E .. Morris, R.E. Hypcracute rcjection of orthotopic cardiac allo-
grafts in dogs following solubilized antigen prc-treatment. Transplantation. 16, 252,
1973.
11. Guttmann, R.D. Genetics of acute rejection of rat cardiac allografts and a model of hy-
peracute rejcction. Transplal1tation. 17,383,1974.
12. Kuwahara, 0 .. Kondo, y, Kuramochi. T .. Grogan. 1.B .. CoekrclL J.V .. Hardy. J.D.
Organ specificity in hyperacute rejection of canine heart and kidney allografts. Ann.
SlIrg. 180.72,1974.
13. Cattell, V., Jamieson, S.W. Hyperacute rejcction of guinea-pig to rat cardiac xenografts.
I. Morphology..!. Patho/. 115.183,1975.
References 241
14. Corry, RJ., Kelly, S.E. Survival of cardiac xenografts. Effect of antithymocyte serum
and enhancingheteroserum. Arch. Surg. 110,1143,1975.
15. Jamieson, S.W. Modification ofthe hyperacute reaction in the rat by sulphinylpyrazone.
Thromb. Diath. Haemorrh. 30,349,1975.
16. Whittum, J.A., Lindquist, RR Mechanism of cardiac allograft rejection in the inbred
rat: the effect of complement depletion by cobra venom factor on hyperacute cardiac al-
lograft rejection. Transplantation. 24,226,1977.
17. Forbes, RD., Guttmann, RD., Bazin, H. Hyperacute rejection of cardiac allografts in a
rat strain with a hereditary platelet function defect. Lab. Invest. 37, 158, 1977.
18. Guttmann, RD. In vitro correlates of rejection. I. Suppressive effect and specificity in
mixed lymphocyte interaction of alloantiserum producing hyperacute rejection.
Transplant. Proe. 9,755,1977.
19. Forbes, RD., Guttmann, RD., Kuramochi, T. Controlled studies of the pathogenesis of
hyperacute cardiac allograft rejection in actively immunized recipicnts. Transplant.
Proc.9,301,1977.
20. Forbes, RD., Guttmann, RD., Pinto-Blonde, M. A passive transfer model of hyper-
acute rat cardiac allograft rejection. Lab. Invest. 41,348,1979.
21. Guttmann, RD., Bazin, H. Lack of significance of allograft differences in hyperacutc rc-
jection ofrat cardiac allografts. Transplantation. 28, 155, 1979.
22. Coleman, D.A., Eichwald, E.J. Hyperacute rejection of allografted murine hearts and
the white graft reaction. Transplantation. 26,355,1978.
23. O'Regan, CC, Robitaille, P .. Pinto-Blonde, M., Chartrand, C Delayed rejection of car-
diac xenografts in C6-deficicnt rabbits. Immunology. 38,245,1979.
24. Lcxer. G., Cooper. D.K.C, Rose, A.G., Wicomb, W.N., Rees, J., Keraan, M., du Toit, E.
Hyperacute rcjection in a discordant (pig-to-baboon) cardiac xenograft model. 1. Heart
Transplant. 5,411,1986.
25. Lexer, G., Cooper, D.K.C, Wicomb, W.N., Rose, A.G., Rees, J., Keraan, M., Reichart,
B., du Toit, E. Cardiac transplantation using discordant xenografts in a non-human pri-
mate model. Transplant. Proe. 19,1153,1987.
26. Cooper, D.K.C, Lexer, G., Rose, A.G., Rees, J., Keraan, M., du Toit, E., Oriol, R
Cardiac allograft survival in ABO blood group incompatible baboons. Transplant. Proc.
19,1036,1987.
27. Cooper, D.K.C, Human, P.A., Rcichart, B. Prolongation of cardiac xenograft (vervet
monkey-to-baboon) function by a combination of total lymphoid irradiation and im-
munosuppressive drug therapy. Transplant. Proe. 19,4441,1987.
28. Cooper, D.K.C, Human, P.A., Rose, A.G. Is ABO compatibility essential in xenograft-
ing between closely related species? Transplant. Proe. 19,4437,1987.
29. Reemtsma, K., Pierson, RN .. Marboe, CC, Michler, R.E., Smith, CR, Rose, E.A.,
Fenoglio, J.J. Will atherosclerosis limit clinical xenografting? Transplant. Proc. 19,108,
1987.
30. Cooper, D.K.C, Human, P.A., Lexer, G., Rose, A.G., Rees, J., Keraan, M., du Toit, E.A
Effects of cyclosporine and antibody adsorption on pig cardiac xenograft survival in the
baboon. 1. Heart Transplant. 7,238, 1988.
31. Cooper, D.K.C, Rose, A.G., Lcxer, G., Keraan, M., Rees, J., du Toit, E., Oriol, R
Cardiac allotransplantation across major blood group barriers in thc baboon. 1. Med.
Primatol. 17,333,1988.
32. Reichenspurner. H., Human, P.A., Boehm, D.M., Rose, A.G., May, R .. Cooper, D.K.C,
Zilla, P., Reichart, B. Optimalization of immunosuppression after xenogeneic heart
transplantation in primates. 1. Heart Transplant. 8,200,1989.
33. Cooper, D.K.C, Human, P.A., Rose, A.G., Rees, J., Keraan, M., Reichart. B., du Toil.
E., Oriol, R The role of ABO blood group compatibility in heart transplantation be-
tween closely related animal species. 1. Thorae. Cardiovasc. Surg. 97,447,1989.
34. Rose, A.G., Cooper, D.K.C, Human, P.A., Reichenspurner, H., Reichart, B.
Histopathology of hyperacute rejection of the heart - experimental and clinical observa-
tions in allografts and xenografts. 1. Heart Transplant. In press.
35. Forbes, RD., Pinto-Blonde, M., Guttmann, RD. The effect of anticomplementary co-
bra venom factor on hyperacute rat cardiac allograft rejection. Lab. Invest. 39. 463.
197K
36. Reichenspurner, H .. Human. P.A.. Rose. A.G .• Reichart. B .. Cooper. D.K.C. Effect of
pharmacologic immunosuppression on donor heart survival in a closely related nonhu-
man primate xenograft model. Transplant. Proc. 22. 1086. 1990.
37. Knopf. W.D .. Gravanis. M.B. Cardiac transplantation. In: Cardiovasclliar Patho-
physiology. M.B. Gravanis (Ed.). New York. McGraw Hill. 1987. p. 298.
38. Trento. A .. Hardesty. R.L.. Griffith. B.P .. Zerbe. T.. Kormos. R.L.. Bahnson. H.T. Role
of the antibody to vascular endothelial cells in hyperacute rejection in patients undergo-
ing cardiac transplantation.J. Thorae. Cardiovasc. Sllrf? 95.37. 1988.
39. Hammond. E.H .. YowclL RL.. Numoda. S.. Menlove. RL.. Renlund. D.G .. Bristow.
M.R .. Gay. W.R.JR.Jones. K.W .. O·Connell.J.B. Vascular (humoral) rejection in heart
transplantation: pathologic observations and clinical implications.i. Heart Transplaill. 8.
430.1989.
Chapter 14
Ultrastructure of Hyperacute Rejection

in Cardiac Xenografts
A.G. ROSE AND D.K.C. COOPER
Introduction
Little is known about the light microscopic appearances of hyperacute (vascu-

lar, humoral) cardiac rejection (Chap. 13). Data available are mainly based
upon animal experimentation [1-5] and experience with the occasional hyper-
acute rejection of human cardiac allografts [6-8]. Even less is known about the
ultrastructural features [1,2,9-13].
To our knowledge, there is no published information regarding the ultra-
structural features of this form of rejection in porcine cardiac xenografts im-
planted in dogs or baboons. This study examines these features in six porcine
xenografts implanted in three canine and three baboon recipients (discordant
xenografts). We also report briefly on the electron microscopic features in ba-
boon and chimpanzee donor hearts which were implanted in two human pa-
tients (concordant donors).
Hyperacute rejection ofxenografts is thought to be due to species-specific
humoral antibody which is present in the recipient at the time of transplanta-
tion, and which binds to histocompatibility determinants within the graft. This
initiates a complement -dependent form of rapidly progressive tissue destruc-
tion [13]. Since hyperacute rejection is believed primarily to involve the vascu-
lature of the graft, in this study particular attention was paid to changes in the
microcirculation.
Materials and Methods
Table 14.1 summarizes the donor-recipient combinations that were studied.

Outbred Landrace pigs, weighing 10-18 kg, were used as donors in the six ani-
mal experiments. Mongrel dogs, weighing 20-25 kg, were used as recipients in
three experiments, and Chacma baboons (Papio ursinus), weighing 15-30 kg,
as recipients in the other three experimental cardiac transplants. The opera-
tive technique was a modification ofthat described by Mann et al. [14]; hetero-
topic heart transplantation was performed in the neck. The donor heart was
excised after infusion of cardioplegic solution (15 mllkg body weight) at 4°C.
Donor heart ischemic periods varied between 30 and 60 min.
244 Ultrastructure of Hyperacute Rejection in Cardiac Xenografts
Table 14.1. Ultrastructural findings in cardiac xenografts
Donor/recipient Duration of Platelet Fibrin Capillaries Myocardium

graft function thrombi thrombi
Pig/dog 8min ++ 0 Intact Contraction

bands
Pig/dog 10min +++ 0 Intact Contraction
bands
Pig/dog 13 min + + Intact Contraction
bands
Pig/baboon 2h 0 + Intact Normal
Pig/baboon 3h 0 0 Endothelial Abnormal
cell swelling mitochondria
Pig/baboon 4h 0 0 Necrosis Necrosis
Baboon/human 6h 0 0 Endothelial Edema
cell swelling
Chimpanzee/human 4 days 0 0 Intact Edema
+, few; ++, moderate; +++, extensive
The cardiac xenografts (one baboon, Papio ursinus, and one chimpanzee,
Pan troglodytes) in the human patients were heterotopically transplanted in
the right side of the chest [15] (Chap. 34). Only the patient who received the
chimpanzee heart lived long enough to receive high doses of immunosuppres-
sive drugs (azathioprine, corticosteroids, and antithymocyte globulin).
Samples for light and electron microscopy were taken from the two clinical
xenografts shortly after the death of the two human recipients (at 6 hand 4
days, respectively) and from the experimental xenografts at the time of cessa-
tion of donor pig heart function (mean of 10 min in the dog and 3 h in the ba-
boon) [16-18]. Samples for electron microscopy were collected in 5%
buffered glutaraldehyde, postfixed in osmic acid, and embedded in araldite.
Ultrathin sections were stained with uranyl acetate and lead citrate and exam-
ined in a Hitachi electron microscope. The light microscopic appearances in
the pig-to-baboon studies and in the two human patients have been reported
elsewhere [17] (Chap. 13).
Results
Pig Cardiac Xenografts in Dogs and Baboons
The experimental xenografts showed the following ultrastructural changes

(Table 14.1). One pig donor heart in a baboon was totally necrotic, possibly
due to hyperacute rejection. The general cytoarchitecture was so badly dam-
aged and poorly preserved in this case that no specific changes could be detect-
Results 245
Fig. 14.1. Electron photomicrograph showing early platelet aggregation within a capillary of
a hypcracutely rejecting cardiac xenograft (pig-to-dog). (Original magnification, xIS (00)
ed. The remaining five hearts showed varying combinations of capillary ob-
struction due to occlusive platelet thrombi (Figs. 14.1-14.4), fibrin deposition
(Fig. 14.5), and massive endothelial cellular swelling in one graft. Three hearts
showed contraction banding of myocytes, one demonstrated swollen, dis-
torted mitochondria, some of which exhibited loss of matrix, and in one the
myocardium was normal.
246 Ultrastructure of Hyperacute Rejection in Cardiac Xcnografts
Fig. 14.2. Electron photomicrograph showing vascular (hyperacute) rejection of a pig heart
10 min after transplantation into a dog. Aggregated platelets can be seen within a capillary.
which also shows a prominent endothelial cell nucleus. (Original magnification. x12 000)
The appearances were similar irrespective of the host species, though the
changes developed more rapidly in dogs (mean 10 min) than in the baboon
(mean 3 h) . Platelet thrombi were a more marked feature in the porcine
xenografts in the dogs than in those in baboons.
Results 247
Fig. 14.3. Electron photomicrograph showing severe capillary lumenal obstruction due to
aggregation of platelets, many of which appear degranulated (pig-to-dog). (Original magni-
fication , x 12 000)
Baboon and Chimpanzee Hearts in Man
The baboon donor heart that was transplanted into an adult female human
patient proved to be too small to support the recipient's circulation and fibril-
lated after 6 h. Light microscopy revealed evidence of contraction band ne-
crosis due to the effects of catecholamine excess and partial ischemia.
248 Ultrastructure of Hyperacute Rejection in Cardiac Xenograrts
Fig. 14.4. Electron photomicrograph showing degranulated , aggregated platelets occluding

most of the lumen of a capillary in a hyperacutcly rejecting porcine cardiac xenograft in a
canine recipient 13 min after transplantation. (Original magnification , xl2 000)
Ultrastructural examination revealed intact capillaries with basophils within

some of the capillaries examined in the samples. Apart from contraction band-
ing in some of the myocytes, the myofilaments and Z-bands were well pre-
served. Mitochondria showed some autolytic changes.
The chimpanzee heart that was transplanted into an adult male human pa-
tient supported the circulation for 4 days before mild hyperacute rejection of
Results 249
Fig. 14.5. Electron photomicrograph showing the lumen of a capillary to be occupicd by

dark-staining fibrin strands which lie between two erythrocytes. Vascular rejection of
porcine cardiac xenograft in a baboon at 3 h. (Original magnification, x15 000)
this relatively small donor heart led to death of the patient from heart failure.
Light microscopy was characterized by evidence of focal capillary destruction
with extravasation of erythrocytes (Chap. 13). The myocytes appeared well
preserved ultrastructurally, and no significant changes were noted in the small
vessels included in the ultrastructural sample.
250 Ultrastructure of Hyperacute Rejection in Cardiac Xenografts
Discussion
The ultrastructural features of hyperacute rejection appear to be dominated

by occlusion of the capillary circulation by platelet microthrombi, though this
was more marked in the dog recipients than in the baboon. In addition, fibrin
was present within some of the capillaries. Massive endothelial cellular
swelling produced significant lumenal obstruction in one graft. Destruction of
capillaries, which is a dominant feature of hyperacute rejection on light mi-
croscopy in grafts examined once contractile function has ceased (Chap. 13),
was surprisingly not detected by ultrastructural examination. This may be a re-
flection of the timing of sampling, or be related to focal differences, since light
microscopy of the chimpanzee heart revealed scanty capillary platelet thrombi
which were not detected on electron microscopy. The later destruction of
capillaries with development of interstitial hemorrhage would appear to be a
separate event to the intralumenal obstruction produced by platelets and/or
fibrin.
References
1. Forbes, RD., Guttmann, RD., Bazin, H. Hyperacute rejection of cardiac allografts in a

rat strain with a hereditary platelet function defect. Lab. Invest. 37, 158,1977.
2. Mullerworth, M.H., Lixfield, W., Rachkewich, RA., Goldman, B.S., Silver, M.D.,
Shumak, K.H., Crookston, J .H. Hyperacute rejection of heterotopic heart allografts in
dogs. Transplantation. 13,570,1972.
3. Kuwahara, 0., Kondo, Y., Kuramochi, T., Grogan, J.B., Cockrell, J.V., Hardy, J.D.
Organ specificity in hyperacute rejection of canine heart and kidney allografts. Ann.
Sllrg. 180,72,1974.
4. Sharma, H.M., Rosenweig, J., Chatterjee, S., Moore, S., De Champlain, M.L. Platelets in
hyperacute cardiac allografts in presensitized dogs. An experimental study. Am. 1. Path.
70,155,1973.
5. Rose, A.G. Pathology of xenograft rejection. In The Tramplantation and Replaeementof
Thoracic Organs. D.K.C. Cooper and D. Novitzky (eds.), Kluwer; Dordrecht, Boston,
London, 1990, p. 479.
6. Weil, R, Clarke, D.R, Iwaki, Y., Porter, K.A., Koep, L.J., Paton, B.C., Terasaki, P.I..
Starzl. T.E. Hyperacute rejection of a transplanted human heart. Transplantation. 32,71,
1981.
7. Trento, A., Hardesty, RL., Griffith, B.P., Zerbe, T., Kormos, RL., Bahnson, H.T. Role
of the antibody to vascular endothelial cells in hyperacute rejection in patients undergo-
ing cardiac transplantation. 1. Thorae. Cardiovase. Surg. 95,37, 1988.
8. Hammond, E.H., Yowell, RL., Numoda, S., Menlove, RL., Renlund, D.G., Bristow,
M.R, Gay, W.A., Jones, K.W., O'Connell, J.B. Vascular (humoral) rejection in heart
transplantation: pathologic observations and clinical implications. 1. Heart Transplant. 8,
430,1989.
9. Forbes, R .. , Kuramochi, T., Guttmann, R.D., Klassen, J., Knaack, J. A controlled se-
quential morphologic study of hyperacute cardiac allograft rejection in the rat. Lab.
Invest. 33,280, 1975.
10. Forbes, RD., Guttmann, RD., Kuramochi, T., Klassen, J., Knaack. J. Nonessential role
of neutrophils as mediators of hyperacute cardiac allograft rejection in the rat. Lab.
Invest. 34,229. 1976.
References 251
11. Cattell, Y., Jamieson, S.W. Hyperacute rejection of guinea-pig to rat cardiac xenografts.
I. Morphology. J. Pathal. 115,183,1975.
]2. Forbes, R.D., Guttmann, R.D. Evidence for complement-induced endothelial injury in
vivo: a comparative ultrastructural tracer study in a controlled model of hyperacute rat
cardiac allograft rejection. Am. J. Pathal. 106,378, 1982.
13. Forbes, R.D.e., Guttmann, R.D., Pinto-Blonde, M. A passive transfer model of hyper-
acute rat cardiac allograft rejection. Lab. Invest. 41,348, 1979.
14. Mann, F.e., Priestley, J.T., Markowitz, J., Yater, W.M. Transplantation of the mam-
malian heart. Arch. Surg. 26,219,1933.
15. Barnard, e.M., Wolpowitz, A., Losman, J.G. Heterotopic cardiac transplantation with a
pass. S. Afr. Med. J. 52,1035,1977.
16. Lexer, G., Cooper, D.K.e., Rose, AG., Wicomb, W.N., Rees, J., Keraan, M., du Toit, E.
Hyperacute rejection in a discordant (pig-to-baboon) cardiac xenograft model.
J. Heart Transplant. 5,411,1986.
17. Cooper, D.K.e., Human, P.A, Lexer, G., Rose, AG., Rees, J., Keraan, M., du Toit, E.
Effects of cyclosporine and antibody adsorption on pig cardiac xenograft survival in the
baboon. J. Heart Transplant. 7,238, 1988.
18. Rose, AG., Cooper, D.K.e., Human, P.A, Reichenspurner, H., Reichart, B.
Histopathology of hyperacute rejection of the heart - experimental and clinical observa-
tions in allografts and xenografts. J. Heart Transplant. In press.
Chapter 15
Histopathology of Liver Xenograft Rejection

D.G.D. WIGHT
Introduction
Following the NIH Consensus Development Conference Statement [1],

worldwide interest in liver transplantation has increased dramatically. Recent
published figures suggest that about 2000 grafts have been performed in North
America [2] and over 1200 in 32 centers in Europe [3], with the current rate
probably about 5 per million population in 69 centers in Europe and 9 per mil-
lion in 66 centers in the USA. Transplant programs have also commenced in
Australia [4], and parts of Eastern Europe and South America so that the total
world experience may well have already exceeded 10 000 cases. This huge in-
crease in demand, in the face of a finite supply of human donor organs, is one
of the main reasons for a renewed interest in xenografts.
Most organ xenografts have been performed with hearts and kidneys, the
literature on liver grafts being very small. There has also been much debate
about the susceptibility ofliver allografts to antibody-mediated rejection. For
these reasons the nature of the allograft rejection reaction in the liver will be
considered in some detail before going on to consider the position with
xenografts.
Morphology of Allograft Rejection
Organ grafts differ from the classical skin grafts in that they are vascularized
from the time of insertion, allowing both the process of sensitization and sub-
sequently that of rejection to occur promptly. Much of the early work defining
patterns of liver rejection, both with and without immunosuppressive treat-
ment, was done with experimental animals (reviewed in [5,6]). Initially, large
outbred animals such as the dog and the pig were used, but now, following
Kamada's development of a simplified technique [7], a great majority of ani-
mal experiments are performed on the laboratory rat. This animal also has the
great advantage of the ready availability of pure inbred strains which help to
minimize the many variables involved in such a complex procedure.
Because of the complexity of the procedure, other complications of liver
transplantation are also quite common [8] and must be distinguished from re-
jection. These may damage both the graft. the most important of which are
254 Histopathology of Liver Xenograft Rejection
technical factors related to liver preservation and the surgical procedure itself,
or the patient as a whole, the most important of which are bacterial and viral
infections.
Liver grafts in the pig often survive indefinitely, even when there is a com-
plete major histocompatibility complex (MHC) mismatch between donor and
recipient [9, 10-12]. Similarly, grafts may survive indefinitely between certain
strain combinations in the laboratory rat despite incompatibilities at the RT1
locus [6]. There was for many years a widespread feeling that rejection did not
playa major role in liver graft failure [13-15], probably at least in part due to
the high death rate from infectious complications in the presence of a function-
ing graft. Now, with greatly improved management of infections, rejection is
regarded as a major complication, requiring careful manipulation of immuno-
suppressive drugs.
Although there are some differences of emphasis, the morphological
changes of rejection of the liver are similar in all species so far studied. In cer-
tain strain combinations of rats [16] and in outbred pigs [10, 12], rejection may
be self-limited and does not shorten graft survival. Indeed, in these animals, a
simultaneous or subsequent liver graft may even abort what would otherwise
be a second-set rejection or a skin graft from the same donor [17]. Although
there were early hopes that human liver grafts might behave in a similar fash-
ion, it is now clear that rejection is a major cause of graft and patient morbidity.
Classification of Rejection
Rejection can be defined as graft damage caused by an immunological re-

sponse by the recipient and in the kidney is traditionally classified as hyper-
acute, acute, and chronic, based mainly on the speed of onset, and measured in
minutes with hyperacute, days with acute, and weeks or months with chronic.
In the case of the liver these terms, whilst generally understood, are not wholly
approximate since, as discussed below, the timing may be very different from
the classical descriptions of renal grafts [18].
Hyperacute Rejection
Hyperacute, a term coined by Kissmeyer-Nielsen et al. [19], and accelerated

rejection are due to the presence of preformed circulating antibodies directed
against donor-specific antigens within the graft and was first recognized as
such by Terasaki et al. [20]. Those of the ABO blood groups are of course in-
nate, whilst presensitization to human leukocyte antigens (HLA) can occur
not only from a previous graft, but also as a result of blood transfusion or preg-
nancy. Their incidence in kidney grafts has been greatly reduced by careful
matching of the blood group and use of an in vitro cross-match assay which
uses donor lymphocytes to detect antibodies in the recipient's serum. The few
remaining cases are due either to antibodies directed against antigens not ex-
Morphology of Allograft Rejection 255
pressed on lymphocytes, and, therefore, undetected by the cross-match [21],
or due to errors, for example of blood grouping.
Although very well known following renal grafts, hyperacute rejection is
rare in liver grafts, even when, in extremis, grafts are performed across ABO
blood group barriers [22,23], an observation which has led to the widely held
belief that the liver is relatively resistant to such antibody-mediated injury
[24].
Presensitized Rat Model

This belief was supported by the early experiments with rat liver graft models
where sensitization was followed by tolerance induction rather than hyper-
acute rejection [25]. However, Knechtle et al [26] were able to induce hyper-
acute rejection by presensitizing Lewis rats (RTl1) with three successive 2-cm
skin grafts at 14-day intervals from ACI animals (RTI a). Two weeks later the
sensitized rats received an ACI liver. Nine of ten recipients died within 4 h with
bleeding from the liver surface. In contrast, nine unsensitized recipients sur-
vived a mean of 1O.7±0.5 days before succumbing with cellular rejection.
Death of the sensitized animals was not due to coagulopathy or to technical
failure.
Histological studies of hyperacutely rejected livers revealed marked hem-
orrhage, edema, congestion, and necrosis within the parenchyma, with bound
immunoglobulin G (lgG) and complement demonstrable immunochemically.
Serum from presensitized, but not control, recipients showed a high titer of
donor-specific complement-dependent cytotoxic activity. These authors [27]
were able to produce the same degree of sensitization by the passive transfer
of serum from their hyperimmunized rats. They were subsequently able to
show that the principal target antigen in this model was the major class I anti-
gen, RTlA [28].
Presensitized Rhesus Monkey and Pig Model

Accelerated rejection has also been induced in rhesus monkeys by a similar
process of presensitization with donor-specific skin grafts and blood transfu-
sions [29] and now in pigs [30]. The latter authors showed that within 30 min of
revascularization, liver inserted into presensitized subjects became swollen
and dusky, then at 1-2 h the animals became acidotic and hypotensive, and
died within the next 1-2 h. Histologically, there was parenchymal hemor-
rhage, endophlebitis with endothelial injury, platelet deposition, and neu-
trophil infiltration (Fig. 15.1). In contrast, the control animals which had not
been sensitized showed typical acute cellular rejection. Levels of lymphocyto-
toxic antibody rose progressively after each of the sensitizing skin grafts and
then dropped dramatically immediately after liver grafting as the antibody
was consumed in the hyperacute rejection process.
Fig. IS. 1. Hyperacute rejcction. Lewis-to-BN rat liver transplant following three presensitiz-
ing Lewis skin grafts . The liver was removed 5 h after transplantation and already shows
complete loss of zone III (centrilobular) liver cells and replacement by hemorrhage. Note
also necrosis of the wall of the te rminal hepatic venule (T). A few surviving liver cells can be
seen in the lower left part of the field , although they show extensive fatty change (steatosis),
H& E, x200
Clinical Reports
These experiments have, therefore , shown that antibody can indeed by re-
sponsible for destruction of liver grafts, and have prompted a fresh look at
clinical grafts. Hyperacute rejection was suspected in one of the first clinical
attempts at liver transplantation [31], but, following the successes noted above
with ABO-incompatible grafts, its existence subsequently came to be doubt-
ed, particularly since several of the early reports seemed to lack credibility and
because of the distraction provided by many other complications, particularly
thromboses of the vascular anastomoses (but see below). There are , however,
now several reports of apparently well-documented antibody-mediated rejec-
tion [32- 35], although in general, as well as being much rarer than following
kidney grafts, the speed of onset is also slower and thus it should perhaps more
properly be called accelerated, or simply humoral, rejection.
This point was particulary well shown in two cases described by Starzl et al.
[35]. Both patients received combined liver-kidney transplants, and in both
cases the kidney underwent immediate hyperacute rejection with coagulo-
pathy occurring within a few minutes of liver revascularization. One of the liv-
ers developed widespread necrosis, requiring retransplantation after 3 days,
whilst the other was severely damaged (although not biopsied), but recovered.
Starzl likened the transplanted kidney in these two cases to the canary once
used by miners to provide early warning of a hostile environment.
Nevertheless, although liver cell necrosis did not appear for some hours, there
was clear evidence of functional damage much earlier, since the bile flow
which began immediately after vascularization ceased again within minutes.
Although individual livers might survive grafting across ABO barriers,
Demetris et al. [36] showed clearly that the risk of graft loss within the first
30 days was substantially higher (11 out of 24, 46%) than in ABO-matched
controls (4 of 38, 11 %). Detailed analysis of individual cases [37] led the au-
thors to believe, on the basis of immunostaining for immunoglobulins and
complement, and elution studies, that in four patients the preformed isoagglu-
tinins were wholly responsible for the graft loss, and a major contributory fac-
tor in the remaining eight patients.
In retrospect, many of the other cases of sudden acute graft failure de-
scribed by a number of different authors under a variety of names may also be
examples of antibody-mediated rejection. Hubscher et al. [38] defined a group
of six cases in which the liver underwent massive hemorrhagic infarction in the
presence of normal patent vessels. In each case the patient developed fulmi-
nant hepatic failure suddenly and unexpectedly between 3 and 20 days after
surgery. Ludwig described a similar case [39]. Starzl et al. [40] described a sim-
ilar syndrome which he initially ascribed to kinking of the hepatic artery to the
right lobe. This is a plausible explanation for several of our own cases which
occurred in children given an adult liver which in retrospect was probably too
large. The condition also may correspond to septic hepatic gangrene [41], the
gram-negative organisms being secondary invaders of dead tissue rather than
causative. However, Fagan et al. [42] felt that in another similar case the or-
ganisms might have been responsible for the liver cell necrosis as a result of a
single organ Schwartzmann reaction. Wall et al. [43] attributed their case, in
which the patient developed acute massive necrosis of the graft suddenly on
day 7, to recurrence of non-A, non-B hepatitis.
Finally, it is possible that some cases of vascular thrombosis may be at-
tributable to severe rejection rather than technical factors [44,45]. Gugenheim
et al. [46] found arterial thrombosis to be more common in ABO-incompatible
than compatible grafts.
Histopathology
The appearances of the liver are similar in all these instances to those observed
in the experimental animals with proven antibody-mediated rejection [26,29].
The liver may be swollen, dark in color, and increased in weight. The major
vessels are generally patent, yet microscopically the appearances closely re-
semble acute ischemic damage caused by major vessel occlusion, with
eosinophilic necrosis of both parenchyma and portal tracts accompanied by
hemorrhage and a light neutrophilic infiltrate [33,46] (Figs. 15.2-15.4). In the
earliest cases sampled histologically, Demetris et al. [36,37] saw clusters of
Fig. 15.2. Antibody-mediated rejection. ABO-incompatibl e human orthotopic liver trans-

plant removed after 6 days. Low magnification view showing a hemorrhagic portal tract ( P)
surrounded by necrotic hepatocytcs. Hemorrhage is also clearly visible in zone ITI around
the terminal hep atic venules (f). H & E, x 130
neutrophil polymorphs with fibrin deposition and red blood cell sludging in si-
nusoids, followed on the subsequent days by the appearance of hepatocyte
necrosis. In contrast to the findings in kidneys, fibrinoid necrosis of arteries
was unusual , but focal masses of fibrin attached to a partly disrupted vessel
wall, usually of veins, were quite commonly seen. Distinction from severe
preservation injury or from ischemic damage may not be easy [37].
Immunofluorescent studies, however, should detect immunoglobulin , Clq,
and C3 in arterial walls [37,47], although the distribution of positive staining
may be very focal compared with the picture in the kidney.
Relative Resistance of' Liver to Antibody-Mediated Rejection

The mechanism of the relative resistance of the liver to antibody-mediated re-
jection is not entirely understood. However, when one analyses the cause of
the instantaneous loss of a heart or kidney graft, it perhaps becomes clear [48].
Following combination of preformed antibody with target antigens in the graft
(either blood group or HLA) there is activation of platelets as well as of the
complement and coagulation cascades. These lead to plugging of the micro-
vasculature with fibrin and platelet aggregates, and probably also to vasocon-
striction. Since both the heart and the kidney contain many end-arteries, or-
gan ischemia inevitably follows.
Fig. 15.3. Antibody-mediated rejection. Same case as Fig. 15.2. This higher magnification
shows red cells and neutrophil polymorphs in the portal tract to the right. The major part of
the field shows complete necrosis of all the hepatocytes, which have therefore lost all cyto-
plasmic detail, with the intervening sinusoids heavily infiltrated by neutrophil polymorphs,
H& E,x320
The bulk of the liver, in contrast, has a rather different blood supply with no
true end-arteries. Instead, the major part of the hepatic microvasculature con-
sists of sinusoids, which are themselves fenestrated and without a basement
membrane, and which derive their blood from two sources. When either the
hepatic artery or portal vein flow is compromised, the other vessel can proba-
bly compensate by an increased flow, although the mechanism by which this is
controlled is not known. In addition, the sinusoids are lined by a vast number
of macrophages, the Kupffer cells, which have a great capacity for removing
complexes, and also, again by unknown mechanisms, the liver can release
large quantities of soluble Class I HLA antigen [49,50] capable of mopping up
large quantities of antibody. Thus, for all these functional and anatomical rea-
sons, the liver might be expected to be relatively resistant to antibody-mediat-
eddamage.
Acute (Cellular) Rejection
Rat Models
The morphological changes of acute rejection have been well documented in a
variety of experimental animals. Much of the early work was performed in the
Fig. 15.4. Antibody-mediated rejcction . Same casc as Figs. 15.2 and 15.3. This large septal
portal tract shows extensive intcrstitial hemorrhage, but note thc completely normal artcr-
ies without any evidencc of fibrinoid changc (as compared to the findings in antibody-medi-
atcd rejection ofthc kidn ey ), H & E , x 130
dog [51 , 52], the pig [9-11] , and the baboon [53], but although the pathology
was well documented [10, 54, 55], the picture was often complicated by sec-
ondary factors such as infection and drug toxicity. Also, in these early studies
little was known of the HLA status of donor or recipient. With the advent of
the rat model, made possible by the development of the cuff technique for the
vascular anastomoses [7], it has been possible to define and quantitate acute
rejection using pure inbred strains of known HLA (RTIA) status, with appro-
priate controls [5, 6] .
When livers from DA rats (RTl a) are grafted into BN recipients (RTl U),
acute rejection predictably leads to the death of the animals in about 15 days
[56] . In these vigorous reactions there is extensive edema and mononuclear
cell infiltration, not only of expanded portal tracts but also of the central parts
of lobules [6]. At both sites mononuclear cells infiltrate the walls of venules.
There is also widespread liver cell loss, affecting both periportal and centrilob-
ular zones, with occasional foci of coagulative necrosis which are mainly peri-
portal. Mononuclear cells are also numerous in the sinusoids of surviving
parenchyma, often apparently in direct contact with hepatocytes. The cells are
a mixture of macrophages, lymphocytes, many undergoing blast cell transfor-
mation, and usually significant number of plasma cells. Polymorphonuclear
leukocytes are sparse. Arterial lesions and bile duct lesions are not seen (no at-
tempt is made , in this model, to reanastomose the artery).
DA livers inserted into PVG recipients (RT1 C), in contrast, survive indefi-
nitely. Graft biopsy at weekly intervals, however, reveals that there is a self-
limiting cellular rejection response which is maximal at 2 weeks [6]. At its peak
it is much less intense than in the BN rats. Inflammatory cells, qualitatively
similar to those in BN recipients, are concentrated in the portal tracts, and
there is little spillover into the parenchyma. Cells are present in the walls of
portal and hepatic venules, and from the latter may extend for a short distance
into neighboring centrilobular parenchyma. Hepatocyte necrosis is restricted
to occasional hepatocytes undergoing eosinophilic necrosis.
The rats described above received no immunosuppression but these
changes are closely similar to those seen in human liver grafts in patients given
standard immunosuppression (see below). DA livers inserted into
(DAxBN)F1 hybrids are subject to rejection which is intermediate in inten-
sity, and the animals generally survive.
The majority of patients have been treated with a standard immunosup-
pressive regimen; that most favored currently is a combination of cyclo-
sporine, steroids, and azathioprine, so-called triple therapy.
Morphology
Snover et al. [57] defined three cardinal features of acute rejection: (i) mixed
portal tract inflammation, (ii) bile duct damage, and (iii) attachment oflympho-
cytes to the endothelium of portal and/or hepatic venules (Fig. 15.5). Although
the relative importance of these three features has varied from series to series,
all authors have stressed their importance in the diagnosis of acute rejection.
The earliest change seen is generally an inflammatory cell infiltration of
portal tracts (Fig. 15.5). Frequently the intensity is quite variable from one
part of the liver to another, and thus interpretation can be quite misleading un-
less a series of levels is examined from each biopsy. The cells are mostly lym-
phocytes but always include significant numbers of activated or blast cells.
These are large cells with large open nuclei with one or more prominent nucle-
oli, occasionally in mitosis, and correspond to the pyroninophilic cells de-
scribed in the older literature [58]. Blast cells were rather neglected by a num-
ber of authors before being rediscovered by those groups practising fine
needle aspiration biopsy, largely pioneered in Helsinki [59]. In addition, there
are macrophages and often eosinophils and/or neutrophils.
The second feature of the triad, bile duct damage, which is sometimes
called rejection cholangitis [39], is very variable. The most common finding is
vacuolation of biliary epithelium with associated infiltration by lymphocytes
and/or neutrophil polymorphs; only very rarely are cells found in the lumen.
Occasional cells are in mitosis. The small interlobular ducts and the ducts of
Hering are the principal targets of this type of injury, which may lead to the dis-
appearance first of individual nuclei and then subsequently, in severe cases, to
loss of ducts altogether.
The third of Snover et ai's. triad, endothelialitis is found in both portal and,
less commonly, hepatic venules, although it may be easier to evaluate in the lat-
Fig. 15.5. Acute cellular rejection. ABO-compatible huma n liver graft. Biopsy taken a t
7 days for minor liver dysfunction. The appearances are typical of acute rejection of mode r-
ate degree. The portal tract occupying most of the field is densely infiltrated by mononucle-
ar cells, which can be seen infiltrating the endothelium of the portal venule (below) and the
walls of small bile ducts (arrows) , H & E , x320
ter because of the intensity of the portal infiltrate. Mononuclear cells adhere to
the endothelium, but also characteristically infiltrate beneath the endothelium
lifting it from the underlying media (Fig. 15.5). In portal tracts this can result in
the vessel being totally obscured. Acute arterial changes are common.
Time of Onset
Most episodes of acute rejection occur in the first few weeks after transplanta-
tion with a peak incidence between day 5 and day 14. In Klintmalm's series [60]
the first episode of rejection occurred within 21 days in no fewer than 60 of 63
patients who experienced any acute rej ection. Occasionally, otherwise typical
changes can be seen as early as day 4. Much less commonly, acute rejection
may appear for the first time weeks or months after transplantation, when it
may be related to incomplete or faulty immunosuppression .
Chronic Rejection
Late or chronic rejection of human liver grafts occurs in 10%-20% of patients,

mostly within the first few months after transplantation, and has two principal
manifestations: (i) foam cell endovasculitis (which corresponds to the trans-
Hepatic Xenografts 263
plant atherosclerosis seen in the heart transplant literature [61]), and (ii) van-
ishing bile ducts [6]. The precise mechanism remains a matter for debate but,
since it has been described in neither experimental allografts nor xenografts, it
will not be considered further.
Hepatic Xenografts
Compared with the now very considerable literature on the pathology of ex-
perimental and human allografts, that on hepatic xenografts is very paltry.
There was a little activity between 1968 and 1974, at a time when human liver
transplantation was in its infancy but donors were very difficult to obtain.
Following the introduction of the concept of "brain-stem death" in the mid-
1970s [62], there was for a number of years a relative excess of potential donors
over potential recipients, until the NIH consensus conference [1] which led
first to a greatly increased number ofliver transplant centers and subsequently
to a new shortage of donors.
Perhaps that is one of the reasons for the revival, in 1987, of interest in ex-
perimental hepatic xenografts. The previous work, therefore, naturally di-
vides into that published before 1987, which is largely anecdotal and unsys-
tematic or uses numbers too small for significant conclusions, and that since
1987, which is exclusively in rodents.
There is, however, an alternative method of classification. As discussed else-
where in this volume (Chap. 1), in 1970 CaIne introduced the concept ofconcor-
dant and discordantxenografts [63]. From common usage concordant has come
to mean that in a particular pair of species, there is no demonstrable preformed
antibody in the recipient serum which is directed against donor antigens.
Generally, although not exclusively, this applies to species which have a fairly
close evolutionary relationship, such as hamster and rat or chimpanzee and hu-
man. Rejection of solid organs in such cases tends to be less vigorous and, in the
liver, resembles more closely the acute cellular rejection described above.
Discordant species, on the other hand, such as the guinea pig and rat or pig
and baboon, tend to have pre-existing antibodies which lead to rapid or hyper-
acute rejection (although, in a guinea pig-to-Lewis rat heart graft model,
Miyagawa et al. [64] recently showed that complement can be activated direct-
ly through the alternative pathway without the participation of antibody).
Tables 15.1 and 15.2 list the published series classified according to 'era', and
also whether between concordant or discordant species.
Early Experience
Although there was a great deal of experimental and clinical work with
xenograft kidneys in the early 1960s [65,66], there was very little work with the
liver, at least in part because many of the technical problems associated with
Table 15.1. Early experience with experimental and clinical liver xenografting"
Author Year Donor Recipient II Treatment Survival

[Reference]
A. Discordant grafts
Caine [67] 1968 Pig Baboon 7 Nii/steroids/ 6 h-3.5 days
steroids+
azathioprine
Caine [71] 1970 Pig Rhesus 3 Previous ALS 12 h
Pig Chimpanzee 1 Nil 8h
Terblanche [69] 1970 Baboon Pig 7 Nil 5h
Jerusalem [70] 1971 Pig Dog ? ? ?
Dog Pig ? ? ?
B. Concordant grafts
Starzl [31] 1969 Chimpanzee Human ALG, 9 days
azathioprine,
steroids
Giles [68] 1970 Chimpanzee Human Prior kidney 26 h
transplant
Caine [71] 1970 Cynomolgus Rhesus 3 ALS 19h-20 days
Starzl [22] 1974 Chimpanzee Human ? 14 days
ALS, antilymphocyte serum; ALG, antilymphocyte globulin

aHistopathology reported in all cases.
such a complex operation had yet to be solved in liver grafts of any type (Table
15.1). CaIne was the first to attempt the experimental transfer of the liver be-
tween species [67], whilst Starzl three times attempted to treat patients with a
chimpanzee's liver [22, 31, 68], and Terblanche et al. [69] transplanted a ba-
boon liver into the pig. The only other authors to attempt xenotransplantation
of the liver at this time were Jerusalem et al. [70] who used heterotopic dog-to-
pig and pig-to-dog combinations. However, the latter authors' results are im-
possible to interpret since the xenografts are not reported separately from al-
lografts and isografts. Even the numbers of animals transplanted are not
given.
Few conclusions seem possible from these small and uncontrolled series.
However, all CaIne's animals transplanted with livers from a discordant
species [67, 71] died within days, the majority in a matter of hours (Table
lS.lA). All but one died of hemorrhage, disseminated intravascular coagula-
tion, or liver failure. The livers all showed centrilobular necrosis with little cel-
lular infiltration, with or without hemorrhage and fibrin deposition, appear-
ances very much in keeping with antibody-mediated rejection. The sole but
interesting exception was a baboon which received a pig's liver [67] and which
died from bronchopneumonia with a well-preserved liver which showed only
mononuclear cell infiltration of portal tracts.
Similarly, Terblanche's seven cases all died within a few hours with shock
and profound metabolic acidosis. Although the livers became revascularized
immediately, and were initially normal, by 30 min sinusoidal congestion with
scattered neutrophil polymorphs were seen. Over the next few hours changes
became progressively more marked until in the longest survivors there was ob-
vious patchy liver cell necrosis with disintegration of sinusoidal walls and of
liver cell plates. No fibrin or platelet thrombi were identified, but the appear-
ances are entirely consistent with antibody/complement-mediated rejection,
and almost identical pathology to Merion's hyperacutely rejected pig allo-
grafts [30].
In contrast, the concordant grafts fared better. Two of Starzl's chimpanzee
livers showed no signs of rejection when the recipient patients died at 26 h [68]
and at 14 days r221, respectively, whilst the third showed features suggestive of
severe acute cellular rejection. Similarly, one of CaIne's three cynomolgus-to-
rhesus grafts showed changes suggestive of cellular rejection when the animal
died at 19 days [71]. However, these authors did feel that one of the three ani-
mals died of hyperacute rejection at 19 h.
Recent Experience
Discordant Grafts (Table 15.2A)

One group has worked with guinea pig-to-Lewis rat grafts [72,73], using well
controlled experiments. Settaff et al. [72] showed that livers had a mean sur-
vival of 1002 min as compared with only 26 min for hearts across the same
species, thus confirming the relative resistance of the liver to this form of rejec-
tion. However, even at surgery the liver behaved quite differently from allo-
grafts because it took some 30 min to recolor, and even then revascularization
remained partial in 11 of 14 animals.
At autopsy, the livers showed relatively minor changes with congestion of
terminal hepatic venules and of the centrilobular region, but no hepatic necro-
sis. In addition, one animal had a portal vein thrombosis, five others showed
congestion of portal venules and portal tract edema. Although the authors
were uncertain as to the precise cause of death in these animals there was
clearly impaired blood flow through the liver in most, if not all, instances. This
suggests that there may well have been fibrin deposition and sludging of red
cells in sinusoids, as described in hyperacutely rejected human allografts by
Demetris et al. [37], not always easy to distinguish from artefact, especially at
autopsy.
The authors showed that Lewis rats have a naturally occurring anti-guinea
pig antibody in their blood, and they were able to demonstrate large deposits of
complement C3 in sinusoids, around large vessels, and, in three cases, on hepa-
tocytes. Interestingly, no staining was obtained with antibodies to IgM, IgG or
IgA, which perhaps reaffirms the importance of complement in discordant re-
jection [64]. The distribution of findings correlates well with the theories of Platt
Table 15.2. Recent experience with experimental liver xenografting
Author [Reference] Year n Treatment Survival
A. Discordant grafts"
Settaff [72] 1987 14 Nil 1002±624 min
Filipponi [73] 1989 10 Nil 205± 38.9 min
10 BN 52063 306± 36.5 min
B. Concordant graftsh
Monden [76] 1987 14 NilorCsA 7.1± 0.4 days
Valdivia [78] 1987 7 Nil 7.3± 0.5 days
5 CsA20 mg 7.4± 0.5 days
5 CsA40mg 7.6± 0.8 days
5 Spl 7.2± 0.4 days
10 Spl+CsA 30 mg 17.6± 5.6 days
Yamaguchi [79] 1988 10 Nil 6.7± 0.9 days
8 Spl 6.9± 0.8 days
10 CsAlOmg 7.1± 0.9 days
10 Spl+CsA 10 mg 12.5± 1.0 days
4 Spl+CsA 20 mg 13.0± 0.7 days
Yamaguchi [80] 1990 9 Nil 6.8± 0.9 days
8 Spl 6.9± 0.8 days
10 CsA IOmg 7.1± 0.9 days
5 CsA20mg 6.8± 0.7 days
6 CsA30mg 6.7± 1.1 days
10 Spl-CsA 10 mg 12.5± 1.0 days
5 TLI5 Gy 6.4± 0.5 days
5 TLII0 Gy 8.8± 2.3 days
5 TLI 15 Gy 6.6± 1.0 days
5 TLI 10 Gy+CsA 10 mg 13.6± 4.1 days
6 TLI 15 Gy+CsA 10 mg 6.6± 2.0 days
6 TLI 10 Gy+Spl+CsA 10 mg 41.7±19.8 days
CsA, cyc1osporine; Spl, splenectomy; TLI, total lymphoid irradiation

" All guinea-pig-to-Lewis rat. Histopathology only reported by Settaff [72].
h Either hamster-to-Wistar rat (Monden, Valdivia) or hamster-to-Lewis rat (Yamaguchi).
Histopathology not reported by Yamaguchi [79].
et al. [74], who believe that endothelial activation is responsible for most of the
consequences of both hyperacute and discordant xenograft rejection.
The major problem preventing engraftment between discordant species is,
of course, the presence of preformed antibody and complement, whose pres-
ence is uninfluenced by conventional immunosuppression. Many attempts
have been made to address this problem by various therapeutic manipula-
tions, such as plasmapheresis, successive organs from the same donor, and
treatment with cobra venom factor (reviewed in [75]). Although prolongation
of graft survival was quite commonly achieved, in no case was this clinically
meaningful since death of the organ was almost always still measured in min-
utes.
The same team from Paris was the first to attempt this with liver grafts [73],
and they found that the platelet activating factor BN 52063 approximately
doubled the survival time to 306.2±36.5 - a similar prolongation was also noted
with heart grafts, to 18.3±1.4 minutes - but an analogue of prostacyclin 12 had
no effect.
Thus, although experience is somewhat limited, it seems clear that rejec-
tion of the hepatic xenograft between discordant species is morphologically
closely similar to the hyperacute rejection seen in hyperimmunized experi-
mental allografts [27], and rarely in presensitized patients [36]. Furthermore,
the hepatic xenograft appears to hold the same privileged position as the
hepatic allograft in being more resistant to antibody-mediated rejection than
other organs, although this difference is a long way from being clinically appli-
cable.
Concordant Grafts (Table 15.2B)

Two groups have worked with concordant xenografts. Both have used the
golden hamster as the donor and rats as the recipient, although the workers
from Osaka have used Wistar rats [76-78], whilst those from North Carolina
have used Lewis rats [79,80]. Nevertheless, both sets of experiments are well
controlled and the results are comparable.
Both groups of workers have shown that, without treatment, livers are re-
jected predictably and inevitably at about 7 days after surgery. The liver re-
mains histologically normal as late as 5 days, but at the time of the animal's
death there is diffuse cellular infiltration, in which polymorphonuclear leuko-
cytes outnumber mononuclear cells, combined with interstitial hemorrhage
and extensive hepatocyte necrosis, sparing only a narrow rim of cells around
the portal tracts and terminal hepatic venules [76, 78, 80]. It is of interest that
cyclosporine alone, even given in doses as high as 40 mg/kg per day, had no ef-
fect whatever on survival. This suggests a negligible contribution from T lym-
phocytes to this rejection process. In support of this interpretation, Valdivia et
al. [78] showed that lymphocytotoxic antibody, absent before surgery, rose to
very high titers on day 7. Finally, although the timing of the rejection is remi-
niscent of that due to acute rejection of heart or skin grafts, histologically, with
dominance of hepatocyte necrosis, hemorrhage and neutrophil polymorphs, it
remains the picture of antibody-mediated rejection. It does, of course, corre-
spond quite closely to the definition of the term accelerated rejection, i.e., the
antibody is synthesized following engraftment.
Both groups showed further that strategies which reduced the antibody lev-
els (which cyclosporine mono therapy does not) were able to prolong graft and
host survival. Both groups noted marked splenomegaly in grafted but other-
wise untreated controls, but splenectomy alone had no effect on survival [78,
79], although both groups found that cytotoxic antibody levels were lower.
Liver damage, assessed histologically, was less severe and much more like
acute cellular rejection, with mononuclear cell infiltration of portal tracts and
no hemorrhage or liver cell necrosis, although still fatal. The combination of
splenectomy with cyclosporine, however, lcd to significant prolongation of

survivaL still lower levels of cytotoxic antibody, and greatly improved hepatic
morphology.
Yamaguchi et al.l80] went further and added total lymphoid irradiation to
the combination of splenectomy and cyclosporine, and obtained dramatic im-
provement in survival to 41.7±19.8 days. All of the six animals in this group
were thought to have died of infection as were six of ten in Valdivia's splenec-
tomy plus cyclosporine group [78]. It seems probable that, in addition to sup-
pression of antibody formation, these latter animals also experienced inhibi-
tion of helper T cell function.
These experiments have shown that. using rather drastic measures it is pos-
sible to prevent, or at least delay, the synthesis of new antibody directed
against graft antigens following engraftment. The same maneuvers, although
primarily directed against antibody synthesis, seem also to have pre-empted
the appearance of cellular rejection. This suggests that if antibody-mediated
rejection can be controlled, then cellular rejection may well be, at least in the
liver, no more difficult to control than in allografts.
Comment
Experience with hepatic xenografts remains limited. However, certain conclu-

sions seem possible. The major barrier to successful transplantation is anti-
body directed against recipient graft antigens. It is beginning to become pos-
sible to prevent the post-transplant synthesis of cytotoxic antibody in
concordant grafts, at least in the guinea pig-to-rat modell80], but there is as
yet little evidence of substantial progress in the prevention of graft damage by
preformed antibody as found in most discordant grafts. This is also, of course,
a continuing and unsolved problem with allografts.
The liver differs from the other organs in the speed at which it is rejected, an
observation as true of xenografts as of allografts. It also differs in that it is
much more metabolically active, having a great range of catabolic and synthet-
ic activities. Will the liver xenograft. once rejection can be prevented, be able
to metabolize the various host proteins and peptides presented to it, and will
the host be able to handle the various products, some of which are bound to be
antigenic, synthesized by the new liver? These questions were discussed by
Caine [63], now 20 years ago. and again recently [81], but we are still very little
nearer today to providing any answers.
Acknowledgements. I am most grateful to Professor Michel Reynes who kind-

ly gave the histological slides from which Figs. 15.1-15.4 were prepared.
References 269
References
1. National Institutes of Health Consensus Development Conference Statement.

Hepatology. 4, 107S, 1984.
2. Van Thiel. D.H., Makowka, L.. Starzl, T.E. Liver transplantation: where it's been and
where it's going. Gastroenterol. Clin. N.A. 17, 1, 1988.
3. Bismuth, H., Castaing, D., Ericzon, B.G., Otte, J.B., Rolles. K.. Ringe, B., Sloof, M.
Hepatic transplantation in Europe. First report of the European Liver Transplant
Registry. Lancet. 2,674,1988.
4. Sheil, A.G., Thompson, A.F., Gallagher, N.D., Dorney, S.F., McCaughan, G.W.,
Stephen M.S .. Eyers, A.A., Graham, J.c., Bookallil, M., Harrison, V.L. Australian
National Pilot Liver Transplantation Program: the first year's report. Transplant. Proc.
20,31,1988.
5. Wight. D.G.D. The morphology of rejection of liver transplants. In: Transplant
Immunology. Caine, R.Y. (cd.), Oxford, Oxford liniversity Press, 1984, p. 53.
6.Wight. D.G.D., Portmann, B. Pathology of liver transplantation. In: Liver
Transplantation. Caine, R.Y. (ed.) (2nd edn.), London, Grune & Stratton, 1987,
p.435.
7. Kamada, N., Caine, R.Y. Orthotopic liver transplantation in the rat. Technique using
cuff for portal vein anastomosis and biliary drainage. Transplantation. 28,47,1979.
8. Portmann, B., Wight, D.G.D. Pathology of liver transplantation. In: Liver
Transplantation. Caine, R.Y. (ed.) (2nd edn.), London, Grune & Stratton, 1987,
p.435.
9. Cordier, G., Garnier H., Clot, J.P., Camplez, P., Gorin, J.P., Clot, P., Bassinier, J.P.,
Nizza, M., Levy, R. La greffe de foie orthotopique chez Ie porco Premiers resultats. Mem.
Acad. Chir. 52,799,1966.
10. Caine, R.Y., White, H.J.O., Yorfa, D.E., Binns, R.M., Maginn, R.R., Herbertson, B.M.,
Millard, P.R., Molina, V.P., Davis, D.R. Prolonged survival oflivertransplants in the pig.
B~Med.~4,645, 1967.
11. Peacock, J.H., Terblanche, J. Orthotopic homotransplantation ofthe liver in the pig. In:
The Liver. Read, A.E. (cd.), London, Butterworth, 1967, p. 333.
12. Meijer, S., Hoitsma, H.F., Visser, J.J., de Lange, J.J. Long-term survival following ortho-
topic liver transplantation in pigs; with special reference to gastric ulcer complications.
Neth.l. Surg. 36, 168, 1984.
13. CaIne, R.Y., Williams, R., Lindop, M., Farman, J.V., Tolley, M.E. Rolles, K..
MacDougall, B., Neuberger, J., Wyke, R.J., Raftery, A.T., Duffy. T.J., Wight, D.G.D.,
White, D.J.G. Improved survival afterliver grafting. Br. Med.l. 283, 115, 1981.
14. Fennel, R.H., Roddy, H.J. Liver transplantation. The pathologist's perspective. In:
Pathology Annual. Vol. 14, part 2, Sommers, S.c. (cd.), New York, Appleton-Century-
Crofts, 1979,p. 155.
15. Starzl, T.E., Iwatsuki, S., Klintmalm, G., Schroter, G.PJ., Weil, R., Koep, L.J., Porter,
K.A. Liver transplantation 1980, with particular reference to cyclosporin A. Transplant.
Proc. 13,281,1981.
16. Kamada, N., Davies, H.ff.S., Wight, D.G.D., Culank, L., Roser, B. Liver transplantation
in the rat. Biochemical and histological evidence of complete tolerance induction in non-
rejector strains. Transplantation. 35,304,1983.
17. Kamada, N., Wight, D.G.D. Antigen-specific immunosuppression induced by liver
transplantation in the rat. Transplantation. 38,217,1984.
18. Ludwig, J. Classification and terminology of hepatic allograft rejection: whither bound?
Mayo Clin. Proc. 64,676, 1989.
19. Kissmeyer-Nielsen. F., Olsen, S., Peterson, V.P., Fjeldborg, O. Hyperacute rejection of
Lancet. 2,662, 1966.
20. Terasaki, P.I., Marchioro, T.L., Starzl, T.E. In: Histocompatibility Testing. van Rood, J.J.
and Amos, D.B. (eds.), Washington D.C., National Academy of Sciences, 1965.
21. Banner, B., Makowka, L .. Demetris,J., Tzakis, A .. Griffin, M .. Starzl, T. Hyperacute re-
jection of the kidney in patients with a negative cross-match. Tramplallt. Proc. 20,453,
1988.
22. Starzl. T.E., Ishikawa, M., Putnam, e.W., Porter. K.A .. Picache, R, Husberg, B.S.,
Halgrimson, e.G .. Schroter, G. Progress in and deterrents to orthotopic liver transplan-
tation with special reference to survival. resistance to hyperacute rejection, and biliary
duct reconstruction. Transplallt. Proc. 6, 129, 1974.
23. Gordon, RD., Iwatsuki, S .. Esquivel. e.O .. Tzakis, A, Starzl, T.E. Liver transplantation
across ABO blood groups. Surgery. 100,342, 1986.
24. Gordon, RD., Fung, J .. Markus, B .. Fox, I.. Iwatsuki. S., Esquivel, e.O., Tzakis, A,
Todo, S., Starzl. T.E. The antibody cross-match in liver transplantation. Surgery. 100.
705,1986.
25. Kamada, N., Davies, H.ff.S., Roser, B. Reversal of transplant immunity by liver grafting.
Nature. 292,840,1981.
26. Knechtle, S.J., Kolbeck, P., Tsuchimoto, S.. Coundouriotis, A, Sanfilippo, F., Bollinger,
RR Hepatic transplantation into sensitized recipients. Demonstration of hyperacute
rejection. Transplantation. 43,8,1987.
27. Knechtle, S.J., Kolbeck, p" Tsuchimoto, S., Coundouriotis, A, Sanfilippo, F., Bollinger,
RR Humoral rejection of rat hepatic transplants by passive transfer of serum.
28. Knechtle, SJ., Yamaguchi, Y .. Harland, Re., Coundouriotis, A, Bollinger, RR
Mediation of rat hepatic allografts by RT-l antigens. Transplantation. 48,723, 1989.
29. Gubematis, G., Lauchart, W., Jonker, M., Steinhoff, G., Bomscheuer, A, Neuhaus, P.,
van Es, A.A, Kemnitz, J., Wonigeit, K., Pichlmayr, R. Signs of hyperacute rejection of
liver grafts in rhesus monkeys after donor-specific presensitization. Transplant. Proc. 19,
1082,1987.
30. Merion, RM., Colletti, L.M. Hyperacute rejection in porcine liver transplantation. I.
Clinical characteristics, histopathology, and disappearance of donor-specific lympho-
cytotoxic antibody from serum. Transplantation. 49,861, 1990.
31. Starzl, T.E. Experiences in Hepatic Transplantation. Philadelphia, W.B. Saunders, 1969,
p.269.
32. Hanto, D.W., Snover, D.e., Sibley, R.K. Hyperacute rejection of a human liver allograft
in a presensitized recipient. Clin. Transplant. 1,304,1987.
33, Bird, G., Friend, P., Donaldson, P., O'Grady, J., Portmann, B., Caine, RY" Williams, R
Hyperacute rejection in liver transplantation: a case report. Transplant. Proc. 21,3742,
1989.
34. Gubematis, G" Kemnitz, J., Bomscheuer, A, Kuse, E.R, Pichlmayr, R Potential vari-
ous appearances of hyperacute rejection in human liver transplantation. Langenbeck's
Arch. Chir. 374,240,1989.
35. Starzl, T.E., Demetris, AJ., Todo, S., Kang, Y., Tzakis, A, Duquesnoy, R, Makowka,
L., Banner, B., Concepcion, W., Porter, K.A. Evidence for hyperacute rejection of hu-
man liver grafts: the case of the canary kidneys. Clin. Transplant. 3,37,1989.
36. Demetris, A.J., Jaffe, R, Tzakis, A, Ramsey, G., Todo, S., Belle, S., Esquivel, e.,
Shapiro, R, Markus, B., Mroczek, E., van Thiel, D.H., Gordon, R, Makowka, L., Starzl.
T. Antibody-mediated rejection of human orthotopic liver allografts. A study of liver
transplantation across ABO blood group barriers. Am. 1. Patho!' 132,489,1988.
37. Demetris, AJ., Jaffe, R, Tzakis, A, Ramsey, G., Todo, S., Belle, S., Esquivel, e.,
Shapiro, R, Zjako, A, Markus, B., Morozec, E., van Thiel, D.H., Sysyn, G., Gordon, R,
Makowka, L., Starzl, T.E. Antibody-mediated rejection of human liver allografts.
Transplantation across ABO blood group barriers. Transplant. PRoc. 21,2217, 1989.
38. Hubscher, S.G., Adams, D.H, Buckels, J.A.e., McMaster, P., Neuberger, J., Elias, E.
Massive hemorrhagic necrosis of the liver after liver transplantation. 1. Clin. Patho!. 42,
360,1989.
39. Ludwig, J. Histopathology of the liver following transplantation. In: Transplantation of
the Liver. Maddrey, W.e. (ed.), New York, Elsevier, 1988, p. 191.
40. Starzl. T.E., Groth, e.G., Brettschneider, L., Penn, I, FUlginiti, V.A., Moon, J.B.,
References 271
Blanshard, H., Martin, AJ., Porter, K.A Orthotopic homotransplantation of the human
liver. Ann. Surg. 168,392,1968.
41. Starzl, T.E., Iwatsuki, S., van Thiel, D.H., Gartner, J.e., Zitelli, B.J., Malatack, J.J.,
Schade, RR, Shaw, B.W., Hakala, T.R., Rosenthal, J.T., Porter, K.A Evolution ofliver
transplantation. Hepatology. 2, 614, 1982.
42. Fagan, E.A, Ellis, D.S., Portmann, B., Tovey, G.M., Williams, R, Zucherman, AJ.
Microbial structures in a patient with sporadic non-A, non-B fulminant hepatitis treated
by liver transplantation. J. Med. Virol. 22,189,1987.
43. Wall, W.J., Duff, J.H., Ghent, e.H., Stiller, e.R, Keown, P.A, Kutt, J.L. Liver trans-
plantation: the initial experience of a Canadian center. Can ad. J. Surg. 28,286, 1985.
44. Samuel, D., Gillet, D., Castaing, D., Reynes, M., Bismuth, H. Portal and arterial throm-
bosis in liver transplantation: a frequent event in severe rejection. Transplant. Proc. 21,
2225,1989.
45. Yanaga, K., Makowka, L., Starzl, T.E. Is hepatic artery thrombosis after liver transplan-
tation really a surgical complication? Transplant. Proc. 21,3511,1989.
46. Gugenheim, J., Samuel, D., Fabiani, B., Saliba, F., Castaing, D., Reynes, M., Bismuth, H.
Rejection of ABO incompatible liver allografts in man. Transplant. Proc. 21,2223,1989.
47. Olson, L.M., Klintmalm, G.B., Husberg, B.S., Nery, J. Physiologic aberrations diagnos-
tic of hepatic graft malfunction: a case report. Transplant. Proc. 20,667, 1988.
48. Duquesnoy, RJ. Is there hyperacute rejection of the liver? Transplant. Proc. 21, 3506,
1989.
49. Davies, H.ff.S., Kamada, N., Roser, B. Mechanisms of donor specific unresponsiveness
induced by liver grafting. Transplant. Proc. 15,831,1983.
50. Davies, H.ff.S., Pollard, S.G., Caine, R.Y. Soluble HLA antigens in the circulation of
liver graft recipient. Transplantation. 47,524,1989.
51. Starzl, T.E., Kaupp, H.A, Brock, D.R, Linman, J.R Studies of rejection of the trans-
planted homologous dog liver. Surg. Gynec. Obstet. 112,135, 1961.
52. McBride, RA, Wheeler, H.B., Smith, L.L., Moore, F.D., Dammin, G.J. Homo-
transplantation of the canine liver as an orthotopic vascularized graft. Histologic
and functional correlations during residence in the new host. Am. J. Pathol. 41, 502,
1962.
53. Myburgh, J.A, Abrahams, e., Mendelsohn, D., Mieney, e.J., Bersohn, I. Cholestatic
phenomenon in hepatic allograft rejection of the primate. Transplant. Proc. 3,501, 1971.
54. Hunt, Ae. Pathology of liver transplantation in the pig. In: The Liver. Read, AE. (ed.),
London, Butterworth, 1967, p. 337.
55. Porter, K.A Pathology of the orthotopic homograft and heterograft. In: Experiences in
Hepatic Transplantation. Starzl, T.E. (ed.), Philadelphia, W.B. Saunders, 1969, p. 422.
56. Kamada, N., Brons, G., Davies, H.ff.S. Fully allogeneic liver grafting in rats induces a
state of systemic non-reactivity to donor transplantation antigens. Transplantation. 28,
429,1980.
57. Snover, D.e., Sibley, RK., Freese, D.K., Sharp, H.L., Bloomer, J.R, Najarian, J.S.,
Ascher, N.L. Orthotopic liver transplantation: a pathological study of 63 serial liver
biopsies from 17 patients with special reference to the diagnostic features and natural
history of rejection. Hepatology. 4, 1212, 1984.
58. Williams, R, Smith, M., Shilkin, K.B., Herbertson, B.M., Joysey, V., Caine, RY. Liver
transplantation in man: the frequency of rejection, biliary tract complications, and recur-
rence of malignancy based on an analysis of26 cases. Gastroenterol. 64,1026,1973.
59. Hayry, P., von Willebrand, E. Transplant aspiration cytology. Transplantation. 38,7,
1984.
60. Klintmalm, G.B.G., Nery, J.R, Husberg, B.S., Gonwa, T.A, Tillery, G.W. Rejection in
liver transplantation. Hepatology. 10,978,1989.
61. Stovin, P.G.I. The morphology of myocardial rejection a,nd transplant pathology. In:
Transplant Immunology, Clinical and Experimental. Caine, RY. (ed.), Oxford, Oxford
University Press, 1984, p. 78.
62. Conference of Medical Royal Colleges and the Faculties in the United Kingdom.
Diagnosis of brain-stem death. Br. Med. J. 2, 1187, 1976.
2,550, 1970.
64. Miyagawa, S., Hirose. H., Shirakura, R, Naka, Y., Nakata, S., Kawashima. Y .. Scya, T..
Matsumoto, M., Venaka, A, Kitamura, H. The mechanism of discordant xenograft re-
jection. Transplantation. 43, 745, 1988.
65. Reemtsma, K. Heterotransplantation. In: Human Transplantation. Rappaport, F. and
Dausset, J. (eds.), New York, Grune & Stratton, 1968, p. 357.
66. Starzl, T.E., Marchioro, T.L., Peters, G.N. Renal heterotransplantation from baboon to
man: experience with six cases. Transplantation. 2.752.1964.
67. Caine, RY., White, H.J.O., Herbertson, B.M., Millard, P.R, Davis, D.R, Salaman, J.R,
Samuel, J.R Pig-to-baboon liver xenografts. Lancet. 1,1176,1968.
68. Giles, G.R. Boehmig. H.J .. Amemiya. H .• Halgrimson. e.G., StarzL T.E. Clinical het-
erotransplantation of the liver. Transplant. Proc. 2,506,1970.
69. Terblanche, J .. Dent, D.M., Uys, e.J., Hickman, R, Ackermann, J.RW .. Harrison.
G.G., Biebuyck, J.F .• Saunders. S.J. Baboon into pig liver and kidney xenotransplanta-
tion. S. Afr. Med.l. 44,919,1970.
70. Jerusalem, e., van der Heyde, M.M., Jap, P., Reinking, J.W., Schmidt, W.J.H., Bilski, R,
Tjebbes, F.A. Rejection phenomena in heterotopic liver and homo- and heterografts.
71. Caine, RY., Davis, D.R, Pena, J.R, Bainer, H., de Vries, M., Herbertson, B.M., Joysey.
V.e., Millard, P.R, Seaman, M.J., Samuel, J.R, Stibbe, J., Westbrook, D.L. Hepatic al-
lografts and xenografts in primates. Lancet. 1,103,1970.
72. Setaff, A., Meriggi, F., Van de Stadt, J., Gane, S., Crougneau, S., Reynes, M., Rouger, P.,
Houssin, D. Delayed rejection of liver xenografts compared to heart xenografts in the
rat. Transplant. Proc. 19,1155,1987.
73. Filipponi, F., Michel, A, Houssin, D. Prolongation of guinea pig to rat xenograft survival
with BN 52063, a specific antagonist of platelet activating factor. Ital. 1. Surg. Sci. 19,325.
1989.
74. Platt,J.L., Vercellotti, G.M., Dalmasso,AP., Matas, AJ., Bolman, RM., Najarian. AS.,
Bach, F.H. Transplantation of discordant xenografts: a review of progress. Immunol.
Today. 11,450,1990.
75. Auchincloss, H. Xenogeneic transplantation. A review. Transplantation. 46, 1,1988.
76. Monden, M., Valdivia, L.A, Gotoh, M., Hasuike, Y., Kubota, N., Kanai, T., Okamura,
J., Mori, T. Hamster to rat orthotopic liver transplants. Transplantation. 43,745,1987.
77. Valdivia, L.A, Monden, M., Gotoh, M., Hasuike, Y., Kubota, N., Endoh, J., Okamura,
J .. Mori, T. Hepatic xenografts from hamster to rat. Transplant. Proc. 19.1158,1987.
78. Valdivia, L.A, Monden, M., Gotoh, M., Hasuike, Y., Kubota, N., Ichikawa, T.,
Okamura, J., Mori, T. Prolonged survival of hamster to rat hepatic xenografts using
splenectomy and cyclosporine administration. Transplantation. 44,759,1987.
79. Yamaguchi, Y., Harland, R, Bollinger, RR Prolonged survival of hepatic xenografts in
the hamster to rat combination: efficacy of cyclosporine in combination with splenecto-
my. Transplant. Proc. 20,731,1988.
80. Yamaguchi, Y., Halperin, E.e., Harland, R, Wyble, e., Bollinger, RR Significant pro-
longation of hamster liver survival in Lcwis rats by total lymphoid irradiation, cy-
c1osporine and with splenectomy. Transplantation. 49,13,1990.
81. CaIne, RY. Xenograft: functional definition. In: Xenograft 25. Hardy, M.A (ed.),
Amsterdam, Elsevier, 1989, p. 3.
Section IV
Experimental
Xenotransplantation
Chapter 16
Isolated Pancreas Islet Xenografting

F.T. THOMAS
Introduction
At present, short-term success has been obtained with isolated pancreas islet
allografts [1,2]. Clinical success has not been obtained with islet xenografts, al-
though at least one study suggest that the human immune response to islet
xenografts may be no stronger than that to allografts in some instances [3].
Recent advances in both islet isolation techniques and suppression of xeno-
geneic immune reactivity have provided a measure of optimism regarding the
future of islet xenografting which was not previously appropriate.
Isolated islet xenografts have a multitude of advantages over islet allo-
grafts. At present, islet xenografting is the only realistic hope for the
widespread application of pancreas islet replacement therapy. The limited ap-
plication of whole-organ pancreas allografting to type I diabetes is starkly evi-
dent when one considers that there are currently a million or more diabetics in
this country who could potentially benefit form islet replacement therapy and
that only 400 or so whole-organ pancreas allografts were performed in 1990.
Human organ donor shortage severely limits all areas of human organ trans-
plantation, but is a particular problem with islet transplants, since the shortage
of human pancreases is especially acute. In addition, problems with efficiency
in islet isolation will probably result in a need for a minimum of two human
donors per recipient.
Islet xenografting using discordant donor species, preferably animals
which are freely slaughtered for food, provides an immense potential for ap-
plication of islet replacement therapy to the problem of human type I diabetes.
It seems probable that virtually unlimited donor islets could be made available
and, at the same time, the many thorny ethical and cost-effective issues involv-
ing use of human donors would be obviated. Islet xenografting has recently
been demonstrated to represent a unique paradigm for xenografting in that
isolated islets from discordant species do not apparently suffer a high rate of
hyperacute or accelerated rejection. Discordant xenografts also do not have a
high rate of primary nonfunction and, in fact, appear to have a rate similar to
many allograft models [4]. This important matter will be discussed in more de-
tail later using discordant pig donor islet models as examples.
Historically, clinical islet xenografting is now nearly 100 years old, since the
first recorded clinical islet xenotransplant of a minced sheep pancreas was per-
formed in 1893 [5]. Modern progress in islet xenografting, however, only oc-
276 Isolated Pancreas Islet Xenografting
curred in the last 10 years. In 1975, Weber et al. reported a unique xenograft,
placing piscine (fish) islets into rats, with short-term function in irradiated
hosts [6]. Eloy et al. in 1979 reversed diabetes in rats on a short-term basis with
chicken embryo free pancreas grafts [7]. The first successful islet xenografts
surviving for more than 50 days with good function were reported in 1980, and
this is essentially the beginning of the modern era in islet xenografting [8].
To our knowledge, there are no published articles devoted entirely to pan-
creas islet xenografting, so this review, hopefully, will provide a more com-
plete overview of this developing field.
Pancreas Islet Isolation for Xenografting
Effective and efficient techniques for the isolation of pancreas islets are essen-
tial to both allografting and xenografting. The two major parameters against
which islet isolation techniques are measured are the purity and quantity of
well-functioning isolated islets obtained. In human allografting, the quantity
or efficiency of isolation can be critical because of donor shortfalls. Efficiency
is often less critical in xenograft donors lacking ethical or cost-effective restric-
tions. Purity in isolation would appear to be a virtue in both allotransplanta-
tion and xenotransplantation since the vigor of the rejection response as well
as problems of primary nonfunction appear at least partially related to islet
contamination by exocrine tissue and the resulting inflammation response, in-
sulitis, and islet injury [9, 10]. Thus, this discussion is pertinent to allografting
or xenografting, but it will be focused on isolation factors most germane to
xenografting.
In 1965, Moskalewski reported the isolation of guinea pig pancreatic islets
by collagenase digestion. In 1967, Lacy and Kostianovsky reported on the use
of collagenase and a Ficoll density gradient to isolate intact islets from the rat
pancreas [11]. Lacy was the first investigator to report short-term success in
islet xenotransplantation. He reported that low temperature (24°C) islet cul-
ture (and later culture at 37°C) prolonged survival of xenografts to 50 days
when used in combination with short-course antilymphocyte serum (ALS)
therapy [8]. Previous attempts at xenografting of islets were only successful on
a short-term basis [12].
In 1984, Grey et al. introduced an important variant into islet isolation pro-
cedures involving the initial distention of the pancreatic duct with a warm
(37°C-39°C) collagenase solution together with an initial incubation period
under these conditions [13]. Although these techniques were described for the
human pancreas, they were quickly adapted to pancreas islet isolation from
the nonhuman pancreas.
In 1988, Ricordi described an "automated" (semiautomated) method for
pancreatic islet isolation [14]. The basic features of this procedure are the use
of an islet isolation [14]. The basic features of this procedure are the use of an
isolation-perfusion chamber into which collagenase at 37°C is circulated until
Immunoisolation of Islet Xenografts in the Recipient 277
free islets are seen on microscopy. The isolation machine is then switched to
filtration-dilution-cooling phase to protect the islets from overdigestion. In
1990, Ricordi published the best description of a reproducible high-yield isola-
tion technique for pig islets incorporating a gentle shaking motion of the diges-
tion chamber to avoid disruption of the fragile pig islets [15]. Using this tech-
nique, he showed a yield of 5000-10 000 islets/g of tissue - by far the best
results recorded to date - in a large series of digestion of pig islets. Calafiore
has reported similar yields of pig islets, using a somewhat different technique
with a multienzyme digestion and variants in techniques of islet purification
[16].
There have been few descriptions of techniques for isolation of the other
nonhuman species pancreas islets. Lacy described a technique for bovine pan-
creas islet isolation using velcro strips, which has been improved upon recently
by Hering et al. [17]. The utility of bovine islets is questionable because of their
peculiar insulin secretory parameters. The major stimuli for insulin secretion
from bovine islets are fatty acids, and glucose stimulates insulin release poorly
[18]. Thus, it is doubtful that these islets would closely replicate human insulin
secretion patterns, although a recent description of two separate populations
of bovine islets responding to different physiological stimuli suggest the need
to re-examine this matter [19].
A number of workers have developed efficient techniques for isolating dog
pancreas islets. Alejandro has reported a large and excellent experience with
the dog pancreas [20]. Rajotte et al. have developed techniques for consistent
isolation of large volumes of dog islets (2000-5000 islets/g of pancreatic tissue)
[21]. This group has also developed techniques for reliable cryopreservation
of islets permitting the retained viability of the islets after thawing [22].
Immunoisolation ofIslet Xenografts in the Recipient
Immunoisolation is applied here to denote the separation of the donor tissue

from recipient tissue compartments in which recipient responder cells have
full access and are fully operational.
Immunologically Privileged Sites
One type of immunoisolation has been traditionally termed the "immunologi-

cally privileged site." In the case of xenografts, at least two sites have been
shown to confer some degree of immunoisolation upon islet xenografts - the
brain and/or intrathecal space, and the testes. Tzu showed prolonged survival
of xenografts in the intrathecal space, the subarachnoid space, and in the brain
[23]. Selawry et al. demonstrated the long-term survival of xenograft islets in
the testes [24]. N aji has recently demonstrated that the intra thymic space is im-
munologically privileged for islet allografts, and preliminary studies in his lab-
oratory have suggested that xenografts may also be immunoisolated in the in-
trathymic space [25].
All of these sites pose practical problems for future xenograft application.
Intracerebral injection poses many potential problems of a complex nature.
The intrathecal space is perhaps more attractive but also has potential for mor-
bidity, as does the subarachnoid space. The use ofthe intra thymic space is com-
plicated by the involution of the adult human thymus and the associated ques-
tions concerning its immunological activities in adulthood, any of which could
be involved in active mechanisms of generating immune tolerance in privileged
sites. Nevertheless, these privileged sites could well serve as an excellent repos-
itory for the small-volume islet xenograft and, therefore, deserve careful study.
Bioencapsnlation
The second concept of immunoisolation that has shown promise for

xenografting of islets is the use of so-called bioencapsulation techniques [26].
While a number of techniques of bioencapsulation or mechanical isolation of
islets have been demonstrated, the most attractive and promising technique
would appear to be the use of the bilayered polylysine-alginate membrane.
This technique, devised primarily by Sun and Lim, places a capsule of polyly-
sine-alginate around pancreatic islets [27]. As few as one, and as many as eight
to ten, islets can be enclosed in the capsules, which are usually 300-800 ~m in
diameter. Nutrients diffuse nicely into the capsule and are available to the
islet, while insulin (and presumably the other B cell hormones) diffuse out.
Immune cells, gammaglobulin, and even middle molecules like interleukin-l
(IL-l) can be shown to be blocked from entering the capsule [28]. Rather re-
markable survivals of discordant islets have been recorded with bioencapsula-
tion, and the function ofthe islets has also been clearly demonstrated [29].
There are a host of potential problems involving these bioencapsulation
membranes, including their ability to avoid rupture, proteinaceous collec-
tions, and attachment of immune cells, especially macrophages, on their sur-
face [30]. Nevertheless, they are an attractive and novel device for blocking
islet xenograft rejection. If bioencapsulation has any place in transplantation,
it may well be in xenografting, where there is a clear and present need for im-
munoisolation because of the hyper-reactive xenogeneic immune responses
operative in this situation.
Donor Islet Immunoalteration for Xenografting
Islet Cell Culture
Pioneering work by Lafferty et al. in 1975 demonstrated that cultured en-

docrine tissue enjoyed prolonged survival when allografted [31]. He suggested
that this immunoalteration culture was related to a loss of the so-called passen-
Donor Islet Immunoalteration for Xenografting 279
ger leukocytes, leading to a decrease in what Lafferty has termed "the second
signal to immune reactivity." His culture technique was also shown to prolong
survival of thyroid xenografts [32].
In 1979, Lacy et al. reported prolongation of islet allografts by culturing for
7 days at 24°C, together with a single injection of rabbit ALS [33]. Without the
ALS, prolonged survival was not produced, but with the ALS prolongation
greater than 100 days was achieved. Lacy reported in 1980 a marked prolonga-
tion with over 70% of islet xenografts surviving over 60 days if islets were cul-
tured for 7 days at 24°C, together with a single injection of rabbit ALS [8].
Lacy's group later expanded this work to demonstrate that culture at both
24°C and 37°C was able to produce prolongation of xenografts as well as allo-
grafts in a number of different models.
The precise mechanism by which this immunoalteration occurs is not clear,
and more recent work has suggested that it may be related to either loss of
class II surface antigen cells or, in fact, loss of class I donor antigens, which
could be expected to directly stimulate the CD8 cell [34]. There is much evi-
dence that xenograft rejection occurs via indirect presentation of antigens and
that, therefore, the direct stimulation of the CD8 cell by a class I antigen would
not be a major factor in xenograft immunogenicity [35].
Despite these uncertainties, it is that clear culturing cells does reduce im-
munogenicity. It should be mentioned that the explanation for this could
clearly range from the simple to the complex, and that the known reduction in
exocrine contamination of the cultured islets that occurs within 1-2 weeks af-
ter initiation of culture could well be responsible for the decreased immuno-
genicity seen. Certainly, the cultured cells show none of the hyperimmuno-
genicity seen with fresh islets which results in early rejection (1-3 days) of
grafts. Our group has recently shown prolonged graft survival with culture for
7-10 days at 37°C, and this is our preference for graft culture prior to xeno-
transplantation at the present time [36].
Cryopreservation
Rajotte's group has shown that cryopreservation of islets results in immuno-

alteration of the donor cells [37]. Again the precise mechanism by which this
occurs is not evident. It has been postulated that cryopreservation results in a
ioss of class II antigen cells during the process of freezing and thawing.
Because of this, cryopreservation could be utilized in future xenogeneic ef-
forts to provide extra donor tissue or donor tissue from mUltiple sources for
elective use, and further study ofthis technique for immunoalteration is clear-
ly desirable. Rajotte's work strongly suggests that the cryopreservation tech-
niques need to be precise in terms both of the rapidity of freezing and thawing
and of the techniques utilized (such as rapid-thaw procedures) in order to pro-
vide optimal viability and function of the islets.
Depletion of Antigen-Bearing Cells
A number of techniques have been used to deplete the class Ii antigen-bearing

cells from the islets prior to transplantation [38]. Faustman et al. were able to
demonstrate significant prolongation of islets with depletion of dendritic cells
using monoclonal antibodies [39]. Antidendritic cell antibodies and anti-la an-
tibodies are clearly also useful in depletion of these cells. Recently this matter
has become clouded by demonstrations that dendritic cells in fact proliferate
locally within the tissue and that initial removal of the dendritic cells may be
followed by a prompt reaccumulation of the cells. although these cells are
known to be marrow-derived.
In a novel series of studies recently. Morris' group demonstrated that den-
dritic cells actually migrate out of the allograft and into the spleen and other ex-
tragraft lymph node depots [40]. These studies suggest that previous attempts to
deplete class II cells with antibodies may not have been done in an optimal man-
ner. Nevertheless. the results justify optimism in this area. and future studies de-
signed to remove class II antigen-bearing cells and/or dendritic cells from islet
xenografts may yield valuable donor immunoalteration techniques.
Exposure to Ultraviolet Light
Hardy's group demonstrated that ultraviolet (UV) light is capable of decreas-

ing the immunogenicity of islets in culture [41]. They demonstrated a marked
prolongation of islet xenografts following treatment with UV light. The UV
light treatment is quite simple and, if effective, it could be easily applied to islet
therapy. This is yet another example of the great potential for application of
immunoalteration techniques in xenograft islets. which can be programmed
into precise protocols of immunoalteration due to the elective nature of the
donor islet procurement and the lack of limits on donor pretreatment.
Since UV light is a form of irradiation. it is not unexpected that x-irradia-
tion is also able to reduce islet immunogenicity [42]. In a similar manner x-irra-
diation will markedly decrease the immunogenicity of stimulator (donor) cells
in mixed lymphocyte culture. Radiation is thus another potential means of im-
munoalteration of islet culture.
In summary, immunoalteration of xenograft islets is a technique with great
potential for xenogeneic islet transplantation. This is especially so because, of
all of the islet grafts. xenografting lends itself to the widest variety of im-
munoalteration, since it is the most elective of the islet transplant procedures.
In contrast, islet allografting. involving as it does the emergent procurement of
human islet tissue. creates constraints in the ability to electively pretreat islets
to produce immunoalteration. In addition. the procurement of the pancreas is
often compromised by the need to use techniques for optimal procurement of
other organs which may not complement the pancreas procurement. Thus, im-
munoalteration of islets is most applicable to xenotransplantation. and it is
likely that it will receive its greatest use in this area.
Immunology and Immunosuppression in Islet Xenografting 281
Immunology and Immunosuppression in Islet Xenografting
Islet Graft Survival
Early studies of rodent allografting of pancreas islets indicated that the isolat-
ed islets evoked a strong, early immunological response [6,9]. This response
was manifested primarily as early nonfunction of the islets or as a pattern of
early function followed by rapid rejection of the grafts [10]. Both of these phe-
nomena are quite relevant and pertinent to the matter of pancreas islet
xenografting, since heightened immune reactivity seen in disparate species
can be expected to result in a high incidence of primary nonfunction and a high
rate and tempo of early graft rejection.
Most early studies in this area were quite discouraging, with prolongation
of grafts beyond 30 days representing, in general, a distinct exception [4].
Recent results have provided more optimism concerning the potential for iso-
lated pancreas islet xenografting between concordant species combinations
[8]. Equally encouraging have been the reports such as those of Ricordi et al. in
which certain islet treatments are able to prolong disparate xenografts [43].
The greatest hope is that tolerance to islet xenografts could be induced in a
manner similar to reported induction of tolerance to islet allografts [44].
Early studies with transplantation of islets were discouraging. However,
Sutherland's group was able to obtain significant function of porcine grafts
[12]. Results at the present time are better, perhaps because of better knowl-
edge of pancreas islet isolation techniques as well as the availability of better
immunosuppressive drugs [13]. More recent studies in the concordant rat-to-
mouse and mouse-to-rat combinations have demonstrated survivals of 50 or
more days in a number of models, using both islet pretreatment as well as im-
proved immunosuppression. The most significant results have been obtained
with the use of the L3T4 monoclonal antibody or polyclonal ALG, which seem
to block the T4-induced immune reactivity in these models, and the tech-
niques of anti-Ia antibody treatment and low-temperature culture of islets,
which appear to reduce the immunogenicity of the islets [45].
Importance of Experimental Methodology
Many studies of islet xenografting are compromised by experimental tech-

niques and reporting peculiarities. A number of studies have looked at islet
xenografting, but have not serially measured the blood glucose levels follow-
ing xenotransplantation. This is regarded by many investigators as an inappro-
priate omission, especially since it is such an easy thing to do. In many of these
xenograft models, reversal of hyperglycemia with proven lack of function of
native islets previously treated with streptozotocin (STZ) has not been
demonstrated clearly. It is sincerely hoped that future studies in this area will
all include a clear demonstration of islet function by serial monitoring of blood
glucose.
282 Isolated Pancreas Islet Xcnografting
The matter of the possible return of islet function following STZ treatment
is a highly problematic one [4, 46-48]. There seems to be no question that
many of the animals do have return of their native islets and, therefore. it is
necessary to demonstrate that the euglycemia is related to function of the islet
transplant and not the native islets. In some studies, it has been clearly shown
that this is precisely what has happened to maintain hypoglycemia in the late
period post transplant [48]. Often. islet function returns at about 100-120 days
after transplantation, following STZ treatment. The demonstration of clear-
cut rejection of pancreas islet transplants by the development of hyper-
glycemia for a period of time, which may later revert to euglycemia. is favor-
ed evidence that the transplanted islets are indeed functioning. However,
euglycemia in the late post-transplant period does not rule out the possibility
of return of function of native islets later after STZ treatment [49].
Reach's group have recently demonstrated a nice technique using high-
performance liquid chromatography (HPLC) to separate pig insulin from rat
insulin and to demonstrate that late insulin secretion is related to the trans-
planted islets (pig insulin) and not due to the native rat islets (rat insulin) [50].
Our group has accomplished similar studies using rat C-peptide, which mea-
sure only a small portion of pig insulin secretion. Together with the pig and rat
insulin levels, the rat C-peptide studies can be combined to establish from
what source the insulin is being secreted.
Immunosuppression
Immunosuppression for pancreas islet xenografts is a poorly developed area.

A number of studies have shown that cyclosporine (CsA) only poorly pro-
longs xenograft islet survival [51,52]. Nakajima et al. showed that totallym-
phoid irradiation (TU) would prolong pancreatic xenografts to the range of
30 days [53]. Lacy's group have demonstrated prolongation of concordant
xenografts with the use of an LT34 monoclonal antibody [54]. Simeonovic et
al. showed modification of the histopathology of pig islet rejection by suppres-
sion with the GK1.5 monoclonal antibody [55]. FK-506 prolongs islet grafts,
but, like CsA, has inherent islet cell toxicity [56].
Thomas et al. recently reported a significant prolongation of survival of pig
islet xenografts in strong responder Lewis rats [57]. Islet cell function was ob-
served for 12-14 weeks in these recipients which were given a combination of
rat antithymocyte globulin (RA TG) and 15-deoxyspergualin (DSG), with or
without splenectomy. This combination looks to be the most promising one
for current treatment of pancreatic islet xenografts. DSG has been previously
shown by our group and others to be a quite effective suppressive agent for
xenografts [58].
The lack of knowledge of the immune mechanisms in islet xenograft rejec-
tion is a major impediment to development of better immunosuppression. The
role of humoral antibody in islet destruction has not been well established.
Naji, Barker, and co-workers, however, have shown that islets appear to be
Practical Considerations in Islet Xenografting 283
more sensitive to antibody destruction than comparable grafts of skin, for ex-
ample [59]. Delmonico's findings, however, do not support this concept [60].
Our own results, showing a low rate «40% ) of primary nonfunction and also a
low rate (<10%) of early graft rejection of pig islets grafted to Lewis rats,
which have high levels of antidonor preformed antibodies, support
Delmonico's findings, but this area requires further, more detailed study.
A number of studies have suggested that the macrophage may be impor-
tant in islet xenograft rejection. This would explain the effectiveness of DSG
in preventing islet rejection, since this agent is known to have antimacrophage
activity [58]. Studies have also shown that the CD4+ cells in islet allograft, as
well as islet xenograft, rejection [54]. Studies of nonislet xenografts have sug-
gested a possible role for natural killer (NK), killer (K), antibody-dependent
cytotoxicity (ADCC), or lymphocyte-activated killer (LAK) effector cells in
xenograft rejection, but little is known of the role of these cells in islet
Islet cells, both allografts and xenografts, are apparently injured and de-
stroyed by inflammatory reactions consequent to contamination of the islet
preparation by pancreatic exocrine cells, leading to a sever inflammatory
response [10]. Hence, the importance of purity in islet preparations is
paramount. The ability of depletion of class II antigens to prolong islet
xenografts is in keeping with the suggestion by Auchincloss that indirect anti-
gen presentation is a major mechanism in xenograft rejection [35]. Hopefully,
future studies will develop better conceptual schemes of islet xenograft rejec-
tion.
Practical Considerations in Islet Xenografting
Site of Placement oflslet Xenografts
Currently, there is a preference for the intraportal placement of islet

xenografts, but the intrasplenic placement, as well as the renal subcapsular
placement, have also received a great amount of attention. The renal subcap-
sular placement, despite its unphysiological nature (drainage into the systemic
venous circulation), has produced consistently good results in xenografting
and, in fact, in some authors' studies, the results are superior to those seen with
intraportal placement [61].
Intraportal placement, however, has much to recommend it, including the
physiological nature of the placement, with drainage of secreted insulin into
the portal bed and, hence, to the liver, where it would normally be secreted
[62]. By adding a small laparotomy incision in which the omentum is brought
up into the wound, the islets can be placed intraportally by injection into an
omental vein or a mesenteric vein through the same incision used for a kidney
transplant. The intraportal placement can also be accomplished by cannula-
tion ofthe obliterated umbilical vein, which drains into the left portal vein, or
the percutaneous placement of islets into the portal system under fluoroscopic
or ultrasonic guidance.
The intrasplenic placement is a procedure midway between portal place-
ment and a more distal splanchnic placement. There is evidence that many of
the islets placed into the spleen, especially if placed into a clamped arterial
pedicle, will reflux into the portal system or later migrate to the liver [63]. The
precise reason why this placement works better than direct portal placement
is uncertain. It has been suggested by Kaufman et al. that the spleen, with its
expansible pulp, provides an accommodating area into which the islets can
grow, and also the toxic exocrine contaminants can spread and be reabsorbed
rapidly to reduce the inflammatory reaction and resulting rejection of the
islets [64].
Placement of the islets directly under the kidney capsule of the transplant-
ed kidney could potentially permit the biopsy of these islets clinically. This
may be an important consideration because little thought has been given at
present to the severe problem of rejection of islets and diagnosis of rejection
and the need to modulate immunosuppression to reverse rejection. It should
be remembered that, in the allograft situation where immune reactivity is
even less than that in the xenograft, survival of kidney transplants, for exam-
ple, could be as low as 10% if we did not have the capability to reverse rejec-
tion crises after diagnosing them and administering large doses of steroids
and other immunosuppressive agents. In short, there are multiple factors
which may provide important considerations in the placement of islet
xenografts.
Repeated Administration
Another technical consideration which seems destined to come to the fore-

front in islet xenografting, because of the ease of application to xenografting,
is the matter of administration of large numbers of donor islets or repeated in-
jection of donor islets [65], In a number of clinical cases to date, advantage has
been taken of the easy opportunity to simply do another islet injection, should
the first injection prove to be of borderline adequacy for maintenance of nor-
moglycemia, or to be simply inadequate, or if it is rejected. In the longest sur-
viving patents in the St. Louis and Miami series, second islet injections were
given after prolonged survival of the first islet injection, and these appeared to
restore normoglycemia [66,67].
If one reasons from the parallelism drawn by Terasaki's group between
the length of function of the first and the second allograft, one could reason
that long-surviving first xenografts might well presage a long-surviving sec-
ond, repeat xenograft [68]. In the case of xenografts, this principle could be
easily exploited because of the essentially unlimited availability of donor
islets.
Use of Multiple Donors
Another matter which deserves future consideration is the theoretical consid-

eration of multiple donors brought forth by Monaco's group [69]. This group
has achieved superior results in rodent xenografting, using islets from multiple
donors. The theoretical basis for this is not certain [70]. It has been suggested
that the use of multiple donors will permit a lower antigen load of any given
antigen grouping and/or the possibility that multiple antigen stimulation by
different antigens could "diffuse" the immune system from a strong primary
attack against a small, defined set of antigens. Regardless of the conceptual
scheme, the experimental facts indicate this system may be valuable.
This system, again, would have unique application to xenografting, since it
would be possible to simultaneously graft islets with antigens of marked anti-
genic disparity, such as a bovine graft with a pig graft or a sheep graft, etc. This,
again, illustrates one of the major advantages of xenografting over allografting
in that the amount and variety of donor tissue available for grafting is rather
large and provides many potential options.
Possibility of Ex Vivo Pretreatment
As a clinical surgical procedure, islet xenografting has a number of unique prop-

erties also. In contrast to a cadaver allograft, an islet xenograft is an elective sur-
gical procedure. The procedure can be scheduled well in advance and, in the
usual situation where donor islets are maintained in culture, one would sched-
ule the procedure some 5-10 days in advance, when the availability of islets was
anticipated. The entire procedure would be quite leisurely and orderly and
would even involve lengthy final testing of the islet preparation over perhaps a
2- or 3-day period before the final decision is made to transplant the islets.
This completely elective nature of the xenograft procedure also makes it
possible to engage in novel and unusual ex vivo pretreatment of the donor
islets, as well as recipient pretreatment by techniques designed to induce toler-
ance. Induction of tolerance across xenogeneic barriers is currently in a pre-
liminary developmental phase. The work of Sachs and others suggests that
xenogeneic chimerism and xenogeneic tolerance is indeed possible, at least in
relatively concordant species [71].
Choice of Donor Species
The first necessary prerequisite for human xenografting would be the isolation
in viable and rather pure form of animal islets which would be suitable for hu-
man implantation. Furthermore, these islets should preferably be from a dis-
parate species, nonprimate in type, in which no ethical constraints exist re-
garding the obtaining of adequate pancreas donor tissue. Animals satisfying
these criteria are almost all disparate to the human. The animals which logical-
ly come to mind are the pig, the cow, and other species of animals freely used
for human food and freely slaughtered. In general, there are few ethical con-
straints to the use of virtually any of the disparate species, including sheep,
goats, horses, or other farm animals. Rodent donors are impractical, since the
number of islets obtained is so small that up to thousands of individual rodents
might be required for a single human transplantation.
A broad prerequisite for adequacy of a species for human xenotransplanta-
tion is that the physiology of the pancreas islets in these species ought to be
near that of the human. The physiology of islets varies throughout the animal
kingdom, and patterns of maintenance of specific levels of blood sugar, pat-
terns that influence secretion, counter-regulatory mechanisms, etc., do vary
between the species [72, 73]. There is evidence that quite distant species in-
cluding fish and chickens will maintain a carbohydrate metabolism relatively
close to that of the human.
The classic species which is highly analogous to human islets is the pig islet.
Pig insulin has been used for years as a replacement for human insulin, and it
has a remarkably similar chemical structure with a significant difference in
only two amino acid residues between the two insulins. The human antibody
response to pig insulin is weak in most cases. The pig maintains fasting blood
sugar levels in the range of that seen in the human, and pig islet secretion in
vivo can be shown to be relatively similar to the human, including the biphasic
insulin response.
Caution concerning these physiological differences is evident from the ex-
perimentalliterature, however. For example, Ricordi et al. reported on ham-
ster islets transplanted into mice and found that the hamster islets maintained
a fasting blood sugar level of around 66 mg%, which corresponds to blood
sugar levels in hamsters, but is considerably different from the average 145
mg% in fasting mice [74]. Thus, the hamster islets maintained a level unique
to the hamster, and it is possible that variations in islet physiology between
species may render some species inappropriate donors of islets for the hu-
man. Over all, however, one suspects there is a much higher degree of similar-
ity than difference between the various species in terms of their carbohydrate
metabolism, which undoubtedly had a common evolutionary basis.
As mentioned, in general, the goal in islet isolation for xenotransplantation
is purity and not necessarily efficiency of extraction, since the amount of donor
xenograft tissue is often unlimited. Because immunogenicity is such a major
problem, the goal should be to increase the purity of cells in order to reduce re-
jection potential as much as possible.
Practical aspects are bound to be major considerations in choosing the
xenograft donor species. Animals such as the goat, the sheep, and numerous
other species could be seen as potentially useful xenograft donors. The avail-
able number of these donors, the ease in breeding them, and the simple matter
of their availability, may well playa role in the feasibility of use of a given
species. Needless to say, the overriding medical considerations in human dis-
ease will usually result in extraordinary measures being taken to provide an
ample supply of animals for critical human use.
Practical considerations and outright cost-effectiveness, however, are a
salient feature of our current medical environment. Thus, it is highly likely
that the pig and/or cow might prove to be the most practical animals for
xenograft donation if the physiology of these animals' islets is appropriate and
the difficulty of preventing rejection is not overwhelming. Both pigs and cows
grow to quite large size, and it is not unusual to find pig pancreases in the range
of 300 g and cow pancreases in the range of 500-600 g, containing millions of
islets, which could potentially provide adequate donor islets for five to ten hu-
man transplants from a single animal. Add this to the situation in which these
animals are widely and freely slaughtered for human food and other human
uses, and you have many compelling reasons for the choice of these species.
In addition, the pig, in particular, and, to a large degree, the cow are free of
pathogens that would endanger the human species insofar as we know. Pig and
cow products are widely consumed by the human without ill effects, including
fresh products, such as cow's milk, which are the result of lactation processes
and, therefore, might be expected to contain any of the shedding viruses and
other pathogens found in such tissues.
In contrast to this situation, for example, the primates represent problem-
atic donors. It is clear that these animals harbor a number of viruses and other
pathogens which can be quite dangerous and even fatal to the human, such as
the monkey B virus, etc. [75]. Some monkey species have an human immuno-
deficiency virus (HIV), and it is quite possible that monkeys also have many
other viruses pathogenic to man, including some potential leukemogenic
viruses, which are unknown at the present time [76]. These considerations will
most certainly form the basis for future directions in xenografting.
Insulitis and the Recurrence of Diabetes
In the case of islet transplantation for diabetes, an interesting and probably

important area is the recurrence of the original disease process (diabetes) in
the transplanted islets. Recurrence of diabetes in islet transplants has been
well documented by the Minnesota group in human leukocyte antigen
(HLA)-identical siblings [77]. The insulitis that precedes type I diabetes is an
immune attack upon the islets, which seems, at least to some degree, to be
related to the major histocompatibility antigens. Thus, it seems likely that rep-
etition of these histocompatibility antigens will lead to the possibility of the re-
currence of disease.
In this respect, the xenograft, especially from a disparate species, could be
expected to be relatively immune to recurrence of disease, since the mem-
brane antigens which would form the target of an immune insulitis are quite
dissimilar to those which were the targets of the original disease process. In
fact, there is some experimental evidence for this in the nonobese diabetic
(NOD) mouse, in which isografts are shown to suffer the heaviest early insuli-
tis after transplant, with allografts in between, and xenografts showing the
least amount of early immune damage following transplantation [78].
All of these matters point up the increasing understanding that xenografts,

while they have unfavorable characteristics, also have some highly favorable
characteristics for transplantation in the future.
Modification of Donor Tissue
In this respect, techniques of immunoalteration of donor tissue will be critical-

ly important. At the present time, it would seem that the mostly potentially ef-
ficacious techniques for decreasing islet immunogenicity would be focused
primarily upon islet cell xenograft culture for a minimum of 5-7 days. Studies
to date would suggest that culture at either 24°C or 37°C would be useful in re-
ducing immunogenicity. The value of treatment of the cultured islet cells with
anti-Ia monoclonal antibody seems problematical at present. Elimination of
contaminating exocrine tissue is probably the most important desired goal of
islet culture. UV irradiation may well form an excellent adjuvant in immuno-
alteration of cultured islets. Irradiation of islets is another potential for im-
munoalteration which needs to be more completely studies.
Xenografting permits the widest use of immunoalteration techniques on
donor tissue because of our ability to carefully program the donor procure-
ment and to define its time frame and parameters, and also because there is
lack of ethical constraint on treatment of donors. It is entirely possible that
treatment such as total body irradiation of the donor, prohibitive in human
donors, could be a well-accepted procedure in xenograft donors.
Modification ofthe Recipient Immune Response
Second only to immunoalteration of the xenograft donor is the modification of

the recipient immune response, which is currently the Achilles heel in
xenografting. Approaches to immunosuppression for xenografting could take
many different avenues. The blockage of humoral antibody is felt by some to
be an important goal for islet xenografting. The macrophage has been clearly
shown by Kaufman et al. [79] to be an important effector cell in both primary
nonfunction and early graft rejection. The CD4+ cell is important in islet
xenografting and has previously been shown by Auchincloss to be important
in nonislet xenografts [80]. Indirect antigen processing has also been shown by
Auchincloss to be an important mechanism of xenograft rejection [35], and
this was corroborated by islet studies [34].
Development of tolerance to islet xenografts, like allografts, is a very im-
portant goal. Xenogeneic tolerance is very difficult to achieve in discordant
species, although it has been achieved in concordant species [81]. Finally, one
of the most attractive goals for islet xenografting would be the development of
immunoisolation techniques either using privileged sites or bioencapsulation
techniques. Such systems might permit the elimination of immunosuppression
which must continue to be a goal for all of transplantation, including islet
transplantation, where suppressive agents are toxic to the islet.
Promising Islet Xenograft Donors 289
Technical Options
A major potential of pancreas islet xenografting will be to provide for impor-

tant technical options in the placement of these grafts. These options would in-
clude the use of multiple donors, the use of multiple islet injections done on a
sequential basis, the possibility of multiple placement sites, the possibility of
utilizing large amounts of tissue from the single donor to induce tolerance over
a period of time, as well as many other considerations that will probably come
to light with early xenograft experience. An example of this would be the ap-
plication of the innovative technique of Monaco's group utilizing multiple or-
gan donors [65]. In the case ofxenografts, the free availability of donor islets
and the use of multiple donors of a given species or, in fact, the simultaneous
use of islets from different species make multiple donor techniques quite at-
tractive. Studies in Monaco's lab suggest that hypoimmunogenicity may occur
related to the use of multiple donors of different antigen make-up.
In short, islet xenografting will foster the development of a number of im-
portant technical options, both in terms of immunoalteration of donor tissue,
timing and technique of transplantation, the timing and technique of immuno-
suppression, as well as techniques which would modify the recipients in man-
ners which could favorably alter the xenograft survival. Induction of xenograft
tolerance is yet another example of the large potential for immunomodulation
in islet xenografting. It seems entirely conceivable that some of the major ad-
vances in islet transplantation will occur with innovative technical modifica-
tions possible with the use of donor xenografts for islet cell replacement in the
future.
Promising Islet Xenograft Donors
Pig
Because of the large potential for pancreas islet xenografting in treating the
huge number of diabetics who might require this procedure, our group began
investigations to develop techniques for the isolation of pancreatic islets ap-
proximately 3 years ago. Early in our experience, encouraging results were ob-
tained with the use of the porcine pancreas obtained at animal slaughter for
food, and we have, therefore, continued our work primarily in this animal. The
pig has classically been the source of insulin used for human diabetics. The pig
pancreas islet has a physiology very much like that of the human [82].
The leading role in the development of techniques for isolation of pig islets
has been taken by Camillo Ricordi, working in Lacy's laboratory and, later, in
Milan and Pittsburgh [14, 15, 83, 84]. Ricordi's description of isolation of pig
islets in April 1990 represents perhaps the first comprehensive demonstration
of a consistently successful technique for pig islet isolation, using a semiauto-
mated technique [15].
Using Ricordi's techniques, his results have been reproduced by our labo-
ratory recently. We have also had an islet yield of about 8000-10 000 islets per
gram prior to Ficoll separation, and 4000-5000 islets per gram after Ficoll sep-
aration. with a high degree (>90%) of purity. In addition, recent studies in our
laboratory have demonstrated the ability to transplant these pig islets with a
low rate (10% or less) of primary nonfunction and a survival with recipient
euglycemia in a difficult pig-to-Lewis rat discordant model for 10-14 weeks
with some newer forms of immunosuppression.
These results are quite promising since many of the results of islet allografts
have been less impressive. Finally, the expected high rate of primary nonfunc-
tion relating to high immune reactivity across discordant species barriers has
not been borne out in these early studies, nor has there been a large rate of ear-
ly islet xenograft loss to rejection.
Thus, the critical factors in pig islet isolation from the intact pig pancreas
seem to have been worked out, since they are reproducible from laboratory to
laboratory. Our group has been able to verify the concepts set out by Ricordi.
Technique of Pig Islet Isolation

Perhaps the most critical thing about pig islets is their fragility and ease of dis-
ruption. It is. therefore, necessary to utilize a gentle technique - far more gen-
tle than that used in islet separation from the human or other animals. The pig
pancreas is best digested with a static ductal perfusion after removal of the
pancreas, with a short warm ischemic time. This is currently a limitation since
the pigs are used primarily for slaughtered meat products and full cooperation
in obtaining the pancreas is usually not obtained. We have been able to obtain
satisfactory pancreases from both old breeding sows, which give large pan-
creases up to 300 g, as well as market-weight pigs.
The pancreatic duct is perfused with a 2% collagenase solution at 37°C to
achieve full distention of the gland. The gland is usually cannulated proximally
and distally and a catheter sewn in place for perfusion under pressure. We uti-
lize a volume of collagenase which is twice the weight of the pancreas (i.e., 200
ml for a 100-g segment of pancreas). Following a static perfusion for about
5 min, the porcine pancreas is placed in the digestion chamber, described by
Ricordi (Fig. 16.1), consisting of a stainless steel chamber of approximately
350 ml volume. The chamber is capped with a filter of 200 !lm diameter and is
perfused with a collagenase solution containing 2% fetal bovine serum at
37°C. The collagenase solution is heated in a water bath, beginning at a tem-
perature of about 29°C and gradually warmed to 37°C. The perfusate enters
the chamber and comes into contact with the pancreas; the digested portions
of the pancreas pass through the filter exit at the top of the chamber. An in-line
roller pump maintains a flow of 50 ml per minute.
After approximately 10 min of digestion, with sampling of the recirculating
fluid, free islets can be seen with a dithizone (DTZ) stain supplemented with a
2% dimethyl sulfoxide (DMSO) solution. Once free islets are seen, the efflu-
ent from the chamber is directed into a beaker in a 4% water bath. The gland is
Promising Islet Xenograft Donors 291
Peristaltic
Recirculation-Dilution Pump Heating Circuit Healing
Coil
-
Bypass
Switch
~
- ~
1
Hank's
t
Collecting
t
Recirculation
t
Cooling
t
Pa ncreas
- Vertical
1
Isolatio n Chamber
Sol ution Flask Cylinder Circuit Shaker
Fig. 16.1. Automated procedure for isolation of pancreatic islets (by courtesy of Dr. C.
Rieordi [14])
then perfused at 150 ml per minute with a collagenase solution containing 10%
fetal calf serum, with usually a 3-41 volume. The resulting dispersed pancreat-
ic fragments are purified on a Ficoll density gradient to a purity of 80%-95%
islets. The final preparation is carefully monitored by DTZ staining, concen-
trated, and usually placed in culture prior to transplantation.
At present, yields of 5000 islets/g of pig tissue, after Ficoll separation, have
been routinely achieved by Ricordi's laboratory, and our laboratory has been
able to corroborate his reports using this technique. This may be the most
promising donor islet preparation for future xenografting.
Fetal Islets
Hellerston et at. [81] recently published data on isolation of fetal pig islets.
These islets were obtained from fetal pigs of gestational age 60-70 days. One
litter produced about 100 000 islets, and these islets performed well in nude
mice. Fetal islets have the advantage of ease of extraction due to a low amount
of fibrous tissue in the fetal pancreas, a potential for expansive growth and
differentiation, and possibly some degree of hypoimmunogenicity [85].
Although human fetal tissue use is currently very restricted due to ethical con-
cerns over fetal tissue use and abortion, the use of animal donors would proba-
bly not suffer similar ethical quandaries, and fetal xenograft tissue may well be
a useful source of donor tissue for islet xenografting.
292 Isolated Pancreas Islet Xcnografting
Teleost Fish
In certain teleost fishes, islet tissue is aggrcgated into visible organs, called
Brockmann bodies. They are easily identified and have a high purity of islets.
Weber performed the original xenografting of piscine islets to rats [6].
Recently, Schrezenmeir et aI. identified certain teleost species with blood sug-
ars in the human range (86-89 mg%) [86]. In addition, they showed these islets
released insulin and glucagon in a glucose-dependent manner, tolerated mam-
malian temperatures, and survived encapsulation for weeks. These islets,
available from such common fish as flounder and trout, would seem to be an
exciting new approach to potential islet donation for xenografting [87].
Dog
Techniques for isolation of islets from dogs have been well established [88,89].
In general, the dog islet is characterized as easier than the pig to isolate, but
harder than the rodent. The size of the dog makes dog islets attractive for hu-
man use. There is apparently good physiological compatibility between dog
islets and human islets. The major problem with the use of the dog may well be
ethical concerns.
It will be interesting to see whether the ethical constraints advocated by
some will be pursued with equal vigor when they result in a direct interference
with a life-saving procedure (e.g., animal-to-human heart xenografting). Most
animal rights disagreements to date have involved situations where the use of
animals only indirectly impact upon human care (experimentation, training of
surgeons and doctors, etc.)
In summary, the dog is a potentially attractive source of islets for human
transplants, but ethical considerations will be paramount in the decision on
whether to use dog donors for islet xenografting.
Comment
Although this chapter is perhaps the most comprehensive publication solely on
islet xenografting to date, it must necessarily be considered preliminary. The
chapter seeks to summarize the state of clinical and, especially, experimental
islet xenografting. In addition, it seeks to plan some future strategies for appli-
cation and expansion of islet xenografting. Hopefully, it more fully outlines the
large potential for islet xenografting that has been developed by workers in the
pancreas islet isolation field those who developed techniques for donor islet im-
munoalteration, those publishing on recipient immunoisolation techniques,
and those seeking to achieve successful islet xenografting by the traditional
manner of immunosuppression of the host response. In the future, these efforts
may be complemented by induction of host tolerance to islet xenografts.
Perhaps the highest goal of current work in this area will be to develop the
optimal "chimeric" approach to islet xcnografting, borrowing the best quali-
References 293
ties of these individual areas and molding an elegant composite creature, re-
flecting the noblest features of them all. Hopefully, the information in this
chapter will provide the chimera creator with strategic information which will
aid him or her in their quest.
Acknowledgements. Some of the author's work in relation to this chapter was

supported by NIH Grant #ROl A122293 and Diabetes and Education Fund
Grant #B39-II89.
References
1. Scharp, D.W., Lacy, P.E., Santiago, J.V., McCullough, CS., Weide, L.G., Falqui, L.,
Marchetti, P., Gingerich, RL., Jaffe, A.S., Cryer, P.E., Anderson, CB., Flye, M.W.
Insulin independence after islet transplantation into type I diabetic patient. Diabetes. 39,
515,1990.
2. Tzakis, A.G, Ricordi, C, Alejandro, R, Zeng, Y., Fung, J.J., Todo, S., Demetris, A.J.,
Mintz, D.H., Starzl, T.E. Pancreatic islet transplantation after upper abdominal exenter-
ation and liver replacement. Lancet. 336,402, 1990.
3. Kondratiev, Y.Y., Sodovnikova, N.V., Petrova, G.N., Fedotov, V.P., Bljumkin, Y.N.,
Ignatenko, S.N., Pankov, Y.A. Islet cell transplantation in type I diabetes mellitus: eval-
uation of humoral immune response. Exp. Clin. Endocrinol. 93,147,1989.
4. Weber, C, Zatrigi, A., Weil, R, McIntosh, R, Hardy, M.A., Reemtsma, K. Pancreatic
islet isografts, allografts, and xenografts: comparison of morphology and function.
Surgery. 79,144,1976.
5. Williams, P.W. Notes on diabetes treated with grafts of sheep's pancreas. Br. Med. 1. 2,
1303,1893.
6. Weber, C, Weil, R, McIntosh, R, Hogle, H., Warden, G., Reemtsma, K. Xenotrans-
plantation of piscine islets into hyperglycemic rats. Surgery. 77,208, 1975.
7. Eloy, R, Haffen, K., Kedinger, M., Grenier, J.F. Chick embryo pancreatic transplants
reverse experimental diabetes of rats. 1. Clin. Invest. 64,361, 1979.
8. Lacy, P.E., Davie, J.M., Finke, E.H. Prolongation of islet xenograft survival without con-
tinuous immunosuppression. Science. 209,283,1980.
9. Reckard, CR, Stuart, F., Schulak, J.A. Immunologic comparisons of isolated pancreatic
islet and whole-organ allografts. Transplant. Proc. 11,563,1979.
10. Kaufman, D.B., Rabe, F.L., Platt, J.L., Dunn, D.L., Sutherland, D.E.R Effect of host im-
munomodulation on the prevention of islet allograft primary non-function in a murine
model. Transplant. Proc. 22,857,1990.
11. Lacy, P.E., Kostianovsky, M. A method for the isolation of intact islets of Langerhans
from the rat pancreas. Diabetes. 16,35,1967.
12. Sutherland, D.E.R, Matas, A.J., Najarian, J.S. Pancreas islet cell transplantation. Surg.
Clin. N. Amer. 58,365,1978.
13. Morris, P.J., Gray, D.W., Sutton, R Pancreatic islet transplantation. Br. Med. Bull. 49,
224,1989.
14. Ricordi, C, Lacy, P.E., Finke, E.H., Olack, B.J., Scharp, D.W. Automated method for
isolation of human pancreatic islets. Diabetes. 37,413,1988.
15. Ricordi, C, Socci, C, Davalli, A.M., Staudacher, C, Baro, P., Vertova, A., Sassi, I.,
Gavazzi, F., Pozza, G., DiCarlo, V. Isolation of the elusive pig islet. Surgery. 107,688,1990.
16. Calafiore, R, Basta, G., Calcinaro, F., Falorni, A., Pietropaolo, M., Avellini, L.,
Brunetti, P. Islands of Langerhans transplant in the treatment of diabetes mellitus: cur-
rent status, indications, future perspectives. Recenti. Prog. Med. 81, 94,1990.
17. Hering, B.J., Romann, D., Clarius, A., Brendel, M., Bretzel, RG., Federlin, K. Bovine
islets of Langerhans: potential source for transplantation? Diabetes. 38,206, 1982.
18. Hering. B.1 .. Romann. D .. Clarius. A .. Brendel. M .. BretzeL R.G .. Federlin. K. Islands of
Langerhans of cattlc - potential donor tissue for clinical islet transplantation? Z. Exp.
Chir. Transplanr. Klinstliche Organe. 22.138.1989.
19. Bonner-Weir. S.. Fike. A.A. A dual population of islets of Langerhans in bovine pan-
creas. Cell Tissue Res. 206. 157. 1980.
20. Alejandro. R .. Strasser. S.. Zucker. P.F .. Mintz. D.H. Isolation of pancreatic islets from
dogs. Semi-automated purification on albumin gradients. Transplantation. 50. 207.
1990.
21. Finegood. D.L.. Warnock. G.L.. Kneteman, N.M., Rajotte, R.Y. Insulin sensitivity and
glucose effectiveness in long-term islet autotransplanted dogs. Diabetes. 38. 189. 1989.
22. Rajotte. R.Y .. Warnock. G.L.. Kneteman. Erickson. C. Ellis. D.K. Optimizing cryop-
reservation of isolated islets. Transplant. Proc. 21.2638. 1989.
23. Tzu. W.J .. Tai, J. Xenotransplantation of rat pancreatic endocrine cells in spontaneous
and streptozotocin-induced diabetic monkeys. Transplanr. Proc. 21,2736,1989.
24. Selawry. H.P .. Whittington, K.B., Bellgrau, D. Abdominal intratesticular islet-xenograft
survival in rats. Diabetes. 38,220, 1989.
25. Naji, A. Personal communication. December. 1990
26. Ricker. A.T.. Stockberger. S.M., Halban, P.A. In: The Immunology of Diabetes Mellitus.
1aworski, M.A. (ed.) Amsterdam, Elsevier. 1986, p. 193.
27. O·Shea. G.M .. Sun. A.M. Encapsulation of rat islets of Langerhans prolongs xenograft
survival in diabetic mice. Diabetes. 35,943, 1986.
28. Siehers, U., Zekorn, T., Bretzel, R.G., Rcnardy. M .. Zschocke. P., Planck. H .. Federlin.
K. Bioartificial pancreas: islet survival and interleukin-I action. Transplant. Proc. 22.
2035,1990.
29. Wu. Z.G., Shi, Z.O., Lu, Z.N., Yang, H., Shi, F.Y., Zheng, X.R., Sun, A.M. In vitro cul-
ture and transplantation of encapsulated human fetal islets as an artificial endocrine
pancreas. Trans. Am. Soc. Artit Inrern. Organs. 35,736,1989.
30. Weber, C, Zahinski, S., Koschitzky, T.. Wieher, L.. Rajotte. R.. D·Agati. Y .. Peterson.
L., Norton, 1.. Reemtsma. K. The role of CD4+ helper T cells in the destruction of mi-
croencapsulated islet xenografts in NOD mice. Transplanration. 49,396, 1990.
31. Lafferty, K.1., Cooley, M., Woolnough,l., Walker, K.Z. Thyroid allograft immunogcnic-
ity is rcduccd after organ culturc. Science. 188,259,1975.
32. Sollinger. H.W., Burkcnholder, P.M., Rasmus, W.R., Bach, F.H. Prolonged survival of
xenografts aftcr organ culture. Surgery. 81,74,1977.
33. Lacy, P.E., Davie. 1.M., Finke. H .. Scharp. D.W. Prolongation of islet allograft survival.
Transplantation. 27. 171, 1979.
34. Stock, P.G., Ascher, N.L., Chen, S., Bumgardner, G., Field, M.1., Suthcrland, D.E. The
alloimmune response to murinc islets occurs via indirect antigen presentation to L YT2+
and UT4+ lymphocytes. Transplant. Pmc. 22,841, 1990.
35. Auchincloss, H. Xenotransplantation - a review. Transplant. Rev. 46, I, 1990.
36. Thomas, F., Pittman, K. Factors in effective isolation and culture of adult pig islets
(API). Submitted for puhlication.
37. Coulombe, M., Warnock, G., Rajotte, R. Prolongation of islet xenograft survival by cry-
opreservation. Diabetes. 36, 1086,1987.
38. Lafferty, K., Davie, 1.M., Finke, E. Prolongation of islet xenograft survival (rat-to-
mouse). Diabetes. 30,285,1981.
39. Faustman, D.L., Stienman, R.M., GabeL A.M. Depletion of anti-Ia + dendritic cells.
Pmc. Natl. Acad. Sci. USA. 81,3864. 1984.
40. Larsen, C, Austyn, 1., Morris, P. The role of graft-derived dendritic leukocytes in the re-
jection of vascularized organ allografts. Ann. Surg. 212.308, 1990.
41. Chahot. 1 .. Weber. C, Hardy, M., Rivera, S., Bailey-Braxton, D., Strausberg, L.. Wood,
M, Chow, 1., PI-Sunyer, F.x., Reemtsma, K. Synergy of ALS and UY-B in prolongation
of primate-to-mouse islet xenograft survival. Transplant. Pmc. 19. 1160, 1987.
42. Walter. P., Lesmeister. F.. Leetz. H .. Schnabel, K.. Feifel, G., 1aeger, S. Influence of and
UYB irradiation on graft survival and function of islets of Langerhans. Diabetes. 38,
Suppl. 1,288, 1989.
References 295
43. Ricordi, c., Lacy, P.E., Sterbenz, K., Davie, J.M. Low temperature culture of human
islets or in vivo treatment with L3T4 antibody produces a marked prolongation of hu-
man-to-mouse islet xenograft survival. Proc. Natl. Acad. Sci. USA. 84,8080,1987.
44. Has, L., Lafferty, K., Allison, A., Eugui, E.M. RS-61443 allows islet allografting and spe-
cific tolerance induction in adult mice. Transplant. Proc. 22,876, 1990.
45. Lacy, P.E. Islet transplantation. Clin. Chem. 32/lOB, B76, 1986.
46. Kruszinska, Y., Hone, D., Morley, A., Alberti, A. Preservation of islet function and mor-
phology in high dose streptozotocin diabetic rats. Diab. Res. 3, 175, 1986.
47. Trimble, E., Karakash, c., Malaisse-Lagae, R. Effects of intraportal islet transplantation
on the transplanted tissue and recipient pancreas. Diabetes. 29,341,1980.
48. Richardt, M., Menden, A, Bretzel, R., Federlin, K. Islet transplantation for experimental
diabetes of the rat. B cell regeneration following transplant. Horm. Metab. Res. 16,551,
1984.
49. Slipjepcevic, M., Helmke, K., Federlin, K. Islet transplantation in experimental diabetes
ofthe rat. Horm. Metab. Res. 7,20,1975.
50. Chicheportiche, D., Darquy, S., Lepeintre, J., Capron, F., Halban, P.A., Reach, G. High-
performance liquid chromotography analysis of circulating insulins distinguishes be-
tween endogenous insulin production (a potential pitfall with streptozotocin diabetic
rats) and islet xenograft function. Diabetologia. 33,457, 1990.
51. Terasaka, R., Lacy, P.E., Bucy, R.P., Davie, J.M. Effect of cyc1osporine and low tempera-
ture culture on rejection ofisletxenografts (rat -to-mouse). Transplantation. 41,661,1986.
52. Stegall, M., Chabot, J, Weber, c., Reemtsma, K., Hardy, M.A. Pancreatic islet transplan-
tation in cynomolgus monkeys. Transplantation. 48,944,1989.
53. Nakajima, Y., Nakano, H., Nakagawa, H., Segawa, M., Shiratori, T. Effect of totallym-
phoid irradiation on pancreas islet xenograft survival in rats. Transplantation. 34, 98,
1982.
54. Lacy, P.E. Present status of islet transplantation. Clin. Inv. Med. 10,496,1987.
55. Simeonovic, c., Ceredio, R., Wilson, J.D. Effect ofGK1.5 antibody dosage on survival of
pig pro islet xenografts in CD4+ Tcell depleted mice. Transplantation. 49,849,1990.
56. Yosunami, Y., RYU, S., Kamei, T. Characterization of immunosuppressive activity of
FK506 in rat islet allografts. Transplant Proc. 22,875,1990.
57. Pittman, K., Thomas, F.T., Patselas, T., Thomas,J.M. Long-term (12-14 week) survival of
pig islet xenografts (PIX) in Lewis rats (LR) treated with splenectomy (SPL), deoxysper-
gualin (DSG) and rabbit anti thymocyte globulin (RATG). Submitted for publication.
58. Thomas, F. Comparative immunosuppression for xenografting. In: Xenograft 25. Hardy,
M.A. (ed.) Elsevier; Amsterdam, N ew York, Oxford, 1989, p. 55.
59. Naji, A., Barker, C. Differential susceptibility of endocrine tissues to humoral immunity.
Surg. Forum. 29, 349, 1978.
60. Delmonico, F., Chase, c., Russell, P. Transplantation of rat islets of Langerhans into dia-
betic mice. Transplant. Proc. 9, 367, 1977.
61. Sutton, R., Greay, D.W., Burnett, M., McShane, P., Turner, R., Morris, P. Metabolic
function of intraportal and intrasplenic islet autografts in cynomolgus monkeys.
Diabetes. 38, 182, 1989.
62. Alejandro, R., Cutfield, R.G., Steinvold, F., Polonsky, K.S., Noel, J., Olson, L.,
Dillberger, J., Miller, J., Mintz, D.H. Natural history of intrahepatic canine islet cell
autografts. Clin. Invest. 78,1339,1986.
63.Warnock, G.L., Rajotte, V.R., Procyshyn, A.W. Normoglycemia after reflux of islet-con-
taining pancreas fragments into the splenic vascular bed in dogs. Diabetes. 32,452, 1983.
64. Kaufman, D., Morel, D., Field, M., Tzardis, P., Munn, S.R., Sutherland, D.E. Canine islet
autografts. Functional outcome as influenced by islet number and purity. Transplant.
Proc. 22,771,1990.
65. Kanai, T., Porter, J., Monaco, A.P., Maki, T. Sequential multiple transplantations of
multiple-donor pancreaticislet allografts. Diabetes. 38, Suppl. 1,286,1989.
66. Alejandro, R., Mintz, D.H., Noel, J., Latif, Z., Koh, N., Russell, E., Miller, J. Islet cell
transplantation in type I diabetes mellitus. Transplant. Proc. 19,2359,1987.
67. Scharp, D.W., Lacy, P., Ricordi, c., Boyle, P., Santiago, J., Cryer, P., Gingerick, R.,Jaffe,
A, Anderson, C, Flye, W. Human islet transplantation in patients with type I diabctes.

Transplant. Pmc. 21,2744.1989.
68. Cicciarelli. 1.. Cho, RV. Regraft kidney transplant survival. In: Clinical Transplants.
Terasaki, P.I. (cd.) Los Angelcs, Ucla Tissue Typing Lab, 1986. p. 285.
69. Gotoh, M .. Porter, J., Kanai, T, Monaco, A.P., Maki. T. Effcct of specific and nonspecif-
ic alloantigen stimulation on islet allograft survival. Tramplalli. Proc. 21.2746.1989.
70. Kanai. T, Porter. 1.. Maki, T. Monaco, AP. Possible mechanisms underlying markcd
graft survival following multiple donor islet allotransplantation. Transplant. Proc. In
press.
71. Sachs, D .. Bach, F. Immunology of xenograft rejection. Hum. Immllnol. 28,245,1990.
72. Sun. A.M .. O·Shea. G.M .. Yuan. Z.Y., Ma., F., Cai, Z. Allo- and xenotransplantation of
microencapsulated islets of Langerhans (a bioartificial pancreas). In: Advanced Models
for Therapy of Inslliin Dependent Diabetes. Brunetti. P .. WaldhausL W.K. (cds.) New
York, Raven Press, Scrono Symposia, 37. 303.1987.
73. Manns, J.G., Boda, J.M. Insulin release in lambs and adult sheep. Am . .I. Physiol. 212,
747,1967.
74. Lacy, P., Ricordi, C, Finke, E. Effect of transplant site and alpha L3T4 treatment on sur-
vival of rat, hamster and rabbit islet xenografts in mice. Transplantation. 47.761, 1989.
75. King, N.W., Chalifoux, L.V., Ringler, DJ., Wyand. M.S .. Sehgal. P.K., Daniel. M.D.,
Letvin, N.L. Comparative biology of natural and experimental SIVmac infection in
macaque monkeys: a review.f. Med. Prima/ol. 19. 109,1990.
76. Daniel, M.D., Letvin, N.L., Sehgal, P.K., Schmidt, O.K., Silva, D.P .. Solomon. K.R.
Hodi, F.S. Jr .. Ringler, D.J., Hunt, RD., King, N.W., et a!. Prevalence of antibodies to
3 retroviruses in a captive colony of macaque monkeys. Int. f. Cancer. 41,601,1988.
77. Sibley, R.K.. Sutherland, D.E.R, Goetz, F., Michael. AF. Recurrent diabetes in the
pancreas iso- and allograft. Lab. Invest. 53, 132, 1985.
78. Mandel, T. Koulmanda. M., Loudovaris, T. Bacelj, A Islet grafts in NOD mice - com-
parison of iso- allo- and pig xenografts. Transplant. Pmc. 21,3813, 1989.
79. Kaufman, D., Platt, J., Rabe, F., Dunn, D., Bach, F., Sutherland, D.E.R. Differential
roles of Moc 1+ cells and CD4+ and CD8+ T lymphocytes in primary non-function and
classic rejection of islet allografts. f. Exp. M ed. 172,291, 1990.
80. Moses, R., Pierson, R.I1I, Winn, H., Auchincloss, H. Xenogeneic proliferation and lym-
phokinc production are depcndent upon CD4+ helper T cells and self-antigen present-
ing cells in the mouse.J. Exp. Med. 172,567,1990.
81. Ildstad, S., Wren, S., Stephany, D., Sachs, D.H. Effect of selective T cell depletions in
mixed xcnogeneic reconstitution on species hyporeactivity to xenogeneic transplanta-
tion. Transplantation. 41,372,1986.
82. Crowthcr, N., Gotfreadsen, C, Moody, AJ., Green. I.C Porcine islet isolation cellular
digestion and secretory response. Harm. Met. Res. 21.590.1989.
83. Ricordi, C, Finke, E., Lacy, P.E. A method for the mass isolation of islets from the adult
pig pancreas. Diabetes. 35,649. 1986.
84. Ricordi, C, Socci, A, Davilli, C, Staudacher, C, Vertova, A, Baro, P., Sassi, I.. Braghi.
S., Giuzzi, N., Pozza, G., DiCarlo, V. Application of the automated method to islet isola-
tion in swine. Transplant. Proc. 22,784, 1990.
85. Hellerstrom, C, Andersson, A., Korsgren, 0., Jansson, L., Sandler, S. Aspects of pan-
creatic islet transplantation in diabetes. Balliere's Clin. Gastroenteml. 3,851,1989.
86. Schrezenmeir, J., Faust, P., Laue, C, Kirchgessner, J., Stallmach, T, Muntefering, H ..
Beyer, J. Immunoprotection in biocompatibIc membranes permits transplantation of
piscine islets to rats. Transplant. Proc. 21, 2730,1989.
87. Falkmer, S. Ostberg, Y. Comparative morphology of pancreatic islets in animals. In: The
Diabetic Pancreas. Yolk, B.W., Arquilla, E.R (cds.) New York. Raven Press. 1977. p. 15.
88. Alejandro, R, Cutfield, R, Schienvold. F., Polonsky, K.S., Noel, J., Olson, L.,
Dillberger, J .. Miller, J., Mintz, D.H. Natural history of intrahepatic canine islet cell
autografts.J. Clin. Invest. 78. 1339. 1986.
89. Warnock, G., Rajotte, R Critical mass of purified islets that induce normoglycemia after
implantation in the dog. Diabetes. 37,467,1988.
Chapter 17
Experimental Xenotransplantation
of Encapsulated Cells
R.P. LANZA AND P. SOON-SHIONG
Introduction
Transplantation of xenogeneic tissues of widely divergent species is difficult

due to vigorous humoral and cellular immune responses. A possible solution
to this problem might be the use of microencapsulation as an immunoisolation
procedure. The unique ability of cellular grafts to be enclosed in artificial
membranes that permit crossover of low molecular weight substances such as
nutrients and oxygen, but not of cells or high molecular weight components,
provides a distinct advantage of isolated xenogeneic tissue over whole engraft-
ed organs, and many, in the long run, prove the only way to establish pro-
longed survival of xenografts in patients with diseases caused by the loss or
malfunction of specific cells in the body.
The concept of preventing the immune system from gaining access to cells
by means of encapsulation evolved more than 35 years ago [1]. Using the diffu-
sion-chamber technique, different cell strains were immunoisolated inside a
cellulose-derived membrane. Since that time numerous immunoisolation sys-
tems have been investigated in order to achieve transplant tolerance. These in-
clude diffusion-based hybrid organ devices, especially the early diffusion
transplantation chambers [2-5], hollow-fiber bioartificial devices [6-9], cell
microencapsulation [10-13], diffusion vascular hybrid devices [14-16], and
ultrafiltration systems [17]. Membrane materials include polyvinylchloride,
polyamides (nylon), polypropylene, polycarbonate, cellular nitrate, cellulose
triacetate, and polysaccharide-polyamino acids [4,18,19].
Most of these systems have been successfully employed to reverse experi-
mental diabetes in animals. However, clinical application of the diffusion
chambers and hollow fiber units are hindered by problems such as fragility
[20], the limited surface area [10], and, in the case of the chamber system, the
surgery required for implantation or shunt connection with the risks of throm-
bosis [15, 21, 22], calcification [23], and infection [10, 11], and the relatively
high diffusion resistance inherent in the filter membranes and hollow fibers
[4]. The microencapsulation technique bypasses many of these problems and
provides more options for the method and site of implantation. Capsules can
simply be injected into the peritoneal cavity [10, 11,24-27], or even placed in
vascular prostheses in the systemic circulation [28].
298 Experimental Xenotransplantation of Encapsulated Cells
Microencapsulation of Tissues and Cells
Alginate-Polylysine Capsules
Although several techniques for microencapsulating cells have been investi-

gated [26-34], only the alginate-poly-L-lysine (PLL)-alginate system has re-
sulted in long-term xenograft function. The microencapsulation system is
widely used to immobilize microbial cells for industrial applications [35, 36].
The procedure involves extruding a mixture of cells and sodium alginate using
a droplet generation device into a CaCh solution (Fig. 17.1).
Using this procedure to entrap rat islets of Langerhans in calcium alginate
gels, Lim and Sun [10] then coated the negatively charged gel droplets with
positively charged PLL, followed by a layer of polyethyleneimine (PEl).
However, because of the poor biocompatibility of the PEl, which produced an
inflammatory response when implanted into animals [37,38], this final layer
was subsequently eliminated and the capsule coated with alginate after the un-
bound intracapsular alginate gell was liquified with sodium citrate.
Both in vitro and in vivo studies in our laboratory have confirmed that the
insulin secretory capacities and functional viability of the encapsulated islets
are comparable to those of free control islets [39]. The encapsulated islets are
morphologically intact, and respond to static glucose stimulation with a
twofold (canine) or fivefold (human) increase of insulin secretion over basal
levels. Perfusion of the human islets with glucose elicited a biphasic release of
insulin with the mean response (U/islet per minute) rising to a first and second
peak of 54.9±12.4 and 59.5±17.0, respectively, followed by a return to baseline
(9.6±2.3). When human (n=7) or canine (n=12) islets were encapsulated and
xenotransplanted into streptozotocin (STZ)-induced diabetic rats, normo-
glycemia (plasma glucose <200 mg/dl) was restored in the recipient rats within
1-2 days, confirming islet-cell viability.
That the alginate-poly lysine microcapsule system can prevent cellular con-
tact and thus prevent rejection was demonstrated by Darquy and Reach [40] in
terms of specific humoral immunoprotection, and by Soon-Shiong et al. [12] in
terms of the cellular mechanism involved in the effector limb or destructive
phase of the immune response, including specific cell-mediated destruction in-
volving cytotoxic T lymphocytes and nonspecific killing by natural killer (NK)
cells. However, in the latter study, the microcapsule did not protect NK-sensi-
tive (Y AC-l) cells from the destructive effect of NK cytotoxic factors
(NKCF), which presumably are small enough to pass through the pores in the
capsule membrane. Furthermore, in view of accumulating evidence that cy-
tokines and other low molecular weight substances, including tumor necrosis
factor (TNF) [41-43], gamma-interferon (IFN-gamma) [41-44], and inter-
leukin-l (IL-l) [41,45,46], induce the degeneration of cells, encapsulated tis-
sue may well remain susceptible to destruction by the immune process.
Therefore, the microcapsule membrane may not provide total protection
from the host immune system, especially in the case of xenogeneic cells pos-
sessing strong histocompatibility antigens, nor wholly eliminate the need for
an immunosuppressive adjuvant.
[.. .
Microencapsulation of Tissues and Cells 299
1. FORMATION OF •••
ISOLATED
+ ALGINICACID
(Long-Chain
+ SALINE
CELL SUSPENSION
PANCREATIC Polyamonl
ISLETS
CELL SUSPENSION
DROPLET
+ FORMING
DEVICE
2. FORMATION OF
CALCIUM ALGINATE SPHERE
'-"+ FORMATION OF
~ DROPLET
~ CALCIUM ALGINATE
~ GELLED DROPLETS
~
~
®.A':"'" LONG-CHAIN
POLYCATION SOLUTION
(Poly-L-Lyslnel
3. FORMATION OF
SEMI-PERMEABLE MEMBRANE ~ Poly-L-Lysin.
~ ~
ALGINATE
MEMBRANE
I' ~ISLET IN GELLED

, INTERIOR
t'i~CALCIUM CITRATE
4. REMOVAL OF
INTRACAPSULAR Ca ALGINATE ~ ./Poly-L-LYSln.
, ALGINATE
~\ MEMBRANE
~~ ISLET IN AOUEOUS
INTERIOR
Fig. 17.1. Microencapsulation process.] The cells are suspended in a solution of sodium algi-
nate.2 Alginate droplets are formed by syringe-pump extrusion under an air jet, and collect-
ed in a solution of CaCh to form spherical cell-containing calcium alginate gels. 3 The gells
are coated by exposure to poly-L-lysine solution followed by reaction with sodium alginate.
4 The intracapsular alginate gell is liquefied by washing with sodium citrate
The major problem preventing further advances in this encapsulation tech-

nology is fibrotic overgrowth of the extravascularly implanted microcapsules
[18,47,48], resulting in compromised nutrition and diminished oxygen tension
within the cell compartment. The fundamental pathological basis for fibro-
blast proliferation in alginate-based microcapsules has eluded investigators
for the past decade. Recently, we have demonstrated an immunological basis
for the fibrotic response, and have reported that the mannuronic acid (M)
monomer within alginate plays an important role in the induction of the fib-
rotic response [49].
Fibroblast proliferation has been shown to be regulated by immune cell-de-
rived mediators. Early studies revealed that monocytes in vitro released fac-
tors that stimulate fibroblast proliferation. IL-l is mitogenic for fibroblasts
and plays a role in collagen synthesis and pannus formation. Other cytokines
which share the ability with lL-1 to stimulate fibroblast proliferation include
platelet derived growth factor (PDGF). Soon-Shiong et al. [49] have recently
identified an alginate component which evokes a strong IL-1 and TNF stimu-
lation and fibrotic reaction, and more significantly, a formulation which does
not elicit either of these responses. This basic understanding may, in part, ex-
plain the phenomenon of hybrid fibrosis. Furthermore, by exploiting this find-
ing that soluble alginate or alginate gells of low mannuronic acid content
evoke a minimal cytokine response, it may be possible to finally overcome this
obstacle associated with the alginate-PLL-alginate system. Using a novel for-
mulation low in mannuronic acid, we have demonstrated for the first time suc-
cessful long-term reversal of both surgically induced and spontaneous dia-
betes in the canine model by implantation of immunoprotected islets
(manuscript in press).
Other Encapsulation Technologies
Over the past decade, several other cell microencapsulation procedures have
been developed. These include the chitosan-alginate system of Rha [33, 34],
the polyacrylate capsules of Sefton [31,50], and the agarose gell technique of
Iwata [51]. However, problems severely limit the usefulness of these proce-
dures in the treatment of diseases such as diabetes.
Chitosan- Alginate Microcapsule Membranes

Chitosan is a natural polycationic polymer, and is extremely abundant in na-
ture (e.g., crustacea shells, insects, and fungi). The polysaccharide is highly
versatile and can be used for cell immobilization [52,53]. Chitosan capsules
can be made by direct gelling of sodium alginate droplets in a solution contain-
ing chitosan and calcium, or formed in a liquified core while avoiding capsule
core gelation by adding cell-containing drops of a solution of either an anionic
polymer composition or a cationic polymer composition to a solution of an
ionic polymer of opposite charge. The interface of the ionic polymers form a
semipermeable membrane surrounding the liquid drops.
This process has been used successfully for the encapsulation of viable cells
with little cellular damage reported [54]. Qualitative results indicate that cap-
sules prepared with chitosan derivatives are consistently stronger and more
flexible than those prepared with PLL [53]. Cell viability, however, cannot be
retained in the long-term with this capsule, possibly as a result of small con-
Microencapsulation of Tissues and Cells 301
taminating molecules, short chit os an chains or unreacted nitrite diffusing

through tathe cells.
Polyacrylate Microcapsule Membranes

Cells can be encapsulated in water insoluble hydrophilic polyacrylates (such
as Eudragit RL) by coaxial extrusion and interfacial precipitation [31, 50].
Despite exposure to organic solvents (diethyl phthalate) and nonsolvents
(hexadecane, corn oiL mineral oil) erythrocytes [31], fibroblasts [55], and pan-
creatic islet cells [56] have been shown to survive for up to 6 months in vitro,
with the latter cells secreting insulin and responding to glucose in both static
glucose challenges and perfusion assays as well and as long as control islets
which were not encapsulated.
Unlike the alginate-polylysine and alginate-chitosan systems, the in vivo
stability of a polyelectrolyte complex is of no concern. However, the Eudragit
RL polymer used for these studies is not biocompatible and induces an inflam-
matory response when implanted into the intraperitoneal cavity of animals. A
new polymer, HEMA-MMA, a biocompatible copolymer of hydroxyethyl
methacrylate and methyl methacrylate is currently being investigated [57].
Agarose Microcapsules
Agarose is the gelling component in agar, and is a binary linear copolymer that
forms strong transparent thermoreversible gels at concentrations of polymer
>0.2% [58]. It has been successfully used for gel entrapment or encapsulation
of a wide range of cells, either by mixing the cells with a warm aqueous solution
of agarose and letting the gel set by cooling [59], or by emulsifying the polymer
cell suspension in paraffin oil, or various nontoxic seed oil, and then solidified
by cooling in an ice bath [60].
Using this latter technique, Iwata et al. [51] examined whether xenotrans-
plantation of microencapsulated islets into diabetic animals could reverse the
diabetic state. Hamster islets encapsulated in microbeads containing from
11 %-14 % of low molecular weight agarose were xenogeneically transplanted
into the peritoneal cavity of diabetic mice. All of the mice promptly became
normoglycemic with plasma glucose levels of <200 mg/dl, and maintained this
level for> 11 days, the longest normoglycemic period being 53 days. In con-
trast, the longest normoglycemic period obtained by nonencapsulated control
islets was 10 days.
While there are still many problems to be solved, such as lack of control of
microcapsule morphology and permeability, agarose appears to be a stable
immobilization material, and is currently being explored as a matrix for em-
bedding the islets coated with a protective polyacrylamide outer layer [61].
Xenotransplantation of Encapsulated Islets of Langerhans
Despite insulin therapy, diabetes remains a devastating disease, afflicting an

estimated 80 million people worldwide. The disease leads inexorably to one or
more of the secondary complications, including renal failure, blindness, and
coronary and peripheral vascular occlusive disease. It is for this reason, and
the amenability of islets to manipulation in vitro, that most of the work to date
with encapsulation and transplantation of mammalian cells has been carried
out in the field of pancreatic islet transplantation. In view of the limited avail-
ability and yield of viable. isolated human islet tissue, the use of this technolo-
gy is especially attractive, since it might allow the use of donor islets harvested
from a variety of animal sources.
Small Animal Model
Concordant Islet Xenografts

In 1986, O'Shea and Sun [62] demonstrated that concordant islet xenografts
(that is, islets from a closely related animal source) can be protected from re-
jection for up to 144 days, with a mean xenograft survival of 80 days, in nonim-
munosuppressed STZ-induced diabetic mice by encapsulation in alginate-
polylysine membranes. In all cases, diabetes was reversed within 3 days -
fasting glucose levels fell from a mean of 485.8±13.7 (±SE) mg/dl to 95.8±8.7
mg/dl. Eighteen ofthe 22 (82%) grafts functioned for >50 days.
At 2-3 weeks after the reappearance of hyperglycemia, 13 animals received
a second xenograft of encapsulated islets. These xenotransplantations again
were able to restore normoglycemia for periods ranging from 16 to 110 days.
At the time of euthanasia, the polylysine capsules were generally found free
flowing throughout the peritoneal cavity, though varying degrees of cell over-
growth (including fibroblasts, neutrophils and macrophage-like cells) were
noted around some of the capsules. By contrast, intraperitoneal implants of
free unprotected xenografts functioned for <14 days. Other studies have cor-
roborated these results [63-65].
Encouraging data with survival of encapsulated islet xenografts have also
been obtained in a recent study of xenogeneic islets grafted into spontaneous-
ly diabetic nonobese diabetic (NOD) mice [66]. Alginate-polylysine micro-
capsules containing 1800-2000 rat islets were xenografted intraperitoneally
into NOD mice, with or without recipient treatment with GK 1.5 (anti-CD4
monoclonal antibody, MAb) (20-30 I i.p. every 5 days, begun on day 07). The
encapsulated islets normalized blood glucose (<150 mg/dl) in the animals
within 24 h. Without GK 1.5 treatment, the NODs destroyed the microencap-
sulated islets within 10±2 (X±SE) days. Graft biopsies showed an intense cel-
lular reaction composed of lymphocytes, macrophages and giant cells, and no
viable islets. With GK 1.5 therapy, however, the xenografts continued to func-
tion for 65-200 days (mean 127±30 days). Graft biopsies demonstrated viable
islets and no perimicrocapsule cellular reaction.
Xenotransplantation of Encapsulated Islets of Langerhans 303
Discordant Islet Xenografts
In the same study, Weber et al. [66] examined the metabolic function of totally
unrelated (discordant) islet xenografts. They found that microencapsulated
dog islets (4000-12000) functioned for 51±8 (X±SE) days in STZ-induced dia-
betic C57BLl6J mice, with minimal cellular reaction and viable donor islets
within microcapsules at graft biopsy. This was in marked contrast to encapsu-
lated dog-to-NOD islet grafts, which functioned for only 11.5±3 days (range
5-17 days). With anti-CD4 MAb treatment, however, long-term functional
survival (>100 days) was observed in four of eight (50%) recipients. Free,
nonencapsulated dog islets xenografted into the spleen and peritoneal cavity
of unmodified NODs failed to function, even on day 1, or resulted in fluctuat-
ing reductions of blood glucose for up to 28 days, respectively.
The enhanced survival of encapsulated dog islets in NOD mice given GK
1.5 MAb, compared with those xenotransplanted into unmodified mice, and
compared with the poor survival of free control islets xenotransplanted into
unmodified and into GK 1.5-treated mice, suggests that the effects of the mi-
crocapsule and MAb treatment are synergistic. In our laboratory, we have
studied dog-to-rat microencapsulated islet xenografts, and demonstrated that
this kind of bioartificial pancreas (BAP) can achieve satisfactory in vivo glu-
cose-insulin kinetics. This latter finding is important since the success of this
approach is contingent on the rapid transfer of the glycemic signal across both
an extravascular compartment and a semipermeable membrane, and of in-
sulin from the BAP to the recipient.
Preclinical Large Animal Model
While encapsulated islet xenografts have been used to reverse diabetes in ro-
dents, studies in large animals will be required before clinical trials can be con-
templated. We have successfully treated surgically induced diabetes in dogs by
the intraperitoneal implantation of microencapsulated islet allografts [67,68].
There are, however, no reports to date of proof of principle of this technology
in Type I diabetes in the large animal model.
Clinical trials of intraperitoneal encapsulated islet allografting in sponta-
neously diabetic dogs are in progress. The preliminary results (in press) are
highly encouraging - euglycemia has been achieved in seven out of seven dogs
transplanted, with the longest graft survival being 160 days at the time of this
report. Based on these studies, a pilot human-to-dog islet xenograft was per-
formed in a totally pancreatectomized dog. The animal promptly reached fast-
ing euglycemia, which was sustained for >6 days post-transplant (at which time
the dog was euthanized for complications unrelated to graft function). These
are encouraging, and provide evidence of the feasibility of encapsulated islet
xenotransplantation as a possible source for the treatment of diabetes mellitus
in humans.
Potential Application to Other Diseases Requiring Enzyme

or Hormone Replacement Therapy
Xenotransplantation of cells encapsulated in protective microcapsules may

have broad application, not only to the diabetic population, but also for the
treatment of a wide range of diseases requiring enzyme or hormone replace-
ment therapy. These include the use of hepatocytes for the treatment of liver
failure [69-74], the use of dopamine neurons in Parkinson's disease [75], nerve
growth factor (NGF) in Alzheimer's disease [76-78], factors VIII and IX in
hemophilia [79,80], and endocrine cells in disorders resulting from hormone
deficiency [81-83].
Liver Failure
There is no adequate therapy for fulminant hepatic failure (FHF) in man [84,
85]. Although whole liver transplantation has proven clinically useful in a lim-
ited group of patients [86.87], the procedure is formidable. given the problems
of rejection and the limited availability of donor livers. As an alternative, in-
vestigators are now studying hepatocyte transplantation, not only for irre-
versible hepatic failure, but for several disease processes including (i) heredi-
tary enzyme abnormalities, (ii) acute hepatic failure, where the ability of the
liver to regenerate may still exist, and (iii) as a bridge to whole liver transplan-
tation in patients who develop sudden hepatic failure, either because of medi-
cal progression or because of rejection-related complications [88].
Encouraged by the successful allografts and xenografts of encapsulated
islets in diabetic animals, several investigators have attempted to develop this
technique for transplanting hepatocytes [71, 73, 89]. Encapsulated hepato-
cytes transplantation is potentially a simpler, less hazardous treatment for
FHF than whole liver grafting, in that it requires minimal or no surgical inter-
vention to implant the capsules, and it may actually permit the use of donor
hepatocytes harvested from a variety of animal sources.
Indeed, Wong and Chang [69] have demonstrated the viability and regen-
eration of microencapsulated rat hepatocytes transplanted into mice. Viable
hepatocytes were microencapsulated in alginate-polylysine membranes and
implanted intraperitoneally into normal and galactosamine-induced liver fail-
ure mice. No significant changes were observed in the number of hepatocytes
recovered from the microcapsules. Eight days after xenotransplantation in the
mice with induced liver failure, the viability of the encapsulated hepatocytes
increased from 42% to nearly 100%; after 29 days, the viability of the encapsu-
lated hepatocytes implanted in normal mice also increased from 42% to near-
ly 100%. By contrast, in mice implanted with free hepatocytes, no viable cells
were observed 4 or 5 days after xenotransplantation.
Other investigators have shown that encapsulated hepatocytes continue
the synthesis and secretion of many specific proteins and enzymes [71-73]. Cai
et al. [71] developed and evaluated a system of microencapsulation of primary
Potential Application 305
rat hepatocytes. Urea formation, prothrombin and cholinesterase activity, the
incorporation of tritiated leucine into intracellular proteins, and the im-
munolocation of synthesized albumin were monitored in culture. Despite
gradual decreases in some of these activities, the encapsulated hepatocytes
continued to function throughout the 35-day observation period, producing
and excreting urea, and demonstrating prothrombin and cholinesterase activi-
ty into the medium.
Bruni and Chang [89] demonstrated the possibility of using encapsulated
hepatocytes to lower bilirubin levels in hyperbilirubinemia. Micro-
encapsulated hepatocytes were injected into the peritoneal cavity of Gunn
rats. Bilirubin dropped from 14 mg/IOO ml to 6 mg/IOO ml, and remained de-
pressed after 90 days. These and other in vitro and in vivo studies are promis-
ing, and represent the first essential steps required to determine the potential
clinical applications of microencapsulated hepatocytes.
Parkinson's Disease
Parkinson's disease can be considered an example of neuronal system disease,

involving mainly a degeneration of the nigrostriatal dopaminergic system [90].
Despite therapeutic measures, ultimately a point seems to be reached where
pharmacotherapy can no longer compensate for the loss. The major difficul-
ties consist of fluctuations or sudden variations in the response to the drugs
used, the development of weakness or immobility, and dyskinesias, which in
the late stages of the disease alternate with paralyzing akinesia. Recent neuro-
surgical treatment has focuses on adrenal medullary transplants to the stria-
tum and, perhaps more effective, fetal tissue transplants containing substantia
nigra neurons.
Experimental work in both rodents and nonhuman primates has shown
that transplantation of fetal dopaminergic neurons from ventral mesen-
cephalon to dopamine-depleted striatum reinstates near normal dopamine in-
nervation and reduces motor abnormalities. Widner et al. [75] recently report-
ed evidence of fetal nigra 1allograft survival and function up to 10 months after
transplantation in a patient with Parkinson's disease. Immunosuppression
with cyclosporine, azathioprine, and prednisolone was begun 2 days prior to
the operation. Beginning from the 2nd month after the transplantation, they
observed a progressive decrease in limb rigidity. increased movement speed in
a number of arm, hand and foot movements, and prolonged "on" periods
(>80% increase) after a single dose of L-dopa. Positron emission tomography
(PET) scans performed at 5 and 8 months revealed increased uptake of fluo-
rodopa in the grafted putamen.
These results support the concept that transplantation of fetal neural tissue
may have a great potential as a new therapeutic approach in patients with neu-
rological disorders. However, in the case of transplanted xenogeneic donor
tissue, rejection would pose a serious problem, even by the combined ap-
proach of using an immunoprivileged site and by employing immunosuppres-
sive drugs [91,92]. The technology described in this chapter may permit a nov-
el approach to this problem - the delivery of dopamine for the treatment of
Parkinson's disease using microencapsulated donor tissue harvested from ani-
mals.
Alzheimer's Disease
It is estimated that from 2.5 to 3.0 million Americans are afflicted with
Alzheimer's disease, and that this number will increase dramatically, as the
proportion of the population over the age of 65 is increasing faster than any
other age group [77]. The disease is characterized by a progressive loss of cog-
nitive function associated with degeneration of basal forebrain cholinergic
neurons. Studies in animals indicate that (NGF) and other neurotropic factors
may normally act to support the viability and function of these cells, and that
continuous infusion ofNGF into the ventricles can prevent injury-induced de-
generation of cholinergic neurons [93, 94], which, in turn, correlates with im-
proved cognitive function in rodents with memory impairment [95].
Moreover, following NGF treatment, the size of basal forebrain cholinergic
neurons appears to increase toward control values in these animals. These
studies suggest that by using recombinant methods [96], or encapsulated grafts
ofNGF-secreting tissue such as astroglial cells [97,98] or developing skin [99],
it may prove possible to treat patients suffering from Alzheimer's disease.
Hemophilia
Hemophilia is an X-linked hereditary bleeding disorder caused by factor VIII

or factor IX deficiency and is characterized clinically by hemarthrosis,
hematomas, ecchymoses, gastrointestinal and genitourinary bleeding, and ex-
cessive traumatic bleeding. Although plasma products enriched in these fac-
tors have revolutionized the care of hemophilia patients, their widespread use
has also produced serious complications including viral hepatitis, chronic liver
disease, and the acquired immunodeficiency syndrome (AIDS) [100, 101].
Studies have now demonstrated VIII:ag and VIII mRNA in a number of tis-
sues [102]. In fact, the finding that hemophilic humans and dogs can be cured
by liver transplantation [103, 104] suggests that the liver is an important site for
production of this coagulation protein. There is hope that encapSUlation and
transplantation of different types of liver and extrahepatic cells will be a step
toward an ideal replacement surgery for hemophilia.
References 307
Hormone Replacement
Hyperparathyroidism
Severe hypocalcemia remains an unsolved therapeutic problem in patients un-

dergoing parathyroid operations secondary to parathyroid adenoma or hyper-
plasia [105, 106]. Transplantation of encapsulated parathyroid cells has been
proposed as a potential method of overcoming this problem without immuno-
suppression [107].
To examine this possibility of microencapsulated parathyroid cells as a
bioartificial parathyroid, Lu et al. [lOS] encapsulated parathyroid cells from
Lewis rats in alginate-poly lysine membranes, and transplanted them into the
peritoneal cavity of parathyroidectomized Sprague-Dawley rats. Hypo-
calcemia was promptly reversed, with blood calcium concentrations increas-
ing from a pretransplant level of 5.7±0.S mg/dl (±SEM) to 9.S±1.2 mg/dl
(p<0.01) at 1 week post-transplant. The allografts continued to function for up
to almost 10 weeks (median survival 44 days) without immunosuppression. In
contrast, nonencapsulated parathyroid cells functioned for <7 days (p<0.05).
These results indicate that encapsulated parathyroid cells can serve effec-
tively as a bioartificial parathyroid in the treatment of hypocalcemic rats, and
that further studies are warranted to investigate whether this methodology
can be applied to xenograft recipients.
Other Hormone Deficiency States

With a few exceptions hormone deficiency results in pathological manifesta-
tions [109]. The use of encapSUlation technology in clinical disorders that re-
sult from hormone deficiency or absence may play an important future role in
the treatment not only of diabetes mellitus, but of pituitary and adrenal insuf-
ficiency, hypothyroidism, and hypogonadism. This therapeutic measure
opens up the possibility for xenotransplantation, and for a solution to the over-
whelming problems of insufficient amounts of endocrine tissue.
References
1. Prehn, RT., Weaver, J.M., Algire, G.H. The diffusion-chamber technique applied to a
study of the nature of homograft resistance. 1. Nat. Cancer [nst. 15,509, 1954.
2. Albisser, A.M., Leibel, B.S., Ewart, G., Davidovac, Z., Botz, c.K., Zinger, W. An artifi-
cial endocrine pancreas. Diabetes. 23,389,1974.
3. Strautz, RL. Studies of hereditary-obese mice (obob) after implantation of pancreatic
islets in Millipore filter capsules. Diabetologia. 6,306, 1970.
4. Theodorou, N.A., Vrbova, H., Tyhurst, M., Howell, S.L. An assessment of diffusion
chambers for use in pancreatic islet cell transplantation. Transplantation. 27,350,1979.
5. Gates, RJ., Lazarus, N.R Reversal of streptozotocin-induced diabetes in rats by in-
traperitoneal implantation of encapsulated neonatal rabbit pancreatic tissue. Lancet. 2,
1257,1977.
6. Chick, W., Like, AA, Lauris, V. Beta cell cultures on synthetic capillaries: an artificial
endocrine pancreas. Science. 187,847,1975.
7. Archer, J., Kaye, R, Mutter, G. Control of streptozotocin diabetes in Chinese hamsters
by cultured mouse islet cells without immunosuppression: a preliminary report. 1. Slirg.
Res. 28,77, 1980.
8. Altman, J.J., Manoux, A., Callard, P., Houlbert, D., Desplanques, N., Bruzzo, F.,
Galletti, P.M. Successful pancreatic xenografts using semipermeable membranes.
Artit Organs. 5 (Supp\.), 776,1981.
9. Tze, W.J., Wong, F.e., Chen, L.M., O'Young, S. Implantable artificial endocrine
pancreas unit used to restore normoglycemia in the diabetic rat. Nature. 264, 466,
1976.
10. Lim, F., Sun, AM. Microencapsulated islets as bioartificial endocrine pancreas.
Science. 210,908,1980.
11. Sun, A.M., Goosen, M.F., O'Shea, G. Microencapsulated cells as hormone delivery sys-
tems. Crit. Rev. Ther. Drug Carrier Syst. 4, I, 1987.
12. Soon-Shiong, P., Lu, Z.N., Greqal, I., Lanza, RP., Clark, W. An in vitro method of as-
sessing the immunoprotective properties of microcapsule membranes using pancreatic
and tumor cell targets. Transplant Proc. 22,754,1990.
13. Soon-Shiong, P., Heintz, R, Yao, Z., Yao, Q.x., Lanza, RP. Glucose-insulin kinetics of
the extravascular bioartificial pancreas. Diabetes. 39 (Supp\. 1),311, 1990.
14. Chick, W.L., Perna, J., Lauris, W., Low, D., Galletti, P.M., Whittemore, A, Like, A,
Colton, e.K., Lysaght, M. Artificial pancreas using living beta cells: effects on glucose
homeostasis in diabetic rats. Science. 197,780, 1977.
15. Sun, AM., Parisius, W., Healy, G., Vacek, I., Macmorine, H.G. The use, in diabetic rats
and monkeys, of artificial capillary units containing cultured islets of Langerhans (arti-
ficial endocrine pancreas). Diabetes. 26, 1136, 1977.
16. Tze, W.JH., Tai, F.e., Davis, H.R Studies with implantable artificial capillary units
containing rat islets on diabetic dogs. Diabetologia. 19,541,1980.
17. Reach, G., Poussier, P., Sausse, A, Assan, R, Itoh, M., Furman, B.L., Gerich, J.E.
Functional evaluation of a bioartificial pancreas using isolated islets perfused with
blood ultrafiltrate. Diabetes. 30,296,1981.
18. Yderstroede, K.B. Pancreatic islet transplantation: experimental and clinical aspects.
Dan. Med. Bull. 34,323,1987.
19. Goosen, M.F.A Insulin delivery systems and the encapsulation of cells for medical and
industrial use. CRe. Crit. Rev. Bio-compat. 3, 1, 1987.
20. Buchwald, H., Corry, RJ., Langer, R, Sun, AM. Insulin replacement: bionic or natu-
ra\. Trans. Am. Soc. Artit Intern. Organs. 33,806,1987.
21. Tze, W.J., Tai, J., Wong, F.e., Davis, H.R Studies with implantable artificial capillary
units containing rat islets in diabetic dogs. Diabetologia. 19,541,1980.
22. Whittemore, AD., Chick, W.L., Galetti, P.M. Effects of the hybrid artificial pancreas in
diabetic rats. Trans. Am. Soc. Arlit Intern. Organs. 23,336,1977.
23. Klomp, G.F., Hashiguchi, H., Ursell, P.e., Takeda, Y., Tadahiro, T., Dobelle, W.H.
Macroporous hydrogel membranes for a hybrid artificial pancreas. II. Biocompa-
tibility.J. Biomed. Mat. Res. 17,865,1983.
24. Altmann, J.J., Houlbert, D., Chollier, A., Leduc, A, Mcmillan, P., Galletti, P.M.
Encapsulated human islet transplants in diabetic rats. Trans. Am. Soc. Artit Intern.
Organs. 30,382, 1984.
25. Gerlis, L. Islets in envelopes could beat diabetes. Science. 6,30, 1986.
26. Sun, AM. Encapsulated versus modified endocrine cells for organ replacement. Trans.
Am. Soc. Arlit Intern. Organs. 33,787,1987.
27. Soon-Shiong, P., Lanza, RP. Pancreas and islet-cell transplantation. Postgrad. Med. 87,
133,1990.
28. Calafiore, R, Brotzu, G., Basta, G., Falorni, A, Piermattei, M., Avellini, L., Brunetti, P.
Successful transplantation of microencapsulated islets within artificial vascular pros-
thesis. Diabetes. 39 (Supp\. 1), 158A, 1990.
29. Chang, T.M.S. Semipermeable microcapsules. Science. 146,565,1964.
References 309
30. Chang, T.M.S., Mcintosh, F.C, Mason, S.G. Semipermeable aqueous microcapsules. I.
Preparation and properties. Can. J. Physiol. Pharm. 44, 115, 1966.
31. Sefton, M.V., Broughton, RL. Microencapsulation of erythrocytes. Biochem. Biophys.
Acta. 717,473,1982.
32. Lamberti, F.V., Wheatley, M.A., Evangelista, R.A., Sefton, M.V. The use ofpolyacry-
lates in the microencapsulation of viable tissue cells. Polym. Prepr. Am. Chern. Soc.
Div. Polym. Chern. 24,75,1983.
33. Rha, C, Rodriguez-Sanchez, D. Process for encapsulation and encapsulated active ma-
terial system. US Patent. N. 766,214,1988.
34. Rha, C Chitosan as a biomateria!. In: Biotechnology in the Marine Sciences. Colwell,
RR,Parish,E.R, Sinskey,A.J. (eds.) John Wiley & Sons, New York, 1984, p.l77.
35. Hackel, V., Klein, J., Megret, R, Wagner, F. Immobilization of microbial cells in poly-
meric matrices. Eur. J. Appl. Microbiol. 1,291,1975.
36. Kierstan, M., Bucke, C The immobilization of microbial cells, subcellular organelles
and enzymes in calcium alginate gels. Biotechnol. Bioeng. 9,387,1977.
37. Sun, A.M., O'Shea, G., Van Rooy, H., Goosen, M. Microencapsulation d'ilots de
Langerhans et pancreas artificia!. J. Ann. Diabetol. (Hotel- Dieu). 20, 161, 1982.
38. Sun, A.M., O'Shea, G.M., Goosen, M.F.A. Injectable microencapsulated islet cells as a
bioartificialpancreas.Appl. Biochem. Biotechnol.1O,87, 1984.
39. Lanza, RP., Heintz, R, Merideth, N., Yao, Z., Yao, Q., Zavitz, D., Chen, C, Soon-
Shiong, P. Large-scale canine and human islet isolation using a physiological islet pu-
rification solution. Diabetes. 39 (Supp!.l), 309A, 1990.
40. Darquy, S., Reach, G. Immunoisolation of pancreatic B cells by microencapsulation.
Diabetologia. 28,776,1985.
41. Pukel, C, Baquerizo, H., Rabinovitch, A. Destruction of rat islet cell monolayers by cy-
tokines. Synergistic interactions of interferon- , tumor necrosis factor, lymphotoxin,
and TNF. Diabetes. 39 (Supp!. 1), 242A, 1990.
42. Miyajima, E., Yamada, K., Nonaka, K. Cell selectivity in MHC antigen induction and
islet cell cytotoxicity by IFN- and TNF. Diabetes. 39 (Supp!.l), 242A, 1990.
43. Kover, K., Moore, W. V. Expression of class II antigen on neonatal rat islets by interfer-
on and tumor necrosis factor. Transplant. Proc. 22,853, 1990.
44. Campbell, I.L., Kay, T.W.H., Oxbrow, L., Harrison, L.C IFN- and IL-6 are involved in
the pathogenesis of autoimmune diabetes in NOD mice given cyclophosphamide.
Diabetes. 39 (Supp!. 1), 68A, 1990.
45. DeClercq, L., Remacle, C, Reusens-Billen, B., Hoet, J.J. Does IL-6 mediate the cyto-
toxic effect ofIL-1 on cultured neonatal rat islets? Diabetes. 39 (Supp!. 1), 100A, 1990.
46. Mandrup-Poulsen, T., Bendtzen, K., Nerup, J., Dinarello, CA., Suenson, M., Nielsen,
J .H. Affinity-purified human interleukin-1 is cytotoxic to isolated islets of Langerhans.
Diabetologia. 29,63,1986.
47. Rone!, S.H., D'Andrea, M.J., Hasiguchi, H., Klomp, G.J., Dobelle, W.H. Macroporous
hydrogel membranes for a hybrid artificial pancreas. I. Synthesis and chamber fabrica-
tion. J. Biomed. Mat. Res. 17,855,1983.
48. Scharp, D.M., Mason, N.S., Sparks, RE. Islet immuno-isolation: the use of hybrid arti-
ficial organs to prevent islet tissue rejection. WorldJ. Surg. 8,221,1984.
49. Soon-Shiong, P., Otterlie, M., Skjak-Braek, G., Smidsrod, 0., Heintz, R, Lanza, R.P.,
Espevik, T. An immunological basis for the fibrotic reaction to implanted microcap-
sules. Transplant. Proc. 23,758,1991.
50. Sefton, M.V., Dawson, RM., Broughton, RL., Blysnivk, J., Sugamori, M.E. Micro-
encapsulation of mammalian cells in a water insoluble polyacrylate by coextrusion and
interfacial precipitation. Biotechnol. and Bioeng. 29, 1135, 1987.
51. Iwata, H., Amemiya, H, Matsuda, T., Takano, H., Hayashi, R, Akutsu, T. Evaluation of
microencapsulated islets in agarose gells as bioartificial pancreas by studies of hormone
secretion in culture and by xenotransplantation. Diabetes. 38 (Supp!. 1),224,1989.
52. Shiotani, T., Shiiki, Y., Kimura, T., Kako, M., Hayashi, H. Encapsulation of unstable
cells and substances with polyanionic polysaccharides and chitin derivatives. Japanese
Patent No. 6111, 139, 1986.
53. Mcknight, CA., Ku, A .. Goosen. M.F.A .. Sun. D., Penney. C Synthesis of chitosan-al-
gin ate microcapsule membranes. 1. Bioact. Compat. Polymers. 3.334, 1988.
54. Rha. C. Rodriguez-Sanchez, D .. Kienzle-Sterzer. C In: Biotechnology of Marine
Polysaccharides. RR. Colwell. E.R Pariser. AJ. Sinskey (eds.) Hemisphere,
Washington, 1984, p. 283.
55. Boag. A.H., Sefton, M.V. Microencapsulation of human fibroblasts in a water insoluble
polyacrylate. Biotech. Bioeng. 30,954,1987.
56. Sugamori, M.E., Sefton. M.V. Microencapsulation of pancreatic islets in a water insolu-
ble polyacrylate. Trans. Am. Soc. Artif Intern. Organs. 35.791, 1989.
57. Dawson. RM .. Broughton. R.L.W .. Stevenson, T.K.. Sefton. M.V. Microencapsulation
of CHO cells in a hydroxyethyl methacrylate-methyl methacrylate copolymer.
Biomaterial. 8,360, 1987.
58. Skajak-Braek, G., Martinsen. A. Application of some agal polysaccharides in biotech-
nology. Norwegian Institute of Technology. Trondheim, 1989.
59. Matsunaga, T., Karube, I., Suzuki, S. Some observations on immobilized hydrogen-pro-
ducing bacteria: behavior of hydrogen in gel membranes. Biotechnol. Bioeng. 22, 2607,
1980.
60. Nilsson, K., Scheirer, W., Merten, O.W., Ostberg, L., Liehl, E., Katinger, H.W.S.,
Mosbach, K. Entrapment of animal cells for production of monoclonal antibodies and
other biomolecules. Nature. 302,629,1983.
61. Dupuy. B., Gin, H., Cadic. C, Baquey, Ch., Ducassou. D. Polyacrylamide encapsula-
tion of agarose microbeads. An Alternative to alginate polylysine microcapsules.
Proceedings of the 2nd International Congress on Pancreatic and Islet Transplantation.
Minneapolis, Minnesota, 1989.
62. O'Shea, G.M., Sun, A.M. Encapsulation of rat islets of Langerhans prolongs xenograft
survival in diabetic mice. Diabetes. 35,943,1986.
63. Ricker, A, Stockberger, S., Halban, P. Hyperimmune response to microencapsulated
xenogeneic tissue in non-obese diabetic mice. In: The Immunology of Diabetes
Mellitus. Jaworski, M. (ed.) Elsevier; Amsterdam, N ew York, Oxford, 1986, p. 1193.
64. Norton, J., Weber, C, Reemtsma, K. Microencapsulation: prevention of islet graft re-
jection. In: Transplantation of the Endocrine Pancreas in Diabetes Mellitus. Van
Schilfgaarde, R, Hardy, M. (eds.) Elsevier; Amsterdam, New York, Oxford, 1988,
p.308.
65. Weber, C, Zabinski, S., Norton, J., Koschitzky, T., D'Agati, V .. Reemtsma, K. The fu-
ture role of microencapsulation in xenotransplantation. In: Xenograft 25. Hardy, M.
(ed.) Elsevier; Amsterdam, New York, Oxford. 1989, p. 297
66. Weber, CJ., Zabinski, S., Koschitzky. T., Wicker, L., Rajotte, R, K'Agati, V., Peterson,
L.. Norton, J., Reemtsma, K. The role of CD4+ helper T cells in the destruction of mi-
croencapsulated islet xenografts in NOD mice. Transplantation. 49,396,1990.
67. Lanza, RP., Heintz, R, Merideth, N., Yao, Z., Yao, W., Soon-Shiong, P. Successful
long-term transplantation of a bioartificial pancreas in the large and small animal mod-
el. Annual Meeting Western Association of Transplant Surgeons. Oakland, California,
1990.
68. Soon-Shiong, P., Heintz, R, Merideth, N., Yao, Z., Yao, O.z., Zheng, T., Lanza, RP.
Reversal of diabetes in pancreatectomized dogs by transplantation of micro en cap suI at -
ed rat hepatocyte xenograft transplants in mice. In preparation.
69. Wong, H., Chang, T.M.S. The viability and regeneration of artificial cell microencapsu-
lated rat hepatocyte xenograft transplants in mice. Biomat. Art. Cells Art. Org. 16,731,
1988.
70. Makowka, L., Falk, RE., Rostein, L.E., Fald, J.A, Nossal, N., Langer. B., Blendis,
L.M., Phillips, M.J. Cellular transplantation in the treatment of experimental hepatic
failure. Science. 210,901,1980.
71. Cai, Z., Shi, Z., Sherman, M., Sun, AM. Development and evaluation of a system of mi-
croencapsulation of primary rat hepatocytes. Hepatology. 10,855,1989.
72. Sun, AM .. Cai, Z., Shi, Z., Ma, F., O'Shea, G.M. Microencapsulated hepatocytes: an in
vitro and in vivo study. Biomat. Art. Cells Art. Org. 15.1483,1987.
References 311
73. Sun, A.M., Cai, Z., Shi, Z., Ma, F., O'Shea, G.M., Gharapetian, H. Microencapsulated
hepatocytes as a bioartificialliver. Trans. Am. Soc. Artif Intern. Organs. 32,39, 1986.
74. Hori, M. Will artificial liver therapy ever become a reality? Artif Organs. 12,293,1988.
75. Widner, H., Brundin, P., Rehncroma, S., Gustavii, B., Frackowiar, R, Leenders, K,
Sawle, G., Rothwell, J., Marsden, e., Bjorklund, A., Lindvall, O. Transplanted allo-
geneic fetal dopamine neurons survive and improve motor function in idiopathic
Parkinson's disease. Transplant. Proc. 23,793, 1991.
76. Phelps, e.H., Gage, F.H., Growdon, J.H., Hefti, F., Harbaugh, R, Johnston, M.V.,
Khachaturian, Z.S., Mobley, W.e., Price, D.L., Raskind, M., Simpkins, J., Thai, L.J.,
Woodcock, J. Potential use of nerve growth factor to treat Alzheimer's disease.
(Letter) Science. 243,11,1989.
77. Phelps, e.H., Gage, F.H., Growdon, J.H., Hefti, F., Harbaugh, R, Johnston, M.V.,
Khachaturian, Z.S., Mobley, W.C, Price, D.L., Rakind, M., Simpkins, J., Thai, L.J ..
Woodcock, J. Potential use of nerve growth factor to treat Alzheimer's disease.
Neurobiol. ofAging. 10,205,1989.
78. Hefti, F., Knusel, B. Chronic administration of ncrvc growth factor and other neu-
rotropic factors to the brain. Neurobiol. ofAging. 9,689,1988.
79. Roberts, H.R The treatment of hemophilia: past tragedy and future promise. N. Eng/.
1. Med. 321, 1188, 1989.
80. Hellman, L., Smedsrod, B., Sandberg, H., Pettersson, U. Secretion of coagulant factor
VIn activity and antigen by in vitro cultivated rat liver sinusoidal endothelial cells. Br.
1. Hematol. 73,348, 1989.
81. Lu, Z., Zheng, T., Lanza, RP., Soon-Shiong, P. Reversal of hypocalcemia in parathy-
roidectomized rats by transplantation of microencapsulated parathyroid cells. In
preparation.
82. Fu, X.W., Sun, A.M. Microencapsulated parathyroid cells as a bioartificial parathyroid:
in vivo studies. Transplantation. 47,432,1989.
83. Darquy, S., Sun, A.M. Microencapsulation of parathyroid cells as a bioartificial
parathyroid: in vitro studies. Trans. Am. Soc. Artif Intern. Organs. 33,356, 1987.
84. Trey, e. The fulminant hepatic failure surveillance study. Brief review of the effects of
presumed etiology and age of survival. Can. M ed. Assoc. 1. 106, 525, 1972.
85. Rueff, B., Benhamou, J.P. Acute hepatic necrosis and fulminant hepatic failure. Gut.
14,805,1983.
86. Krom, R.A., Gips, G.H. Skills and resources needed for liver transplantation.
Hepatology. 4, 72S, 1984.
87. Scharschmidt, B.F. Human liver transplantation: analysis of data on 540 patients from
four centers. Hepatology. 4, 95S, 1984.
88. Aiken, J., Cima, L., Schloo, B., Mooney, D., Johnson, L., Langer, R, Vacanti, J.P.
Studies in rat liver perfusion for optimal harvest of hepatocytes. 1. Ped. Surg. 25,140,
1990.
89. Bruni, S., Chang, T.M.S. Hepatocytes immobilized by microencapsulation in artificial
cells: effects on hyperbilirubinemia in Gunn rats. Biomat. Art. Cells Art. Org. 17, 403,
1989.
90. Beal, M.F., Richardson, E.P., Martin, J.B. Degenerative diseases of the nervous system.
In: Harrison's Principles of Internal Medicine. 12th Edition. Wilson, J.D., Braunwald,
E. Isselbacher, K.J., Petersdorf, RG., Martin, J.B., Fauci, A.S., Root, RK (eds.)
McGraw-Hill, New York, 1991, p. 2060.
91. Tze, W.J., Tai, J. Xenotransplantation ofrat pancreatic endocrine cells in spontaneous
and streptozotocin-induced diabetic monkeys. Transplant. Proc. 21,2736,1989.
92. Chabot, J.A., Stegall, M.D., Weber, e., Reemtsma, K., Hardy, M.A. Pancreatic islet allo-
andxenotransplantation in cynomolgus monkeys. Transplant. Proc. 21,2739,1989.
93. Hefti, F. Nerve growth factor promotes survival of septal cholinergic neurons after fim-
brial transections.l. Neurosci. 6,2155,1986.
94. Williams, L.R, Varon, S., Peterson, G.M., Wictorin, K, Fisher, W., Bjorklung, A.,
Gage, F.H. Continuous infusion of nerve growth factor prevents basal forebrain neu-
ronal death after fimbria fornix transection. Proc. Natl. Acad. Sci. USA. 83,9231,1986.
9S. Fisher, W., Wictorin, K.. Bjorklund, A., Williams, L.R, Varon, S., Gage, F.H.
Amelioration of cholinergic neuron atrophy and spatial memory impairment in aged
rats by nerve growth factor. Ncurobiol. Aging. 10,89, 1989.
96. Bruce, G., Heinrich, G. Production and characterization of biologically active recombi-
nant nerve growth factor. Ncurobiol. Aging. 10,89, 1989.
97. Takeuchi, R, Murase, K, Furukawa, Y, Furukawa, S., Hayashi, K Stimulation of nerve
growth factor synthesis/secretion by I, 4-benzoquinone and its derivatives in cultured
mouse astroglial cells. Febs Lett. 261. 63,1990.
98. Furukawa, S., Furukawa, Y., Satoyoshi. E., Hayashi, K Regulation of nerve growth fac-
tor synthesis/secretion by catecholamine in cultured mouse astroglial cells. Biochem.
Biophys. Res. Commlln. 147,1048,1987.
99. Rohrer, H., Heumann, R, Thoenen, H. The synthesis of nerve growth factor (NGF) in
developing skin is independent of innervation. Dev. Bioi. 128,240, 1988.
100. Goedert, J.J., Kessler, e.M., Aldenont, L.M., Biggar, RJ., Andes, W.A., White, G.e.,
Drummond, J.E., Vaidya, K, Mann, D.L., Eyster, M.E., Ragni, M.V., Lederman,
M.M., Cohen, A.R, Bray, G.L., Rosenberg, P.S., Friedman, RH., Hilgartner, M.W.,
Blattner, W.A., Kroner, B., Gail, M.H. A prospective study of human immunodeficien-
cy virus type 1 infection and the development of AIDS in subjects with hemophilia. N
Engl. J. Med. 321,1141,1989.
101. Handin, R.I. Disorders of coagulation and thrombosis. In: Harrison's Principles of
Internal Medicine. 12th Edition. Wilson, J D., Braunwald, E. Issellbacher, KJ.,
Petersdord, RG., Martin, J.B., Fauci, A.S., Root, RK (eds.) McGraw-Hill, New York,
1991,p.1S0S.
102. Wion, KL., Kelly, D.A., Summerfield, J.A., Tuddenham, E.G.D., Lawn, RM.
Distribution of factor VIII mRN A and antigen in human liver and other tissues. Nature.
317,726,198S.
103. Webster, W.P., Zukoski, e.F., Hutchin, P., Reddick, RL., Mandel, S.R, Penich, G.D.
Plasma factor VIII synthesis and control as revealed by canine organ transplantation.
Am. J. Physiol. 220, 1147,1971.
104. Lewis, J.H., Bontempo, F.A., Spero, J.A., Ragni, M.V., Starzl, T.E. Liver transplanta-
tion in a hemophiliac. N. Engl. J. Med. 312, 1189, 1985.
lOS. White, J.V., Logerfo, P., Feind, e., Weber, e. Autologous parathyroid transplantation.
(Letter) Lancet. 2,461,1982.
106. Wells, S.A., Gunnells, J.e., Shelburne, J.D., Schneider, A.B., Serwood, L.M. Trans-
plantation of the parathyroid gland in man: clinical indications and results. Surgery. 78,
34,197S.
107. Aebischer, P., Russell, P.e., Christensen, L., Panol, G., Monchik, J.M., Galletti, P.M.
A bioartificial aprathyroid. Trans. Am. Soc. Artif Intern. Organs. 32, 134, 1986.
108. Lu, Z., Zheng, T., Lanza, RP., Soon-Shiong, P. Reversal of hypocalcemia in parathy-
roidectomized rats by transplantation of microencapsulated parathyroid cells. In
preparation.
109. Wilson, J.D. Hormones and hormone action. In: Harrison's Principles of Internal
Medicine. 12th Edition. Wilson, J.D., Braunwald, E., Isselbacher, KJ., Petersdorf,
RG., Martin, J.B., Fauci, A.S., Root, RK (eds.) McGraw-Hill, New York, 1991,
p.I647.
Chapter 18

I: Plasma Exchange and Splenectomy
R.REDING
Introduction
A variety of microsurgical transplantation models that are available in rodents

for organ allografting [1] have been used in xenograft research, particularly
with rats as recipients of organs from size-matched xenogeneic donors.
Among the 1687 species classified as rodents in the zoological taxonomy, only
four have been used for xenotransplantation - the hamster, rat and mouse
belong to the same suborder (Myomorpha), whereas the guinea pig
(Hystricomorpha) is more distantly related [2]. Theoretically, a rough predic-
tion of xenograft survival time can be made according to disparity in the zoo-
logical system - the more distantly related the donor and the recipient, the
more aggressively and rapidly will the xenograft rejection process occur.
CaIne's classification of xenografts distinguishes "concordant" and "dis-
cordant" species combinations [3]. Xenogeneic organ rejection occurs
through cellular mechanisms in the former (as in nonpresensitized allograft
systems) and humoral injury in the latter (which mimics hyperacute antibody-
mediated vascular rejection in hyperimmunized or ABO-incompatible recipi-
ents of allografts). However, when analyzing experimental xenograft models
currently available in rodents, it appears that the division between concordant
and discordant combinations is not as absolute as suggested above, since both
humoral and cellular mechanisms may be associated in variable proportions in
the unmodified rejection process of so-called concordant xenografts [4,5].
Control survival times in primarily vascularized rodent xenograft models
(excluding tissue and skin transplants) vary considerably, depending on the
species combination and the organ transplanted (Table 18.1). Interpretation
and relevance of these graft survival data depend mainly on the criteria of re-
jection - cessation of palpable beating for heterotopic (cervical or abdominal)
cardiac transplants versus death of the animal for kidney and orthotopic liver
xenografts. The species combination as well as the type of organ transplant
must, therefore, be taken into account when analyzing xenograft data, particu-
larly in rodents.
Despite its limitations and imperfections, however, the concordant-discor-
dant classification remains a practical terminology in rodent combinations and
will be used in this review.
314 Experimental Xenotransplantation in Rodents
Table 18.1. Experimental xenograft models in rodents - mean survival times in untreated
recipients
Model Heart Liver Kidney

( donor/recipient) (heterotopic) (orthotopic)
Guinea pig/rat 15 min [9,21]" 16 h [21]" 9.2 h [4]"

Mouse/rat 4 days [22] <2 days [27]
Hamster/rat 3.6 days [8, 26] 7.3 days [8, 26]
Hamster/mouse 7.5 days [22]
Rat/mouse 4.9 days [22]
a Numbers in brackets indicate references
Liver Xenografting
Among the models reviewed in Table lS.1, orthotopic liver xenografts offer
particular interest. Xenogeneic hepatic transplants seem to be much less sus-
ceptible to rejection than other organs, even in the discordant guinea pig-to-
rat model. Cardiac transplants are invariably destroyed by hyperacute vascu-
lar rejection (HVR) within 20 min following revascularization whereas, in the
same combination, mean rejection time for liver grafts is 40 times longer [6].
The dual vascularity of the liver and (particularly in rodents) the lack of hepat-
ic artery reconstruction, as well as the potential removal of immune complexes
by Kupffer cells, among other factors, may be responsible for the privileged
behavior of hepatic xenografts.
An additional characteristic of liver xenografts is their capability of
metabolic function in a xenogeneic environment. This has been investigated
by Monden et al. [7], who, using an immunological assay, demonstrated the
presence of hamster proteins in rat serum, indicating that the hamster liver
was working. Moreover, in this combination, xenogeneic hepatic grafts have
sustained life for up to 25 days, providing that rejection was delayed by ade-
quate immunosuppression [S],
Mechanisms of Discordant Xenogeneic Rejection
Guinea pig-to-rat heterotopic cardiac transplantation, initially described by

Jamieson [9], constitutes the easiest model ofxenogeneic HVR. It has subse-
quently been used by investigators as the prototype of a discordant xenograft
in small animals as the time course of rejection is highly reproducible, At the
cessation of graft function histopathological examination of the graft shows
typical humoral rejection, with intravascular platelet aggregates occluding ar-
terioles, and interstitial hemorrhage [6, 10-13] (Fig, lS.1).
Nevertheless, the initiating event of xenogeneic HVR in the guinea pig-to-
rat model remains a matter of debate. Rat serum contains preformed (natural)
Mechanisms of Discordant Xenogeneic Rejection 315
Fig. 18.1 a, b. Light micrographs of hyperacute vascular rejection in a heterotopic guinea

pig-to-rat cardiac xcnograft , at cessation of heart beating (15 min following revasculariza-
tion). a Congested venule (V); intravascular platelet aggregates (PA) completcly occluding
an arteriole. b Interstitial hemorrhage (arrow) and intravascular platelet aggregates (PA).
Neither mono nuclear cell infiltration nor extensive myocardi al necrosis are observed
(H & E)
antiguinea pig hemolyzing or cytotoxic antibodies [12, 13]. A specific immuno-

logical phenomenon (antigen-antibody recognition on graft vascular endothe-
lium) was considered until recently to trigger activation of various nonspecific
inflammatory effector cascades, leading to a single-organ disseminated in-
travascular coagulation, vascular obstruction, and xenograft ischemic necrosis
within minutes or hours after revascularization of the transplant. Such a mech-
anism has been well documented for xenograft combinations in large animals
[14-16). Moreover, immunohistological studies confirm the presence of rat
immunoglobulin M (IgM) and complement within the rejected xenografts [6,
12].
Antibody-Depletion by Plasma Exchange
The importance of the role of specific antigen-antibody immune recognition

was tested in rodents using pre transplant antibody depletion by plasma ex-
change (PE)(Table 18.2). PE performed immediately before guinea pig-to-rat
cardiac xenografting allowed prolongation of graft survival up to 4 h [13).
However, in this experiment, the lack of selectively of the PE procedure,
which removed coagulation and complement factors and platelets as well as
preformed antibody, did not provide a definite answer to the exact nature of
the initial activating event of HVR.
In the PE experiment of Van de Stadt and colleagues [12), plasma removal
performed the day prior to xenografting did not prolong xenograft survival.
This was explained by early and rapid (postdepletional) antibody resynthesis,
a phenomenon well documented in rats for immune and preformed antibody
[13, 17]. In contrast, combining PE on the day prior to xenografting with cy-
Table 18.2. Combined effect of plasma exchange and splenectomy, cyclosporine or cyclo-
phosphamide on guinea pig-to-rat heart xenografts"
Immune modulation Mean survival timc Statistical

(minutes) significance
(versus control)
Control 16
Plasma exchange (day -I)a 13 NS
Plasma exchange (day -I) 302 p<O.OOI
+splenectomy (day -7)"
Plasma exchange (day -I) 418 p<O.OOI
+CsA (days -3, -2, -I)"
Plasma exchange (day -I) 200 p<O.OOl
+CP (days -2, -I)"
Plasma exchange (hour -2)b 235 p<O.OOI
CsA, cyclosporine 30 mg/kg per day: CP, cyclophosphamide 20 mg/kg per day: NS. not sig-
nificant
" From [121
h From [13]
Mechanisms of Discordant Xenogeneic Rejection 317
THE CENTRAL ROLE OF C 3 ACTIVATION IN
HYPERACUTE VASCULAR REJECTION OF XENOGRAFTS
XENOGENEIC C Activation of coagulation

I---~ 3 and inflammatory cells
ANTIGEN-ANTIBODY c1aSSlca~
COMPLEXES athway '"
(Vand.stadt .t al 1988) " ~ ICsa ",--'--V-A-S-C-U-L-A-H-
C 3a )
I ~
C 3b .... Cs_C9
Lndothelial
alternative - - - - 1..
_ cell damage
XENOGENEIC pathway Membrane attack
TARGET complex
MEMBRANES I~-
'---------'
(Miyagawa et dJ.,1988)
Fig. 18.2. The protcins of the complement system form two interrelated enzyme cascades,
termed the classical and the alternative pathways. The former is activated specifically by
antigen-antibody complexes, through activation ofCl, C4, and C2, whereas the latter is non-
specifically triggered on target foreign membranes, without mandatory antibody involve-
ment. These two routes lead to the conversion of C3 to C3a and C3b. Subsequent activation
allows C5 to C9 to form tunnel-like structures (membrane attack complex), which penetrate
the endothelial cell membrane, leading to osmotic cellular lysis, endothelial wall disruption,
platelet activation and interstitial hemorrhage. At the same time, low molecular weight pep-
tides (C3a, C5a) are released during firing of the complement cascade, and they exert pow-
erful effects on inflammatory cells, leading to further tissue injury (release oflysosomal en-
zymes by leukocytes and of histamine by mast cells, enhancement of capillary permeability,
edema and contraction of arterial smooth muscle). Cobra venom factor inhibits the whole
cascade by causing massive conversion of C3 to C3b and C5-9 consumption by discharging
the feedback loop to exhaustion
closporine (CsA) administration or splenectomy (on day-7), allowed a signifi-

cant prolongation of xenograft survival. This was correlated with inhibition of
post-PE resynthesis of preformed antiguinea pig cytotoxic antibodies which
was attributed to splenectomy (which removes antibody-producing cells) or to
CsA treatment (resulting in inhibition of T cell help to antibody-producing
cell activation).
From these results, and by analogy with hypotheses formulated to explain
xenograft data from large animals, it was assumed that the antigen-antibody
recognition that occurs on the endothelial surface of the xenograft constitutes
the activating factor of the complement cascade in xenogeneic HVR. This fol-
lows the classical pathway, leading to the cleavage of C3 into C3a and C3b, the
central event in the complement system (Fig. 18.2).
However, the importance of the specific antibody-antigen recognition as
the initiating event of complement activation in xenogeneic HVR has recently
been questioned [11]. During rejection of guinea pig-to-rat cardiac xenografts
Miyagawa et al. observed a significant reduction of complement hemolytic
titers in serum, without consumption of C4 and C2, suggesting complement ac-
tivation through the alternative pathway. Moreover, in vitro experiments
showed that rat complement attacked guinea pig erythrocytes via activation
through the alternative pathway. In contrast with the PE experiments, these
results suggested a minor role for xenogeneic immune complexes in triggering
the HVR cascade.
Cobra Venom Factor
Whatever the precise mechanism of inflammatory cascade activation, deple-

tion of the complement system of the recipient by administering cobra venom
factor (CVF) has been most efficient in prolonging xenograft survival in the dis-
cordant rodent model (guinea pig-to-rat heart transplant) up to 3 days [11, 18].
CVF contains, among its many toxins and enzymes, cobra C3b which ap-
pears to be resistant to the action of human and rat factors H and I, the C3b in-
activating factors. This C3b (cobra) combines with recipient cleavage prod-
ucts of the alternative pathway of complement, forming a highly stable
enzyme which has a half-life of 37 h, and causes massive consumption of com-
plement components by discharging the feedback loop to exhaustion [19]. To
date, CVF has induced the most efficient and durable inhibition of HVR in ro-
dents.
Pharmacologic Agents
The nonspecific effector inflammatory cascade that ensues once complement

activation has initiated vascular rejection has served as a target for a variety of
therapeutic interventions. These therapeutic modalities have included inhibi-
tion of blood coagulation [4,20], as well as antiplatelet agents [18]. However,
these protocols only marginally prolonged discordant xenograft survival.
From the theoretical point of view, attempts to control the effector mecha-
nisms involved in HVR suppose that xenogeneic recognition has already taken
place, indicating that immune injury has been initiated. Moreover, there are nu-
merous alternative mediator pathways that can back up the same set of func-
tions - inhibition of one arm of the inflammatory reaction can easily be replaced
by intervention of another effector cascade. These reasons may explain the rel-
atively poor results obtained with such approaches. Nonetheless, the argument
forpolypharmaceutical therapy of humoral rejection appears strong [16].
Concordant Xenografts
In contrast to the typical xenogeneic HVR observed in guinea pig hearts trans-
planted into rats, cardiac xenografts in the hamster-to-rat combination are re-
jected more slowly (mean rejection time 3 days), through cellular and delayed
humoral mechanisms [5] (Table 18.1). Prolongation of xenograft survival in
Concordant Xenografts 319
this concordant model has been achieved by forms of immunosuppression that
suppress the cell-mediated allogeneic response.
CsA was reported to significantly enhance hamster heart graft survival in
rat recipients, but nearly toxic dosages (35 mg/kg per day) were required [21].
In two similar closely related species combinations (hamster-to-mouse and
rat-to-mouse) cardiac xenograft survival was significantly extended with a
horse antimouse antithymocyte globulin (ATG), previously shown to be very
immunosuppressive when tested in allografts [22] (Table 18.1). In addition,
these authors showed that, in both combinations, A TG administration for 4
weeks (rather than 2 weeks) resulted in longer graft survival (mean survival
times 65 days versus 38 days, respectively in the hamster-to-rat cardiac
xenograft model compared with 4.9 days in controls).
Moreover, in the hamster-to-rat combination, Knechtle and co-workers
[23] observed a synergistic immunosuppressive effect of CsA combined with
total lymphoid irradiation. According to their data, which have been con-
firmed [24], delayed humoral rejection (occurring 3 days post-transplant in
control recipients) was abrogated, and graft survival extended to more than
100 days without histological evidence of rejection.
The same animal model (hamster-to-rat heart) was used to assess the im-
munosuppressive activity of the newly developed antimetabolite 15-de-
oxyspergualin (15-DS), which inhibits antibody resynthesis as well as the
monocyte/macrophage system. At a nontoxic dosage of 2.5 mg/kg per day, 15-
DS combined with splenectomy significantly prolonged cardiac xenograft sur-
vival by up to 30 days, with a predominant histological picture of mononuclear
cell infiltration at the time of rejection [25].
As discussed above, orthotopic liver xenograft models have shown their
own characteristics, particularly in the hamster-to-rat combination, where
death of the untreated recipient from rejection was reported within 8 days
post-transplant [7] (Table 18.1), the histological picture being of humoral in-
jury. At the time of graft failure, marked splenomegaly was noted in these re-
cipients, without signs of portal hypertension. This was considered to be part
of the immune response against circulating xenogeneic hamster antigens se-
creted by the liver xenograft.
Combined treatment with CsA and splenectomy allowed a significant pro-
longation of liver graft survival up to 17 days [8]. This synergistic effect has
Table 18.3. Combined effect of cyc1osporine and splenectomy in hamster-to-rat xenografts a
Donor organ Control CsA
Liver (Orthotopic) 7.3 days 7.6 daysc 7.2 days 17.6 daysd
Heart (Heterotopic) 3.4 days 4.2 daysd 5.2 days 41.3 daysd
a From [7,8].
b Splenectomy performed at the time of xenografting.
c 40 mglkg per day.
d 30 mg/kg per day.
been subsequently confirmed for heterotopic cardiac xenografts using the

same species combination (Table 18.3), with essentially preserved histological
structure in both long-standing hamster liver and heart xenografts [26].
Splenectomy alone significantly decreased antidonor antibody production in
the recipient, and greatly modified the histological pattern of rejection. The
hepatic necrosis and interstitial hemorrhage observed in unmodified
xenograft rejection was replaced by cellular infiltration with relatively pre-
served parenchyma in splenectomized animals.
Comment
From the rodent xenograft data detailed above, it appears that the xenogeneic
immune response constitutes a highly complex and heterogenous rejection
mechanism, differing qualitatively and quantitatively from allograft systems,
and involving cellular as well as specific and nonspecific humoral rejection
pathways. Moreover, species combinations and organ-related differences
should be carefully considered before extrapolating the conclusions reached
from rodent models to large animal models (especially when considering man
as the recipient).
Nevertheless, rodent models represent useful tools for increasing our
knowledge of the biologic mechanisms involved in xenogeneic immune re-
sponses. Organ xenotransplant systems in rodents are convenient, relatively
cheap, and easily reproducible, particularly in inbred strains. They can serve
for the investigation of new therapeutic interventions in recipients as well as in
donors (donor pretreatment) of cross-species transplantation, and can pro-
vide valuable information on the efficacy and mechanisms of action of such in-
terventions, and their potential synergy with established immunosuppressive
therapies.
References
1. Thiede, A., DeItz, E., Engemann, R., Hamelmann, H. (eds.) Microsurgical Models in Rats
for Transplantation Research. Springer-Verlag; Berlin, Heidelberg, New York, 1985.
2. Rothschild, Lord (ed.) A Classification of Living Animals. 2nd edition, Longmans
Green; London, 1965, p. 46.
3. CaIne, RY. Organ transplantation between widely disparate species. Transplant Proc. 2,
550,1970.
4. Sakai, A., Kountz, S.L. Dissociation of humoral and cellular immune reactions of the
rabbit, guinea pig and dog kidneyxenografts in the rat. J. Surg. Res. 21,159,1976.
5. Hardy, M.A., Oluwole, S., Fawwaz, R, Satake, K., Nowygrod, R, Reemtsma, K.
Selective lymphoid irradiation. III. Prolongation of cardiac xenografts and allografts in
presensitized rats. Transplantation. 33, 237,1982.
6. Settar. A., Meriggi, F., Van de Stadt, J., Gane, P., Crougneau, S., Reynes, M., Rouger, P.,
Houssin, D. Delayed rejection of liver xenografts compared to heart xenografts in the
rat. Transplant.Proc.19, 1155, 1987.
References 321
7. Monden, M., Valdivia, LA, Gotoh, M., Hasuike, Y., Kubota, N, Kanai, T., Okamura, J.,
Mori, T. Hamster-to-rat orthotopic liver xenografts. Transplantation. 43, 745,1987.
8. Valdivia, L.A, Monden, M., Gotoh, M., Hasuike, Y., Kubota, N., Ichikawa, T.,
Okamura, J., Mori, T. Prolonged survival of hamster-to-rat liver xenografts using
splenectomy and cyclosporine administration. Transplantation. 44, 759,1987.
9. Jamieson, S.W. Xenograft hyperacute rejection. A new model. Transplantation. 17,533,
1974.
10. Cattell, V., Jamieson, S.W. Hyperacute rejection of guinea pig-to-rat cardiac xenografts.
I. Morphology. J. Path. 115, 183, 1975.
Matsumoto, M., Venaka, A., Kitamura, H. The mechanism of discordant xenograft re-
jection. Transplantation. 46, 825, 1988.
12. Van de Stadt, J., Vendeville, B., Wiell, B., Crougneau, S., Michel, A., Filipponi, F., Icard,
POo Renoux, M., Louvel, A, Houssin, D. Discordant heart xenografts in the rat.
Additional effect of plasma exchange and cyclosporine, cyclophosphamide or splenecto-
my in delaying hyperacute rejection. Transplantation. 45, 514,1988.
13. Reding, R, Davies, H.ff.S., White, DJ.G., Wright, L.J., Marbaix, E., Alexandre, G.PJ.,
Squifflet, J.P., CaIne, RY. Effect of plasma exchange on guinea pig-to-rat heart
xenografts. Transplant. Proc. 21, 534,1989.
14. Mejia-Laguna, J.E., Martinez-Palomo, A., Biro, e.E., Chavez, B., Lopez-Soriano, F.,
Garcia-Cornejo, M. Morphologic study of the participation ofthe complement system in
hyperacute rejection of renal xenotransplants. Am. J. Patho!. 69, 71,1972.
15. Hammer, e., Chaussy, e., Brendel, W. Preformed natural antibodies in animals and
man. Outlook on xenotransplantation. Eur. Surg. Res. 5, 162, 1973.
16. Makowka, L., Miller, e., Chapchap, P., Podesta, L., Pan, e., Pressley, D., Mazzaferro, V.,
Esquivel, e.O., Todo, S., Banner, B., Jaffe, R, Saunders, R, Starzl, T.E. Prolongation of
pig-to-dog renal xenograft survival by modification of the inflammatory mediator re-
sponse. Ann. Surg. 206, 482,1987.
17. Reding, R, White, D.J.G., Davies, H.ff.S., Latinne, D., Delepaut, B . Lambotte, L.,
CaIne, RY. Effect of splenectomy on antibody rebound after plasma exchange.
Transplantation. 48,145, 1989.
18. Adachi, H., Rosengard, B.R., Hutchins, G.M., Hall, T.S., Baumgartner, W.A., Borkon,
AM., Reitz, B.A. Effects of cyclosporine, aspirin and cobra venom factor on discordant
cardiac xenograft survival in rats. Transplant. Proc. 19,1145,1987.
19. Muller-Eberhard, H.J., Fjellstrom, K.E. Isolation of the anti-complementary protein
from cobra venom and its mode of action on C3. J. Immuno!. 107,1666,1971.
20. Zhang, J., Munda, R, Glas-Greenwalt, P., Weiss, M.A, Pollak, V.E., Alexander, J.W.
Prolongation of survival of a heart xenograft by defibrination with ancrod.
21. Homan, W.P., Williams, K.A, Fabre, J.W., Millard, P.R, Morris, P.J. Prolongation of
cardiac xenograft survival in rats receiving cyclosporin A Transplantation. 31,164,1981.
22. Sakakibara, N., Click, RE., Condie, RM., Jamieson, S.W. Rejection/acceptance of
23. Knechtle, S.J., Halperin, E.e., Bollinger, RR Xenograft survival in two species combi-
nations using total lymphoid irradiation and cyclosporine. Transplantation. 43, 173,
1987.
24. Bouwman, E., de Bruin, RW.F., Marquet, RL., Jeekel, J. Prolongation of graft survival
in hamster-to-rat xenografting. Transplant. Proc. 21,540,1989.
25. Valdivia, L.A., Monden, M., Gotoh, M., Kubota, N., Hasuike, Y., Nakano, Y., Okamura,
J., Mori, T. Prolonged cardiac xenograft survival by 15-deoxyspergualin combined with
splenectomy. Transplant. Proc. 21,532,1989.
26.Valdivia, L.A., Monden, M., Gotoh, M., Hasuike, Y., Kubota, N., Ichikawa, T., Nakano,
Y, Okamura, J., Mori, T. An important role of the spleen in rejection of hamster-to-rat
27. Owen, E.R Xenograft protection by low-dose, antigen-induced tolerance. Transplant.
Proc.3,562,1971.
Chapter 19

II: Skin Versus Heart Grafts
E.BoUWMAN, M.C.J. WOLVEKAMP,R.W.F. DE BRUIN,J.JEEKEL,
AND R.L. MARQuET
Introduction
When studying xenotransplantation in a small animal model, one has a choice

between two different types of models. In one, rejection of the graft takes
place within minutes after transplantation - so-called hyperacute rejection. In
the other type, rejection takes more time - usually 3-5 days - and is generally
referred to as accelerated acute rejection as it occurs some days earlier than
the acute graft rejection seen in allogeneic transplantation.
Hyperacute rejection is generally believed to be caused by preformed "nat-
ural" antidonor antibodies [1-3] with activation of the classical route of the
complement system, although recently a role of the alternative route of the
complement system has been suggested. According to some authors [4], acti-
vation of this arm of the complement system can lead to hyperacute graft re-
jection without the involvement of antibodies.
The mechanism of accelerated acute graft rejection is not fully understood
at the present time and seems to be limited to xenografting in donor-recipient
combinations where no, or only very low, titers of antidonor antibodies are
found.
The aim of our studies was to develop immunosuppressive protocols per-
mitting long-term graft survival in a donor-recipient combination where the
graft is rejected in an accelerated acute fashion. To that end, the hamster-to-
rat heterotopic heart transplantation model was chosen. The immunosuppres-
sive schedules tested were all based on cyclosporine (CsA), the most effective
immunosuppressant available at the time these investigations were begun.
Since reports in the literature [5, 6] showed that in some combinations
xenogeneic skin grafts could be maintained relatively easily for prolonged pe-
riods of time, we tested this in the hamster-to-rat combination We were fur-
ther interested to find out whether such long-standing hamster skin grafts,
if attainable, were beneficial to the survival of subsequently transplanted
hamster hearts.
Animals
Male Syrian hamsters were used as organ donors and male Lewis rats (RTI 1)
of 250-400 g body weight were used as recipients.
Surgical Techniques
Heterotopic heart transplantations were performed using the technique de-

scribed by Ono and Lindsey [7]. A graft was considered rejected when regular
heart beating could no longer be palpated. Skin transplants of about 2 cm
diameter were taken from the trunk and, after carefully being cleaned of the
subcutaneous tissues, grafted on to the back of the recipient. The grafts were
kept in place by means of a tight bandage until day 7. A skin graft was consid-
ered rejected when (i) no hair growth was observed and (ii) there was a greater
than 75% reduction in the original graft size.
Immunosuppression
CsA dilutions were made in olive oil from commercially available batches, and
given intramuscularly in aliquots of 0.05-0.2 m!. Total body irradiation (TBI)
was performed in one session, using a 137 Cs gamma source.
Results
Heart Transplantation
When transplanted into untreated recipients, hamster heart grafts were reject-
ed in 3-5 days (Table 19.1, Group 1). Attempts to prolong graft survival by
means of administration of CsA failed, in spite of the very high doses given
(Group 2). In order to address the question of whether effective CsA levels
might be achieved too late because of the very rapid progression of the rejec-
tion process, we pretreated recipients with CsA during the 4 days immediately
prior to transplantation. Again, no prolongation of graft survival was obtained
(Group 3).
When 5 Gy TBI was used as immunosuppression, prolongation of graft sur-
vival was obtained (Group 4). The combination of 5 Gy TBI with high daily
doses of CsA (Group 5) resulted in remarkable additional prolongation of
graft survival, although this protocol caused considerable toxicity, with more
than 50% of the animals dying with a functioning graft. Reduction of the CsA
Results 325
Table 19.1. Survival of hamster heart grafts in Lewis rat recipients
Group Treatment Graft survival (days)'
1 Control (no therapy) 3,3,4,4,4,4,4,5,5

2 CsA 45 mg/kg per day 3,4,4,4,4,5,5,5,5,6
3 CsA 25 mg/kg per day (from day -4) 4,4,4,4
4 TBI5 Gy(dayO) 6,7,7,8,8,8,8
5 TBI5 Gy (day O)+CsA 25 mg/kg per day (30),42, (43),44, (45,45),59
6 TBI 5 Gy (day O)+CsA 25 mg/kg 7,10,13,14,17,36,38,39,40,40,
(alternate days) 51,63, (117)
7 Splenectomy (day 0) 5 (xl0)
8 Splenectomy (day O)+CsA 25 mg/kg per day 5,5,5,7,7,8,8,42 b
9 Splenectomy (day -21)+CsA 25 mg/kg per day 3,4,5,6,7,7,13
(from day 0)
CsA, cyc1osporine; TBI, total body irradiation

a Figures in parentheses indicate that the animal died with a functioning graft.
b CsA discontinued on day 14.
dose virtually eliminated this toxicity (Group 6), but the immunosuppressive
effect was significantly reduced as well.
Splenectomy as a single mode of immunosuppression was only marginally
effective (Group 7). The combination of splenectomy with CsA (Group 8) did
not improve graft survival dramatically, and there was similarly little effect
when splenectomy was carried out 3 weeks prior to transplantation (Group 9).
Skin Transplantation
Untreated controls all rejected their grafts within 10 days of grafting (Group
10). In contradistinction to the effect on survival of heart grafts, the combina-
tion of splenectomy and CsA was very effective in prolonging hamster skin
graft survival (Table 19.2, Group 11). Importantly, however, CsA alone was
equally effective (Group 12) in obtaining long-term skin graft survival.
Table 19.2. Survival of hamster skin grafts on Lewis rat recipients
Group Treatment Graft survival (days)
10 Control (no therapy) <10 (x8)

11 CsA 25 mg/kg per day (days 0--10); >100 (x5)
CsA 25 mg/kg 3xweek (>day 10)
+Splenectomy (day 0)
12 CsA25 mg/kg/day(days 0--10); >100 (xl0)
Abbreviations as for Table 19.1.

Table 19.3. Survival of hamster heart grafts in Lewis rat recipients pregrafted with hamster
skin
Group Pretreatment Treatment Graft survival (days)"
13 Long-standing hamster skin CsA 25 mg/kg 3xwcek Hearl, 3, 3. 3. 4. 4. 4:

grafts: 30-50 days: Skin>100(x6)
CsA 25 mg/kg per day (day 0-10):
14 Long-standing hamster skin CsA 25 mg/kg 3xweek Heart. 10,10. (31).
grafts: 30-50 days: >100.>100:
CsA 25 mg/kg per day (day 0-10): Skin. (31). > 100 (x5)
CsA 25 mg/kg 3xweek (>day 10),
plus splenectomy (day 0)
15 Immunosuppression as for CsA 25 mg/kg 3xwcek Heart, >50 (x6)
Group 14, but no previous
skin graft
a Figures in parentheses indicate that the animal died with an intact skin graft and a func-
tioning heart graft.
Abbreviations as for Table 19.1.
Skin and Subsequent Heart Transplantation
Next we tested whether long-standing hamster skin grafts, obtained by both

previous protocols (CsA alone and CsA plus splenectomy), were advanta-
geous to subsequently transplanted hamster hearts. Hamster skin grafts at
30-50 days after transplantation under CsA alone were of no advantage to
subsequently transplanted hamster hearts (Table 19.3, Group 13) - all hearts
were rejected in an accelerated acute fashion. Remarkably, all skin grafts re-
mained in perfect condition and survived over 100 days.
In sharp contrast, when hamster hearts were transplanted in recipients
bearing long-standing hamster skin grafts under CsA plus splenectomy
(Group 14), significant prolongation of heart graft survival was observed.
Again, although two recipients rejected their heart grafts, the corresponding
skin transplants were not rejected, although both underwent a "rejection cri-
sis" - they became edematous and purple, but regained a normal texture after
a few weeks, with persistent hair growth after shaving.
When, as a control, Group 14 was repeated but without a skin graft (Group
15), it turned out that equally good or even better graft survival was obtained,
illustrating that the skin graft itself did not contribute to the observed prolon-
gation of heart graft survival. The superior effect of the protocols used in
Groups 14 and 15, compared to Group 9, was entirely accounted for by the ap-
plication of both CsA and splenectomy prior to transplantation.
Comment 327
Comment
The observation that CsA monotherapy has no effect on heart graft survival in
the model used is in agreement with the majority of the literature [8-11], al-
though prolongation of graft survival with CsA has been reported sporadically
[12, 13]. Rejection in this model is considered by many investigators to be me-
diated mainly by T cells. The fact that CsA is at best only marginally effective
suggests that either we are dealing with an extremely strong T cell-mediated
rejection, or that other factors are involved, such as the humoral part of the im-
mune system.
The small but significant beneficial effect of TBI, affecting both Band T
cells suggests a possible role for B cells. A stronger argument for B cell in-
volvement is the development of high titers of antihamster antibodies by day 3
after transplantation (data not shown). Removal of the spleen, an important
source of B cells, resulted in prolongation of graft survival, especially when
combined with CsA. Similar observations have been made by others with re-
gard to the role of antibodies [8, 12, 14, 15] and the remarkable effect of the
combination of splenectomy and CsA [8, 16, 17]. This combination proved ex-
tremely effective in our hands when it was started before transplantation. This
underscores the importance of a high CsA level at the time of transplantation.
Some important conclusions can be drawn from the skin transplantation
experiments. The remarkable effectiveness of CsA monotherapy in prevent-
ing skin graft rejection indicates that skin grafts (not vascularized) are rejected
in a different fashion compared to heart grafts (vascularized). This is in agree-
ment with the opinion that skin grafts are rejected by cellular mechanisms.
The normal rejection time (3-4 days) of the heart grafts transplanted into skin
graft-bearing recipients further illustrates that two different, and possibly in-
dependent, mechanisms are operating. The cardiac graft rejection observed
here is most likely caused by antibodies, which apparently do not destroy the
skin graft.
This could be explained as follows. Shortly after transplantation a skin graft
is vascularized by connections of recipient vessels with those of the graft.
Gradually, graft vessels are lost, being replaced by recipient vessels. In the pre-
sent study, most skin grafts were probably completely vascularized by recipi-
ent vessels 30-50 days after transplantation, so no donor endothelium re-
mained as a potential target, except in the two grafts undergoing a "rejection
crisis". Data obtained by others in similar experiments support this explana-
tion [18, 19].
Since it can be assumed that antibodies rapidly penetrate the vascular en-
dothelium and enter the extravascular tissues, it is surprising that this does not
cause destruction of the skin graft, at least macroscopically. This might indi-
cate that the relevant antibodies are directed against an antigen exclusively or
predominantly present on vascular endothelium. It might be speculated that,
since grafting of nonvascularized organs such as skin can be accomplished so
relatively easily, this could be of great benefit in xenogeneic pancreatic islet
transplantation, where no vascular endothelium is present.
It can be concluded that long-term heart graft survival can be achieved in

the hamster-to-rat combination. This requires application of combinations of
immunosuppressive modalities in the induction phase, recipient pretreatment
being highly beneficial, whereas maintenance therapy can be with CsA only.
Skin grafting in this model can be achieved easily on CsA monotherapy, but is
of no benefit to the survival of subsequently transplanted heart grafts. These
differences between the immunosuppressive requirements for heart versus
skin grafting probably reflect differences in rejection mechanisms, vascular-
ized grafts being initially rejected by humoral and nonvascularized grafts by
cellular mechanisms.
References
1. Giles, G.R, Boehmig, H.J., Killy, J., Amemiya, H., Takagi, H., Coburg, A.J., Hathaway,
W.E, Wilson, CB., Dixon, F.J., Starzl, T.E. Mechanism and modification of rejection of
heterografts between divergent species. Transplant. Proe. 2,522, 1970.
2. Hammer, C Preformed natural antibodies (Pnab) and possibilities of modulation of
hyperacute xenograft rejection (Hxar). Transplant. Proc. 21,522,1989.
3. Auchincloss, A Xenogeneic transplantation. Transplantation. 46, 1, 1988.
Matsumoto, M., Uenaka, A, Kitamura, H. The mechanism of discordant xenograft re-
jection. Transplantation. 46,825, 1988.
5. Biren, CA, Barr, RJ., Mccullough, J.L., Black, K.S., Hewitt, CW. Prolonged viability
of human skin xenografts in rats by cyclosporine. 1. Invest. Dermatol. 86,611,1986.
6. Cerilli, G.1., Gideon, L. Successful long-term inhibition of xenograft rejection. Surg.
Forum. 20,284,1969.
7. Ono, K., Lindsey, E.S. Improved technique of heart transplantation in rats. 1. Thorae.
Cardiovasc. Surg. 57,225, 1969.
8. Monden, M., Valdivia, L.A, Gotoh, M., Kubota, N., Nakano, Y., Okamura, J., Mori, T.
A crucial effect of splenectomy on prolonging cardiac xenograft survival in combination
with cyclosporine. Surgery. 105,535,1989.
9. Sakakibara, N., Click, RE., Sakakibara, K., Aziz, S., Jamieson, S.W., Wick, M.R
Unconventional lymphocytes involved in rejection of xenogeneic heart grafts. Lab.
Invest. 62,481, 1990.
10. Steinbruchel, D.A, Madsen, H.H.T., Nielsen, B., Larsen, S., Koch, C, Jensenius, J.C,
Hougesen, C, Kemp, E. Treatment with total lymphoid irradiation, cyclosporin A and a
monoclonal anti-T-cell antibody in a hamster-to-rat heart transplantation model: graft
survival and morphological analysis. Transplant. Int. 3,36,1990.
11. Nakajima, K., Sakamoto, K., Ochial, T., Nagata, M., Asano, T., Isono, K. Prolongation of
cardiac xenograft survival in rats treated with 15-deoxyspergualin alone and in combina-
tion with FK 506. Transplantation. 45, 1146, 1988.
12. Homan, W.P., Williams, K.A, Fabre, J.W., Millard, P.R, Morris, P.J. Prolongation
of cardiac xenograft survival in rats receiving cyclosporin A. Transplantation. 31, 164,
1981.
13. Knechtle, S.J., Halperin, E.C, Bollinger, RR Xenograft survival in two species combi-
nations using total-lymphoid irradiation and cyclosporine. Transplantation. 43, 173,
1987.
14. Rosengard, B.R, Adachi, H., Ueda, K., Hall, T.S., Hutchins, G.M., Herskowitz, A,
Borkon, AM., Baumgartner, W.A, Reitz, B.A Differences in the pathogenesis of first-
set allograft rejection and acute xenograft rejection as determined by sequential mor-
phologic analysis. 1. Heart Transplant. 5,263,1986.
Refercnces 329
15. Hardy, M.A., Oluwolc, S., Fawwaz, R, Satake, K., Nowygrod, R, Recmtsma, K.
Selective lymphoid irradiation. Transplantation. 33,237, 1982.
16. Yamaguchi, Y., Halperin, E.C., Harland, R.C., Wyble, c., Bollinger, RR Significant
prolongation of hamster liver transplant survival in Lewis rats by total lymphoid irradia-
tion, cyclosporine, and splenectomy. Transplantation. 49, 13, 1990.
17. Valdivia, L.A., Monden, M., Gotoh, M., Hasulke, Y., Kubota, N, Endoh, W., Okamura,
J., Mori, T. Hepatic xenografts from hamster to rat. Transplant. Proc. 19,1158,1987.
18. Bogman, M.l.l.Th., Berden, J.H.M., Hagemann, J.F.H.M., Maass, C.N., Kocne, RA.P.
Patterns of vascular damage in the antibody-mediated rejection of skin xenografts in the
mouse. Am. 1. Pathol. 100,727, 1980.
19. Jooste, S.V., Colvin, RB., Winn, H.J. Thc vascular bed as the primary target in the de-
struction of skin grafts by antiserum. 1. Exp. Med. 154, 1332,1981.
Chapter 20

III: Total Lymphoid Irradiation, Cyclosporine,
and Monoclonal Antibodies
D.A. STEINBRUCHEL, B NIELSEN, AND E. KEMP
Introduction
A treatment strategy including total body irradiation (TBI) or total lymphoid

irradiation (TU) in combination with cyclosporine (CsA) would seem to be a
promising method for prolonging xenograft survival in the rodent [1-6]. The
most impressive results have been reported by Knechtle et al. where graft sur-
vival after TLI + CsA treatment was prolonged for more than 100 days in a
Syrian hamster-to-Lewis rat model [7,8]. Several attempts by other groups,
however, to reproduce these results have not been successful [4,5,9].
The mechanism of graft rejection in rodent xenotransplantation is unclear.
Antibody- and cell-mediated responses, which mayor may not be identical in
allogeneic and xenogeneic combinations, seem to result in graft destruction,
but alternative mechanisms involving natural killer (NK) cells and antibody-
dependent cell-mediated cytotoxicity (ADCC) cannot be excluded [10].
While anti-CD4 monoclonal antibody (MAB) treatment can effectively
deplete CD4+ cells and prevent allograft rejection in rats [11], data on xeno-
geneic helper T cell responses in vitro are conflicting, describing primary
xenogeneic helper responses of equal, greater, and weaker magnitude or no
response at all compared with results from allogeneic combinations [12]. Anti-
CD4 treatment can prolong xenogeneic skin graft survival, which was recently
shown by Pierson et al. [13].
In our laboratory, we have been focusing on the effect of additional MAB
therapy (anti-CD4 and anti-CDS) in combination with TLI and CsA. The ra-
tionale of this treatment strategy was based on our experience that TLI plus
CsA did not result in permanent graft acceptance and that anti-CD4 treatment
seemed to be effective in preventing graft rejection in our allograft models.
Furthermore, TLI plus CsA therapy conceivably changes the immunological
status of xenogeneic concordant graft recipients, thus additional MAB treat-
ment could possibly control graft rejection.
These investigations, focusing on graft survival and immunological moni-
toring, were supplemented by sequential immunohistological and ultrastruc-
tural studies on the very early, morphological events in the same model (un-
published data).
In a slightly modified heterotopic heart transplantation modeL inbred Syrian

hamsters (albino variant) served as donors, and outbred Sprague-Dawley rats
(or, in some control series, inbred Wistar-Kyoto (WKy) rats) were used as re-
cipients (for details, see [5]).
Total Lymphoid Irradiation
Preoperatively, TLI was performed by a 4 MY linear accelerator (Philips SL

75-5) with pentobarbital-sedated rats in a custom-built perspex box. The head,
most of the lungs and heart, half of the liver, and the tail were shielded by lead
alloys. Treatment was given four times a week for 3 weeks - 1.25 Gy per ses-
sion to a total central dose of 15 Gy. The dose rate was 0.41 Gy/min at a source-
to-animal distance of 211 cm. The dose was calculated using a perspex phan-
tom and continually checked by Baldwin-Farmer air ionization chambers
during the sessions of irradiation.
Monoclonal Antibodies
The following murine antirat MABs were used and given by i.p. injections:
1. MRC OX-19 - immunoglobulin G 1 (IgG,) subclass, directed against all rat
T cells; CD5 equivalent [14].
2. MRC OX-35 and MRC OX-38 - both IgG za subclass, directed against the
rat CD4 equivalent; noncompetitive [15].
Cyclosporine and Cyclosphosphamide
CsA and cyclophosphamide (CP) were given by gastric intubation.
Monitoring
1. Graft function was evaluated by transabdominal palpation.

2. Total and differential white blood cell counts (on days-21, 0,14,28,56,84, or
at the time ofrejection).
3. Flow cytometric evaluation of lymphocyte subsets (CD2+, CD4+, CD5+,
CD8+, interleukin (IL)-2R+, and Ig+ cells) at the same time intervals as
above under point 2.
4. Flow cytometric evaluation of antidonor antibody levels at the same time
intervals [16].
5. Histopathology by light microscopy and immunohistological analysis of re-
jected grafts and lymphoid tissue, with immunofluorescence microscopy
and ultrastructural analysis in selected series.
Results 333
Experimental Groups
Nineteen groups were studied (Table 20.1).
Results
Graft Survival
Graft survival data are shown in Table 20.1. Our results demonstrate that CsA
alone (up to toxic doses) or in combination with anti-CD4 or anti-CDS MABs
and CP does not provide sufficient immunosuppression to control graft rejec-
tion effectively in this concordant cardiac xeno model, although graft survival
in some groups was significantly improved when compared with controls.
Table 20.1. Hamster-to-Sprague-Dawleyrat heart xenograft survival a
Group Treatment Graft survival (days)
I Control (no therapy) 3,4,3,4,4,4,3,5,3,4,4,5,5,3

2 CsA 12.5 mg/kg per day 4,5,4,5,5,5,4,4,5
3 CsA 25 mg/kg per day 5,4,7,5
4 CsA 50 mg/kg per day 4,4,4,5,3,7,6
5 CsA 12.S+0X-19 100 Ilg/kg per day 7,7,7,4,3
6 CsA 12.S+0X-19 500 Ilg/kg per day 5,4,3,3,4
7 CsA 12.S+CP 2.5 mg/kg per day 7,6,6,7,6,9
8 CsA 12.S+CP 5.0 mg/kg per day (from day 0) 4,5,7,7,4,5
9 CsA 12.S+CP 5.0 mg/kgperday (from day -1) 5,8,5,6,6
10 CsA 12.S+0X-3S 2000 Ilg/kg 2xweek+ 5,10,5,6, TF, TF
OX-38 5000 Ilg/kg 2xweck (from day 0)
11 As for group 10 but treatment from day -7 5.5,5,8,6,6,13,5
12 CsA 12.S+CP 2.5 mg/kg per day+OX-38 14,8,8,8, (2), 8
5000 Ilg/kg 2xweek
13 TLi 5,10,15,5,14,5,12
14 TLl+CsA 12.5 (two series) 14,15,15,11,14,13,57,42,22,
22,12,32,12,13,13,28,19,23
15 As for group 14 but with WKy rats as recipients 50,24,19,18,14,14,17
16 TLi +CsA 12.S+0X-19 500 Ilg/kg per day (6), (4), 16, 14, 16,18, (2),
(days 0-7) >100, (2), 13
17 TLi +CsA 12.S+0X-3S 500 Ilg/kg per day 14,36,14,17,42,27,80,48
(days 0-7)
18 TLi +CsA 12.S+0X-38500 Ilg/kg per day 32,14, (6), 13,28,12, (37),
(days 0-7) >100
19 TLl+CsA 12.5+0X-3S+0X-38 15,>100, TF, 14, 15,83, TF,
500 Ilg/kg 2xweek (from day 0) 19,25
TF, technical failure; CsA, cyclosporine; TLl, total lymphoid irradiation, WKy, Wistar Kyoto
a Figures in parentheses indicate that the recipient animal died from sepsis, ileus, or un-
known cause, with a functioning heart graft.
TLI, however, in combination with CsA or Csa plus MABs, seems to be a

promising treatment strategy. Additional therapy with anti-CD4 MABs pro-
vided the best results in terms of graft survival. A remarkable observation was
the heterogeneous effect of the given treatment; some graft recipients showed
long-term graft survival while others did not benefit from the additional MAB
therapy.
Lymphocyte Populations and T Cell Subsets
TLI caused an effective leukocyte depletion, the number of pre-TLI leuko-

cytes in peripheral blood being reduced to 8% and the number of lymphocytes
decreasing to 5%, though the percentage of CD4+ and Ig+ cells remained un-
changed (Fig. 20.1).
Fourteen days after transplantation, the pre-TLI numbers of peripheral
blood lymphocytes was re-established. While the CD4+ fraction was slightly re-
duced (from an average of 47% to 37%), the percentage of Ig+ cells increased
from 10% to 23%. At the time of rejection, CD4+ cells were further reduced to
31 %, but Ig+ cells increased to 36%. These changes are illustrated in Fig. 20.1.
Calculated as absolute figures, CD4+ cells were reduced to 4.6% during
TLI, and at the time of rejection constituted a total of 40% of their initial level.
Ig+ cells decreased to 6.5%, but at the time of rejection the number was dou-
bled (210% of pre-TLI level). The changes mentioned above were statistically
significant at p<0.05.
In summary, the regeneration of peripheral lymphocytes after TLI was
characterized by a disproportional increase of Ig+ cells, an increase which may
be correlated with the mechanism of rejection since B lymphocytes are more
sensitive to irradiation than T cells and recover to a pre irradiation level over a
4- to 6-week period (data from spleen and lymph nodes) [17].
Flow Cytometric Evaluation of Antidonor Antibodies
Flow cytometric cross-match results showed a weakly positive reaction at the

time of transplantation. All animals with short graft survival had a strongly
positive IgM cross-match, where more than 95% of the hamster target lym-
phocytes were found positive. Antibody titers in recipients with graft survival
exceeding 100 days, or in animals who died with functioning grafts, showed no
increase above initial levels. The two animals with graft survival of 80 (Group
17) and 83 (Group 19) days, respectively, were initially weakly positive; IgM
antibody titers subsequently decreased, but at the time of rejection a strong
IgG-positive cross-match was observed.
Our results seem to indicate a correlation between graft rejection and the
level of antidonor antibodies. The data, however, do not demonstrate directly
that these antibodies are actually responsible for rejection. This question is un-
der current investigation. Furthermore, we do not have an explanation as to
Results 335
total WBC, lymphoc., CD4+ and Ig+ c ells
average value s for TLJ+CyA+MAB grou ps
14
12
10
4 tola l WE C
-+- lymphocytes
2 __ 8 -*-
-~ B
CD4+ cells
Ig+ cells
0
pre TLI day 0 day 14 R
Fig. 20.1. Total white blood , lymphocyte, and CD4+ and IgG + cell counts (i) before TLI , (ii)
on the day of heart transplantation (day 0) , (iii) on post-transplant day 14, and (iv) at the
time of rejection (R)
why three recipients with long-term graft survival apparently did not produce
antidonor antibodies.
Histopathology
In rejected grafts, vascular changes were the predominant morphological find-

ing. In the five grafts functioning for >80 days chronic vascular changes were
seen, with endothelial cell proliferation, intimal thickening, and vessel obliter-
ation. In the remainder, acute vascular changes were seen with granulocyte in-
filtration , necrosis in vessel walls, and cardiac infarction with hemorrhage
(Fig. 20.2).
Myocardial infiltration with mononuclear cells was modest; morphologi-
cally the infiltrates consisted of smaUlymphocytes, but less than 5% could be
definitively characterized as T or B cells. This finding was unexpected, but
confirmed the results of Sakakibara et al. [18] in a rat-to-mouse heart trans-
plant model. The role of these ill-defined lymphocytes requires further clarifi-
cation.
Fig. 20.2. Histopathology at the time of rejection of a hamster heart transplanted into a
Sprague-Dawley rat. A central artery is surrounded by pronounced granulocyte infiltration
with hemorrhage in the vessel wall and marked perivascular edema
As a rule, no Ig or C3 deposits could be demonstrated by immunofluores-

cence microscopy, possibly because we used a rather insensitive one-layer
technique.
Histological evaluation of spleen and lymph nodes in TLI-plus-CsA-plus-
MAB-treated animals showed a sustained depletion of T cell areas and, in
some recipients, B cell clusters lined along splenic central arterioles. These ob-
servations need confirmation by further detailed examination but, tentatively.
References 337
these spleen B cells could be responsible for the antidonor antibody produc-
tion.
Sequential ultrastructural examination of rejected grafts showed an early
destruction of myofibrils with mitochondrial damage in myocytes adjacent to
capillaries. The changes were characterized by continuous progression 24 and
48 h after transplantation with increasing cell disintegration in larger areas
and, finally, total tissue necrosis.
In summary, our morphological data demonstrated a primarily humoral
type of rejection, possibly with delayed onset in recipients with long-term graft
function. Neither in recipients with short-term graft survival nor in those with
long-term graft function after TLI plus CsA plus MAB treatment was the
"classical" first-set histopathological allograft response observed, indicating
decisive qualitative and quantitative differences in the mechanism of graft re-
jection.
Conclusions
1. TLI plus CsA plus anti-Tcell MAB treatment improves graft survival of the
hamster-to-rat heart transplant.
2. The morphological and flow cytometric cross-match data suggest that TLI
plus CsA plus anti-T cell MAB therapy postpones a primarily humoral type
of rejection, and that this mechanism of rejection is qualitatively different
from a first-set allograft response.
3. Late rejection seems to correlate with an increase of antidonor antibodies,
while graft infiltration with mononuclear lymphocytes presumably is less
important.
4. Graft survival is independent of the duration of MAB therapy, indicating
that the effect of MABs in this model is an alteration of immunoregulatory
cell-to-cell interactions and not simply a question of cell depletion.
5. Anti-T cell MAB treatment would seem to be of value in concordant car-
diac xenotransplantation, but its optimal use requires a more precise under-
standing of the rejection mechanisms.
References
1. Bouwman, E., de Bruin, R.W.F., Marquet, RL., leekel, 1. Prolongation of graft survival
in hamster-to-rat xenografting. Transplant. Proc. 21 (1),540,1989.
2. Yamaguchi, Y., Halperin, E.C, Harland, RC, Wyble, C, Bollinger, RR A synergistic
effect of total lymphoid irradiation, cyclosporine, and splenectomy in a hamster-to-rat
hepatic xenograft model. Transplant. Proc. 21 (3),3558,1989.
3. Yamaguchi, Y., Halperin, E.C, Harland, RC, Wyble. C, Bollinger. RR Significant
prolongation of hamster liver transplant survival in Lewis rats by total lymphoid irradia-
tion, cyclosporine. and splenectomy. Transplantation. 49,13,1990.
4. Demasi, R., Alqaisi, M., Araneda, D., Nifong, W., Thomas, J., Cross, 0., Swanson, M.,
Thomas, F. Reevaluation of total lymphoid irradiation and cyclosporine therapy in the
Syrian hamster-to-Lewis rat cardiac xenograft model. Transplantation. 49, 63,1990.
5. Steinbruchel, D.A, Madsen, H.H.T., Nielsen, B., Larsen, S., Koch, e, Jensenius, J.e,
Hougesen, e, Kemp, E. Treatment with total lymphoid irradiation, cyclosporin A and a
monoclonal anti-T-cell antibody in a hamster-to-rat heart transplantation model: graft
survival and morphologic analysis. Transplant. lnt. 3,36, 1990.
6. Tufveson, G., Roos-Engstrand, E., Gaunedahl, e, Fellstrom, B., Larsson, E. Hetero-
topic cardiac xenograft transplantation from mouse to rat. Transplant. Proc. 22 (1), 139,
1990.
7. Knechtle, S.J., Halperin, E.e, Tahani Saad, B.S., Bollinger, R.R Prolonged heart
xenograft survival using combined total lymphoid irradiation and cyclosporine. J. Heart
Transplant. 5, 254, 1986.
8. Knechtle, S.J., Halperin, E.e, Bollinger, RR Xenograft survival in two species combi-
nations using total lymphoid irradiation and cyclosporine. Transplantation. 43, 173,
1987.
9. Steinbruchel, D.A, Madsen, H.H., Nielsen, B., Kemp, E., Larsen, S., Koch, e Graft sur-
vival in a hamster-to-rat heart transplantation model after treatment with total lymphoid
irradiation, cyclosporin A, and an anti-T-cell antibody. Transplant. Proc. 22, 1088, 1990.
11. Herbert, J., Roser, B. Strategies of monoclonal antibody therapy that induce permanent
tolerance of organ transplants. Transplantation. 46, 128 S, 1988.
12. Moses, RD., Pierson, RN., Winn, H.J., Auchincloss, H. Xenogeneic proliferation and
lymphokine production are dependent on CD4+ helper T cells and self antigen-present-
ing cells in the mouse. J. Exp. Med.I72, 567, 1990.
13. Pierson, RN., Winn, H.J., Russel, P.S., Auchincloss, H. Xenogeneic skin graft rejection
is especially dependent on CD4+ Tcells. J. Exp. Med. 170,991,1989.
14. Dallman, M.J., Thomas, M.L., Green, J.R MRC OX-19: a monoclonal antibody that la-
bels rat T-lymphocytes and augments in vitro proliferative responses. Eur. J. Immunol.
14,260,1984.
15. Jefferies, W.A, Green, J.R, Williams, AF. Authentic T helper CD4 (W3/25) antigen on
rat peritoneal macrophages. J. Exp. Med. 162, 117, 1985.
16. Garovoy, M.R, Rheinschmidt, M.A, Bigos, M., Perkins, H., Colombe, B., Feduska, N.,
Salvatierra, O. Flow cytometry analysis: a high technology crossmatch technique facili-
tating transplantation. Transplant Proc. 15, 1939, 1985.
17. Farnsworth, A, Wotherspoon, J.S., Dorsch, S.E. Postirradiation recovery of lymphoid
cells in the rat. Transplantation. 46, 418,1988.
18. Sakakibara, N., Click, RE., Sakakibara, K., Aziz, S., Jamieson, S.W., Wick, M.R
Unconventional lymphocytes involved in rejection of xenogeneic heart grafts. Lab
Invest. 62, 481, 1990.
Chapter 21

Closely Related Nonprimate Species
C. HAMMER
Introduction
For almost a century it has been man's dream to replace diseased organs in
end-stage patients by using xenogeneic grafts. Systematic immunological, bio-
chemical, and physiological studies in this context have demonstrated that
evolutionary and zoological factors would limit both the supply of suitable or-
gans and the success rate following transplantation. Organs appropriate in size
and availability are a prerequisite for the successful outcome of clinical xeno-
geneic organ transplantation. However, not many animal species exist world-
wide where the numbers are abundant enough to serve as general organ
donors. Only farm animals like the pig, sheep, goat, and small equine and
bovine breeds would be readily available.
Intensive studies have classified transplantation between different species
as either "discordant" or "concordant" [1-3]. Relative to man, almost all
species are discordant; their organs, if grafted into man, would be irreversibly
destroyed within minutes. Heavy immunosuppressive therapy and other major
immunomodulatory procedures may, under certain conditions, modify the re-
sponse to an organ from a discordant species [4,5]. Transplants suitable forman
that are classified as concordant are mainly derived from primate species [6].
The Canine Family
In the early 1960s, highly informative clinical studies were undertaken involv-
ing transplantation into man of organs taken from monkeys or apes [7, 8]. The
scarcity of these endangered species has forced us to search for similar animal
combinations in other zoological orders (i.e., families with suitable phyloge-
netic relatives) to investigate immunological mechanisms in closely related
species. For experimental purposes in Europe, it has been the family of ca-
nines - dogs, wolves, dingos, and foxes - that have been used to tackle this
problem. Canids are ideal in terms of surgical procedures. A large number of
species with equivalent body size exist and, like primates and rodents, they
have been investigated for their genetic markers.
Using routine tests, no preformed natural antibodies (PNAB) are found
within members of this zoological family, and the rejection mechanism is basi-
340 Experimental Xcnotransplantation Between Closely Related Nonprimate Species
cally of cellular (acute) type. Hormones. enzymes. interleukins. and other pro-
teins of one member of the canid family have structures compatible enough to
interact with the appropriate receptor of the other members of the family.
Membranes and their surface antigens are so closely related that the same
methods can be used for their detection throughout the species.
Other large animals of the order of ungulates have been used for other ex-
perimenta studies [9-13]. More often. however. rodents serve for experimen-
tal work [14]. It is a general principle. though there are exceptions. that such
closely related species of one family of the same zoological order reject grafts
in a fashion comparable to the allogeneic mcchanism. Without any immuno-
suppressive therapy. survival times excecd 48 h and function is maintained
over days or weeks. With suitable pharmacologic immunosuppression. func-
tion may be maintained for several months.
Immunogenetic Markers in Canines
An investigation was made of several immunogenetic markers and physiolog-

ical parameters relating to transplantation in canines. Included in the studies
were the following species: beagle. mongrel dog. dingo. wolf. and fox (Table
21.1 ).
Dog Leukocyte Antigens
The major histocompatibility complex (MHC) of canines is similar to that de-

scribed in other mammalian species [15]. Currently about 14 dog leukocyte
antigens (DLA) have been identified. Histocompatibility testing was carried
out using the microcytotoxicity assay according to Kissmeyer-Nielsen and van
Rood [16]. The test sera used were defined at the Canine Histocompatibility
Workshop [17]. Xenogeneic test sera were prepared in a similar fashion by us-
ing lymphocytes from the different species or skin transplants for immuniza-
tion. The xenogeneic sera were absorbed against the appropriate red blood
cells (RBC) in order to eliminate the common xenogeneic characteristics.
Table 21.1. Species of the zoological order of carnivores used for xenogeneic transplanta-
tion
Canines Felines
Domestic dog (Canis/amiliaris) Domestic cats (Felisfelis)

Wolf (Canis lupus) Tiger (Felis tigris)
Dingo (Canis dingu) Lion (Felis leo)
Red fox (Vlllpes vlllpes)
Immunogenetic Markers in Canines 341
Table 21.2. Survival time of donor dingo, wolf, and fox kidneys transplanted into recipient
beagles and mongrel dogs in relation to canine erythrocyte antigen and dog leukocyte anti-
gen specificities
Recipient Donor Survival time

of kidneys
CEA DLA CEA DLA (days)
Beagle Dingo
KDB 2 3,5,7,8 2- 9/6-12 2,6 3-8/bl-l1 10
DKB 3 8 3- 9/6-12 2,6 1-8/12-14 12
DKB 4 1,2,8 3- 9/6-12 2,6 1-8/12-14 9
KDB 9 1, 4, 8 3- 9/6-12 2,6 3-8/bl-bl 12
KDB 11 1,3,7,8 3-10/6-12 2,6 3-81 5-11 9
KDB12 1,3,7,8 3- 8/5-13 2,6 3-81 5-11 17
Mongrel dog Wolf
KWH 1 ND 3-101 5-13 1,4,8 7-10111-14 31
KWH 2 ND 3- 71 6-11 1,4,8 1- 3/11-13 22
KWH 3 ND 2- 01 4-11+6 ND 1- 3111-13 13
KWH 4 ND 2- 01 4-13+5 ND 1- 3/12- 0 21
KWH 5 ND 3- 71 6-12 ND 1- 3/13- 0 32
KWH 6 ND 3- 7/12- 5+ 13 ND 3- 7113- 0 20
KWH 7 ND 2- 31 4- 5 ND 2- 01 5- 0 10
KWH 8 ND 2- 01 5-12 ND 3- 71 5-12 11
KWH 9 ND 2- 71 6- 5-13 ND 2- 0/12- 0 12
KWHI0 ND 2- 8/12-13 ND 3- 81 6-12 14
KWH 11 ND 2- 8/12-13 ND 3- 81 6-12 13
KWH 12 ND 2- 81 0-13 ND 2- 31 6-12 14
KWH 13 ND 2- 8/12-13 ND 2- 31 6-12 13
KWH 14 ND 1- 01 0-12 ND 3- 7/13- 0 12
Beagle Fox
KFHB2 3,7,8 bl-bllbl-14 3,4,7 bl-bI/5-bl 6
KFHB3 8 3-10/12-14 8 bl- 7/bl-bl 6
KFHB5 1,8 2- 71 5-11 7,8 bl-bllbl-bl 8
KFHB6 1,8 3- 91 6-12 3,7,8 bl-bllbl-bl 5
KFHB7 3,7,8 3- 91 6-12 7,8 bl- 81 5-bl 9
CEA, Canine erythrocyte antigens; DLA, Dog leukocyte antigens; ND, Not done; bl, No
antigen dctected
Dog alloantisera were able to identify 14 DLA specificities with a repro-

ducibility of 90% (Table 21.2). Today 12 alleles of DLA Class I and 9 of Class
II are recognized. In beagles, the results were clear-cut, while mongrels, din-
gos, and wolves showed frequently incomplete haplotypes (blanks). Dogs ex-
pressed all 14 specificities at different frequencies. In 16 dingos, only a limited
spectrum of DLA antigens was found indicating inbreeding. In 14 wolves,
almost all alleles could be detected.
For typing of foxes (n=50), only sera with a broad specificity were success-
ful. A complete haplotype could not be determined. Only fox leukocyte anti-
gens (FLA) 5, 7, and 8 were found. Antifox sera from dogs developed two pos-
itive reactions against DLA 7 and 8 cells. All sera showed a strong toxicity
342 Experimental Xenotransplantation Between Closely Related Nonprimate Species
against fox RBC, which were able to differentiate fox erythrocytes.

Absorption with fox spleen and liver eliminated the xenospecificity. The typ-
ing results had no relevance to or correlation with the survival time of organ
transplants (Table 21.2).
Mixed Lymphocyte Reactions
Mixed lymphocyte cultures between the various canine species were per-
formed as described [18]. Pooled dog serum was used. Mixed lymphocyte re-
actions (MLR) using wolf and fox lymphocytes as stimulator or responder
cells were positive in all cases. Mongrel lymphocytes reacted significantly
more strongly against wolf cells than against dog cells. Fox cells showed a low-
er response and stimulatory capacity than dog cells. Beagle lymphocytes
showed the lowest H3-thymidine uptake (Table 21.3).
Canine Erythrocyte Antigens
Dog RBC express eight different erythrocyte antigens which are not able to in-
duce isoagglutinins. To differentiate the canine erythrocyte antigens (CEA),
12 sera were used. All eight CEA could be detected in all four species. In mon-
Table 21.3. Results from mixed lymphocyte cultures using lymphocytes from different
canine species a
Dog A DogB Doge DogD Dog E

(m) (m) (m) (m) (m)
Dog 3-12/9-4.610 18 26 CD CD 18
WolD-I 111-13 85 124 74 39 64
Fox r 41 50 48 31 18
Fox II 13 11 12 19 5
Dog4 Wolf Dingo I Dingo II Fox I Fox II

(m) (m) (m) (m) (m) (m)
3-12/3-12 3-4/1-13 3-8/8-14 3-bl/8-bl
Dog I 1-13/1-13 56 99 28 25 37 20
Wolf3-111l-13 41 @ 59 45 72 75
Fox I 43 85 42 24 @ 12
Fox II 7 41 5 nd 6 CD
(m). mitomycin-treated (stimulator cells): bl. no antigen detected

a Ratios of stimulation of allogeneic and xenogeneic mixed lymphocyte reactions are indi-
cated. Mixed lymphocyte cultures with dog leukocyte antigen-compatible dogs and/or
autologous combinations showed no response (shown in circles). Xenogeneic mixed lym-
phocyte reactions induced high stimulation rates.
b Refers to dog leukocyte antigens.
grels and beagles, all eight known specificities were present. Using dingo
RBC, only CEA 2,6,7, and 8 were found [19].
Isoenzymes
Patterns of isoenzymes were semiquantitatively distinguished according to

their electrophoretic mobility. Adenosine deaminase, adenylate kinase, phos-
phoglucomutase, and G-phosphogluconate dehydrogenase were investigat-
ed. The questions as to what extent enzymes and enzyme function vary be-
tween different xenogeneic systems and how important this is for satisfactory
graft function remain open. While the substrate specificity seems to be
species-unspecific, the electrophoretic mobility (i.e., amino acid sequence)
shows significant variations within the canine family and domestic cat.
Adenyl kinase and glutamate phosphatase show no differences between
the carnivores. G-phosphogluconate dehydrogenase of dog, dingo and wolf
are identical, but differ in three bands from the fox. Phosphoglucomutase is
different in wolves when compared with dog and dingo.
Serum Proteins
Variations in electrophoretic mobility of serum albumin, transferrin and other

serum proteins were found. The serum electrophoresis showed no differences
in the canine species tested. Immuno- and 2-dimensional electrophoresis us-
ing anti dog and antifox sera showed distinct differences between foxes and
dogs, especially in regard to albumin and gamma globulin. These proteins are,
therefore, easy to distinguish (Fig. 21.1).
Chromosomal Analysis
Chromosome number and karyotype were established from peripherallym-

phoblasts for all species used. Karyotypes of dogs [20], dingos [21], and wolves
[22] do not differ from each other [23]. The typical number of chromosomes
for dogs is 78. The number in foxes varied from 34 to 40, with 36 seeming to be
the most common [24]. The fox karyotype is characterized by 18 chromosomes
with 5 microsomes in the male and only 4 in the female; this fact might be re-
sponsible for the different counts.
Summarizing the results concerning immunogenetic canine markers, the
high grade of identity between dog, dingo, and wolf becomes obvious. All
species hybridize and therefore can be called closely related or even
semixenogeneic. According to the zoological system the fox still belongs to the
canines. This species, however, shows variations in the number of chromo-
somes and karyotype, serum proteins, and enzyme patterns. Phylogenetically,
the fox's close relationship to other canids is proven by the sharing of histo-
compatibility and erythrocyte antigens.
344 Experimental Xenotra nsplantation Between Closely Related Nonprimate Species
Fig. 21.1. Two dimension al electrophoresis of dog

(top) , wolf (center) and fox (bol/om) sera, tested
against rabbit antidog a ntibodies
Using these markers, the classification of xenogeneic transplant combina-

tions in closely related systems can be refined (Table 2].4). These methods al-
low prognosis concerning the type of rejection that will occur and the survival
time after organ transplantation between members of this order, as well as se-
lection regarding optimal immunosuppression.
PNAB are present in all of the canine species that were tested. The mean
titers against members of other zoological orders were high, yet lower than in
primates where isoagglutinins interfere with the other PNAB. Within the zoo-
logical order of carnivores the PNAB titers were low. Individuals of one ca-
nine species contained no PN AB against other mem bers of the same species,
i.e., members of the same family. Preformed hemagglutinins directed against
feline antigens reached log titers of 3.2±O.8. A schematic diagram of survival
time and titers of PN AB is given in Fig. 21.2.
Table 21.4. Systematic summ ary of immunogenetic ma rkers and the ir importance for th e
prognosis of type and mechanism of rejection, survival tim e, and optimal immunosuppres-
sion in systems of different zoological relationship
Zoological relationship
Extra order Intra order

Ungulates Felines Canines
Pig/sheep Tiger, lion , cat Fox, wolf, dingo, dog
Rej ection type Hyperacute Delayed Acute

Rej ection mechanism Humoral Humoral/cellular Cellular
Survival time Minutes Hours Days
Effect of immunosuppression None Slight Good
Identity with dog
Serum protein (+) (+) (+) + +++ +++ +++
D og leukocyte A antigen + ++ ++ +++
Canine Erythrocyte antigens + ++ ++ +++
Red blood cell isoenzymes (+) + ++ +++
Chromosomes/karyotype ++ ++ +++
Prefo rm ed nat ural antibodies +++ ++ ++ ++ (+)
PNAB
6
..
CII)
'l:
12
Q.
CII)
,..
<II
c {l 8 4
~,..
(1
II
c
'0
~ 2
'0
~
>
VI
"'0)0>(-
Fig. 21.2. Schematic diagram of "§ 0 0 ) ...0 ,,,!
survival time (SVT) of various
'" b:§ -
kidney xenografts transplanted family canides
into dogs in relation to th e titer of
family cats
preformed natural antibodies
(Pnab) agai nst the do nor species order ungulates
present in dog serum order carnivores
Preparation ofImmune Sera
Antidog Antisera
Antidog, -wolf, -fox and -cat antisera were produced by immunizing rabbits
with 1.0 ml serum of each species. Complete Freund's adjuvant (CFA) was
added at the first application. After 2 and 4 weeks only antigen was given intra-
muscularly. After 6 weeks the animals were bled and the sera pooled and used
in aliquots.
Sera from individual dogs that had survived skin or kidney transplantation
for a long period of time and had developed antibodies against the donors
species were also collected.
Antidog Antilymphocyte Globnlin
Thoracic duct lymphocytes were used for immunization in a horse.

Lymphocytes (2x109 ) were injected intramuscularly at 2-week intervals. No
adjuvant was used. Six days after the last booster injection, the sera collected
were absorbed against washed dog RBC to a hemagglutinin titer of less than
1:2. Gamma globulins were separated with ammonium sulfate. The final titer
was 1:4096. The sterile compound was stored in aliquots at 4°C [23].
Antidog Antimacrophage Serum
Young dogs with a body weight of 10-15 kg received intraperitoneal injections

of 5% proteose-peptone. Ninety-two hours later the animals were sacrificed
under anesthesia. The peritoneal exudate collected contained 50%-70%
macrophages and 10%-30% lymphocytes. Granulocytes and mast cells com-
prised 3%-5%.
The monocytes were purified by adhesion to plastic surfaces. A first injec-
tion of 15x109 cells plus CF A was followed by further injections of 5x109 cells
biweekly over 11 weeks (i.m. and i.v.). The horse was bled at 4-week intervals.
The pooled serum was heat-inactivated and absorbed against washed dog
RBC (x2) and against spleen lymphocytes (xl). After sterile filtration the anti-
macrophage serum (AMS) had an anti macrophage titer of 1:64 and an anti-
lymphocyte titer of 1:4, but no hemagglutinins or precipitating antibodies
against serum proteins. Titers of 1:16 of complement fixing antibodies against
liver and kidney antigens were detectable [24-26].
Transplantation 347
Transplantation
Donor Cat Kidney Transplantation in Dogs
Controls (First Set Transplantation)

In order to study the survival time of transplants between closely related
species of two zoological families within one zoological order, cat kidneys
were grafted into small mongrel dogs using the en bloc technique [27].
After opening of the vascular anastomosis, all grafts showed a homogenous
hemoperfusion, physiological color and turgor. Urine production was ob-
served in all nine cases. The function time as judged by urine output was
15-24 h. The beginning of rejection was indicated by a decrease of serum com-
plement activity during the first 6 h. At the same time, cat protein and potassi-
um were found in considerable amounts in the urine samples. Urine osmolari-
ty decreased from 358±6 mosmol/l during the first 12 h to 289±2 mosmol/l after
24 h. Urine sodium dropped from 132±7 mmol/l to 125±8.8 mmol/l, and potas-
sium from 15.2±2.4 mmol/l to 5.2±0.5 mmol/l, indicating the loss of concentrat-
ing capacity after about 12 h.
Arteriography of the grafts revealed severe reduction in the caliber of all
vessels especially the interlobular arteries. The cortex was clearly demarcated
from the medulla. Recipients which survived showed a significant increase of
anticat antibodies with a peak of 1:16 after 12 days. The titer started to drop
again after 21 days.
Histology showed slight congestion of veins and intertubular capillaries. In
the glomeruli there was a moderate increase of intracapillary granulocytes
with platelet aggregates. Tubular cells showed a marked vacuolization and
pyknotic nuclei. A systematic semiquantitative evaluation of the histopatho-
logical features is shown in Fig. 21.3.
Second Set Transplantation

Five dogs received a second cat kidney 3 weeks after rejection of the first graft,
at a time when an increase in antibody titers against donor tissue had been
proven in the serum of all the recipients. In this second set group cessation of
renal blood flow (RBF) occurred within 17.4±2 min. Urine production never
started. The transplants became cyanotic after 10 min. The arteriovenous dif-
ference in the hemagglutinins decreased. Complement activity dropped to
values of 50% after 5 min of perfusion to 70% at the end of the experiment.
Thrombocyte and leukocyte counts (expressed in percentage of the arteriove-
nous difference) showed a similar reduction during the period of perfusion.
The microscopical picture of the kidneys, rejected within 15-20 min, showed
marked endothelial lesions with pyknotic nuclei and many occluding platelet
thrombi in arterioles and glomerular capillaries (Fig. 21.3).
PERFUSION
TRANSPLANT ATION PERFUSION "SECOND SET"
SURVIVAL IPERFUSION TIME 2h - 3d (x=1.3d ) 12' - 30' (x=18.2')
VESSELS 7/8
Thrombocyte
Aggregates
III
II
I
.
:::::::::::::::::::::::::.~.;.;.;.;.;.617 ::.:::.:::.:::.:::.:::.:::.:::.:::.:::.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.;•••;5;.~.;5
••• •••.
Granulocyte Increase III

II
intravasal I
Granulocyte Adherence III
II
to Endothelium I
III 3/8
Endothelial Necrosis :::::~••..•.•...
II
I
GLOMERULI
III 5/8
Thrombocyte II
Aggregates I
III
Granulocyte Increase II
I @i~:::::~·.·.·.·.·.·.-.·.
III
Endothelial Necrosis II
I
III
Mesangiolysis II
I
TUBULI 3/7
III
Necrosis II
I
Fig. 21.3. Semiquantitative summary of histopathological findings in the three groups of cat-
to-dog kidney xcnotransplantations or hemoperfusions. Degree of histopathological sevcri-
ty is expressed on a scale of I-III, with I being mild and III being severc. Dotted areas indi-
catc the number of grafts showing such histopathological changcs. Transplanted kidneys
functioned for betwccn 2 hand 3 days with a mean of 1.3 days. Perfused kidneys function cd
for 8-15 h with a mean of 10.7 h. Perfused kidneys in sensitized hosts ("sccond set") func-
tioned for only 12-30 min with a mean of 18.2 min
Ex Vivo Perfusion of eat Kidneys Using Dog Blood

In order to study the function of xenohemoperfused cat kidneys, RBF, arte-
riovenous oxygen difference (A VD-02)' oxygen consumption, urine produc-
tion as well as enzyme and electrolyte release in urine and serum were moni-
tored. Starting at 92±15 m1l100 g kidney per minute, the RBF reached peak
values of 170 ml after approximately 2 h. The A VD-0 2 , oxygen consumption,
and RBF changed significantly during the period of perfusion [28].
The typical increase in cell enzymes and potassium in the venous effluent
was not observed in this system. Serum osmolarity remained unchanged.
Urine osmolarity proved the kidney to have the capacity to concentrate during
the first 2 h. Later, urine osmolarity only slightly exceeded the serum values.
Coincidentally, massive proteinuria was measured, increasing during the
hours of perfusion. The urine/plasma potassium ratio varied only insignifi-
cantly. The titer of preformed anticat tissue antibodies decreased during
hemoperfusion. In contrast, titers of xenohemagglutinating antibodies (X-
Haggl) against pig RBC and rat RBC remained unchanged during rejection
(Table 21.5), indicating a species-specific absorption. Like the X-Haggl, the
Transplantation 349
Table 21.5. Titers of preformed xenohemagglutinating antibodies (X-Haggl) and xenocom-
plement-fixing antibodies (X-CFAB) in the venous effluent during ex vivo perfusion of cat
kidneys using dog blood"
Time X-Haggl X-CFAB X-CFAB X-Haggl X-Haggl

(min) CatRBC Cat kidney Cat liver PigRBC RatRBC
0 2.6±O.6 4.5±0.6 3.0±1.0 4.7±1.0 8.5±O.3

1 2.0±0.5 3.7±0.8 2.5±1.2 4A±1.1 8.5±O.3
5 2.6±OA 3.7±O.8 2.5±O.8 4A±1.1 8.5±O.3
60 2.2±OA 3.2±0.3 2.5±O.9 3.5±0.6 8.8±OA
300 1.0±0A 3.7±0.5 2.2±O.9 4.3±0.6 8.3±OA
600 1.0±0A 3.0±0.5 2.2±O.9 4.3±0.7 7.8±O.6
RBe, red blood cells; SEM, standard error of the mean

" Titers in -2 log; mean values ± SEM.
total complement activity in the venous efflux decreased during the first 5 h of
perfusion. This consumption of total complement activity correlated with the
arteriovenous difference in complement and the loss of C3 during that time.
Comparable to the loss of complement, thrombocytes were retained in the cat
kidneys throughout the period of hemoperfusion though with considerably
more during the first 30 min and again at the very end of the experiment. A
similar, but less severe, decrease was true for leukocytes [28].
On histological examination (Fig. 21.3) the perfused cat kidneys showed
perivascular and interstitial lymphocytic infiltration with increased numbers
of granulocytes in the glomerular arteries and arterioles together with platelet
aggregates. The endothelium showed minimal desquamation of cells with
pyknotic nucleoli. The tubular cells were vacuolized and dilatation of the
tubules was markedly increased proximally. Several protein casts were found
in the tubular lumena. Slight to massive hyperemia and interstitial edema
characterized the medulla of the kidneys.
Effect of Treatment with Antilymphocyte Globulin

Five dogs were prophylactically treated with antidog antilymphocyte globulin
(ALG) (20 mg/kg) starting on day-7. In contrast to untreated animals, the
RBF reached rather high values (120 mlllOO g kidney per minute) within
15 min and almost physiological values (236 mll100 g kidney per minute) after
120 min. No change in hemoperfusion of the anatomical compartments was
seen for 6 h. Typical rejection was not seen within 9 h of perfusion. Oxygen
consumption changed insignificantly. Urine production reached maximal val-
ues (0.26 mll100 g per minute) after 2 h. Protein excretion was low for 2 h
(0.3%). Later, the histuria reached values similar to the control group.
Effect of Preoperative Serum Transfusion

Thirty minutes before hemoperfusion of cat kidneys five dogs received infu-
sions of either species-specific (anticat) or species-unspecific (antipig) im-
mune sera. No differences in oxygen consumption or urine production were
observed. A slight prolongation of the survival time could be attributed to
hemodilution.
Donor Fox Kidney Transplantation in Dogs (Table 21.6)
Controls
The mean survival time (MST) of 6.5± 12 days of eight en bloc fox kidney trans-
plants suggested that the rejection mechanism was of cellular type, possibly
with humoral participation during the final stages of the event. Therefore,
pharmacologic immunosuppressive therapy was effective in prolonging graft
function [23,26,29,30].
Untreated dogs rejected fox kidneys during a narrow time interval of 5-8
days. Intraoperative failures were seen but could not be proven as hyperacute
rejection. Usually the post-transplant course of kidney function was reflected
by creatinine and urea values, which started to increase after 2 days. The
urine/plasma osmolarity ratio steadily decreased after 3 days, indicating dete-
rioration of kidney graft function (Fig. 21.4). Sequential scintigraphs and
reno grams showed postoperative recovery of renal function on day 1 after
transplantation. On day 4 an accumulation pattern became apparent in the
renogram signaling the start of rejection. Fine needle aspirates clearly con-
firmed these observations [31]. The infiltrate consisted predominantly of lym-
Table 21.6. Survival time of donor fox kidneys and hearts in untreated and immunosup-
pressed recipient dogs
Organ Immunosuppressive therapy n Survival time (days)
Mean SD
Kidney Control 8 6.5 1.2

Xenogeneic blood transfusion 6 5.6 0.9
Allogeneic blood transfusion 6 6.3 2.1
ALG 9 13.4 1.0
AMS 5 15.8 0.7
ALG+AMS 7 17.7 0.9
CSA+MP 7 10.9 3.0
15-Deoxyspergualin 6 13.0 3.4
Heart Control 6 8.4 1.9
CSA+MP 6 20.2 4.1
ALG, anti-lymphocyte globulin; AMS, anti-macrophage serum; CSA, cyclosporine; MP,

methylprednisolone; SD, standard deviation
Transplantation 351
CREATININE VALUES OF DOGS
mg% AFTER TRANSPLANTATION OF
12 . - . FOX KIDNEYS n = 7
x-x DINGO· n = 12
. - . WOLF' n = 12
6 x
Fig. 21.4. Post-transplanta-

tion serum creatinine values
(in mg%) in dogs that re-
2 4 6 8 10 12 14 16
ceived xenogeneic (fox, din-
go, or wolf) kidneys DAYS
phocytes and monocytes. With the progress of rejection, gamma globulin-pos-

itive lymphocytes appeared.
Treatment with ALG/AMS

In an attempt to modify this type of rejection, recipient beagles were pretreat-
ed with antidog ALG or AMS or a combination of both. To suppress the pro-
duction of antibodies against horse ALG-immunoglobulin G (IgG), immuno-
logical unresponsiveness was induced in the recipients by IgG pretreatment.
The MST of fox kidneys under ALG therapy (20 mg/kg) was 13.4±1.0 days.
AMS (0.3 ml/kg), if given daily, prolonged the survival of fox kidneys in dogs
to 15.8±0.7 days. The combination of both regimens showed only slight further
improvement, but no additive effect (MST, 17.7±0.9 days). In the group of
dogs pretreated with IgG and then given ALG therapy, the survival time was
nearly doubled (MST, 29.5±5.6). The longest survival of a fox kidney graft was
65 days [23,29] (Fig. 21.5).
352 Expcrimental Xenotransplantation Betwecn Closely Related Nonprimate Specics
mg%
SERUM-UREA
Fig. 21.5. Post-transplantation serum crea-

tinine and urea values (in mg%) in dogs
that received fox kidney transplants and
either no therapy (control) or therapy with
antilymphocyte globulin (A LG) or anti-
4 6 8 10 12 14 16 DAYS macrophage serum (AMS)
CycIosporine Therapy
Survival time offox kidneys in eight dogs receiving cyclosporine (9 mg/kg p.o.)
was 8.9±2.5 days. In combination with methylprednisolone (4 mg/kg tapered
by 1 mg every 3 days) the survival time was 1O.9±3.0 days [31]. Creatinine val-
ues (initially <4 mg/dl) started to increase rapidly after 4 days to reach maxi-
mum values after7 days (> 12 mg/dl). At that time cell infiltration of the kidney
graft (seen in fine needle aspiration biopsy) reached maximal levels. When
compared with fox kidneys grafted in untreated host dogs, no lymphoblasts,
plasma cells or macrophages could be detected. Prednisolone therapy had no
additional impact on this mechanism. It appeared that humoral factors caused
the final deterioration.
15-Deoxyspergualin Therapy
Mean graft survival time was 13.0±3.4 days. Serum creatinine remained low
«8.0 mg/dl) as compared with all other groups, especially the cyclosporine-
treated group. Findings of fine needle aspiration biopsy and histology re-
vealed no significant differences either, though signs of humoral rejection
were modified by slight infiltrates [32].
Xenogeneic and Allogeneic Blood Transfusion

Pretreatment of the recipient dogs with 1.8xl09 washed fox RBC on day-
7 shortened the survival time to 5.6±0.9 days [33]. All animals produced an-
tifox antibodies (hemagglutinin titers 3.0±O.6, Iymphotoxin titers O.6±O.6).
Transplantation 353
Histology of the short-lived grafts showed signs of ischemia due to reduced

hemoperfusion. It appeared that humoral mechanisms were prevailing.
Allogeneic blood transfusions of 6x200 ml of heparinized dog blood start-
ing on day -35 had no influence on the mean graft survival time in ten dogs
(6.3±2.1 days). However, six kidneys showed survival times of 8-12 days, and
four kidneys survived 3-4 days. Production of antibodies was not influenced
by prior allogeneic blood transfusion. DLA typing of donor and recipient as
well as of blood donors had no influence on the outcome. Histology was com-
parable with the xeno blood transfusion group.
Donor Fox Heart Transplantation in Dogs
Xenogeneic fox hearts were transplanted heterotopically into dogs either to

the neck or intrathoracically. Without immunosuppression the survival time
was 8.4±1.9 days (n=6). Cyclosporine (8 mg/kg per day) and prednisolone (1.0
mg/kg per day reducing to 0.25 mg/kg per day) increased graft survival to
20.2±4.1 days (n=6). In contrast to fox kidney transplants, lymphoprolifera-
tion, blastogenesis, and changes in the peripheral blood as shown by the cy-
toimmunological monitoring were found in these heart-transplanted dogs. An
increase in blood monocytes appeared to be related to the development of in-
fection. Signs of humoral rejection were not seen [34-36].
Donor Fox Skin Transplantation in Dogs
Mongrel dogs (average body weight 8.5±1.2 kg) were used for skin transplan-
tation. One fox was selected as the donor for all skin grafts. Skin pieces (2x3 cm
in size) were transplanted on to the lateral thoracic wall under sterile condi-
tions. The grafts were fixed at the four edges with atraumatic sutures, and the
rest of the skin was attached by tissue adhesive. Wire mesh was fixed by suture
and plaster at the edges, leaving a window through which visual monitoring
was possible. Rejection was defined as 50% graft destruction.
In untreated dogs, fox skin grafts were rejected within 5.9±1.4 days. The
survival time ranged from 4 to 9 days. Rejection of fox skin was characterized
by edema, hemorrhage, and induration. The interval between the first signs of
rejection and 50% necrosis of the graft was 1-2 days. Correlation between sur-
vival time and DLA on dog and fox lymphocytes could not be established. The
cytotoxic reaction of DLA antisera with fox lymphocytes was weak [29].
Donor Dingo Kidney Transplantation in Beagles
Dingos differ in many ways from domestic dogs. Fragments of dingo skeletons
exist which are believed to be as old as 8500 years. The whole skeleton of a
male dingo 3000 years old exists at the University of Sydney in Australia. This
relic proves that the dingo morphological pattern has remained unchanged
over 3000 years. Immunogenetic markers verify these observations.
Dingo kidneys transplanted to beagles were rejected after 11.5±3.0 days,

with a range of 7-18 days. Clearance values proved renal function continued
until day 10 in most animals. The rejection was purely cellular. Preformed nat-
ural antibodies (hemagglutinins and complement fixing antibodies) against
dingo tissue did not exist and complement activity remained unchanged. In
most histological specimens glomeruli and capillaries were hyperemic with
fresh local necrosis and fibrin deposits. At the border of cortex and medulla in-
filtrates of mononuclear cells, plasma cells, and monocytes were frequent.
Donor Dingo Skin Transplantation in Beagles
Dingo skin on beagles (n=9) was rejected at 10.6±2.9 days after transplanta-
tion, which is identical to dog allogeneic graft survival time. Examination of
skin biopsies revealed that the ingrowth of capillaries ended at day 6.
Mononuclear cell infiltrates increased until day 6. After 8 days, blood flow de-
creased with dilatation of the vessels. Hemorrhages and necrosis began to
develop. At day 10 rejection was complete.
Donor Wolf Kidney Transplantation in Dogs
Survival times of 14 wolf kidneys ranged between 11 and 32 days in dogs with-
out surgical or other accidental failure. The mean survival time of 12 wolf kid-
neys was 19.4±2.8 days and was significantly longer (p<0.0002) than the sur-
vival time of allogeneic dog kidneys (11.5±3.0 days) that served as controls [37]
(Table 21.7).
Treatment of the recipient with ALG did prolong survival time in four ani-
mals to 24, 27, 34, and 35 days, respectively, with a mean of 30.4±2.7 days.
Although survival time could be prolonged, the effect of ALG was limited and
prolongation of function not statistically significant since only two of four kid-
neys functioned longer than kidney grafts in untreated animals.
Cyclosporine-treated xenografts survived 40, 45, 57, and 90 days, with a
mean of 58.0±22.5 days. These function times were longer than those previ-
Table 21.7. Survival time of donor wolf kidneys and skin in recipient mongrel dogs
Organ Immuno- Survival time (days)

suppressive
therapy Individual times Mean SD
Kidney Control l1,12,13,13,14,20,22,23,2IUL32 19.9 2.4

ALG 24,27,34,35 30.4 2,7
CSA 40,45,57,90 58.0 22.5
Skin Control 7,7.8.9,10.12,16,18,33 13.3 2.9

ALG 22,28,34,38,64.89.96,228 85.6 20.6
ALG, anti-lymphocyte globulin: CSA, cyc1osporine: SD, standard deviation

Transplantation 355
ously observed in either ALG-treated (30.4±2.7 days) or nontreated (19.4±2.4
days) dogs receiving wolf kidneys (Table 21.2) [38]. In all animals tested, no
matter if immunosuppressed or not, blood urea nitrogen (BUN) and creati-
nine increased in correlation with the cellular infiltrate. Inflammation was
comparable to that seen in allografts. Only in one histological case was a hu-
moral component observed. Expression of MHC antigens on the surface of
tubular cells was seen at the time of rejection.
Donor Dog Kidney Transplants in Wolves
The striking difference in survival time between wolf and allogeneic kidney
grafts could not be verified in the opposite direction. When seven dog kidneys
were transplanted into wolves the mean survival time was almost identical,
(11.4±2.7 days) to allogeneic grafts (11.5±3.0 days). The fact that postopera-
tive care in wolves was less intensive than in dogs can hardly be the reason for
this difference.
Donor Wolf Skin Transplantation in Dogs
Wolf skin grafts transplanted on to mongrel dogs survived 13.3±2.9 days with a
range between 7 and 33 days. This was not significantly longer than the sur-
vival time of allogeneic dog skin (8.6±2.4 days). Daily treatment with ALG re-
sulted in a prolongation of skin graft survival of up to 228 days, with a mean
survival time of 85.6±20.6 days. Around 40 days after transplantation, fur was
growing on the grafts [37]. ALG treatment therefore seemed to be more effec-
tive in prolonging skin grafts than kidney grafts (Fig. 21.6).
The histological picture of rejection of the skin grafts was of cellular type
and similar to that seen in allogeneic or dingo transplants. Since neither DLA-
[°/0 ]
100 t-~---,r-+.. .""n CONTROL SKIN n =4
I
~U~
+-+
-
SKIN .ALG
daily 20 mg/kg b.w.
CONTROL KIDNEY
n= 8
n=5
I
...... KIDNEY.ALG n=4
daily 20 mg/kg b.w.
60
I
~
I -'-'j
20
'1 • 1---1/1---
0~~~--~~~~--~ro~--~8~0----~~-----1~~---1~~·: ~
[DAYS ]
Fig. 21.6. Different effect of antilymphocyte globulin (ALG) therapy on wolf skin and kid-
ney graft survival in the dog
356 Experimental Xenotransplantation Between Closely Related N onprimate Species
specificity nor blood groups were identical and MLR was positive in all cases,
this long survival time cannot be explained from close genetic compatibility
[2,39].
Other Experimental Models
Other groups have transplanted different organs in closely related xenogeneic

experimental models. These will be briefly reviewed.
Lung Grafts in the Cat-to-Dog Model
The exact survival time of cat lungs transplanted en bloc into mongrel dogs
was not reported. However, intravital recording of the microcirculation
showed that RBC were immediately involved in dense aggregates. The aggre-
gates did not adhere to the cat capillaries during their 1-min passage through
the graft. With time, increasing amounts of RBC aggregates plugged the circu-
lation.
In addition, dog blood or plasma was infused into intact cats. The changes
were described as identical to those seen in the xenografted lung [40].
Cat kidneys were transplanted into dogs and treated with prostacyclin.
Without treatment RBF ceased within 25 min. No urine was produced.
Venous platelet counts remained stable. Histology showed typical signs of hu-
moral hyperacute rejection. Prostacyclin was able to postpone rejection as
long as it was infused. Discontinuation of prostacyclin infusion initiated an
identical rejection episode as described in the untreated model [41].
Xenotransplantation Between Ungulate Species
Despite the favorable experimental situation within the order of ungulates,

where many large species exist as domestic animals (e.g., sheep, goats, cattle,
and pigs) and many species are under domestication (e.g., deer and antelopes)
few attempts at xenotransplantation have been ventured. In an effort to find a
model for separation of humoral and cellular mechanisms, pig kidneys were
transplanted into goats. Untreated animals rejected the xenografts within
3 days and ALG-treated individuals after 11 days. In this system (as in the
combination cat-to-dog) preformed antibodies of a titer of 1:8 against pig
RBC were found. The antibodies were absorbed on to the xenografts.
The kidneys functioned and produced concentrated urine (>325
mosmollkg). The rejection was both cellular and humoraL and the histological
findings were comparable to those already described. Gamma globulin, bound
to the majority of the glomeruli, was assumed to be of pathognomonic signifi-
cance. Since ALG did eliminate mononuclear cells from the circulation and
Comment 357
prolong survival time, a cellular mechanism was presumed to be responsible

for the accelerated rejection seen in untreated animals.
Survival time of sheep kidneys in unsensitized goats was 11.2 days and al-
most identical to that of allografts (11.8 days). Sensitization reduced survival
time to 2.7 days compared with 2.9 days in allografts. Similar findings were re-
ported concerning skin transplantation where survival time of 7 days com-
pared with 9 days in allografts. Intensive studies in this interesting experimen-
tal model have not been carried out [42-45].
Cardiac and skin transplantation between calves and goats resulted in re-
spective survival times of 5 and 7 days. Antilymphocyte serum (ALS) was able
to prolong skin graft survival to between 28 and 79 days, but prolonged heart
grafts only to 14-30 days [45], comparable findings to those in the wolf-dog
model [37,39].
Orthotopic cardiac xenografting using donor lambs in the newborn goat re-
sulted in mean graft survival of 32 days (range 13-72 days) when treated with
cydosporine. Survival of the controls was only 6 days. The longest surviving
animal died after 165 days from anemia [13,46,47].
Transplantation has been carried out between hare and rabbit, both of the
order Lagomorpha. The two species are closely related as proven by immuno-
genetic markers. Kidney transplantation resulted in an MST of 5 days.
Splenectomy and cyclosporine had little beneficial effect. As described in the
fox-to-dog model, cyclosporine and steroids did not improve the survival time
of xenografts of this phylogenetic distance [41]. Cobra venom factor as an anti-
complement agent failed to delay the rejection. Presensitization led to hyper-
acute rejection [43].
Comment
The systematic evaluation of the presence of PN AB has resulted in the conclu-

sion that transplantation between species of two zoological families within one
zoological order leads to a rejection mechanism in which antibodies are not as
dominant as in widely divergent species. From the literature, little is known
about grafts of such a phylogenetic distance. In 1910, Unger [48] described the
first kidney transplantations between cat and dog and vice versa without giv-
ing details. A vramovici in 1924 [49] transplanted in the same system after hav-
ing exchanged serum between the species. After this conditioning the survival
time was described as reaching 11 days. In 1969, Owen [50], also using "pre-
treatment", achieved survival times of up to 25 days in the rabbit-to-guinea-
pig model. Rejection occurred in controls within 24 h. Looking for a model in
which humoral and cellular mechanisms would be mixed, Perper et al. [44,45]
transplanted between pig and sheep. Survival times of 3 days, and, using ALS,
up to 11 days, were reported.
Dramatically improved results were achieved in the order of primates using
chimpanzees and baboons as donors for clinical transplantation in man.
Extraordinary function times of between 1 and 9 months were described using

the maximal immunosuppressive therapy available at that time.
Pathophysiology of the Delayed Type of Hyperacute Rejection

in Closely Related Systems
Cat-to-Dog System
We used kidneys from three different species of cat -lion, tiger and domestic
cat - as donor organs for dogs. Clear differences in the anatomical and physio-
logical parameters exist between these three cat species and canines.
Immunogenetic markers show only few parameters in common with dogs.
Titers of PNAB in the dog are, however, low in all cases. Participation of
PNAB in the rejection mechanism is suggested by decreases in both comple-
ment activity and in the cellular components of the perfusing blood.
The antigen-antibody interaction, with activation of complement, induces
lesions on the endothelial cells of the graft vessels which involve platelets in
this event. After 30-60 min this primary humoral part of the rejection episode
is completed without totally destroying the graft (in contradistinction to that
seen in widely divergent species). Prophylactic treatment with highly efficient
antidog ALG had no influence on the rejection process.
The response to kidneys from lions and tigers was different from that seen
when organs from domestic cats were transplanted. This could be due to the
reduced hemoperfusion resulting from the greater size (240-400 g) of the
lion/tiger kidneys or from the different phylogenetic background. The initial
events of rejection were increased as compared with domestic cat kidneys. A
sharp decrease in antibodies and complement was associated with an extreme
loss ofthrombocytes and leukocytes from the perfusing blood.
After 6-8 h the rejection of kidneys from the big cats was completed. The
morphological findings correlated well with the RBF measurements.
Endothelial cell swelling and necrosis, and platelet and leukocyte adhesion,
preceded the changes that occur in oxygen consumption in the cortex and
medulla.
The intrarenal redistribution of RBF after about 3 h was a consequence of
secondary mechanisms, such as thrombocyte and leukocyte adherence and
was in the favor of the medullary compartment. It obviously led to cortical is-
chemia. Nephrons were then bypassed so that ultrafiltrate and proteinuria in-
creased abruptly. Ischemia of the tubular cells (as a consequence ofthrombo-
sis of the tubular capillaries) was responsible for the urine/plasma ratios of
osmolarity and of sodium of 1. This reaction was mainly of humoral character,
but was slow enough to allow secondary cellular mechanisms and mediators to
participate.
The accelerated second set reaction and the slight effect of ALG supported
this conclusion. Dogs used to hemoperfuse cat kidneys developed rather high-
er titers of anticat antibodies. The maximum was reached 2 weeks after finish-
ing perfusion. The very fast second set reaction in the presence of these high
Comment 359
titers of antibodies was fully compatible with the hyperacute xenogeneic reac-
tion seen in widely divergent species. ALG was not able to reduce these anti-
bodies or decrease their production. After ALG therapy, and in the presence
of an absolute lymphopenia (but with agranulocytosis), similar rejection and
survival times were seen as in the untreated animals.
Fox-to-Dog System
A preclinical experimental model for the investigation of mechanisms of
xenogeneic organ transplantation was found in the canine system using the
fox-to-dog model. It mimics the clinical situation of baboon-to-man. The im-
munogenetic markers, as well as the survival time of xenotransplants, support
this assumption. Function of the xenogeneic kidney grafts can be observed
over 5 days. Later, the capacity to excrete concentrated urine is lost very rapid-
ly. The immunological damage to the transplant seems to be induced by hu-
moral mechanisms which cannot be accurately documented with the in vitro
and in vivo methods currently available.
The initial and late deficits of RBF, especially to the cortex, must be blamed
on humoral factors. Similar vasoactive mechanisms also occur in allogeneic
situations. This is reversible under immunosuppressive therapy using AMS,
ALG, and possibly 15-deoxyspergualin. Cyclosporine does not improve the
survival time of xenogeneic kidneys significantly.
Sensitization against fox antigens can be detected as early as 4 days after
transplantation. The antibodies are absorbed from the blood by the graft.
Removal of the transplant leads to raised titers of antifox red blood cell anti-
bodies of 1:128. Absorption studies proved the antibodies to be species-specif-
ic. In histological sections, the antibody complexes seem to be mainly of IgG
type. In several dogs hemolysis was detected as long as fox kidneys were func-
tioning; no complement-fixing antibodies were found at that time.
Until day 4, severe signs of rejection are not usually seen on histology. The
negative results of immunofluorescence studies do not support antibody-me-
diated mechanisms. Cellular infiltrates are not as marked as seen in allografts
at comparable times. At the completion of rejection the activated cell type be-
longs predominantly to the B cell line [42,44].
Little is known about immunosuppression with ALG in xenotransplanta-
tion. This is because concordant systems are rare and species-specific ALG
do not exist. We have utilized antidog ALG and AMS together with chemical
immunosuppression with cyclosporine and 15-deoxyspergualin. Concerning
primary vascularized grafts, ALS in discordant systems is without effect since
neither the titers of PNABs nor the production of such antibodies can be re-
duced. Xenogeneic skin transplants (e.g. man-to-mouse), however, can be
prolonged significantly with ALS and antithymocyte globulin (ATG) therapy
[51,52].
In the closely related xenogeneic system, ALG prolongs survival times and
improves graft function significantly. Despite the rather long survival time, in-
dividual variation is relatively little. Histocompatibility in terms of the MHC
antigens seems to have no major impact on xenografting in such models and

can be compared with the situation of totally mismatched allografts. Under
ALG treatment, the few infiltrating cells are mainly small lymphocytes,
thrombocytes, and granulocytes.
The role of monocytes and macrophages in the rejection of xenografts is
not fully understood. Fine needle aspiration and histological examination,
however, suggest that monocytes and macrophages take part in the recogni-
tion of antigens in the afferent phase, and as phagocytosing cells in the efferent
phase, of xenogeneic rejection. The AMS used did prolong kidney function
survival time. Insignificant reduction of PNAB was measured. Rejection was
due mainly to lymphocytes and their activated forms, but fewer plasma cells
and monocytes were seen than when ALG treatment was administered.
DingolW olf-to-Dog Xenotransplantation

The two dog-like species, dingo and wolf, show more or less compatible genet-
ic markers (including the MHC) with the dog. It is therefore surprising that
wolf kidneys and skin survived longer than comparable allografts.
Transplantation in the opposite direction did not reproduce this finding. The
reason for this phenomenon is not known, though one factor may be the rela-
tive size of the donor kidneys. The explanation of this observation needs fur-
ther investigation in xenogeneic models that have clinical relevance.
Summary
The increasing lack of human donor organs necessitates extensive research in

the field of xenotransplantation. Therefore, models should be developed that
allow investigations of relevance to the clinical situation. We can summarize
the studies documented in this chapter as follows:
1. Using the dog as recipient and based on genetic markers, immunological
mechanisms, and the phenomenon of PNAB as well as the evolutionary re-
lationship, a systematic classification of xenogeneic models and types of re-
jection was achieved.
2. Closely related species were investigated in the study. Only organ trans-
plantation between animals of species within one zoological family undergo
cellular rejection and can thus respond to the presently available pharmaco-
logical immunosuppressive agents.
3. Immunosuppressive therapy not only prolongs survival time but also im-
proves the function of these grafts. The interactions of the multiple media-
tors of rejection and interleukins in xenotransplantation are not yet known
and have been sparsely investigated. Such studies might explain some of the
(as yet not understood) favorable and beneficial observations made in these
studies, and also may explain some of the disappointing results document-
ed.
References 361
References
2, 550, 1970.
2. Hammer, c., Chaussy, c., Van Scheel, J., Roscher, E., Pongratz, E., Brendel, W. Survival
time of skin and kidney grafts within different canine species in relation to their genetic
markers. Transplant. Proc. 7,439,1975.
3. Hammer, c., Chaussy, c., Brendel, W. Preformed antibodies in animals and man.
Outlook on xenotransplantation. Europ. Surg. Res. 5, 162, 1973.
4. Alexandre, G.P.J., Gianello, P., Latine, D., Corlier, M., Dewaeale, A., Van Obbergh, L.,
Moriau, M., Marbaix, E., Lambotte, I.L., Lambotte, L., Squifflet, J.P. Plasmapheresis
and splenectomy in experimental renal xenotransplantation. In Xenograft 25. M. Hardy
(ed.) Elsevier, Amsterdam, New York, Oxford, p. 259, 1989.
5. Caine, RY., White, H.J.O., Herbertson, B.M., Millard, P.R, Davis, O.R, Salaman, J.R,
6. Reemtsma, K. Heterotransplantation. Transplant. Proc. 1,251,1969.
7. Starzl, T.E., Marchioro, T.L., Peters, ,G.N., Kirkpatrick, C.H., Wilson, W.E., Porter,
K.A., Ogden, D.A., Hitchkock, C.R Renal heterotransplantation from baboon to man.
Experience with 6 cases. Transplantation. 2,252,1964.
8. Reemtsma, K., Mccraken, B.H., Schlegel, J.U., Pearl, M.A., Pearce, C.W., De Witt,
C.W., Smith, P.E., Hewitt, RI., Flinner, R.L., Creech, O. Renal heterotransplantation in
man. Ann. Surg. 160,384,1964.
9. Danowick, W.J., Shafer, C.F., Dodd, D.C., Buchanan, J.W., Fregin, C.F. Cardiac and
skin heterograft rejection: suppression with antilymphocyte serum. Transplant. Proc. 3,
551,1971.
10. Perper, RJ. Renal heterotransplant rejection. A model for separation of humoral and
cellular mechanisms. Transplantation. 12,519,1971.
11. Perper, RJ., Najarian, J.S. Localization and quantitation of transplantation antibodies.
J. Immunol. 99,619, 1967.
12. Perper, R.J., Maz, J., Waz, L., Najarian,J.S. Experimental renal heterotransplantation in
closely related species. Fed. Proc. 24,573, 1965.
13. Bailey, L.L., Jang, J., Johnson, W., Jolly, W.B. Orthotopic cardiac xenografting in the
newborngoat.J. Thorac. Cardiovasc. Surg. 89,242, 1985.
15. Templeton, J.W., Thomas, E.D. Evidence for a major histocompatibility locus in the
dog. Transplantation. 11,429,1971.
16. Van Rood, J., Van Leeuven, A., Zweerus, R Histocompatibility testing. In P.I. Terasaki
(ed.) Munksgaard, Copenhagen, p. 93, 1970.
17. Vriesendorp, H.M., Westbroek, D.L., D' Amaro, J., Van der Does, J.A., Van der Steen,
G.J., Van Rood, J.J., Albert, E., Bernini, L., Bull, RW., Cabasson, J., Epstein, RE.,
Erikson, V., Feltkamp, T.E.W., Flad, H.D., Hammer, c., Lang, R, Largiader, F., Van
Loringhoven, K., Los W., Meera Khan, P., Saison, R, Serrou, B., Schnappauf, H.,
Swisher, S.N., Templeton, J.W., Uhlschmidt, G., Zweibaum, A. Joint report of 1st inter-
national workshop of canine immunogenetics. Tissue Antigens. 3, 144, 1973.
18. Grosse-Wilde, H., Baumann, P., Netzel, B., Kolb, H.J., Mempel, W., Wank, R, Albert,
E.D. One way non-stimulation in MLC to DL-A homozygosity. Transplant. Proc. 5,
1567,1973.
19. Swisher, S.N., Yang, L.E. The blood grouping system of dogs. Physiol. Rev. 41,495,
1961.
20. Reiter, M., Gilmore, V., Jones, T. Karyotype of the dog. Manual Chromo News. 12,170,
1963.
21. Valentin, c., Levy, C. The karyotype of canis dingo. Manual Chromo News. 18, 147,
1965.
22. Hungerford, D.A., Snuder, R Chromosomes of the European wolf. Manual Chromo
News. 22,72,1966.
23. Brendel. W .. Duswald. K .. Von Scheel. 1.. Chaussy. C, Sollinger, H .. Hammer, C

Prolonged survival time of canine xcnografts using a new schedule of horse anti-dog lym-
phocytic globulin ALG-thcrapy. Transplant. Pmc. 9,379,1977.
24. Lin, CC, Johnson. D.H., Ramsden, R.O. Polymorphism and quinicrine fluorescence
karyotypes of red foxes (v.vulpes). Can..l. Genet. Cytol. 14.573.1972.
25. Land. W., Sebald. R .. Grabs. C .. Brendel. W. Prolongation of rabbit xenograft survival
with antimacrophage serum in rats. Ellrop. Sllrg. Res. 3.28.1971.
26. Chaussy, C, Hammer, C, Pongratz. H .. Von Scheel. J., Pfeiffer. K.J., Land. W.,
Sollinger. H.W .. Pielsticker. K .. Brendel. W. Prolongation of graft survival in rats and
dogs by a specific antimacrophage serum. Transplant. Pmc. 7,779,1975.
27. Chaussy, C, Hammer. C, Von Scheel, J., Eisenberger, F., Pielsticker. K., Brendel. W.
Experimental xenogeneic kidney transplantation in closely related species.
Transplantation of cat kidneys to untreated and sensitized dogs. Res. Exp. Mal. 159.266.
1973.
28. Hammer, C, Chaussy, C .. Krebs, G., Von Scheel. 1.. Brendel. W. Experimental xeno-
geneic kidney transplantation in closely related systems. Ex-vivo hemoperfusion of kid-
neys in the system cat to dog. Res. Exp. Med. 160.32,1973.
29. Chaussy. C .. Hammer. C, VON Scheel. J. Pielsticker, K., Sollinger, H.W., Pfeiffer, K.,
Pongratz, H., Brendel. W. Xenogcneic skin and kidncy transplants in closely related ca-
nine species, fox-dog. Transplantation. 20. 150, 1975.
30. Hammer. C .. Saumweber. D, Krombach, F. Xenotransplantation in canines. In Xeno 25.
M. Hardy (ed.) Elsevier Amsterdam, New York, Oxford,1989, p. 67.
31. Bbhm, D., Krombach, F, Hammer, C, Gebhard. F., Brendel. W. Fine needle aspiration
cytology in cyclosporine treated xenogeneic kidney rejection. Transplant. Proc. 17,2128,
1985.
32. Saumweber, D., Singer, T., Hammer. C, Krombach. F .. Bbhm. D .. GokeL J. 15-
Deoxyspergualin - a new perspective of immunosuppressive therapy in experimental
xenogeneic kidney transplantation. Transplant. Pmc. 21,542, 1989.
33. Welter. F .. Krause, H., Hammer, C, Brendel, W. Xenogeneic and allogeneic blood
transfusion prior to xenogeneic kidney grafting in the fox-dog model. Langenbecks
Arch. Suppl. 199, 1981.
34. Gams. E .. Chaussy, C, Eckersdorf, B., Hagl, S" Hammer, C, Land, W., Pielsticker, K.,
Schraut, W., Sebening, F. Xenogeneic heterotopic heart transplantation in a closely re-
lated species system fox to dog. Res. Exp. M ed. 157,228, 1972.
35. Hagl, S., Beck, G., Brendel, W .. Land. W. Mayr. N .. Pielsticker, K .. Sebening, F.
Xenogeneic heart transplantation in a closely related system fox-dog. Med. Res.
Progress. 1. 3,1971.
36. Ertel. W., Reichenspurner, H., Hammer, C, Welz, A., Uberfuhr, P., Hemmer, W ..
Reichart. B .. Gokel. M .. Brendel. W. Heart transplantation in closely related species: a
model for humoral rejection. Transplant. Pmc. 16,1259,1984.
37. Hammer, C, Chaussy, Weber, H., Wembaeher, J., Hobel, G., Brendel, W. Exceptional-
ly long survival time in xenogeneic organ transplantation. Transplant. Proc. 13.881.
1981.
38. Krombach. F .. Hammer, C .. Gebhard. F.. Danko. I.. Schulz, S., Gokel, M. The e[[ect of
cyclosporine on wolf to dog xenografts. Transplant. Proc. 17. 1436. 1985.
39. Duswald, K., Von Scheel. J., Hammer. C, Brendel. W. Long-term graft survival in the
xenogeneic system wolf to dog. Res. Exp. Med. 167,255.1976.
40. Cook, W., Stanly, K., Klausner, K., Sinha, S., Kikkawa, Y., Vieth, F. A new look at hyper-
acute rejection. Ann Thomc. Surg. 13,388,1972.
41. Mundy, A. Prolongation of cat to dog renal xenograft survival with prostacycline.
Transplantation. 30,226,1980.
42. Perper, R. Renal heterograft rejection. Transplantation. 12,519,1971.
43. Dieperink, H., Steinbruchel, D., Starklint, H., Larsen. S., Kemp. E. Improvement in
hare-to-rabbit kidney transplant survival. Transplant. Pmc. 19,1114,1987.
44. Perper. R .. Najarian, J. Experimental renal heterotransplantation II closely related
species. Transplantation. 4,700,1966.
References 363
45. Perper, R., Najarian, J. Experimental renal heterotopic transplantation. III: Passive
transfer of transplantation immunity. Transplantation. 5,514,1967.
46. Bailey, L.L., Lacour-Gayet, F., Perier, P., Killeen, J.D., Perry, J.E., Cotie, R.W.,
Schmidt, c.A., Denmark, S.H., Mahoney, A.D., Jolley, W.B. Orthotopic cardiac trans-
plantation in the neonate. Survival studies in a goat model. Proceedings of Beijing
Symposium on Cardiothoracic Surgery 1, New York. Co-published by China Academic
and John Wiley & Sons, Inc., 1982, p. 342.
47. Bailey, L., Nehlssen-Cannarella, S. Observation on cardiac xenotransplantation.
48. Unger, C. Nierentransplantation. Ber!' Klin. Wschr. 47,573,1910.
49. Avramovici, A. Le transplantation du rein. Lyon Div. 21,734,1924.
50. Owen, E.R. Prolonged survival in heterografted kidneys with transplantation antigen
pretreatment. Nature. 219,970,1968.
51. Lance, E.M., Medawar, P.B. Survival of skin heterografts under treatment with antilym-
phocyte serum. Lancet. June 1, 1174, 1968.
52. Pass, F., Niimvra, M., Kreider, W. Prolonged survival of humoral skin xenografts in
anti thymocyte serum treated mice. I. Invest. Dermatol. 61,371,1973.
Chapter 22

Closely Related Primate Species
J.A. SANCHEZ, R.E. MICHLER, E.A. ROSE, AND D.K.C. COOPER
Introduction
The current success of clinical solid organ transplantation has resulted in a

shortage of suitable donors; this is particularly true for intrathoracic organs.
This shortage has prompted many to consider the use of nonhuman organs for
transplantation in man. In order to minimize the vigorous immunological re-
sponses that have been observed in experimental xenotransplantation, inves-
tigators have studied transplantation between phylogenetically closely related
animal models. This has resulted in an increased understanding of the mecha-
nisms of rejection and has also led to the development of models in which im-
munomodulative strategies are tested prior to their use in human allotrans-
plantation.
The first cross-species organ transplant utilizing primates was performed
by Unger in 1910 [1] (Chap. 2). He implanted a chimpanzee kidney into a hu-
man recipient resulting in thrombosis of the graft and death of the patient.
Thereafter the field of xenotransplantation remained relatively dormant ex-
cept for sporadic experiments using nonprimate species.
Success was achieved in clinical xenotransplantation in 1964 when
Reemtsma and his colleagues transplanted chimpanzee kidneys into a series
of patients with renal failure, achieving a maximal graft survival of 9 months [2,
3] (Chap. 33). In this group of patients, rejection did not prove to be a major
barrier to graft survival, with three of six grafts having no histological evidence
of rejection at necropsy. Subsequent attempts at clinical renal xenotransplan-
tation by Starzl et aI., utilizing baboon grafts, resulted in early graft loss despite
an immunosuppressive protocol similar to that employed by Reemtsma [4,5].
This difference in xenograft survival was attributed to a greater phylogenetic
disparity between baboon and man when compared with the chimpanzee and,
in addition, to the lack of ABO compatibility in this group [6]. A limited expe-
rience with clinical hepatic xenotransplantation was also reported by Starzl's
group during this early period [7-10] (Chap. 35).
To date, there have been seven attempts at clinical cardiac xenotransplan-
tation (Chap. 34), four of which have involved the use of primate donors. In
1964, Hardy transplanted a chimpanzee heart into a patient in cardiogenic
shock who was unable to be separated from cardiopulmonary bypass [11].
Subsequently, Barnard heterotopically implanted chimpanzee and baboon
hearts into two patients [12]. More recently, Bailey transplanted a baboon
366 Experimental Xcnotransplantation Between Closely Related Primate Species
heart into a newborn infant with the hypoplastic left heart syndrome [13].
Although the donor and recipient in this latter case were ABO-incompatible,
the graft functioned well until the development of a low cardiac output state
and death of the patient 20 days later. At necropsy. the graft demonstrated in-
terstitial myocardial hemorrhage, suggestive of vascular rejection, with no
lymphocytic infiltrate.
This experience with clinical cross-species transplantation has led many in-
vestigators to persist in their studies of the immunological mechanisms operat-
ing in xenotransplantation. Given the increasing ability to regulate various as-
pects of the immune system in man and the persisting shortage of suitable
human donors, it is likely that further attempts at clinical xenotransplantation
will be undertaken in the near future.
Why Study Primates?
As a result of the vigorous humoral immune response observed between wide-

ly disparate species, nonhuman primates have been considered immunologi-
cally appropriate organ donors for humans. For this reason, experimental
nonhuman primate models have been developed in which the donor-recipient
phylogenetic disparity attempts to parallel that between man and the higher
primates. Whether clinical transplantation using primate donors is ethically
acceptable and logistically possible remains controversial and is discussed
elsewhere in this volume (Chap. 32).
Additional advantages of the nonhuman primate experimental model,
however, include anatomical and physiological similarities to man, the ability
of primates to tolerate cardiopulmonary bypass, and the technical capacity to
provide consistent surgical success in an animal this size, as well as the capacity
to monitor and control hemodynamic responses during the perioperative peri-
od. Furthermore, increasing sophistication in the ability to measure and regu-
late specific components of the immune system in man has made it essential to
study animal models such as primates in which there is a high likelihood that
antigens on the surfaces of immunologically relevant cells are shared with
man. For example, monoclonal antibody therapy directed against various lym-
phocyte surface targets will likely provide an important means of controlling
experimental xenograft rejection in the future [14].
When selecting nonhuman primate combinations as experimental models
of clinical transplantation the following factors should be considered. The
phylogenetic relationship between the two species should ideally correspond
to the relationship between higher-order anthropoid apes and man. In addi-
tion, antigens which are important in transplantation, such as the major histo-
compatibility complex (MHC) and ABO antigens, should be identifiable in
the study primates.
While MHC antigen systems have been analyzed only in selected primate
species, further developments in primate tissue typing may be able to identify
Why Study Primates? 367
optimal donor-recipient pairs [15]. A recent study by Neethling et al. [16]
demonstrated that, if species-specific antibodies are absorbed out, baboons
could be typed for some human histocompatibility antigens, particularly B
and DR, using known antisera (Table 22.1). It appears possible to match ba-
boons with humans for at least some ofthese antigens. DNA analysis would al-
most certainly prove more informative. Using human sera and baboon T cells,
10 of 19 human recipients were negative against all possible baboon donors.
Of the B cell cross-matches, 6 of 19 proved negative. Both Band T cell cross-
matches were negative in five recipients. Lymphocytotoxic antibody levels in
human recipients having either B or T cell-positive cross-matches with baboon
cells varied from 2% to 91 %.
Furthermore, the expression of ABO blood groups in polymorphic forms
in various tissues along with their reciprocal antibodies are characteristic of
simian primates. These allelic gene products are expressed in the erythrocytes
of the anthropoid apes and man but are detectable only in tissues and saliva of
old world monkeys [17]. The role of these blood groups in primate xenotrans-
plantation is unclear. While hyperacute or accelerated rejection might be ex-
pected to occur as a result of the presence of anti-A or anti-B antibodies, it has
been observed by the Cape Town group that in nonimmunosuppressed ani-
mals ABO incompatibility may not influence overall cardiac graft survival
[18]. This may be a reflection of the variable expression of the corresponding
hemagglutinins in primates.
In this study by the Cape Town group [18], heart grafts from vervet mon-
keys in ABO-compatible baboons functioned for a mean of 1O.3±5.2 days,
which was not significantly different from that in ABO-incompatible pairs
(7 .3±5.6 days). In the ABO-incompatible group, however, three of nine hearts
were rejected hyperacutely within 60 min, whereas in the ABO-compatible
Table 22.1. B and DR antigens clearly recognized in

30 Chacma baboons after testing with human antisera"
B antigens n DR antigens n
B 7 12 2,7 1
B 13 1 3,- 1
B 14 1 3,7 3
B 15 9 4,- 2
B17 1 4,7 16
B21 4 5,7 2
B27 3 5,8 1
B51 1 7,- 2
B54 2 7,8 2
Bw63 6
Bw70 6
None 14
Total 60 Total 30
" From [16].

368 Experimental Xenotransplantation Between Closely Related Primate Species
group only one of nine hearts was rejected within 24 h. The results of this study
suggest that although ABO-incompatibility would not appear to be a major
factor in cardiac xenograft survival when transplantation is performed be-
tween closely related primate species, early hyperacute rejection would seem
more likely to occur when blood group incompatibility is present.
Immunosuppressive therapy in the form of cyclosporine, methylprednisolone
and azathioprine did not significantly prolong xenograft survival in either
ABO-compatible or incompatible pairs (Table 22.2), but again early (day 1)
hyperacute rejection was only seen in the ABO-incompatible group [19].
A number of other blood group systems are present in primates [20-23].
Simian-type blood groups can be identified utilizing specific immune hemag-
glutinating reagents. Some are believed to be analogous to known human
blood group antigens and others even have shared specificities with man. The
absence or weakness of heterospecific reactivity between the sera of certain
primate pairs, including the cynomolgus monkey and baboon as well as the
chimpanzee and man, reflect their close evolutionary proximity [22,23]. The
characterization of these "minor" antigens have permitted taxonomic reclas-
sification of several subspecies previously grouped by phenotypic considera-
tions. Although the significance of these antigens in transplantation is not
clear, Michler et al. have shown that fewer than five mismatches of these anti-
gen groups is desirable [21]. While it may not be currently feasible to identify
and match these and other antigenic systems routinely when selecting donor-
recipient pairs in experimental models, it is possible that they will playa role in
the long-term success ofxenografts.
Table 22.2. Cardiac xenograft survival in a vervet monkey-to-baboon model"
Group Immunosuppressive therapy n Graft survival (days)
Mean SD
1 No immunosuppression (control) 9 10 S
2 ABO incompatible, no IS 9 7 6
3 CsA,AZA,MP 6 13 g
4 ABO incompatible; CsA, AZA, MP S 11 11
Sb CsA,AZA,MP S 19 22
6b CsA, AZA, MP, ATG 6 43 19
7b IS-DS, CsA, AZA, MP 7 20 12
gb lS-DS, CsA, MP S 36 14
9 TLI, CsA, AZA, MP S 16 10
10 ABO incompatible; TLI, CsA, AZA, MP S 18 II
IS, immunosuppressive therapy; lS-DS, IS-deoxyspergualin; TLI, total lymphoid irradia-

tion; CsA, cyclosporine; AZA, azathioprine; MP, methylprednisolone; ATG, anti-thymo-
cyte globulin
a From [32].
b These groups received intravenous bolus methylprednisolone therapy for histologically
proven rejection episodes.
Techniques 369
Techniques
One model for studying the effectiveness of various immunosuppressive

strategies employs a surgical technique where the donor heart is transplanted
into the neck of the recipient animal (Fig. 22.1). This technique, originally in-
troduced by Carrel and Guthrie [24] and Mann et al. [25], has been used exten-
sively, often in a modified form, by several groups in recent years; it has been
described in detail by Michler et al. [26].
The benefits of this model include the ability to assess graft function readily
and the capacity to biopsy the graft frequently using a simple percutaneous
technique. Cessation of activity is easily determined by inspection or palpa-
tion and serves as a reliable end-point. As the grafted organ is supported with
blood and oxygen by the recipient animal, the technique reduces other factors
that might result in impaired graft function, particularly in the peri operative
period; it is, therefore, simpler and has some advantages over orthotopic heart
transplantation. Although electrocardiographic signals from these grafts dis-
play prominent voltages, the loss of signal may be variable in the setting of re-
jection, and well-formed QRS complexes have on occasions been recorded
from frankly necrotic grafts.
Orthotopic implantation of xenografts has resulted in markedly dimin-
ished graft survival when compared to heterotopic implantation [27], presum-
ably as a result of the increased functional and metabolic demands placed on
the orthotopic graft. This observation suggests that potential therapies aimed
at prolonging graft survival must ultimately be assessed in the setting of ortho-
topic transplantation.
Cardiopulmonary bypass and orthotopic heart transplantation are well tol-
erated in larger recipient primates such as baboons. These animals and their
xenogeneic primate grafts respond well to intravenous cardiotonic agents such
as dopamine and other pharmacologic catecholamines in the immediate post-
operative period. The recovery period is characterized by early endotracheal
extubation and return of the animals to their cages within hours of transplan-
tation.
Conventional immunosuppression can be delivered to primates daily via
intramuscular injections administered in atraumatic squeeze cages.
Fig. 22.1. Technique for th e

heterotopic (cervical) im-
plantation of xenogeneic
cardiac grafts in the baboon
Cyclosporine in crcmophor [28] or intra lipid [29], administered intramuscu-

larly at an approximate dose of 15 mg/kg per day, achieves satisfactory thera-
peutic levels. Baboons do not absorb cyclosporine well when it is givcn by na-
sogastric tube. Mcthylprednisolone (possibly in depot form, Oepo-Medrol)
and azathioprine can also be delivered intramuscularly. The intravenous ad-
ministration of agents such as antithymocyte globulin (ATG) requires pro-
found sedation unless one employs implantable subcutaneous venous
catheters.
As discussed elsewhere in this volume, the future use of primates for both
experimental and clinical transplantation may be restricted by recognition of
the potential human health risks. The identification of lymphotropic rctro-
viruses and Ebola-like filovirus affecting several primate species has been re-
ported [30, 31]. The potential for transmission of these and other agents to
man is currently being appraised.
Immunomodulative Strategies
Two large series of studies of heart transplantation between closely rclated pri-
mate spccies have been conducted - at Columbia Presbyterian Medical Center
inNewYork [27] and the University of Cape Town in South Africa [32].
Pharmacologic Immuuosuppressive Therapy
Michler and his colleagues in New York demonstrated a greater than tenfold
prolongation of graft survival using cyclosporine-based immunosuppression
when compared with untreated groups [27, 33-35]. In these studies, cynomol-
gus monkeys (Macacafascicularis) served as donors and olive baboons (Papio
anubis) served as recipients (Table 22.3). The combination of cyclosporine
Table 22.3. Cardiac xenograft survival in a cynomolgus monkey-to-baboon model"
Group I mmunosuppressive therapy II Graft survival (days)
Mean SO
I No immunosuppression (control) t) 7 :;
2 CsA.MP () 77 67
3 CsA.AZA.MP 7 61 4K
4 CsA.AZA (, 77 6K
5 CsA, AZA, MP, ATe; 5 KI 22
6 PTx.CsA 10 42 61
CsA, eyclosporine: AZA. azathioprine; MP. methylprednisolone; ATe;. anti-thymocyte

globulin; PTx. prctransplant blood transfusion
" From [27].
Immunomodulative Strategies 371
and steroids alone was as effective as the addition of other agents such as aza-
thioprine and ATG. Pre transplant blood transfusions (shown to prolong by
threefold the survival of baboon renal grafts in the rhesus monkey [36]) were
not effective in these studies [37]. Although hyperacute rejection was not en-
countered in this model, the development of cytotoxic antibodies to donor
lymphocytes correlated chronologically with graft loss [27].
In a separate series of studies performed by the Cape Town group, utilizing
vervet monkeys (Cercopithecus aethiops) as donors and chacma baboons
(Papio ursinus) as recipients, graft survival was not prolonged when conven-
tional immunosuppression was employed (Table 22.2). Vascular (humoral)
rejection of the donor heart occurred in a high percentage of cases [32, 38].
However, increased graft survival was achieved by the addition of rabbit ATG
and 15-deoxyspergualin and the treatment of documented rejection episodes
with bolus methylprednisolone [39]. Pre transplant total lymphoid irradiation
of the recipient appeared to extend graft survival but at the expense of unac-
ceptably high morbidity and mortality [40].
Photochemotherapy
Photo chemotherapy, a novel therapeutic approach which appears to achieve

donor-specific suppression of the host immune system, has recently been stud-
ied by the Columbia group in their nonhuman primate model [41]. This tech-
nique has been shown to be highly effective in the treatment of patients with
cutaneous T cell lymphomas and involves the extracorporeal treatment of re-
cipient leukocytes with ultraviolet A light (UVA) after the administration of
8-methoxypsoralen (8-MOP) [42]. Exposure of this agent to UVA results in
covalent bonding between complementary strands of DNA, thus preventing
cellular replication. Lymphocytes which are activated against specific graft
antigens are preferentially treated by this method. Upon reinfusion, these
cells become targets for immunological attack, resulting in their down-regula-
tion [37,43]. Preliminary results have demonstrated a modest prolongation of
xenograft survival [44]. More importantly, however, the specificity ofthe im-
munological attack provided by this method may allow for the addition of
other potent immunosuppressive strategies. This would result in maximal
xenograft survival while preserving immunological competence to challenges
by microbial antigens.
Monoclonal Antibody Therapy
Monoclonal antibodies directed against surface activation antigens on lym-

phocytes are currently being employed to reverse recalcitrant rejection in clin-
ical transplantation. This form of immunotherapy has great potential in atten-
uating the immunological responses in xenotransplantation. The conjugation
or chelation of these antibodies to biologic toxins and radioisotopes could pro-
vide extremely powerful tools in controlling severe forms of rejection. One

such antibody directed against the interleukin-2 receptor (IL-2R) of activated
killer T cells, when conjugated with yttrium-90, has resulted in prolonged graft
survival in a cynomolgus (Macaca fascicularis) to rhesus (Macaca mulatta)
model [14]. The enthusiasms generated by these agents may be tempered by
their lack of reactivity in certain species and by the potential hazards posed by
their conjugates. Monoclonal antibodies directed particularly against B lym-
phocytes would be beneficial in reducing antibody production and thus, hope-
fully, in preventing the development of vascular rejection.
Comment
The importance of humoral mechanisms in the development of both fatal car-

diac allograft rejection and accelerated graft atherosclerosis has recently been
appreciated [45]. These antibody-mediated responses probably playa more
prominent role in xenogeneic than allogeneic rejection and are believed to be
major determinants of diminished graft survival in cross-species transplanta-
tion. Furthermore, this type of rejection is probably more resistant to reversal
than that which is produced by cell-mediated mechanisms [46].
Immunosuppressive therapies directed at limiting these humoral responses
are essential for the success of xenotransplantation. Strategies such as pho-
tochemotherapy, which appear to regulate both cell-mediated and humoral
mechanisms, in conjunction with monoclonal antibodies directed against acti-
vated B cells, may lead to control of such humoral immune responses. In addi-
tion, the use of more potent immunosuppressive agents such as rapamycin and
FK-506 and, particularly, 15-deoxyspergualin and mycophenolic acid deriva-
tives (e.g., RS-61443), will hopefully result in prolongation of graft survival af-
ter xenotransplantation while at the same time limiting the amount of pro-
found generalized immunosuppression necessary to achieve successful
engraftment.
The current success of clinical organ transplantation is unfortunately tem-
pered by the desperate shortage of donor organs. This has led many investiga-
tors to consider nonhuman organs as an attractive option for human trans-
plantation. Although clinical experience with xenotransplantation is
extremely limited, experimental models have been developed which are pro-
viding an opportunity for the study of cross-species transplantation. Studies
performed to date have demonstrated that certain xenogeneic immunological
barriers may not be insurmountable, and that clinical success will depend upon
the appropriate application of current and more potent immunosuppressive
strategies. These strategies must incorporate the attenuation of humoral
mechanisms, which appear to playa more important role in xenograft rejec-
tion than in allograft rejection.
References 373
References
1. Unger, E. Nierentransplantationen. Klin. Wschr. 47,573,1910.
2. Reemtsma, K., McCracken, B.H., Schlegel, J.u., Pearl, M.A., Pearce, CW., DeWitt,
CW., Smith, P.E., Hewitt, RL., Flinner, RL, Creech, O. Jr. Renal heterotransplanta-
tion in man. Ann. Surg. 160,3384,1964.
3. Reemtsma, K, McCracken, B.H., Schlegel, J.U., Pearl, M.A., DeWitt, CCW., Creech,
O. Jr. Reversal of early graft rejection after renal heterotransplantation in man. 1. Amer.
Med. Assoc. 187,691,1964.
4. Starzl, TE., Marchioro, TL., Peters, G.N., Kirkpatrick, CH., Wilson, W.E.C, Porter,
K.A., Rifkind, D., Ogden, D.A., Hitchcock, CR, Waddell, W.W.R Renal heterotrans-
plantation from baboon to man: experience with six cases. Transplantation. 2, 752,
1964.
5. Kirkpatrick, CH., Wilson, W.E.C Immunologic studies of baboon-to-man renal hetero-
transplantation. In Starzl, TE. (Ed.) Experience in Renal Transplantation. Philadelphia,
W.B. Saunders Co., 1964, p. 284.
6. Hardy, M.A., Todd, G., Reemtsma, K. Xenotransplantation. In Slavin, S. (Ed.) Bone
Marrow and Organ Transplantation. Elsevier, Amsterdam, New York, Oxford 1984,
p.519.
7. Starzl, TE. Experience in Hepatic Transplantation. WB Saunders, Philadelphia, 1969,
p.408.
8. Giles, G.R, Boehmig, H.J., Amemiya, H., Halgrimson, CG., Starzl, TE. Clinical het-
erotransplantation of the liver. Transplant. Proc. 2,506,1970.
9. Giles, G.R, Boehmig, H.F., Killy, J., Amemiya, H., Takagi, H., Coburg, A.J., Hathaway,
W.E., Wilson, CB., Dixon, FJ., Starzl, TE. Mechanism and modification of rejection of
heterografts between divergent species. Transplant. Proc. 2,522, 1970.
10. Starzl, TE., Ishikawa, M., Putnam, CW., Porter, K.A., Picache, R, Husberg, B.S.,
Halgrimson, CG., Schroter, G. Progress in and deterrents to orthotopic liver transplan-
tation, with special reference to survival, resistance to hyperacute rejection, and biliary
duct reconstruction. Transplant. Proc. 6, 129, 1974.
11. Hardy, J.D., Chavez, CM., Kurrus, FD., Neely, W.A., Webb, W.R, Eraslan, S., Turner,
M.D., Fabian, L.W., Labecki, J.D. Heart transplantation in man: developmental studies
and report of a case. 1. Amer. Med. Assoc. 188,1132,1964.
12. Barnard, CN., Wolpowitz, A., Losman, J.G. Heterotopic cardiac transplantation with a
pass. S. Afr. Med. 1.52,1035,1977.
13. Bailey, L.L., Nehlsen-Cannarella, S.L., Concepcion, W., Jolley, W.B. Baboon-to-human
cardiac xenotransplantation in a neonate. 1. Amer. Med. Assoc. 254,3321, 1985.
14. Cooper, M.M., Robbins, RC, Goldman, CK., Mirzadeh, S., Brechbiel, M.W., Stone,
CD., Gansow, O.A., Clark, RE., Waldmann, TA. Use ofyttrium-90-labelled anti-Tac
antibody in primate xenograft transplantation. Transplantation. In press.
15. BaIner, H. The major histocompatibility complex of primates: evolutionary aspects and
comparative histogenetics. Phil. Trans. R. Soc. Lond. B292, 109, 1981.
16. Neethling, F.A., Nortman, P., Cooper, D.K.C Histocompatibility matching between hu-
mans and baboons. Transplant. Proc. 22,1067,1990.
17. Socha, W.W., Marboe, CC, Michler, RE., Rose, E.A., Moor-Jankowski, J. Primate an-
imal model for the study of ABO incompatibility in organ transplantation. Transplant.
Proc.19,4448,1987.
18. Cooper, D.K.C, Human, P.A., Rose, A.G., Rees, J., Keraan, M., Reichart, B., Du Toit,
E., Oriol, R. The role of ABO blood group compatibility in heart transplantation be-
tween closely related animal species. 1. Thorae. Cardiovasc. Surg. 97,447,1989.
19. Cooper, D.K.C, Human, P.A., Rose, A.G., Rees, J., Keraan, M., Du Toit, E., Oriol, R
Can cardiac allografts and xenografts be transplanted across the ABO blood group bar-
rier? Transplant. Proe. 21,549,1989.
20. Michler, R.E., Socha, W.W., Marboe, CC, Smith, CR, Reemtsma, K., Moor-
Jankowski. J .. Rose. E.A. Macaque-to-baboon cardiac transplantation: models of choice

for xenotransplantation in humans. Transplant. Proc. 20,327.1988.
21. Michler. RE .. Marboe. CC, Socha. W.W .. Moor-Jankowski. L Reemtsma. K.• Rose.
E.A Simian-type blood antigens in non-human primate cardiac xenotransplantation.
Transplant. Proc. 19,4456.1987.
22. Socha. W.W .• Moor-Jankowski. J .. Ruffie. J. The BP blood group system of the baboon:
its relationship with macaque red cell antigens. Folia. Primatol. 40.205, 1983.
23. Socha, W.W., Moor-Jankowski. J. Chimpanzee R-C-E-F- blood group system. A coun-
terpart of the human Rh-hr blood groups. Folia Primatol. 33. 172. 1980.
24. Carrel. A, Guthrie, CC The transplantation of veins and organs. Am. Med.
(Philadelphia). 10. 1101. 1905.
25. Mann, F.C, Priestley. J.T.. Markowitz. J., Yater. W.M. Transplantation of the intact
mammalian heart. Arch. Surg. 26.219.1933.
26. Michler. RE., McManus, RP., Smith, CR, Sadeghi, AN .. Rose, E.A Technique for
primate heterotopic cardiac xenotransplantation. J. Med. Primatol. 14.357,1985.
27. Michler, RE., McManus, RP., Smith, e.R, Sadeghi, A.M., Marboe. e.C, Reemtsma,
K, Rose, E.A. Prolongation of primate cardiac xenograft survival with cyclosporine.
28. Kurlansky, P.A, Sadeghi, AM., Michler, RE., Coppey, L.J., Re, L.P., Thomas, W.G.,
Smith, e.R, Reemtsma, K, Rose, E.A Role ofthe carrier solution in cyclosporine phar-
macokinetics in the baboon. J. Heart Transplant. 5,312,1986.
29. Novitzky, D .. Cooper, D.Ke., Wicomb, W.N. A successful method of administering cy-
closporin A to the Chacma baboon. S. Afr. Med. J. 68,737,1985.
30. Van der Reit, F. de St. J., Human, P.A, Cooper, D.KC, Reichart, B., Fincham, J.E.,
Kalter, S.S., Kanki, P.J., Essex, M., Madden, D.L., Lai-tung, M.T., Chaiton, D., Sever,
J .T. Virological implications of the use of primates in xenotransplantation. Transplant.
Proc. 19,4068,1987.
31. Centers for Disease Control. Ebola-related filovirus infection in nonhuman primates
and interim guidelines for handling nonhuman primates during transit and quarantine.
Morbidity and Mortality Weekly Report. 39,22,1990.
32. Cooper, D.Ke., Rose, AG. Experience with experimental xenografting in primates. In
Hardy, M. (Ed.) Xenograft 25. Elsevier, Amsterdam, New York, Oxford, 1989,p. 95.
33. Reemtsma, K, Pierson, RN., Marboe, Ce., Michler, RE., Smith, e.R, Fenoglio, J.J.
Will atherosclerosis limit clinical xenografting? Transplant. Proc. 19,108,1987.
34. Kurlansky, P.A, Robbins, Re., Smith, CR, Marboe, e.e., Thomas, W.A, Coppey, L.,
Rose, E.A. Comparable survival of intra-species and cross-species primate cardiac
transplants. Transplant. Proc. 19,1067,1987.
35. Sadeghi, AM., Robbins, RC, Smith, CR, Kurlansky, P.A, Michler, RE., Reemtsma,
K, Rose, E.A. Cardiac xenograft survival in baboons treated with cyclosporine in com-
bination with conventional immunosuppression. Transplant. Proc. 19,1149,1987.
36. Marquet, RL., Van Es, AA, Heystek, G.A, Van Leersum, RH, Bainer, H.
Prolongation of baboon to rhesus monkey kidney xenograft survival by pretransplant
rhesus blood transfusion. Transplantation. 25, 165, 1978.
37. Michler, RE., McManus, RP., Sadeghi, AN., Kurlansky, P., Smith, e.R, Marboe, e.e.,
Thomas, W.B., Reemtsma, K, Rose, E.A Pretransplant blood transfusion in a primate
cardiac xenograft model. Curro Surg. 44,42, 1987.
38. Rose, AG., Cooper, D.KC, Human, P.A, Reichenspurner, H., Reichart, B.
Histopathology of hyperacute rejection of the heart: experimental and clinical observa-
tions in allografts and xenografts. J. Heart Transplant. In press.
39. Reichenspurner, H., Human, P.A, Boehm, D.H., Rose, AG., May, R, Cooper, D.KC,
Zilla, P .. Reichart, B. Optimalization of immunosuppression after xenogeneic heart
transplantation in primates. J. Heart Transplant. 8,200,1989.
40. Cooper, D.KC, Human, P.A, Reichart, B. Prolongation of cardiac xenograft (vervet
munosuppressive drug therapy. Transplant. Proc. 19,4441.1987.
41. Fuzesi, L.. Pepino, P., Berger, e.L., Panza, A. Chiang, Y.C. Marboe, Ce., Pierson, R,
References 375
Smith, CR, Reemtsma, K, Rose, E. Immunomanipulation of the response to cardiac
allo and xenoreaetive leukocytes. Transplant. Proc. 21,537,1989.
42. Edelson, RL., Berger, CL., Gasparro, F.P. Treatment of cutaneous T-cell lymphoma by
extraeorporal photoehemotherapy. N. Eng!. 1. Med. 316,297,1985.
43. Edwards, N.M., Rose, E.A. The use of non-human primates in xenotransplantation re-
search. In Hardy, M.A. (Ed.) Xenograft 25. Elsevier, Amsterdam, New York, Oxford,
1989, p. 79.
44. Sanchez, l.A., Edwards, N.M., Berger, CL., Ott, G.Y., Coppey, L., He, X., Fong, l.C,
Reemtsma, K, Rose, E.A. Photoehemotherapy provides donor-specific immunosup-
pression in cardiac transplantation. Surg. Forum. 41,421, 1990.
45. Rose, E.A., Smith, CR Petrossian, G.A., Barr, M.L., Reemtsma, K Humoral immune
responses after cardiac transplantation: correlation with fatal rejection and graft
atherosclerosis. Surgery. 106,203,1989.
46. McManus, RP., Kinney, T., Komorowski, R Reversibility of primate xenograft rejec-
tion (abstract). 1. Heart Transplant. 9,75,1990.
Chapter 23

Distantly Related Nonprimate Species
E.KEMP
Introduction
Although this chapter is limited to a description of discordant xenografting be-

tween animals other then primates and rodents (which are described else-
where in this volume), the picture of discordant xenograft rejection in all
species is very similar. The transplanted organ is invariably damaged within a
short period of time (minutes to hours). Morphological changes in the organ
are also similar in all species studied.
Several overviews have been written concerning xenografting, and these
include comments on transplantation between widely differing, nonprimate,
nonrodent species [1-5].
Studies of xenografting between two distantly related species have shown
that a transplanted, vascularized organ retains a normal appearance and a nor-
mal function for some minutes. However, the recipient's immune system soon
brings about a sequence of events that leads to destruction of the organ.
Within a few minutes function ceases and cell destruction (necrosis) begins.
This is termed discordant xenograft rejection, and is a form of hyperacute re-
jection.
Rabbit-to-Cat Experimental Model
As an example ofxenotransplantation between species that are not closely re-

lated, kidney transplantation between a rabbit (donor) and a cat (recipient)
will be described.
Before the rabbit's kidney is dissected free, the donor animal is treated with
a vasodilator drug (e.g., chlorpromazine) to prevent or reduce spasm in the re-
nal vessels. The kidney is carefully removed along with the renal blood vessels
and the ureter. The isolated kidney is immediately perfused with a cold
(2-4°C) fluid, which is somewhat hyperosmolar and has a high potassium con-
tent. The metabolism of the donor kidney is reduced by perfusing the solution
at a low temperature, thus protecting the tissues until revascularization and
reperfusion is initiated.
The cat's own left kidney is removed. Its aorta and inferior vena cava are
then dissected free, and the rabbit kidney's artery and vein are anastomosed to
378 Experimental Xenotransplantation
Fig. 23.1. Kidney transplantation [rom rah-

hit to cat has been performed. Prior to re -
establishing blood flow to the donor kid-
ney, the kidney is a pale gray color, and
there is no production of urine
Fig. 23.2. Immediately after the restitution

of renal blood flow the rabbit kidney has a
healthy red appearance, and the production
of urine, which is normal in appearance, has
begun
Rabbit-to-Cat Experimental Model 379
these vessels (Fig. 23.1). After the vessels are coupled, blood is allowed to flow
through the transplanted xenograft kidney.
Within a few seconds, the kidney adopts a fine red color (from reperfusion
with blood) and the tissue regains a normal consistency. Momentarily the kid-
ney looks normal, having the same appearance as before removal from the
donor (Fig. 23.2). The initial appearance of a rabbit kidney after allotransplan-
tation or autotransplantation is very similar. Blood flow through the trans-
planted kidney during the first minute or so after xenotransplantation is found
to be normal, but within a few minutes there is a dramatic reduction, illustrat-
ed by the flow measurements shown in Fig. 23.3. In auto- or allotransplanta-
MI.
90
Fig. 23.3. A reduction in renal

blood flow occurs within 1- 2 min
after rabbit-to-cat kidney trans- ,
plantation MINUTES
Fig. 23.4. After 5 min reperfusion, the

rabbit kidney becomes cyanotic in patches,
and hematuria can be observed
Fig. 23.S. After 15 min , the xenotransplant-

ed rabbit kidney is uniformly cyanotic, and
urine production has ceased
Fig. 23.6. At 10 min after xcnotransplantation , the rabbit renal vessellumcna are found to he
packed with aggregated platelets and other blood cells
Rabbit-to-Cat Experimental Model 381
tion, this reduction in flow is not seen. Immediately following complete cessa-
tion of blood flow, the kidney first becomes cyanotic in patches (Fig. 23.4), and
then completely blue (Fig. 23.5). The production of urine, which usually be-
gins rapidly after reperfusion, gradually ceases in association with the reduc-
tion in renal blood flow. This happens in the course of 5-15 min after trans-
plantation and indicates the organ has been rejected irreversibly.
The exact mechanisms that contribute to this hyperacute xenograft rejec-
tion remain unclear, though the major steps are now well known and are out-
lined in Chap. 4. In outline, 5-10 min after transplantation is completed,
thrombi have formed in the smaller vessels of the transplanted organ - the so-
called vascular catastrophe. The sequence of events that lead to the formation
of thrombi is uncertain. Blood platelet aggregation is one of the first changes
that can be observed, beginning as early as the 2nd min after transplantation in
guinea pig-to-rat heart transplants, as confirmed in the electron microscopic
studies of Cattell and Jamieson [6] and of Larsen and Starklint [7]).
Fig. 23.7. The appearances of a blood vessel in the rabbit kidney by scanning electron-
microscopy performed 5 min after xenotransplantation. The endothelial surface of the
blood vessel is paved with thrombocytes
The average length of time for hyperacute rejection to take place in the dis-
cordant xenotransplant model described above (rabbit-to-cat) is 10 min.
Histologically, the vessellumena are found to be obstructed by aggregated
blood platelets, which contain occasional red blood cells (Fig. 23.6). The capil-
lary loops in the glomeruli are greatly distended with thrombocyte aggregates,
and many afferent arterioles are totally occluded. At 5 min after the onset of
renal blood flow, electron microscopy shows the endothelial surface of the
blood vessels to be paved with thrombocytes (Fig. 23.7).
Other researchers have documented a similar sequence of events to that
described here when performing xenografting between other distantly related
species. Different organs (e.g., heart) and different pairs of experimental ani-
mals (e.g., pig-to-dog) have been used, but the reported results have been very
similar. A comprehensive list of previous and present researchers in this field
and the organs and experimental animals (excluding rodent pairs and studies
in primates) that have been studied is included in Table 23.1. Organ survival
has almost uniformly been for only minutes or a few hours at most, and no
therapeutic measures have to date led to even moderately long-term graft
function.
Table 23.1. Xenotransplantation between discordant (nonprimate, nonrodent species)
Year Investigators a Species
1902 Ullmann Dog-to-goat

1908 Unger Dog-to-goat
1910 Villard Dog-to-goat
1926 Avramovici Cat-to-dog
1966 Clark et al. Unknown-to-dog
Nelson etal. Pig-to-dog
Perper and Najarian Pig-to-dog and dog-to-pig
Perperetal. Pig-to-dog
Snyderet a!. Pig-to-dog
1967 Perper et al. Sheep-to-goat
1968 Linn eta!. Pig-to-dog
Winnetal. Pig-to-dog
1969 Gunnarson et al. Pig-to-goat
Jensen et al. Pig-to-dog
Rosenberg et al. Pig-to-dog
1970 Bieret al. Pig-to-dog
Gilesetal. Pig-to-dog and dog-to-pig
Hammeretal. Pig/sheep-to-dog
Kuxetal. Pig-to-dog
Linn etal. Pig-to-dog
Rattazzi et al. Cat-to-dog
Shonsetal. Pig-to-dog
1971 Bieretal. Pig-to-dog
Chavez-Peon et al. Rat-to-rabbit
Ghilchik et al. Sheep-to-dog
Hawkins et al. Pig-to-dog
Jerusalem et al. h Pig-to-dog and dog-to-pig
Pathogenesis of Discordant Xenograft Rejection 383
Year Investigatorsa Species
Kuxet al. Pig-to-dog

Linn et al. Pig-to-dog
Merkel et al. Pig-to-dog
Messmer et al. Pig-to-dog
Mobergetal. Pig-to-dog
Mobergetal. Pig-to-dog
Perperet al. Pig-to-goat
1972 Chaussy et al. Cat/pig-to-dog
Hammer et al. Pig-to-dog
1973 Bittscheidt et al. Pig-to-dog
Chaussy et al. Catlpig-to-dog
Chaussy et al. Cat-to-dog
Habaletal. Pig-to-dog
Hammeretal. Cat-to-dog
Hammeretal. Pig/sheep-to-dog
Hammeretal. Sheep-to-dog
Kashii etal. Pig/rabbit -to-dog
Olszewski et aJ.b Rabbit -to-dog
Shons et aJ. Pig-to-dog
Slapak et aJ. Piglrabbit -to-dog
1974 Belitsky et al. Pig-to-dog
GhiJchik et aJ. Sheep-to-dog
(Shons and Najarian) Sheep-to-man
ShonsetaJ. Pig-to-dog
1975 Anfossi et aJ. Dog-to-sheep
Haberal et al. Pig-to-dog
1976 Kempet al. Rabbit-to-cat
Toth et aJ. Pig-to-dog
1977 Kemp et aJ. Cat-to-rabbit
Yoshioka et aJ. Pig-to-dog
1980 Mundy et aJ. Cat-to-dog
1983 Cameron et aJ. Dog-to-sheep
Jorgensen et aJ. Pig-to-dog
1987 Makowka et aJ. Pig-to-dog
1989 Henryet aJ. Pig-to-dog
a Forreviewssee[1-3,5]
b The liver was the organ transplanted or hemoperfused here; in all other cascs, thc kidney
was studied.
Pathogenesis of Discordant Xenograft Rejection
Two major theories have been put forward as to the possible mechanisms
leading to hyperacute rejection when organ transplantation is performed be-
tween distantly related species. Evidence for neither is yet conclusive. Both
will be briefly outlined.
Activation of the Complement System
Activation of the complement system, triggered by the foreign endothelial

surfaces of the xenografted organ, may probably be sufficient alone to elicit
xenograft rejection in discordant models. In turn, the complement cascade
triggers thrombocyte aggregation, and cell lysis ensues (Fig. 23.8). This view
has been put forward both by the Odense [8, 9] and Munich [10] groups, and
has recently been beautifully demonstrated by a Japanese group [11]. Others
share this view [12]. All of the events of discordant xenograft rejection can be
explained by this pathogenic mechanism.
In support of this view, our group has been impressed by experiments
where complement inactivation has led to a prolonged organ survival time.
When the complement system in the cat is inactivated by administration of co-
bra venom factor, the rejection time can be extended to 1 week (Fig. 23.9).
After such therapy, in 80 discordant xenotransplants, no specific immune de-
posits were demonstrated at all [9].
This is in contrast to experiments with presensitized recipients of both allo-
transplants and xenotransplants where large amounts of immune deposits
were identified. Therefore, hyperacute allograft rejection, which occurs only
in sensitized hosts, and natural discordant xenograft rejection are different in
this respect. It may be that hyperacute allograft rejection caused by the pres-
ence of antibodies, with the antigen-antibody reaction activating the comple-
ment system, is not elicited in the same way as hyperacute discordant
xenograft rejection, where complement activation may be stimulated solely by
the appearance of foreign surface molecules.
Once the complement system is activated, several other events follow.
Other cascades such as those of the coagulation system, the fibrinolytic sys-
tem, and the kallikrein-kinin system are activated. Thrombocyte aggregation
and thrombocyte adherence to the vessel wall, and the formation of fibrin and
plugging of the lumen of the smaller vessels of the organ follow. Attempts to
inactivate these systems may also lead to prolongation of the survival of the
discordant xenografted organ, but not to the extent that can be achieved by in-
activation of the complement system [13].
ACTIVATION OF COMPLEMENT CASCADE
THROMBOCYTE
~ ENDOTHELIAL
AGGREGATION CELL DAMAGE
_______ VESSEL WALL

THROMBOCYTE
LYSIS DAMAGE
Fig. 23.8. Basic steps in the patho-

TOTAL OCCLUSION genesis of discordant xenograft re-
L--_ _ _ _---1~ OF VESSELS jection
Pathogenesis of Discordant Xenograft Rejection 385
Fig. 23.9. Light microscopic appearance of two glomeruli from a rabbit kidney 1 week after
transplantation into a cat treated with cobra venom factor. The normal appearances of the
glomeruli demonstrate the beneficial effect of cobra venom factor in preventing hyperacute
rejection . x400
Antigen-Antibody Reaction
The traditional explanation of the mechanism of discordant xenograft rejec-

tion is the antigen-antibody reaction that develops when the organ comes into
contact with preformed natural antibodies directed against the donor species.
These preformed antibodies have been popular as an explanation of this form
of rejection for decades despite the facts that they have been poorly defined
and that we have a very meager knowledge of them. This topic has recently
been discussed by Milgrom [14] and Auchincloss [2]. Whether each species has
a large number of different antibodies, each of which is able to react with anti-
gens from one other species (i.e., species-specific antibodies) or whether these
antibodies cross-react with several foreign antigens remains uncertain.
Preformed antibodies may well be involved in discordant xenograft rejec-
tion in some species combinations but not in others. On the basis of perfusion
experiments using rabbit kidneys and human blood, it seems likely that pre-
formed antibodies may playa role in this pair. In such experiments, after a few
minutes perfusion of rabbit kidneys with human blood, we found deposition of
immunoglobulin (Chap. 25).
There exist many discrepancies concerning the appearance of preformed
antibodies. When rabbit kidneys are transplanted into cats, we found no de-
posit of immunoglobulin [9] . In contrast, an Italian group that transplanted pig
kidneys into rabbits, found immunoglobulin deposition in the transplanted or-

gan [16], thus supporting the traditional view.
A Third Mechanism?
As it has been so difficult to pinpoint the exact mechanism of xenograft rejec-

tion (complement activation alone or complement activation via an antigen-
antibody reaction), one could speculate that such hyperacute rejection could
be due to the existence of a third mechanism. Table 23.2 provides a simplified
review of different attempts at overcoming this form of rejection and con-
siders whether they support the first, the second, or this third hypothesis.
For instance, the third hypothesis could be that recipient thrombocytes are
directly activated for aggregation and adherence by the foreign surfaces of the
donor vasculature, which are known to produce platelet-activating factor.
This mechanism may be similar to that which occurs following trauma or the
division of blood vessels. Thrombocytes aggregate and coagulum forms after
damage to the vessel wall when collagen is exposed to the blood. Neither the
complement system nor the presence of preformed antibodies are required to
explain such events. Such a pathway has not been totally excluded in the hy-
peracute rejection of discordant xenografts.
Table 23.2. Factors of influence in different experimental situations
Situation Antigcn- Complement Unknown Comment

antibody activation mechanism
reaction
Hyperacute (sensitized), + (+) Humoral-

allografting (unmodified) rejection
Concordant xenografting (+) (+) ? ?Cellularl?
(unmodified) humoral-
rejection
Discordant xenografting (+) (+) ? Humoral-
(unmodified) rejection
Cobra venom factor therapy + ?
Plasmapheresis (+) ? ? Removes
antibodies
(serum proteins)
Repetitive transplantation (+) ? ? Removes
antibodies
(serum proteins)
Piglet recipients (+) ? Born without
( nonsuckling) antibodies
References 387
Comment
Xenografting has been under experimental study for many years, yet we have
not made much progress in preventing discordant xenograft rejection.
Possible ways forward have been suggested by several authors [17, 18], but
many problems remain to be solved before we can reach the desired goal.
Recently, more immunologists have been entering this field of study, and
this will certainly help extend our knowledge in this discipline. Furthermore,
all of the classical experiments that have enabled us to understand allografting
have not yet been reproduced in xenograft research; the relationship between
xenografting and allografting needs to be explored further. At the moment,
for example, we still do not know how great are the differences between con-
cordant xenografting and allografting.
In future xenograft research, more in vitro experiments will almost certain-
ly throw light on many of the problems. In addition to the proposals already
put forward, much help would be gained from the development of monoclonal
antibodies directed against the various human complement factors. The ulti-
mate goal for the future, however, must be to develop a method of creating tol-
erance to xenografts. At the present time this remains a distant goal, but the
advances in "tolerance research" in recent years provide us with some hope
for the future.
References
1. Kcmp, E. Xenografting - the Future of Organ Transplantation. Odense University Press,

Odense, 1978.
2. Auchincloss, H. Xenogeneic transplantation. A review. Transplantation. 46, 1, 1988.
3. Dubernard, J.M .• Bonneau. M .• Latour, M. Heterografts in Primates. Simep Editions,
Villeurbanne. France, 1974.
4. Hardy, M.A. Xenograft 25. Elsevier, Amsterdam, New York, Oxford, 1989.
5. Prange, C.H .• Kliems. G. Bisherige Erfahrungen und aktuelle Ergebnisse xenogener
Nierentransplantation. Med. Welt. 27,42,2003,1976.
6. Cattell, V., Jamieson. S. W. Hyperacute rejection of guinea-pig to rat cardiac xenografts.
1. Pathol. 115,183,1975.
8. Kemp. E., Munk-Andersen, G., Andersen, N., Barfort. P., Jorgenscn, K.A., Larsen, S.,
Otte. K.E .. Steinbruchel, D .. Starklint, H. Experimental xenotransplantation: appropri-
ateness of model. In Hardy. M.A. (ed.) Xenograft 25. Elsevicr. Amsterdam. New York,
Oxford. 1989. p. 31.
9. Larsen. S" Stark lint. H, Diepcrink. H .. Kemp. E. Immunofluorescence microscopy in ex-
perimental renal allo- and xenografts. Transplant. Proc. 22. 1061,1990.
10. Schilling. A., Land, W .. Pratschkc, E., Pielsticker. K .. Brendel. W. Dominant role of
complement in the hyperacute xenograft rejection rcaction. Surg. Gynecol. Obstet. 142.
29,1976.
11. Miyagawa, S .. Hirose. H .. Shirakura, R., Naka, Y.. Nakata, S., Kawashima. Y., Seya. T..
Matsumoto. M .. Uenaka, A., Kitamura, H. The mechanism of discordant xenograft re-
jection. Transplantation. 46,825.1988.
12. Simonsen, M. Personal communication.
13. Kemp. E.. Steinbruehel. D .. Starklint. H .. Larsen. S.. Henriksen. I.. Dieperink. H. Renal
xenograft rejection: prolonging effect of eaptopril, ACE-inhibitors. prostaeyclin. and
cobra venom factor. Transplant. Proc. 19.4471.1987.
14. Milgrom. F. Natural antibodies and xenograft rejection. In Hardy. M.D. (ed.) Xenograft
25. Elsevier. Amsterdam. New York. Oxford. 1989. p. 149.
15. Kemp. E .• Larsen. S .. Jorgensen. K.A .. Dieperink. H .. Starklint. H. Flush perfusion of
rabbit kidneys with auto. allo and xenogeneic blood. Scand. 1. Ural. Nephrol. In press.
16. Marino. I.R.. Feria. G .. Celli. S .. Steiber. A.. Mutillo. I.. Maggiano. N .. Musiani. P ..
Perrelli. L. Hyperacute rejection of renal discordant xenograft (pig-to-rabbit): model as-
sessment and rejection mechanisms. Transplant. Proc. 22. 1071. 1990.
17. CaIne. R. Xenograft: functional definition. In Hardy. M.A. (ed.) Xenograft 25. Elsevier.
Amsterdam. New York. Oxford. 1989. p. 3.
18. Palmer. A.. Welsh. K.. Gjorstrup. P.. Taube. D .• Bewick. M .. Thick. M. Removal of anti-
Lancet. 1.10. 1989.
Chapter 24
Experimental Xenotransplantation in Nonhuman

Primates Using Distantly Related Donor Species
Y. YE AND D.K.C. COOPER
Introduction
As the ultimate goal of those involved in xenotransplantation is to be able to

transplant organs from commonly available animals such as the pig or sheep
into man, the experimental model of pig-to-nonhuman primate, probably most
closely mimics the desired clinical situation. The nonhuman primate such as the
baboon or rhesus monkey, represents the recipient patient and the donor ani-
mal is identical to that which would be used in the clinical arena, namely, the pig.
The results of allografting in nonhuman primates correlate closely with
those obtained in man, and there is little reason to doubt that xenografting in
nonhuman primates and man would follow similar patterns. Indeed, what lit-
tle evidence that is available of both concordant and discordant xenografting
in man (Chaps. 33-35) correlates well with experimental data from the nonhu-
man primate. The experimental work relating to xenografting between closely
related nonhuman primate species has been briefly reviewed in Chap. 22. In
this present short chapter, we shall review the limited experience that has been
documented using distantly related species as organ donors for nonhuman pri-
mates. Three studies in this model will be briefly reviewed.
Pig-to-Nonhuman Primate Hepatic Xenografts
These studies are also reviewed in Chap. 15. In 1968, CaIne et al. in the United
Kingdom reported seven pig-to-baboon orthotopic liver xenografts [1] (Table
24.1). All animals recovered consciousness after operation, four subsequently
dying from uncontrollable hemorrhage after 6-30 h. The remaining three ani-
mals, which were given human fibrinogen, did not bleed, but went on to die
from liver failure at 19 and 36 h and from bronchopneumonia at 3.5 days, re-
spectively. Steroids and azathioprine were administered to two baboons, and
steroids alone to two others, including the longest survivor.
Six of the xenograft livers had centrilobular liver necrosis at autopsy. The
liver from the animal which survived 3.5 days showed well-preserved hepato-
cytes, though there was some mononuclear cell infiltration of the portal tracts.
In the authors' opinion, the centrilobular necrosis could have been associated
with poor early perfusion of the liver, and may have been responsible for the
Table 24.1. Experience with pig-to-nonhuman primate organ xenotransplantation
Author/year Donor Recipient Organ n Therapy Survival

[Reference]
Caine et al./1968 [I] Pig Bahoon Liver 7 Nil/steroids/ 6 h-3.5 days

steroids+
azathioprine
Caine et al.!I 970 [2] Pig Rhesus- Liver 3 ALS <12 h
monkey pretreatment
Pig Chimpanzee Liver None 8h
Cooper et al./1988 [19] Pig Bahoon Heart 4 None (control) <8 h
3 Splenectomy <8 h
5 CsA+methyl- <2 h (x4).
prednisolone 5 days(xl)
7 Antibody <20 h (x3).
adsorption 4-5 days (x4)
4 Antibody <24 h (x3).
adsorption 4 days (xl)
+Csi\+MP
Alexandre et al.l Pig Baboon Kidnev 5 Plasmapheresis 1-22 days
19R9 [5] +ALG+CsA+MP
ALS. antilymphocyte serum; ALG. antilymphocyte glohulin; CsA. cyclosporine; MP.

methylprednisolone
hemorrhagic state seen in four animals and the liver failure that occurred in
two of the others.
Calne's group subsequently continued their studies. but used either the
rhesus monkey or chimpanzee as the recipient of the pig liver [2] (Table 24.1).
Three pig-to-rhesus liver grafts were performed. all of the recipients being
pretreated with antilymphocyte serum with differing regimens beginning 5 or
6 days before transplantation. Survival was for 12 h in all three cases. One pig
liver was also implanted into an unmodified chimpanzee. which survived only
8 h. Centrilobular necrosis was present in two livers, focal necrosis in one (the
chimpanzee liver), and necrosis, hemorrhage and fibrin deposits in the arteries
and veins in the remaining liver. Although there was no proof in any of these
four cases of consumptive coagulopathy, there was microscopic evidence,
both in the livers and elsewhere, of diffuse intravascular coagulation. This was
so extensive that the authors considered that there must have been severe co-
agulation disturbances before death.
Pig-to-Baboon Cardiac Xenografts
The Cape Town group performed heterotopic heart grafting in this model us-
ing a variety of immunosuppressive protocols [3] (Table 24.1). In brief. recipi-
ent baboons received no immunosuppressive therapy, with or without pre-
Pig-to-Baboon Renal Xenografts 391
transplant splenectomy, or therapy with cyclosporine, azathioprine and
methylprednisolone. Two final groups underwent pretransplant antipig anti-
body adsorption with or without post-transplant immunosuppressive therapy.
Baboon antipig antibody adsorption was achieved by perfusing the baboon
blood through the donor pig's kidneys before heart transplantation was car-
ried out. Each kidney was perfused for 2 h or until hyperacute rejection oc-
curred.
Neither splenectomy nor pharmacologic immunosuppression increased
mean survival time of the grafts, which in the control group was a mean of 3 h.
Histopathological features of vascular rejection were seen in every heart [3]
(Chap. 13). Pretransplant hemoperfusion of donor kidneys by the recipient
baboon, however, increased graft survival to 4 or more days in four of seven
cases; nevertheless, all four hearts showed features of vascular rejection at the
time that function ceased. The addition of pharmacologic immunosuppression
to pre transplant antibody adsorption did not lead to any further prolongation
of graft function.
The histopathological features of vascular rejection did not show the previ-
ously described classical features seen in allografts transplanted into sensi-
tized canine recipients [4]. Edema and interstitial hemorrhage were promi-
nent, but there was less evidence of intravascular coagulation than reported
previously.
Pig-to-Baboon Renal Xenografts
Alexandre's group in Belgium has to date made most progress in this difficult
experimental field (Table 24.1). Using a course of pre transplant plasmaphere-
sis, performed on a daily basis for 3 days before transplantation, to reduce or
eliminate antipig hemagglutinins, coupled with heavy immunosuppressive
therapy, they achieved survival times of 1,1,10,13, and 22 days, respectively,
in five baboons receiving minipig kidney grafts. Soluble blood group sub-
stances A and B were administered to the potential recipient at the end of the
last plasmapheresis in two baboons. The recipient's immunosuppression was
started on day-2 with rabbit antihuman antithymocyte serum (Fresenius,
Germany) 18 mg/kg per day given intravenously for 10 days. Cyclosporine (10
mg/kg per day i.m.), azathioprine (1 mg/kg per day i.v.), and methylpred-
nisolone (10 mg/kg per day i.v. during the operation, with decreasing daily i.m.
doses thereafter) were administered post-transplantation.
The experiment was terminated in two cases after 24 h due to gastric dilata-
tion and anuria respectively; in the first case the transplanted kidney was mi-
croscopically almost normal, but in the second all of the features of acute vas-
cular rejection were present. In this latter animal it had not been possible to
reduce the preoperative xenoantibodies (agglutinin) titers below 1:32.
The three remaining baboons survived with satisfactory renal function.
Two died from irreversible vascular rejection on post-transplant days 13 and
22, respectively. An increase of the heteroagglutinin titer had been observed,

however, at the time of rejection crises that were seen in both of these animals
on the 6th post-transplant day. There was also a reduction in the platelet
counts, possibly correlating with the development of microarterial throm-
boses of vascular rejection. In one animal a renal biopsy was performed at this
stage, revealing features of acute vascular rejection. Both of these early rejec-
tion episodes were successfully reversed by steroid therapy (as indicated by
normalization of the serum creatinine levels), though irreversible vascular re-
jection developed subsequently.
In the remaining baboon, the serum creatinine remained within normal
limits until the recipient's death from bronchopneumonia on the 10th post-
transplant day. Graft histology was basically normal. In this case heteroagglu-
tinins remained low during the entire postoperative course.
Comment
These three small series constitute much of our very limited experience of dis-
cordant xenografting in nonhuman primates. The results of liver xenotrans-
plantation were generally poor - perhaps surprisingly in view of the tendency
for hyperacute rejection to be milder and delayed in the liver (Chap. 15) - but
this may well have been due to technical and other factors in this early series,
performed at a time when liver allografting was in its formative stages. It
would seem timely for a further series of pig-to-nonhuman primate liver
xenografts to be studied.
The pig-to-baboon cardiac xenograft experience emphasized the lack of ef-
fect of the presently available pharmacologic immunosuppressive drugs, and
the potential of pretransplant antibody adsorption. The technique used for
such adsorption was, however, relatively crude, and many more sophisticated
possibilities exist (as discussed in Chap. 6). This study confirmed in the pri-
mate the well-documented observation in other species, e.g., in the dog, that
hemoperfusion of one donor organ can lead to significant prolongation of
function of a subsequent organ from the same donor. This principle offers po-
tential in reducing the xenoantibody titer pretransplantation, and thus induce
what has been termed "accommodation" (Chap. 6) or "adaptation" (Chap. 9)
or "anergy" (G.P. Alexandre, personal communication).
The work of Alexandre's group in which plasmapheresis was used to re-
duce the antipig antibody titers - again a relatively unsophisticated method of
attacking this problem - encourages us further that such methods, coupled
with other forms of immunosuppressive therapy, offer a reasonable prospect
of allowing long-term xenograft function. This goal has yet to be attained, and
much further experimental work is clearly required, but the studies outlined
above are three small steps towards this end.
References 393
References
1. Caine, RY., White, HJ.O., Herbertson, B.M., Millard, P.R., Davis, D.R, Salaman, J.R.,
2. Caine, RY., Davis, D.R, Pena, J.R, Bainer, H., De Vries, M., Herbertson, B.M., Joyscy,
V.c., Millard, P.R, Seaman, MJ., Samuel, J.R., Stibbe, J., Westbroek, D.L. Hepatic allo-
grafts and xenografts in primates. Lancet. 1,103,1970.
3. Cooper, D.K.C., Lexer, G., Rose, A.G., Keraan, M., Rees, J., du Toit, E. Effects of cy-
ciosporine and antibody adsorption on pig cardiac xenograft survival in the baboon.
1. Heart Transplant. 7,238,1988.
4. Caves, P.K., Dong, E., Morris, RE., Shumway, N.E. Hyperacute rejection of orthotopic
cardiac allografts in dogs foliowing solubilized antigen pretreatment. Transplantation. 16,
252,1973.
5. Alexandre, G.P.J., Gianelio, P., Latinne, D., Carlier, M., Dewaele, A., Van Obbergh, L.,
Moriau, M., Marbaix, E., Lambotte, J .L., Lambotte, L., Squifflet, J.P. Plasmapheresis and
splenectomy in experimental renal Xenotransplantation. In: Xenograft 25. Hardy, M.A.
(cd.), Elsevier, Amsterdam, New York, Oxford, 1989, p. 259.
Chapter 25
Ex Vivo Organ Perfusion Studies

in Xenograft Research
K.E. OTTE, D. STEINBRUCHEL, AND E. KEMP
Introduction
Xenotransplantation experiments can be performed either by experimental

interspecies organ or tissue transplantation in animals, or by using an artificial
circulation where donor organs can be perfused with xenogeneic blood or
blood components.
The hyperacute rejection seen after xenotransplantation probably involves
preformed natural antibodies (PNABs), enzymatic and protein incompatibili-
ty, activation of the coagulation and complement systems with platelet aggre-
gation and leukocyte activation [1,2]. Compared with in vivo models, the iso-
lated organ perfusion system is valuable in establishing the relative
significance of the various factors involved in the rejection process. Systematic
selection of different perfusates is possible and could result in accurate step-
by-step determination of the factors that lead to graft rejection and, hopefully,
define optimal treatment protocols to achieve long-term graft survival.
Experimental approaches involving human recipients with animal organs
are difficult, if not impossible, for obvious reasons. By using isolated organ
perfusion systems, however, it is possible, at least to some extent, to mimic
the rejection mechanisms in an animal-to-human organ transplantation situa-
tion.
Methods
Xenoperfusion experiments can be divided into two basically different

groups:
1. Isolated organs are connected to the circulation of an experimental animal
(Fig. 25.1).
2. Isolated organs are perfused by an artificial circulatory system (Fig. 25.2).
The first model mimics a transplantation situation and allows visualization
of the clinical condition of the "grafted organ". Specimens for histological in-
vestigations can be taken, and arterial and venous blood as well as urine (if the
organ is a kidney) are continuously available during the perfusion experiments.
Recipient pretreatment is possible, but the use of selected and standardized
perfusates is excluded, and only animal-to-animal models can be used.
396 Ex Vivo Organ Perfusion Studies in Xenograft Research
a and v
blood samples
v a saline
pressure
monitoring
animal recipient donor kidney
urine
sam pies
Fig. 25.1. Diagram of organ perfusion using an animal recipient
The second model requires an artificial circulatory system that can provide
normothermia and physiological perfusion pressure and oxygenation condi-
tions. The most optimal apparatus will remain an approximation to a clinical
situation. but is worthwhile because of the opportunitics of experimenting
with animal-to-human models and of selecting different and well-defined per-
fusates.
Historical Background
The following is a brief review of the experiments performed in this field. It is

not comprehensive. but provides an illustration of the issues and a survey of
thc findings.
Marceau ct a1. (1965) [3] uscd dogs to perfuse kidneys from pigs. monkeys.
and rabbits. Dog kidneys were similarly perfused by domestic pigs and by a
strain of miniature pig. Generally. all perfusion experiments were terminated
after a very short time (2.6-21 min). The authors assumed that the "rejection"
was of an immunological nature. and postulated that this response resulted
from the interaction of donor antigen with recipient antibody. which had to be
preformed or natural in order to explain the rapid rejection.
Mozes et a1. (1971) [4] performed a series of apparatus xenoperfusion ex-
periments in various species combinations. such as pig and shecp kidneys per-
fused with dog or human blood. Immunofluorescent microscopy (IMF) re-
vealed human immunoglobulin G (IgG). IgM. and ~-1 C deposits located in
glomerular basement membranes and blood vessels within 30-40 min in both
pig and sheep kidneys. They concluded that they were able to mimic renal
Historical Background 397
a. and v.
blood samples
saline
37°
pressure
contro l
I r _ p u r i n e samples
heated
water 37°
'--------+-----:--11 blood
reservoir
roller
pump
hollow fiber dialyzer
95%°2
5%(°2
Fig. 25.2. Diagram of organ perfusion system with pulsatile flow, control of the different
compartments, oxygenation of residual blood volume, pressure control, and access for arte-
rial and venous blood and urine samples
xenograft rejection by perfusion, and prompt rejection was the result in all the
combinations studied. Their work supported the concept that the mechanisms
involved in the rejection process were initiated by the formation of antigen-
antibody complexes with secondary activation of complement factors, result-
ing in vasospasm and endothelial destruction.
Land et al. [5] studied rat kidneys perfused in an apparatus using whole
blood from rabbit, guinea pig, cat, sheep, pig, and man. The perfusate consist-
ed of 10 ml blood mixed with 14 ml Ringer's solution. Hyperacute xenogeneic
rejection (using both hemodynamic and histological rejection criteria) oc-
curred within a few minutes using blood from rabbits, guinea pigs and cats; a
more delayed type of rejection (40-80 min) occurred when blood from sheep
and pigs was used. No signs of rejection were noted during the 2-h observation
period when they used human blood. The authors investigated the titers of
preformed antibodies against rat erythrocytes in all the species involved and
found positive - but different - titers throughout. No correlation was found
between the titer magnitude and perfusion time.
Hammer et al. [6] perfused cat kidneys by dogs and found cat kidney func-
tion time to range from 10-15 h. They concluded that PNABs triggered hu-
moral and cellular factors leading to progressive organ destruction. The hy-
peracute rejection in this model within one zoological order. but different
zoological families. was more delayed than that found between more distantly
related species.
Pratschke et al. [7] perfused isolated rat kidneys with adult dog whole
blood. neonatal dog blood without PNABs. and adult dog blood after absorp-
tion of antibodies. Hyperacute xenogeneic rejection was at least partially in-
duced by nonimmunological complement activation. independent of PNABs.
Schilling et al. [8]. using the same model but further fractionating the dog
blood. found that PNABs only produced rejection when leukocytes and
platelets were present.
Rossmann and Matousovic [9] addressed the issue of whether a human "re-
cipient" was capable of rejecting highly discordant xenogeneic tissues as effec-
tively as lower animals do. Rabbit kidneys were perfused with human whole
blood and changes in the renal ultrastructure were studies. Moderate focal
platelet aggregation appeared after only 30 min perfusion. Massive disruption
of the vessel endothelium and intracapillary blood elements was present after
15 and 30 min. respectively. Immediate vascular fixation of human antibodies
and complement was also demonstrated. and the authors concluded that man
appears to be endowed with potent destructive tools to reject xenogeneic tis-
sues.
Jorgensen et al. [10] perfused rabbit kidneys with human whole blood and
found deposition of IgG in glomerular capillaries. degenerated and detached
endothelial cells, and leukocyte infiltration. Besides the inflammatory cells.
tightly packed platelets and red blood cells were found both in the glomerular
capillaries and in the parenchymal vessels. The main factor in the hyperacute
rejection. which led to a total cessation of blood flow within 30 min in all the
experiments. could not be ascertained. These results led to a further series of
experiments [11] using (i) leUkocyte-poor, platelet-rich blood, (ii) Icukocyte-
rich, platelet-poor blood. or (iii) leUkocyte-poor. platelet-poor blood. The ef-
fect of removing leukocytes was insignificant. but removal of platelets delayed
rejection. Adding prostacyclin to human blood gave similar results in terms of
improved graft survival [12], but rejection, nevertheless, occurred.
Summing up these experiments. we can conclude that discordant organs
appear to be rejected rapidly as a result of the presence of preformed antibod-
ies in the serum of the blood used for the artificial perfusion. IgG and IgM anti-
bodies adhere to the endothelial surface of the donor organ and activate the
complement system which, in turn. promotes platelet aggregation and leuko-
cyte activation.
These experiments clearly demonstrate the impact of the multitude of reac-
tions that occur during xenograft perfusion. and indicate the difficulties that
Construction of Xenoperfusion Apparatus 399
lie ahead before animal organs can be used successfully in man. Use of closely
related species seems to offer the greatest possibility of overcoming these re-
jection mechanisms. Higher nonhuman primates, such as the gorilla or chim-
panzee, are in short supply themselves, and the use of organs from these ani-
mals for xenoperfusion experiments or transplantation in man is not realistic.
Our attention and research must, therefore, be directed towards more distant-
ly related species. Donor organs must have a suitable size and anatomy for hu-
man beings - the pig, sheep, and goat are obvious possibilities.
Construction ofXenoperfusion Apparatus
The problems of using an apparatus for xenoperfusion experiments are nu-

merous. Blood circulation through the apparatus may itself lead to a number
of reactions which interfere with - and, indeed, simulate - the rejection mech-
anisms, and must, therefore, be taken into consideration.
One of the major obstacles of this type of study is the bioincompatibility of
the artificial inner surfaces of the apparatus. In order to maintain a sufficient
perfusion pressure in the organ, a mechanical pumping device must necessari-
ly be incorporated within the circulatory circuit. The temperature should be
physiological throughout the system in order to facilitate normal metabolism
in the perfused organ. Sterility and freedom from pyrogens are essential to
maintain normal kidney function. Sterilization of all plastic tubing and reser-
voirs is, therefore, necessary. Antibiotics are undesirable because of possible
side effects.
In our laboratory we have carried out a series of experiments without in-
volving an organ. Human blood, under approximately physiological condi-
tion, was circulated in the apparatus in order to establish the damage and side
effects of the artificial circulation itself. The results are shown in Table 25.l.
Roller pumps were used to maintain a physiological organ perfusion pres-
sure. This led to a certain degree of destruction of red blood cells, demonstrat-
ed by an increase in plasma hemoglobin and serum lactate dehydrogenase
(LDH). The plasma hemoglobin level dose over the course of3 h to values that
probably impair kidney function. The plastic tubing constituted another haz-
ard. We used polyvinyl chloride and silicone rubber; the surface of these mate-
Table 25.1. Changes in blood chemistry during apparatus perfusion using human blood
Perfusion Plasma Serum lac- Leukocytes Platelets Comple- Comple-

time hemoglo- tate dehydro- (x1Q9/l) (x10 9/1) mentC3d mentC4
bin (U/I) genase (U/I) (U/I) (U/I)
Control 0.9± 1.1 360.2± 90.5 5.64±0.84 237.8± 7.6 31.0± 4.9 0.78±0.14
90 min 28.9±17.5 564.6±176.5 5.22±O.78 203.6±11.2 39.8± 8.0 0.70±0.14
180 min 67.7±31.8 736.6±178.5 4.83±O.92 209.0±19.4 64.0±18.3 0.77±O.17
rials does not equal that of the "'gold standard", namely vascular endothelium,
resulting in a number of adverse reactions. The leukocytes were activated and
liberated enzymes. This phenomenon has also been demonstrated previously
[13]. In our experiments, a slight loss of leukocytes took place (Table 2S.1),
and flow cytometric analysis demonstrated a proportional loss of B cell and
T cell subsets.
Complement activation also occurs when blood is exposed to artificial sur-
faces [14]. We demonstrated no loss of C4, but a significant increase in C3d,
which led to the conclusion that the complement system is mainly activated by
the alternative pathway in our apparatus. Platelets aggregate when exposed to
artificial surfaces [IS]. Platelet aggregation tests were performed by methods
described earlier (Whole-Blood Aggro-Meter ModeISOO) [16]. But we could
not demonstrate any significant loss of platelets during perfusion (Table 2S.1).
An example from one of our perfusion experiments with human blood in
the absence of an organ is shown in Fig. 2S.3. Before perfusion was started, a
normal response to the addition of adenosine diphosphate was observed, with
aggregation of 6S% of the platelets within 90 s (Fig. 2S.3a). After 90 min of per-
fusion, the aggregation pattern had changed; 3S% of the platelets were aggre-
gated within 90 s (Fig. 2S.3b). After 180 min, only 30% aggregated within 90 s;
a total disaggregation was observed after 240 s (Fig. 2S.3c). A substantial loss
of platelet aggregability during perfusion in our apparatus has, thus, been es-
tablished.
To illustrate the use of the apparatus in the perfusion of an organ, we will
describe two experimental series from our laboratory. The first investigation
protocol addressed the issue of whether ABO blood group compatibility is im-
portant in xenoperfusion experiments using human whole blood and pig kid-
neys [17] (and, therefore, whether ABO compatibility is important in discor-
dant xenografting). Pig kidneys were perfused with human blood of red cell
type AB (n=S) or type 0 (n=S). Perfusion was maintained for 60 min or until
the blood flow decreased to below 2 mllmin. Flow characteristics were equal in
the two groups. No differences in LDH, gamma-glutamyltransferase (GGT),
or plasma hemoglobin were observed. The predominant histological finding
in all kidneys in both groups was platelet aggregation, indicating hyperacute
rejection independent of ABO compatibility.
Pig blood was sampled and epiotyped using highly selected human alloan-
tisera. Pig red cells could be generally characterized as A-, B-, or AB-like. By
contrast, ABO typing using highly specific monoclonal anti-A and anti-B anti-
bodies was clearly negative, leading to the conclusion that the reactions seen
with alloantisera were not due to A- or B-specific antibodies per se, but rather
to unspecific human antipig xenoantibodies. Screening of sera from all pigs
against a panel of human red cells, using techniques to disclose completely (as
well as incompletely) reacting antibodies, was clearly positive throughout, in-
dicating the presence in pig serum of xenoantibodies against human red cells.
The study, therefore, indicated no relevance of the human ABO blood
group antigens, nor of human alloantibodies of this system, in xenoperfusion
experiments using human blood and pig kidneys.
Construction of Xenoperfusion Apparatus 401
% Transmittance % Transmittance
o o
10 10
20 20
30 30
40 40
50 50
60 60
70 70
80 80
90 ADP 90 ADP
3"mol/l 100 3"mol/l
100
30 60 90 120 150 180 210 240 30 60 90 120 1SO 180 210 240
a Ti me , sec. b Time , sec.
0/0 Transmittance
0
10
20
30
40
50
60
70
60
ADP
90
3"mol/ l
100
30 60 90 120 150 180 210 240
c Time , sec.
Fig. 25.3 a-c. Adenosine diphosphate (ADP)-induced platelet aggregation performed on
human blood a before apparatus perfusion (control), b after 90 min perfusion , and c after
180 min perfusion
The second series of experiments involved the addition of a specific

platelet-aggregating factor antagonist (BN 52021 , a natural product extracted
from the leaves of the temple tree, Ginkgo Biloba) to human blood perfusing
rabbit kidneys [18] . Five rabbit kidneys were perfused by human blood with
the addition of BN 52021, and five rabbit kidneys were perfused without this
added extract. Renal blood flow decreased to less than 2 mllmin within 25 min
in all experiments, with no difference between the two groups. Platelet aggre-
gation was normal in the five control experiments both before and after perfu-
sion. When BN 52021 was added, platelet aggregation occurred neither before
nor after perfusion. However, light microscopy revealed so-called "platelet
aggregates' in the glomeruli of all perfused kidneys regardless of the addition
ofBN 52021.
Thus, it would seem that very strong platelet-aggregating factors must be
present in the kidneys during the rejection process, and that rejection could
not be prevented or delayed by the addition ofBN 52021.
In conclusion, xenoperfusion experiments using an artificial circulatory
system that mimics a clinical transplantation situation between distantly relat-
ed species, e.g., pig organs to human recipients, are valuable because of the
ethical and practical reasons mentioned earlier. The results, however. should
be interpreted with caution because of the numbers of potential adverse reac-
tions that interfere with organ function and simulate rejection mechanisms.
Apparatus perfusion should be continued for only a limited time period. A 1-h
maximum is suggested in order to work with the least "damaged" biologic ma-
terial.
Looking to the future, xenoperfusion experiments are valuable for animal
donor-to-human recipient experiments for the reasons mentioned earlier. In
order to be able to compare and reproduce the results obtained by the differ-
ent centers performing xenoperfusion, uniform apparatus construction and
technical procedures would be advantageous. To overcome the major im-
munological obstacles, immunosuppressive drugs that are far more specific
and potent than the drugs known at this moment must be applied.
References
I. Hammer, C Isehemagglutinins and preformed natural antibodies in xenogeneic organ

transplantation. Transplant. Proc. 19.0,4443.19/1,7.
2. Miyagawa, S., Hirose. H., Shirakura, R., Naka, Y., Nakata. SOo Kawashima. YOo Seya. T.,
Matsumoto, MOo Venaka, A., Kitumara, H. The mechanism of discordant xenograft re-
jection. Transplant. Proc. 40, 0. /1,25.19/1,/1,.
3. Marceau, J.P., Hallenbech, G.A., Zollman, P.E., Butler, H.C. Shorter. R.G. A compari-
son of autoplastic, allogeneic and xenogeneic perfusion of isolated kidneys. J. Surg. Res.
5, II. 492. 1965.
4. Mozes. M.F., Gewurz. HOo Gunnarson, A., Moberg, A.WOo Westberg. N.G., Jctzer, T ..
Najarian, J.S. Xenograft rejection by dog and man: isolated kidney perfusion with blood
and plasma. Transplant. Proc. 3. 1.531. 1l)71.
5. Land, W., Corell. J., Pielsticker, K., Brendel, W. Renal in vitro xenohemoperfusion in
different species combinations (in German). Res. Exp. Med. 15l), 276, 1l)73.
6. Hammer. C, Chaussy. C. Krebs. GOo von Scheel, 1.. Brendel. W. Experimental xeno-
geneic kidney transplantation in closely related systems. Res. Exp. Med. 160.32. 1l)73.
7. Pratschke, E., Land, W .. Schilling. A.S., Pielsticker, K.. Brendel, W. Further in vitro
studies of the hyperacute xenogeneic rejection mechanism (in German). Langenbecks
Arch. Chir. Supp!. Chir. Forum. 12l), 1975.
References 403
8. Schilling. A., Land, W., Pielsticker, K., Aldenhoff, J, Brendel, W. Experimental
xenografting in widely divergent species: interaction of humoral factors in hyperacute
xenograft rejection in the rat-dog system (in German). Res. Exp. Med. 165,79,1975.
9. Rossmann, P., Matousovic, K. The production oflesions in the rabbit kidney by xenoper-
fusion with fresh human blood. Virchows Arch. Path. Anat. Histol. 382, 95, 1979.
10. Jorgensen, K.A., Kemp, E., Barfort, P., et al. Xeno- and autoperfusion ofrabbit kidney.
Acta. Path. Microbiol. Immunol. Scand. Sect. A. 93, 305,1985.
11. Jorgensen, K.A., Kemp, E., Barfort, P., Starklint, H., Larsen, S., Munk-Andersen, G. On
the role of platelets and leukocytes in renal xenoperfusion. Acta. Path. Microbiol.
Immunol. Scand. Sect. A. 94,223,1986.
12. Jorgensen, K.A., Kemp, E., Barfort, P., Starklint, H., Larsen, S., Munk-Andersen, G.
Platelet aggregation is not essential for xenograft rejection. Thromb. Res. 43, 87,1986.
13. Wachtfogel, Y.T., Kucich, U., Greenplate, J., Gluszko, P., Abrams, W., Weinbaum, G.,
Wenger, R.K., Rucinski, B., Niewiarowski, S., Edmunds, L.H., Colman, R.W. Human
neutrophil degranulation during extracorporeal circulation. Blood. 69, 1,324, 1987.
14. Knudsen. F. Assessment of complement activation during extracorporeal circulation by
measurement of complement split C3d. Blood Purification. 5. 162. 1987.
15. Hennesy, V.L. JR., Hicks, R.E., Niewarowski, S., Edmunds, L.H. JR., Colman, R.W.
Function of human platelets during extracorporeal circulation. Am. J. Physiol. 232,
H622.1977.
16. Triplett, D.A. Platelet Function. Laboratory Evaluation and Clinical Application.
Educational Products Division, American Society of Clinical Pathologists. Chicago,
1978.
17. Ottc, K.E., Andersen, N., Jorgensen, K.A., Kristensen. T., Barfort, P., Starklint, H.,
Larsen. S., Kemp, E. Xenoperfusion of pig kidney with human AB or 0 whole blood.
Transplant. Proc. 22, 1090, 1990.
18. Otte. K.E., Jorgensen, K.A., Bonnevie, V., Barfort, P., Larsen, S., Starklint, H., Pirotzky,
E .. Kemp. E. Xenoperfusion of rabbit kidney and the impact of BN 52021, a spccific an-
tagonist of platelet-activating factor. Transplant. Proc. 22, 1088,1990.
Chapter 26
Effects of Formed Elements on Xenograft

Rejection in an Ex Vivo Organ Perfusion Model
B.A. BRYAN, M.L. HENRY, L.K. HAN, D.D. SEDMAK, AND R.M. FERGUSON
Introduction
The modification (or abrogation) of the hyperacute rejection (HAR) process

has been one of the major areas of study by xenotransplantation researchers
since work began in this area in the 1960s. Manipulations of blood cellular and
humoral components as well as the use of pharmacologic agents have all been
studied extensively in the past.
The study of ex vivo xenograft organ perfusion stemmed initially from the
desire of early investigators to break down and isolate the contribution of indi-
vidual blood effector components to the HAR process. Almost all work to
date has dealt with the use of kidneys as the xeno organ. The use of an ex vivo
(in vitro) model allows for a more controllable environment for an individual
organ, where the "host" is usually a mechanical perfusion system. The perfu-
sion parameters of that organ can be modified according to the investigators'
study design and data easily collected.
There are relatively few isolated in vitro organ perfusion studies in the liter-
ature. In 1971, both Mozes et al. [1], using a constant perfusion circuit, and
Land et al. [2], using a flow-rate controllable roller-pump perfusion set-up,
published the first articles utilizing this particular model. Recently, Otte et al.
[3,4] (Chap. 25), reported two studies also dealing with the isolated ex vivo
perfusion of porcine kidneys with an oxygenated pressure-flow feedback de-
vice. Variations of the in vitro organ model, usually involving the use of silastic
catheters connecting the recipient's femoral vessels to the xeno organ's artery
and vein, have been used by others [5-10].
The validity and reliability of the in vitro model have been shown in previ-
ous studies. The gross morphological changes, histological picture, renal
blood flows (RBFs) and urine outputs have all been comparable to the in vivo
models in terms of degree and timing [1,2]. In the donorlrecipient pig-to-dog
model, perfusion of isolated porcine kidneys with canine whole blood (WB)
results in mottling and cyanosis within 5 min and cessation of urine output
and RBF within 10 and 20 min, respectively [1,2]. Renal vascular resistance
also rises dramatically after WB perfusion, reflecting the glomerular and ar-
teriolar thrombosis that is the hallmark of the HAR process [1, 7,8]. The cel-
lular composition of the vascular thrombi is also similar, with polymorpho-
nuclear cells (PMNs) and platelets (PLTs)being the predominant cell types
present [1,2,5].
406 Effects of Formed Elements on Xenograft Rejection
The perfusion of canine plasma alone through porcine kidneys has generat-
ed inconsistent data. Y oshiaka et al. [6], perfusing for up to 330 min, noted
maintenance of good urine outputs and a slow but steady increase in the renal
arterial pressure. Histologically, only focal and segmental glomerular mesan-
gial fibrin deposits were noted and immunofluorescence revealed deposition
of immunoglobulin G (IgG), complement and fibrinogen with glomeruli.
Subsequent addition of formed cellular elements promptly resulted in a mor-
phological and histological picture of HAR. Sakai et al. [10] also demonstrat-
ed in the rat-to-dog model that ex vivo perfusion with plasma alone resulted in
no structural changes. Linn et al. [5], however, using unmodified cell-free sera
noted an immediate and marked rise in the kidney's vascular resistance as well
as capillary endothelial basement membrane damage after 30 min.
Using heat-inactivated (i.e., decomplemented) sera, however, neither func-
tional nor structural changes were found. Mozes et al. [1] found an initial sharp
decrease in RBF to 50% within 1-2 min following canine plasma perfusion of
porcine kidneys; the flows subsequently, however, remained constant or even
increased. Perfusion of washed dog cells suspended in culture medium (i.e .. no
xenoantibody) resulted in interstitial edema but no rejection. Other modifica-
tions, including ethylenediaminetetra-acetic acid (EDT A) chelation and im-
munoadsorbtion of dog xenoantibody with pig red blood cells (RBCs) or kid-
ney antigen have resulted in two- to fourfold prolongation in graft survival.
Land et al. [2] using the rat-dog model, perfused with isolated cellular com-
ponents. They found that canine RBCs in a plasma substitute maintained good
RBFs for 120 min, and caused only mild to moderate endothelial damage in
the glomeruli and arterioles. Addition of either leukocytes or PLTs had little
effect, but addition of supernatant of disrupted PLTs (obtained by centrifuga-
tion) to plasma resulted in prompt cessation of RBF and severe endothelial
damage. Perfusate consisting only of serum and intact RBCs caused a delayed
onset of the rejection process.
Therefore, despite and deposition of xenoantibody (IgG and IgM), com-
plement, and fibrin on glomerular and arteriolar endothelial cells, plasma per-
fusion alone is unable to bring about HAR.
The results presented above are confusing, and deal with a host of different
models. The roles of plasma, complement, and formed blood elements in
HAR are also not well defined. In an effort to resolve the inconsistencies not-
ed in previous literature and further elucidate these specific roles, we designed
a series of experiments, using cellular and humoral components, in an ex vivo
organ perfusion system.
Over the past 5 years, the model used for the ex vivo studies conducted at the
Ohio State University has involved the perfusion of porcine kidneys with ca-
nine blood components. This cross-species, discordant transplant combina-
tion provides for a rapid violent rejection process which has been extensively
Materials and Methods 407
studied [5-7,9,11-24]. The mechanical pump used in this model has been the
Waters kidney perfusion device (Waters Instruments Inc., Rochester, MN).
This self-contained unit provides manometrically monitored, consistent pul-
satile organ perfusion in a sterile cassette. Controllable parameters include
stroke volume, oxygen content, and perfusate temperature. This design has
not only been used for kidneys but also for liver and splenic perfusion studies
[25,26].
Mongrel dogs weighing 25-35 kg. were anesthetized with pentobarbital
and blood was collected into either conicals or syringes containing citrate
(5:1 v/v) , or heparinized conicals (100 U/ml). Heparinized WB was stored at
room temperature and used within 6 h. PLT-free plasma was obtained by cen-
trifugation. PLTs, RBCs, and peripheral blood mononuclear cells (PBMCs)
were isolated from citrated blood using Ficoll-Hypaque differential centrifu-
gation. Dextran (6%) was added to the syringes (1:9 dextran:blood) and these
were allowed to stand upright for 1-3 h, where gravity separation yielded a
PMN-rich plasma. After this plasma was separated by Ficoll-Hypaque cen-
trifugation, the supernatant was removed and whole PMNs were isolated from
the pellet after RBC lysis.
Homogenized PMNs were obtained using freeze-thaw and douncing cell
lysis techniques in the absence of a proteinase inhibitor. PMN membranes
were isolated according to a modification of the technique of Radka [27].
Briefly, whole PMNs were lysed in a hypotonic phosphate buffer containing
phenyl methyl sulfinyl fluoride (PMSF), a proteinase inhibitor, and cen-
trifuged to separate nuclei. The membranes were then isolated from the super-
natant by ultracentrifugation on a sucrose gradient. The initial three-compo-
nent perfusion studies did not include PMNs. These cells were subsequently
added during later perfusion experiments because previous (historical) evi-
dence suggested the importance of PMNs on histological examination. All cel-
lular components were stored either on ice or at room temperature and were
used within 4-6 h of isolation.
Domestic white pigs weighing 30-40 kg. were anesthetized with pentobar-
bital, then heparinized with 10 000-15 000 U and both kidneys harvested un-
der sterile conditions. The first kidney was flushed with 250-500 ml of either
lactated Ringer's (LR) solution or Belzer's U.W. (DuPont, Inc.) solution at
4°C. These kidneys were all used within 6 h. The second kidney was flushed
with LR (4°C) until clear and then immediately placed in the perfusion unit.
Overall warm ischemia time for both kidneys was less than 30 min. Baseline
pre perfusion cortical wedge biopsies were taken.
The arterial perfusion line of the Waters unit was connected to the renal
artery via a metal cannula and the venous effluent was collected in a graduated
reservoir. Once perfusion was initiated, all kidneys were allowed to achieve a
steady state, consisting of stable flows and resistances, prior to the introduc-
tion of additional blood components. Frequently, vasospasm was encountered
upon initial perfusion, as reflected by quickly rising perfusion pressures.
Verapamil, at dosages of 2.5-5.0 mg was added to the upper reservoir, and usu-
ally promptly resulted in lowering of the pressures to near-baseline levels.
Plasma volumes averaged 350-500 ml; cell fractions were added to either
the venous reservoir or the pump reservoir as a bolus. Cell fraction volumes
varied according to the number of cells obtained and the volume of buffer in
which the cells were suspended. The average total cell count for PBMCs was
l.1xlO Y (1.2x10 7 to 0.92xlO Y) and for PMNs was 3.8xlOx (1.1 - 5.0xlOX).
Hematocrits were taken 10-20 min later, and, if less than 20, additional blood
was added. To overcome the calcium-binding effect of the citrate used in the
cell isolation procedures, 500-1000 mg calcium chloride was added to the sys-
tem prior to the addition of PLTs. In previous work, intense fibrin production
was noted after the addition of calcium, therefore heparin (1500 U) was also
added prior to the PLTs.
Those kidneys SUbjected to prolonged warm ischemia time or technical dif-
ficulties that made very little «0.2 m1lmin) or no urine and/or had initial resis-
tances greater than 6 were excluded from the study.
Perfusate temperature was controlled by a water bath and was kept be-
tween 36°C and 38°C. Venous flows were measured directly, and subsequent
calculations of arterial resistance were made by dividing the mean pulsatile
pressure (MPP) by the venous flow (ml/min). The stroke volume, after initial-
ly being set to provide a MPP of 60-70 mmHg, remained constant for the dura-
tion of the experiment. Urine was collected via a small caliber polyethylene
catheter placed in the ureter.
Readings, including mean arterial pressure (MAP) (mmHg), venous flow,
vascular resistance, and urine output (m1lmin) were obtained every 10 min.
Cortical wedge biopsies were obtained preperfusion and at variable times dur-
ing the experiment for trichrome and periodic acid-Schiff (PAS) staining.
Experiments were terminated when the end resistances stabilized or reached
values greater than 10 (usually a flow <15 m1lmin, depending on MAP), or
urine production ceased.
Results
To date, our group has performed over 70 ex vivo single kidney perfusion ex-
periments. Groups are formed on the basis of the types of blood components
used as the perfusate through porcine kidneys and are listed in Table 26.1.
Various time, resistance and color change data for all groups are listed in
Tables 26.2 and 26.3.
Group 1 (n=43) and Group 2 (n= 10) represent controls of kidneys perfused
with both porcine and canine plasma alone (Fig. 26.1). Even after a mean per-
fusion time of 60-70 min, the resistances were comparable and remained sta-
ble at low levels for both types of plasma. The urine outputs also remained
brisk, ranging from 3.5-6 m1lmin for the entire perfusion time. Three kidneys
in the heterologous group were perfused for greater than 195 min, and had end
resistance values of 1.3,1.3 and 1.4. Histologically, mild tubular damage, con-
Results 409
Table 26.1. Porcine kidney perfusion groups based on types of blood components used
Group Autologous blood Heterlogous blood
Plasma Cells Whole Plasma Cells Whole Polymorphs

Blood Blood
1 +
2 +
3a + +
3b + +
4 +
5a + +
5b + +
5c + +
6 + + +
7a + (1) +
7b + (2) +
8a + + (3)
8b + + (4)
(1), without calcium; (2), with calcium; (3), homogenized; (4), membranes
Table 26.2. Various perfusion and addition times in individual kidney groupsa
Group Mean total perfusion Time of addition Time from last addition
Time (range) of components to termination of
experiment
1 66(20-240)
2 59 (35-80)
3a 137 (90-270) 114 23
3b 87 38 49
4 98
5a 226 (135-370) 74,130,177 49
5b 160 (140-200) 53,84,116 44
5c 220(190-250) 70,130,165 55
6 230(200-240) 36,75,115,165 81
7a 200 (160-240) 40,80 72
7b 120 42,89 27
8a 155 (110-195) 43,68,88,108 42
8b 185 (120-250) 118, l33, 148, 173 5
a All time periods are expressed in minutes.
sisting of dilatation and brush border (BB) effacement, was noted after 30 and
60 min of xeno plasma perfusion. The porcine plasma perfusion kidneys
demonstrated only minimal tubular dilatation after 30 min.
Group 3a (n=3) and Group 3b (n=2) were perfused with plasma followed
by WB. The heterologous Group 3a (i.e., xenoplasma and WB) had mean ini-
tial resistances that were stable and ranged from approximately 2 to 2.5
410 Effects of Formed E lements on Xenograft Rejection
Table 26.3. Mean resistance val ues and time of onset of color changes in kidn ey groups
Group Resistances Pe riod before Period before

gross development
Initial End Change co lor change of hematuria
0.8 1.2 +0.4

2 1.4 0.8 - 0.6
]a 1.0 9.2 +S.2 II
3b 1.6 1.0 -0.6
4 1.2 5.3 +4.1 23 45"
Sa 0.9 6.3 +5.4 14 13
5b 1.3 1.0 -0.3 NR 4"
5c 1.3 1.9 +0.6 40 NR
6 0.5 2.4 + 1.9 22 33
7a O.S 1.2 +0.4 S3
7b 0.7 10 +9.3 23 IS
Sa 0.5 7.5 +7 .0 40 NR
Sb 0.5 10 +9.5 5 5
NR. not recorded

a
Remained clear in two kidneys.
b Noted in one kidney on ly.
1o r-----------------------------------~ 10
8 8
R
E
S·
6 6
I .... 0 . .••. .
/~:::::::::::::::::··.:a/~ . · ··· · · . · · . . . >.

S
T
A 4 tf .... .. ·Oil 4
N
C
E 2 2
~~·_··________~~________~__________~ O
Oe-
o 30 60 90
TIME
- Heterologous(N-43) -+- Aulologous(N-10)

.. " .. Heterologous UfO ··G·· Aulologous UfO
Fig. 26.1. Renal artery resistances and urin e outputs (UI O) during the ex vivo perfusion of
porcine kidneys with e ither heterologous or a ut ologous plasma a lo ne
(Fig. 26.2). After addition of WB. however. the mean resistances climbed four-
fold to 9.2. within 23 min. The overall change in resistance (from beginning to
end) was 8.2. The urine output. after reaching a maximum of 8.3 . was 4.0
mllmin prior to WB addition. After WB was added, the urine output dropped
to 0.5 ml/min (an eightfold decrease) within 23 min. These kidneys became
mottled within 11 min upon WB addition. and at the end were black in color.
The urine. however. remained clear yellow. Biopsies taken 10 min after WB
Results 411
10 10
8 ..*- '. 8
R. '.
E ".
S
". .....
I 6 .- 6
U
S
T
. ". f
A 4 .. 4
0
N
C
E
2 2
0
.. 0
0 30 60 90 120 150
TIME
- Resistance(N-3) -+-- UfO
Fig. 26.2. Mean resistances and urine outputs (UIO) when porcine kidneys were perfused
initially with canine plasma followed by canine whole blood; #, the time of addition of whole
blood
10 10
8 8
R
E
#
s 6 6
1
I S ..
S 'n _ U
T ." f
0
A 4 .. Ek... 4
N " "El "EJ
C .• D "
E .'
2 .r::r' 2
0 0
0 30 60 90
TIME
-+- Resistance(N-2) --G--- UfO
Fig. 26.3. Resistances and urine outputs (UIO) of porcine kidneys perfused with autologous
(porcine) plasma and whole blood; #, the mean time of addition of whole blood . Note preser-
vation of low resistances and good urine production (compared with Fig. 26.2)
perfusion demonstrated moderate to severe tubular damage, extensive per-

itubular venule and arteriolar thrombosis with PMN- PLT-RBC clots. but
overall preservation of glomerular architecture.
The Group 3b kidneys (Fig. 26.3) served as controls and were perfused with
autologous plasma and WB. Although demonstrating mild fluctuations after
the addition of WB. resistances remained less than 2 for 50 min, and the
change in resistance values was negative (-0.6) (probably reflecting initial
vasospasm). Urine production remained clear in color and greater than
3 ml/min. The kidney itself did not undergo any gross morphological changes
and histologically there were no structural changes seen.
10 10
8 8
R
E'
S 6 6
I U
S I
T
A
o
4
N
""'-- - ,..-:::
... iI>"=-~......... ,....,.......... .
C
E
....
2 .... ~.~ --- 2
...... ....
O~
....
__L -__L -__L -__ L-~L-~L-~ __ ~ __ ~O
o ro w ~ ~ ~ ~ ro ~ ~
TIME
- Resistance(N-g) ,+ .. UfO
Fig. 26.4. Pe rfusion of porcine kidn eys with canine wholc blood alone, A slow progressive
rise in resistances and overall maint enance of urine output (VI O) are demonstrated
Group 4 (n =9) represents those kidneys perfused with WB alone

(Fig. 26.4); WB was used as the sole perfusate in six kidneys. while a prior crys-
talloid solution was used in three. The use of an initial crystalloid perfusate
was designed to allow for stable conditions prior to WB perfusion . Mean resis-
tances rose initially from 1.2 to 3.5 in the first 20 min. and then slowly reached a
maximum of 5.3 over the remaining 70 min. The change in resistances from be-
ginning to end was 4.1. half of the Group 3a value. and the time to reach th e
maximum resistance was almost four times as long as in Group 3a. Mean urine
outputs for Group 4 were steady at 3.2 and 3.0 ml/min at 45 and 90 min. respec-
tively. Gross color changes were seen in the kidney within 23 min. Hematuria
was noted 45 min after WB perfusion and interestingly did not occur at all in
two experiments.
Some differences exist. however. between th e individual experiments with-
in this group. The first three kidneys perfused with WB as the sole perfusate
underwent apparent classical HAR with a prompt rise in res istance to greater
than 10 and poor urine output «0.2 ml/min) within minutes. whereas the sub-
sequent six kidneys appeared to show a delayed HAR process. with a slow and
steady rise in resistances (maximum 5.5) . and good urine production which
ranged from 1.5 to 9.2 ml/min (data not shown).
There were also differences microscopically between the individual sub-
groups. Those kidneys which displayed marked initial resistance increased
were characterized by diffuse glomerular and arteriolar thrombosis as well as
mild. patchy interstitial hemorrhage (IH). whereas those which underwent a
slow. steady rise in resistance demonstrated a lesser degree of thrombosis.
with 40%-60% involvement of the glomeruli and 30% - 50% arteriolar in-
volvement. As expected. the longer the WB perfusion in the latter kidneys. the
more histological damage was noticed in the tubules and interstitium.
Group 5 includ es all those kidneys perfused with plasma and subsequent
cell components. Group 5a (n = \3) were the experimental kidneys and under-
Results 413
10 10
8 8
R # # #
E
s
S
T
I 6
1 1 1 6
I
U
A 4 /*" ... .......... 4

0
N
C " 'lI! '
'*
:
E
2 2
0 0
0 30 60 90 120 150 180 210 240
TIME
- Resislance(N·13) ..... .. UIO
Fig. 26.5. Resistances and urin e outputs during perfusion of porcine kidneys perfused with
canine plasma followed by random cell component addition (PLTs, PBLs, RBCs) in succes-
sion ; #, the mean times of addition of cell components in various orders. Note the slow rise in
resistance until the last cell fraction is added. and minimal drop in urine output (VI O)
10 r---------- ---------- - - -- - - - - - - - - - - - , 10
8 8
R
E
S
I 6 6
S # # U
-1- _. --- !
I
T
A 4 4
o
N
C
E
:2
~--L---L---L---L---L---L---L---L---LJ O
30 60 90 120 150 180 210 240 270

TIME
- Resislance(N·4) ··*·· UIO
Fig. 26.6. Breakdown of Fig. 26.5 showing those porcine kidneys perfused with the canine
cell component sequence of (i) PBLs, (ii) PLTs, (iii) RBCs; #, the mean times of addition of
each cell type; VlO, urine output
went xeno plasma perfusion and subsequent cell fraction additions in various
orders (Fig. 26.5). As can be seen in Table 26.3, total mean perfusion time was
long for this group. The resistances were stable and less than 1.6 up to point 2,
increasing to 2.6 at point 3, and then increased to 6.3 over the remaining 50 min
- a 2.4-fold (242% ) increase over the resistance prior to the addition of the last
component and an almost sixfold (573%) increase from the initial perfusion
resistances. Color change was noted in these kidneys after the last component
was added. Urine output ranged from 2.6 to 3.8 ml/min over the entire perfu-
sion time. The urine output decreased somewhat (30%) from 3.5 to 2.4 ml/min
10 10
. 1/7. :.-.. - !--

8 8
R.
E
# # #
/~.-
S
I 6 6
U
S I
T
A 4
: '.' .. :!it...
4
o
- .:- ---· ··-O::~.7~ --
N
C
E
2 2
O*-----L-----L-----L---~L---~----~ O
o 30 60 90 120 150 180

TIME
- Resistance(N-6)""" UIO
Fig. 26.7. Those porcine kidneys perfused with the canine cell component sequence of (i)
PLTs. (ii) PBLs. (iii) RBCs: #. the mean time of addition of each cell type: VIa. urine output
I.>/
10 r - - - - -- - - - - -- - - - - - - - - -- - - -- - - -- - - - - . 12
# # # Ii
! ............ .
R
E'
8
1 '*-" .'
,'!'··
-., ..: 10
8
S
I
S
6
."" " --. U
....•
6 I
T
A . /:/"'................. .. - - o
4
N 4
C
E
2
: 2
:
0
*-__ ~ __- L__ ~ __ ~L- __ ~ __J -_ _- L__ ~ O
0 30 60 90 120 150 180 210 240

TIME
- Resistance(N-2) .+ .. UIO
Fig. 26.8. Those porcine kidneys perfused with the canine cell component sequence of (i)
RBCs, (ii) PBLs, (iii) PLTs. Note risc in urine output (VIa) after addition of second and
third cell components; #. the mean times of addition of each cell type
after the addition of the last component, and hematuria was noted 13 min
(mean) after the last component in all but one experiment.
Figures 26.6, 26. 7. and 26.8 show the breakdown of Group 5a kidneys into
three separate combinations according to the order which cells were added.
All three combinations demonstrated the same overall pattern. Resistances
rose slowly during successive cell additions from an initial baseline of less than
2, but increased markedly after the last cell fraction was added. The urine out-
puts were >2 ml/min for all three combinations throughout the entire per-
fusion period, and. although varying slightly, decreased by no more than
1 mllmin from point 3 to the end.
Results 415
, - - - - - - - - - - - - - - - - - - -- - - -- - - - - - - - - , 10
# .~. # #
1< ••••• 1 .i
ii \".J ....~..
•. , .....
1 8
6
U
"'. .../ '110 .
....• ............ * f
'.. j
4
o
~ ____ ~ __ ~ _ _ _ _- L_ _ _ _ ~ ____ ~ __ ~ O
30 60 90 120 150 180

TIME
- Resislance(N-4) .+ .. UfO
Fig. 26.9. Those porcine kidneys perfused with autologous (porcine) plasma followed by
successive porcine cell components (PLTs- PBLs-RBCs). Note low resistances and overall
good urine outputs (UlO) and compare with Figs. 26.5, 26.7; #, the mean times of addition of
each cell type
Histological examination showed few structural changes (besides mild

tubular damage), even after the addition of two cellular fractions (in any com-
bination). However, after the addition of the third component, patchy IH
ranging from mild to severe was seen, as well as moderate tubular damage
characterized by cell sloughing and disruption, and basement membrane hem-
orrhage. The glomeruli were remarkable for the presence of intercapillary fib-
rin and PLT debris and mesangial thickening. Also, approximately 40%-50%
of the afferent and efferent arterioles were thrombosed in these kidneys.
Group 5b (n=4) and Group 5c (n=2) kidneys served as controls for Group
Sa experiments. Group 5b were perfused with autologous plasma and cells
(PLTs/PBMCs/RBCs) and are graphed on Fig. 26.9. Total perfusion time
(mean) was 160 min. Mean resistances were slightly elevated initially, but then
decreased and remained steady for the remainder of the experiment. Gross
color changes were not recorded in these experiments. Urine outputs varied
greatly, reaching a peak of8.6ml/min early, and then stabilizing at 5 ml/min af-
ter the last cell fraction was added. Urine color changed in our experiment -
hematuria was noted 4 min after RBC addition in one experiment, but re-
mained clear after autologous RBCs in another. Other than mild tubular dam-
age (as can be seen with cold storage), no other structural damage was seen on
light microscopy.
Group 5c kidneys were perfused with autologous plasma (i.e., no xenoanti-
body) as the initial perfusate and followed with heterologous (canine) cell
components (Fig. 26.10). Total perfusion time was longer than most other
groups at 220 min. Mean resistances varied during the experiments but were
stable throughout. Interestingly, gross color changes (mottling) did occur in
both these kidneys at a mean of 40 min after the addition of the last compo-
nent. Urine output remained stable and did not change appreciably after addi-
10 10
8 8
R
E
S 6
I 6
U
S I
T o
A 4 - - '-11- - -- .,,- # 4
N
C
E
2
....../ lil········ ..<:!7.·. . ·~ ···· .-.:::!-::./Q.·...l... ·····I'l··· .. ·.... .. ·0 2
~ _ _~ _ _~ _ _L -_ _L -_ _~ _ _~ _ _~~ O
0
0 30 60 90 120 150 180 210 240
TIME
-+-- Resislance(N-2) .-G .. U/O
Fig. 26.10. Perfusion of porcine kidneys with alltologolls plasma followed by successive addi-
tions of heterologolls cell components (in order (i) RBCs. (ii) PBLs. (iii) PLTs). Note main-
ten ance of both low resistances and stable urine outputs (UI O); #. the mean tim es of addition
of each cell type
10 ~----------------------------------, l0
8 8
R # #
-1-- ._.-
E'
1
S 6 6
I U
S I
T
A
1'1 .. "'~ '" o
4 '0 · .... 0 " .•.. [)".
N
C
E
2 2
~ _ _L -_ _~_ _~_ _~_ _~_ _~_ _~~ O
0
0 30 60 90 120 150 180 210 240
TIME
-+-- Resislance(N-6) ·G · U /O
Fig. 26.11. Perfusion of porcine kidneys with canine plasma. then pretreatment with PMNs
prior to subsequent addition of cell types in succession; #. mean times of addition of various
cell types. Note rel ative ly low resistances at termination (240 min) compared with Figs.
26.5- 26.8. and maintenance of good urin e output (VlO)
tion of any or the canine cellular fractions. These kidneys were noted on final
biopsy to have only mild tubular damage; in one experiment. there was also
minimal presence of some peritubular capillary thrombosis . but no IH and
only mild edema was found, even 90 min after the last cell addition.
Group 6 (n=6) kidneys were perfused with plasma, then PMNs as the first
cell component, and followed by the other three cell fractions (RBCs, PBMCs,
PLTs) in succession (Fig. 26.11). Mean total perfusion time (246 min) was
comparable to Group Sa. The mean perfusion time from the last cell type to
Results 417
10
,. 14
12
8
R
E 10
S - #- ........................
6
l . .·········~
I # 8
s U
...•.! ::-
I
T 0
A 6
4
N .... ...
.'
C 4
E :/ 'i'"
2
2
0 0
0 30 60 90 120 150 180
TIME
- Resislance(N-2) "*',, U/O

Fig. 26.12. Perfusion of porcine kidneys with canine (i) plasma. (ii) PMNs. (iii) citrated
whole blood (without calcium) in succession; #, mean addition times. Note stable resistances
and pronounced increase in urine output (UIO); compare with Fig. 26.13
termination was increased at 81 min (60% increase from Group 5a). Onset of
color change was noted 22 min after addition of the last component.
Resistances remained low throughout the perfusion. The overall change in re-
sistance from beginning to end was only 1.9 - a 2.8-fold difference compared
with Group 5. Urine outputs were relatively stable throughout the entire per-
fusion time and ranged from a mean of 3.8 to 4.6 mllmin. There was no change
in urine output after the last cell fraction was added. Hematuria was noted
33 min after the last cell was added (compared with 13 min in Group 5a) and
gross color changes were also seen at a slightly later time compared with
Group 5a (22 min versus 14 min).
The histological findings varied between experiments in this group. The
initial microscopic changes were not different (after addition of PMNs) from
those found in previous groups after the addition of two of three cell types. It
was found that after addition of PMNs, 3-5 cells (PMNs) adhered to the capil-
lary endothelium of each glomerulus. No specific binding was noted in the ar-
terioles, however. The consistent finding on terminal biopsies was the lack of
glomerular or arteriolar thromboses. In two of the experiments, some scat-
tered thrombi were noted, but only 10%-20% of the vessels were involved. In
those kidneys where RBCs were added last, significant interstitial edema was
also noted. Final biopsies showed progressive interstitial changes, but no
thrombosis.
Group 7 kidneys were perfused with plasma, PMNs, then WB. Group 7a
(n=2) were those perfused with citrated (i.e ., without added calcium) and are
shown in Fig. 26.l2. Resistances overall remained low and rose only a minor
amount. Time to color change of the kidney was markedly increased at 83 min
(greater than all other groups) after WB infusion. Urine output ranged from
4.2 to 5.9 mllmin prior to WB, and then, curiously, rose progressively to 12.8
ml/min by the end of the experiment (a 2.6-fold increase compared with the
10 10
8 - 8
R.
E
S 6
I 6
U
S '« I
T o
A
N
C
4
./~\\.~.J - 4
E
2 iIe·..... - - -•.•::0 .•••• •• 2
~ __ ~ _ _ _L -____L -_ _ ~ ____ ~ ____ ~ O
0
0 30 60 90 120 150 180
TIME
- Resis1ance(N·2) *' UIO
Fig. 26.13. Perfusion of porcine kidneys with canine (i) plasma. (ii) PMNs. (iii) whole blood
(w ith calcium) in succession ; #, mean addition times; UfO. urine output. Note dramatic resis-
tance increase despite PMN pretreatment
10 r-----------------------------------, 10
8 8
R
E'
S
I 6 # . - ir - If'''# 6
U
S
7;*'······..:-!..-.. . "". ~. !~;O-*.:. ~-: . -::.!.. .....

I
T
A 4
o
4
N i .. . . ...... '"
C
E
2 2
O*-____ ~ ____ ~ _____ L_ _ _ __ L_ _ _ __ L_ _ _ _ ~ O
o 30 60 90 120 150 180

TIME
- Resistance(N·3) ..... UIO
Fig. 26.14. Porcine kidneys perfused with canine plasma. then pretreatment with homoge-
nized PMNs prior to addition of other cell types; #, mean times of addition of PMN ho-
mogenates and cell types. Note increase in resistance after addition of last cell type; UfO.
urin e output; compare with Fig. 26.11
pre-WB rate). No hematuria occurred with either kidney. Histologically,

these kidneys overall showed marked preservation of tubular and glomerular
integrity on final biopsy. Mild IH was present, but the vascular structures con-
tained no evidence ofthrombosis.
In Group 7b (n=2) kidneys, plasma. PMNs, and WB (with added calcium)
were used as the perfusates (Fig. 26.13) . Resistances initi ally rose to 2.4 during
plasma perfusion. but then stabilized at 1.6-1.7 prior to WB. After WB addi-
tion. the resistances increased sharply. The overall change in resistance was
Results 419
10 ,---------------------------~_r~ 10
_______ -HJ/L
8 8
R
E
S
I 6 6
U
S f
T
A 4 4
o
N __ ._ . . . . .... . . -lI( '
C •••• lI< ••••••.• ----.--
E
2 ........ 2
.... ....
O~--~----~--~----~--~----~~
' O
0 30 60 90 120 150 180
TIME
- Resistance(N-2) --*"-- UfO

Fig. 26.15. Porcine kidneys perfused with canine plasma , PMN me mbranes. then cell frac-
tions; #, mean addition times of various cell types (PMN membranes first) ; UIO , urine out-
put Note similarity to Fig. 26.14 and compare with Fig. 26_11
9.3, which closely resembles the Group 3a value (S.2) , but is in sharp contrast
to the Group 7a value (0.4). Gross color changes were noted within 23 min af-
ter WB, also very similar to Group 3a. Urine output decreased notably after
addition of WB, and hematuria became evident after 1S min. Histological
examination demonstrated considerable occlusion of vascular structures
(50%-60% of vessels) and moderate IH (in contrast to Group 7a).
To further identify the role of PMNs in HAR in our model, three kidneys
were perfused with plasma, homogenized PMNs, and cell fractions (Group
Sa), and two were perfused with plasma, PMN membranes (rather than cell
homogenates), and cell fractions (Group Sb) (Figs. 26.14 and 26.15, respec-
tively).
In the Group Sa kidneys, resistances remained stable initially through the
addition of homogenized PMNs and two other cell types, at which time they
slowly increased. After adding the final cells, the resistance rose rapidly to end
stage. The overall change in resistance was similar to those kidneys with serial
cell-type addition but no PMN pretreatment (Group 5a). Color changes were
noted after the last component was added. Urine outputs ranged from 2.S to
4.1 mllmin, and did not change significantly after addition of any cell fraction
or at the experiment's completion.
The Group Sb kidneys had resistances which remained stable until the final
cell types were added, and then climbed sharply to end stage. Urine output
climbed steadily throughout the perfusion, but fell dramatically after the final
cell group was added. Kidney color changes and hematuria were both noted at
the end of the experiments.
Comment
Investigation in the field of xenotransplantation has become more intensified
in recent years as a result of the current need for a large donor organ pool.
With the advent of more effective immunosuppressive regimens and subse-
quently improved graft survivaL the rate-limiting factor in clinical transplanta-
tion today is donor organ availability. Primates (concordant xenotransplants)
have been used as donors in the past [28-31], and there is a growing body of
data accumulating utilizing both primate kidneys and hearts [32-39].
Because of inherent difficulties in using primates, particularly (i) relatively
low numbers available compared with organs needed, (ii) overall cost, (iii) pro-
longed gestation times, and (iv) ethical issues, other potential donor species
have been (and are still being) investigated. Swine is one such potential species
(Chap. 30). Swine demonstrate rapid breeding times, ease and low dose of care,
similarity in organ size and physiology to humans, and a relative lack of conflict-
ing ethical problems surrounding their use. The fact that humans also possess
relatively low levels of preformed natural antiporcine xenoantibody makes this
species a likely candidate as the source of donor organs [40].
The prohibitive factor which occurs in all discordant transplant combina-
tions is the rapidly destructive and currently untreatable process of HAR. The
pig-to-dog donor/recipient transplant combination provides for an extreme
representation of the HAR model, and allows for investigation of the possible
complex cellular and humoral interactions involved.
We have used the pig-to-dog xeno combination in an ex vivo setting, to iso-
late and break down the role of individual blood components and their rela-
tionship to preformed xenoantibody. The contributions of plasma, containing
xenoantibody, complement, and fibrinogen appear to be extremely impor-
tant.
Plasma alone is unable to bring about the full HAR process. In our study,
over 43 isolated kidneys were initially perfused with xeno plasma only.
Minimal changes in resistance, urine output, and structural architecture were
demonstrated, even after 240 min. These findings confirm the work by some
[6, 10], but are contradictory to findings of others [1,5]. The mild tubular di-
latation seen in these kidneys, although present, was more pronounced in
those that were cold stored - a finding probably secondary to cold storage and
ischemia or perhaps reperfusion injury [41].
Perfusion with autologous plasma was also associated with some degree of
tubular damage, although it was less than that seen with xeno plasma. It ap-
pears, therefore, that an element of perfusion injury exists in this model, and
may be partially responsible for the histological differences seen between the
auto- and xenoperfused kidneys.
Perfusion with both autologous plasma and cells provided control data on
the effect of cells themselves, added separately and in the absence of xenoanti-
gens and antibodies. Resistances actually dropped during the perfusion time,
and urine outputs remained excellent. Other than those changes that may be
seen as a result of pulsatile perfusion, no other microscopic abnormalities
Comment 421
were demonstrated. Perfusion with autologous plasma and xeno cells allowed
insight into the effect of foreign cells on the xeno organ in the absence of pre-
formed antibody. The resistance changes were only slightly greater than those
seen when only autologous components were used. There was also little differ-
ence histologically between the all autologous and xeno plasma/autologous
cell component groups, other than the presence of RBCs in 10%-20% of per-
itubular capillaries in one xenocell experiment. These findings are similar to
those of Land et al. [2].
Perfusion with WB in the ex vivo model should result in a HAR process
which closely simulates that seen in the in vivo setting. However, some con-
flicting data were obtained in this group of kidneys. The differences in perfu-
sion times and the maximum resistance values between the plasma/WB and
WB alone groups, as well as between the individual experiments within the
WB alone group, are difficult to explain. One explanation may be that the rel-
atively small volumes of blood and blood components used (approximately
one-quarter of the in vivo volumes) allow for a submaximal response. Also,
those expected results seen with plasma perfusion followed by WB (i.e., classi-
cal HAR findings) may be secondary to concentrated binding of xenoantibody
on the endothelial surfaces, which overcomes the "smaller volume" problem
and enables the full vascular thrombotic cascade to proceed.
To test the "smaller volume" hypothesis, two kidney were perfused ex vivo,
but a catheter was run from the dog's femoral artery to the pump reservoir to
provide a continuous WB supply. Both kidneys underwent prompt HAR, with
resistances which climbed dramatically, and urine outputs and RBFs which
ceased within 10 min (data not shown). This suggests that a critical, or "neces-
sary" quantity of antibody binding is required for the full thrombotic process
to manifest. There was without a doubt HAR occurring in all these kidneys, al-
beit a modified one. In some, however, the process shifted from larger vessel
thrombosis to one affecting the tubules and interstitium predominantly,
where hemorrhage and acute tubular necrosis were the major manifestations
of this injury. Resistance changes were also slower to develop and were less
severe in these kidneys.
We have shown in the past [42] that all the blood components are necessary
for the full thrombotic HAR process to occur. This is again demonstrated in
this work, where perfusion with plasma and any two of three cell types did not
lead to microvascular thrombosis or any appreciable cellular damage, but ad-
dition of the last cell type resulted in prompt resistance increases and histolog-
ical evidence of HAR. There was, however, also a different pattern noted his-
tologically in these kidneys that was similar to the "shift" in the HAR process
described above. Considerably more IH and edema was found in the sequen-
tial cell-addition kidneys, because of ongoing ischemic and "slower" damage,
than in those that underwent more classical HAR (perfused with plasma fol-
lowed by WB). The microvascular involvement was also less, with only ap-
proximately 50% of the vessels thrombosed as compared with the plasma/WB
kidneys, where nearly complete obliteration of all vascular structures oc-
curred.
The use of citrate as a modifier of the HAR process was first investigated by
Linn in 1970 [43], and then by others [21, 23]. These groups demonstrated sig-
nificant prolongation of renal xenograft survival, especially whcn given intra-
arterially. The effects of citrate on the HAR process are presumably sec-
ondary to its calcium binding properties, and the importance of calcium in a
wide variety of physiological roles. We perfused with citrated blood for 120
min and noted no resistance or flow changes and only minimal interstitial
damage (hemorrhage). These findings are in sharp contrast to the plasma/WB
perfusion groups (both 3a and 7b) which involved the use of heparinized
blood. Addition of calcium to the citrated WB perfusion kidneys at a later time
resulted in immediate HAR (data not shown). The finding of mild IH in the
citrate group may reflect either incomplete calcium sequestration or possibly a
noncalcium-dependent antibody or cell-mediated destructive phenomenon.
The role of neutrophils in the inflammatory process and in experimental
transplantation is rapidly beginning to unfold, and it is becoming evident that
PMNs are extremely important in many aspects of these biologic events.
Historically, the importance of PMNs in HAR has been limited to the simple
observation of a significant number of PMNs in the microvascular circulation
and interstitium in hyperacutely rejected kidneys.
We sought to further define the role of PMNs in our model by pretreating
11 kidneys with PMNs prior to other cell (or WB) additions. In the sequential
cell addition experiments, resistances, RBFs, and urine production were re-
markably well preserved. There was a dramatic lack of involvement of the vas-
cular structures in these kidneys (as would be expected by the low resistances),
with at most 20% of the vessels exhibiting thrombus formation. PMNs adhere
to the glomerular capillaries soon after their addition. A varying degree of in-
terstitial involvement still occurred, with edema and IH being the predomi-
nant findings. In the sequential PMN-WB perfusion experiments, the same
PMN adherence pattern was noted, but the resistances and urine outputs
dropped dramatically after WB addition and widespread thrombosis was evi-
dent. These kidneys overall behaved as if no PMN pretreatment had occurred.
To test whether or not intact PMNs were required for the apparent "pro-
tective" effect, PMN homogenates and PMN cell membranes were used in dif-
ferent experiments, and the data clearly showed that these kidneys behaved
like the three-cell addition groups (i.e., without PMN pretreatment) in regard
to rapidly increasing resistance changes, and diminishing urine outputs indica-
tive of HAR; these kidneys were also similar histologically.
Considering the usual destructive role of PMNs, mediated predominantly
through lysozymes and oxygen-derived free radicals, the apparent "protec-
tive" effect of PMN s in our model is intriguing. The process of HAR involves a
complex combination of antibody and cell-cell interactions, with PMNs play-
ing an undefined role. Any explanation for the role of PMNs in our model
would only be speCUlative. The PMNs may block binding of other cells to the
antibody-antigen complex or endothelial cells, or PMNs may need some type
of second messenger (cell) to be stimulated (i.e., to become destructive). The
role of PMN s in our model warrants intensive further investigation.
References 423
In conclusion, we have utilized the perfusion of porcine kidneys with ca-
nine blood components, in an ex vivo setting, to study HAR. Perfusion with
xeno plasma alone is unable to bring about this process, and the simultaneous
interaction of all blood cellular components is necessary for classical HAR to
occur. A certain amount of antibody binding (presumably to the endothelium)
must occur for widespread activation of the coagulation cascade and diffuse
microvascular thrombosis. PMNs playa different role than usual in our model,
apparently "protecting" the vasculature from thrombosis by some unknown
mechanism. The important participation of PMNs in many pathological and
physiological processes is already known, and further investigation in the area
of experimental transplantation is needed.
Acknowledgements. The research described in this chapter was supported by

NIH grant No. AI 28478 and in part by The William G. Pace, III, M.D.
Endowment Fund.
References
1. Mozes, M.F., Gerwurz, H., Gunnarson, A, Moberg, A.W., Westberg, N.G., Jetzer, T.,
Najarian, J.S. Xenograft rejection by dog and man: isolated kidney perfusion with blood
and plasma. Transplant. Proc. 3,531,1971.
2. Land, W., Schilling, A., Aldenhoff, J., Lamerz, R, Pielsticker, K., Mendler, N., Brendel,
W. In vitro studies on the hyperacute xenograft rejection. Transplant. Prac. 3,888,1971.
3. Qtte, K.E., Andersen, N., Jorgensen, K.A., Kristensen, T., Barfort, P., Starklint, H.,
Larsen, S., Kemp, E. Xenoperfusion of pig kidney with human AB or Q whole blood.
4. Qtte, K.E.,Jorgensen, K.A, Bonnevie, V., Barfort, P., Larsen, S., Starklint, H., Pirotzky,
E., Kemp, E. Xenoperfusion of rabbit kidney and the impact of BN 52021: a specific an-
tagonist of platelet-activating factor. Transplant. Prac. 22, 1089, 1990.
5. Linn, B.S., Jensen, J.A., Pardo, V., Davies, D., Franklin, L. Relationship between struc-
tural and functional changes in rejecting renal xenograft. Transplant. Proc. 3,527,1971.
6. Yoshioka, H., McCalmon, R.T. Jr., Putnam, CW., McIntosh, RM., Terman, D.S.
Attenuation of hyperacute xenograft rejection in unmodified host by extracorporeal
plasma perfusion. Transplantation. 24,78, 1977.
7. Toth, I., Roth, E., Szmolenszky, T., Pacsa, S., Torok, B. Characteristics of hyperacute re-
jection of swine kidney xenografts perfused with canine blood. Int. Ural. & Nephral. 8.
323.1976.
8. Marceau, J.P., Hallenbeck, G.A., Zollman, P.E., Butler, H.C, Shorter, RG. A compari-
son of autoplastic, allogeneic, and xenogeneic perfusion of isolated kidneys. 1. Surg. Res.
5,492,1965.
9. Slapak, M., Greenbaum, M., Bardawil. W., Saravis, C, Joison, J., Mcdermott, W.V.
Effect of heparin. arvin, liver perfusion, and heterologous antiplatelet serum on rejec-
tion of pig kidney by dog. Transplant. Proc. 3,558,1971.
10. Sakai. A, Kountz, S.L. Dissociation of humoral and cellular immune reactions of the
rabbit, guinea pig, and dog kidney xenograft in the rat. 1. Surg. Res. 21. 159. 1976.
11. Auchincloss, H. Jr. Xenogeneic transplantation. A review. Transplantation. 46. 1. 1988.
12. Rosenberg, J.C, Broersma, R.J., Bullemer, G., Mammen, E.F., Lenaghan, R,
Rosenberg, B.F. Relationship of platelets, blood coagulation, and fibrinolysis to hyper-
acute rejection of renal xenograft. Transplantation. 8,152,1969.
13. Belitsky. P .. Popovtzer. M.M .. Smith. J .A .. Katz. F. Renin-angiotensin activation in hy-

peracute rcnal xcnograft rejcction. Pmc. Soc. Exp. BioI. Mal. 145.11922.1974.
14. Rosenberg. J.C. Hawkins. E .. Rector. F. Mechanisms of immunologic injury during anti-
body-mediatcd hypcracute rejcction of renal heterografts. Transplantation. 11. 151. 1971.
15. Shons. AR. Bier. M .. Jctzer. T.. Najarian. J.S. Techniques of in vivo plasma modifica-
tion for the treatment of hyperacute rejection. Surgery. 73. 2S. 1973.
16. Mobcrg. A.W .. Shons. A.R .. Gewurz. H .. Mozcs. M .. Najarian. J.S. Prolongation of renal
xenografts by the simultaneous sequestration of preformed antibody. inhibition of com-
plcment. coagulation. and antibody synthcsis. Transplant. Proc. 3. 53S. 1971.
17. Bier. M .. Beavers. CD .. Merriman. W.G .. Merkcl. F.K .. Eiseman. B .. Starzl. T.E.
Selective plasmapheresis in dogs for delay of heterograft response. Trans. Am. Soc. Art.
fllIem. Organs. 16.325. 1970.
IS. Shons. A.R. letzer. 1.. Moberg. AW .. Najarian. 1.S. Prolongation of heterograft sur-
vival by clectrophoretic extraction of preformed antibody. SlIrg. Fo ru Ill. 21.263. 1970.
19. Shons. A.R. Najarian. 1.S. Modification of xenograft rejection by aspirin. dextran. and
cinanserin: the importance of platelets in hyperacute rejection. Transplant. Proc. 6.435.
1974.
20. Kashii. A. Derry. CO .. Lafortune. L.A .. Sise. H .. P·eng. F.K .. Sayhoun. A .. Misra. M.K ..
Slapak. M. The effect of modification of coagulation factors on xenograft rejection. Surg.
Forum. 22.242. 1971.
21. Kux. M .. Boehring. H.J .. Amemiya. H .. Torisu. M .. Yokoyama. T.. Lannois. B ..
Popovtzer. M .. Wilson. CB .. Dixon. F.J .. Starzl. T.E. Modification of hyperacute canine
homograft and pig-to-dog heterograft rejection by the intra-arterial infusion of citrate.
Surgery. 70. 103. 1971.
22. Hawkins. E .. Mammen. E.. Rosenbcrg. 1.C Effects of hcparin and steroids on hyper-
acutely rejected renal xenografts . .!. Slirg. Res. 11.492. 1971.
23. Kux. M .. Boehmig. H.l .. Torisu. M .. et al. Modification of pig- to-dog renal xenograft re-
jection by the intraarterial infusion of citrate. Soc. Univ. Slirg. 32.33. 1970.
24. Snyder. G.B .. Gallesteros. E .. Zarco. R.M .. Linn. B.S. Prolongation of renal xenografts
by complement suppression. SlIrg. Forum. 17. 47S. 1966.
25. Han. L.K .. Henry. M.L.. Orosz. CG .. Sedmak. D.O .. Ferguson. RM. Modification ofhy-
peracute xenograft rejection by ex vivo xenoantibody adsorption. ClIrr. SlIrg. Jan-Feb.
15.1990.
26. Henry. M.L.. Han. L.K .. Orosz. CG .. Scdmak. D.O .. Ferguson. RM. Modification of
xenograft hyperacute rejection via xenoantihody depletion. Transplant. Proc. 22. 1081.
1990.
27. Radka. S.F.. Amos. D.B .. Quackcnhush. L.J .. Crcsswell. P. lILA-DR specific mono-
clonal antihodies and chimpanzee anti-DR serum detect different epitopes on the same
molecule. fllll7lllllOgenetics. 19.63. 1984.
28. Rcemtsma, K .. McCraken, B.H.. Schlegel. 1.U., Pearl. M.A .. Pearce. CW .. DeWitt.
CN .. Smith. P.E.. Hewitt. RL., Flinner, R.L., Creech, O. lr. Renal heterotransplanta-
tion in man. Ann. SlIrg. 160.384.1964.
29. Reemtsma. K.. McCraken. B.H.. Schlegel. 1.U .. Pearl. M.A. DeWitt. CN., Creech. O.
lr. Reversal of early graft rejection aftcr renal heterotransplantation in man. lAMA.
187.691,1964.
30. Starzl. T.E .. Marchioro. T.L., Peters. G.N .. Kirkpatrick. CH .. Wilson. W.E.C. Porter.
K.A .. Rifkind. D .. Ogden. D.A .. Hitchcock. CR, Waddell. W.R Renal heterotrans-
plantation from baboon to man: experience in six cases. Transplantation. 2,752,1964.
31. Barnard. CN .. Wolpowitz. A. Losman. 1.G. Heterotopic cardiac transplantation with a
pass. SOlllh Aji·. Med..!. 52.1035.1977.
32. Porter. K.A. Pathologic changes in transplanted kidneys. In: Starzl. T.E. (ed.)
Experience in Renal Transplantation. Philadelphia. W.B. Saunders. 1964. p. 345.
33. Hardy. 1.0 .. Chavez. CM .. Kurrus. F.D .. Ncely. W.A. Webb. W.R. Eraslan. S .. Turner.
M.O .. Fahian. L.W., Labecki, 1.0. Heart transplantation in man; developmental studies
and report of a case . .lAMA. 188,1132.1964.
References 425
34. Bailey, L.L., Nehlsen-Cannarella, S.L., Concepcion, Woo Jolley, W.B. Cardiac xeno-
transplantation in a neonate. lAMA. 254,3321,1985.
35. Heystek, G.A, Van Leersum, RH., Bainer, H. Prolongation of baboon to rhesus blood
transfusion. Transplantation. 25,165, 1978.
36. Kurlansky, P.A, Sadeghi, A.M., Michler, RE., Smith, CR., Marboe, CC, Thomas,
W.G., Coppey, L.J., Reemtsma, K., Rose, E.A. Comparable survival of intra species and
cross species primate cardiac transplants. Czar. Surg. 43,413, 1986.
K., Rose, E.A Cardiac xenotransplantation in primates. 1. Thorae. Cardiovasc. Surg. 93,
809,1987.
38. Sadeghi, AM., Robbins, RC, Smith, CR., Kurlansky, P.A, Michler, RE., Reemtsma,
K., Rose, E.A. Cardiac xenograft survival in baboons treated with cyc\osporine in com-
bination with conventional immunosuppression. Transplant. Proc. 19,1149,1987.
39. Lexer, G., Cooper, D.K.C, Wicomb, W.N., Rose, A.G., Rees, J., Keraan, M., Reichart,
B., du Toit, E. Cardiac transplantation using discordant xenografts in a non-human pri-
mate model. Transplant. Proc. 19, 1153, 1987.
40. Kirkman, RL. Of swine and men: organ physiology in different species. In: Hardy, M.A.
(ed.) Xenograft 25. Amsterdam, Elsevier; Amsterdam, New York, Oxford, 1989, p. 125.
41. Southard, J.H, Belzer, F.O. Kidney preservation by perfusion. In: Cerilli, G.J. (cd.)
Organ Transplantation and Replacement. Philadelphia, J .B. Lippincott Co., 1988.
42. Han, L.K., Hcnry, M.L., Ferguson, R.M. The effect of formed cellular clements on ex
vivo xenograft hyperacute rejection Transplant. Proc. 23,574,1991.
43. Linn, B.S., Jensen, J., Pardo, V., Levi, D.F., Hudson, D.G. Prolongation of renal
xenografts caused by citrate . .JAMA. 212,864,1970.
Section V
Aspects of
Xenotransplantation
inMan
Chapter 27
Evolutionary, Physiological,
and Immunological Considerations
in Defining a Suitable Donor for Man
C.HAMMER
Introduction
The dilemma of evolution has been to overcome the conflict between the con-
tradicting requirements, on the one hand, of maintaining what has been
achieved and, on the other, of allowing progress. Total constancy of living ma-
terial would have eliminated any future change of nature, yet unhampered
variation would have jeopardized what had already been achieved. The phy-
logeny of man depends on the possibility of improvement and adaptation from
accidental and unplanned mutations.
Nature did not foresee, however, that man had the intention to perform
transplants or even xenotransplants. When it comes to the possibility of ex-
changing animal organs, therefore, most phylogenetic parameters are in-
hibitory.
A great variety of species, their individuality, changes that occur in one ani-
mal during its life, contact of that species with a specific surrounding, heredity,
and changes in the environment are some of the characteristic phenomena of
evolution. It was Linnaeus who first tried to bring system into the chaos that
existed. He was also the first to classify man and chimpanzee within the order
of primates. Lamarque and Darwin later postulated a common descendence
of man and apes from one predecessor. Their observations forced subsequent
researchers to simplify their experiments and analyses and study only one
variable and its consequences at anyone time.
Today, the principles of achieving successful xenotransplantation are
based on such simple and single observations, and yet still few results exist
which provide clues towards further development and future possibilities in
this extremely complex field.
Some aspects of this new and fascinating field of transplantation research
will be briefly discussed.
430 Evolutionary, Physiological, and Immunological
Suitability of Donor Organs for Man
Anatomical Aspects and Availability
The availability of xenograft organs for man is closely linked with the ana-
tomical characteristics, size, and morphology of the potential donor.
Morphological traits in a species appear to shift significantly within a relative-
ly short time simply under the influence of the environment, for example, un-
der domestication. Although most domestic (and experimental) animals dif-
fer completely in their phenotype from their wild ancestors, their "genetic
skeleton" has not changed much [1]. This indicates that the "farming" of ap-
propriate donor species in the future would allow us, for example, to modify
organ size without major histocompatibility changes within a reasonable time
interval.
Man, primates, and kangaroos are the only species that have adapted com-
pletely to an upright posture. In the short evolutionary time period of homo
sapiens (of approximately 5 million years) not much has changed regarding
the coarse anatomical characteristics of mammals of comparable size to man.
Organs suitable for xenotransplantation show only minor differences unrelat-
ed to whether the animal uses the erect posture. For example, the relative
weight of the liver varies between 1.6% and 2.4 % in all mammals that might be
of interest to us for purposes of xenotransplantation. Herbivores have smaller
livers than omnivores or carnivores [2]. Small individuals (i.e., young) have
relatively larger organs than big individuals (i.e., adult). Domesticated ani-
mals like deer, horses, and donkeys possess no gallbladder. Kangaroos, how-
ever, provide heart valves which resemble those of man more than do those of
pigs or other species with horizontal posture [3].
From the anatomical and immunobiologic points of view, the optimal choice
of an organ donor for man would be the large nonhuman primate. However,
these species are almost extinct, and all of them reproduce and grow slowly.
Despite many unanswered questions, the pig would appear to be the most
attractive species as a potential donor for man. Pigs both reproduce and grow
quickly. Their organs are proven to be usable at any size and age. First at-
tempts to modulate the pig genome have been successful [4]. Future studies
will show whether other readily available animals, such as the sheep, goat, or
even kangaroo, will prove appropriate as alternative or additional choices.
Physiological Considerations
Comparative physiology provides many examples of differences between

species that might be considered as donors for purposes of xenotransplanta-
tion. The physiological parameters of closely related species, such as man and
ape, are almost identical. Once, however, we attempt to cross the barrier of a
zoological family, major differences are described. For example, the function
of all organs and grafts depends primarily on the blood circulation. Relative
Suitability of Donor Organs for Man 431
Table 27.1. Blood parameters of man and animals most commonly used for experimental
xenotransplantation
Species Viscosity Total Numbers of Numbers of Erythrocyte

(cP) protein erythrocytes leukocytes diameter
(g/100 ml) (106/111) (1(J3/111) (11m)
Man 4.7 5.0 7.0 7.2

Horse 4.1 7.4 7.5 9.0 5.5
Cattle 4.6 7.5 6.0 8.0 5.7
Sheep 4.3 8.0 10.0 8.0 5.1
Goat 4.0 7.2 14.0 10.0 4.1
Pig 5.9 7.3 6.5 12.0 6.1
Dog 4.7 6.3 6.0 12.0 7.3
Cat 4.2 6.8 8.5 10.0 5.7
Rabbit 3.4 5.8 5.0 9.0 7.0
Rat 7.1 8.0 14.0 6.2
Mouse 9.3 8.0 5.7
Table 27.2. Number of blood group systems in

man and domestic animals
Species Number Isoantibodies
Man 4 Yes
Horse 8 Rarely
Cattle 12 No
Sheep 8 No
Pig 15 Rarely
Dog 7 Rarely
Cat ? Rarely
blood viscosity in domestic animals varies from 5.9 in the pig, 4.7 in man and
dog, to 3.4 in the rabbit (Table 27.1). These differences relate to various blood
components such as the total protein and red and white blood cells, the rela-
tive numbers of which differ significantly [2]. It has been shown in perfusion
experiments that the size of foreign erythrocytes can be critical. Human ery-
throcytes are the largest when compared with those of animals of interest to us
for clinical xenotransplantation, and thus might interfere mechanically with
the adequacy ofthe microcirculation [5] (Table 27.1).
All animals have blood groups that have been extensively studied in their
domestic cousins (Table 27.2). The differences in blood components of man
and baboon are shown in Table 27.3. Domestic farm animal blood groups,
however, resemble more the Rh-system than the ABO-system seen in man
and other primates. Therefore, no reaction occurs in cattle and sheep after a
first unmatched allogeneic blood transfusion. A second contact with the same
blood antigens, however, leads to an adverse reaction due to the prior immu-
nization. Horses, dogs, and pigs need to be tested for blood group compatibili-
ty before transfusion.
Table 27.3. Differences in certain blood group systems and components between man and
baboon
Man Baboon
Human ABO system O,A.B,AB A,B,AB

Human Lewis substances Les and nL Lesand nL
Human I and i factor I andi o-i
Human MNS system MNS Ap. Bp. ABp, Op
Human Rhesus system A.B,C.D A,B,C
Haptoglobin sUbtypes
a-Antitrypsin types
6-Phosphogluconate dehydrogenase
Transferrin All different
Peptidase
Malate dehydrogenase
Caeruloplasmin
Blood clotting system Remarkably similar
All animals, including man, produce so-called preformed natural antibody

(PNAB) without deliberate prior antigen contact. Such PNABs are believed
by many to be an essential element in triggering hyperacute xenogeneic rejec-
tion [6]. In more than 8000 single tests using sera from 111 animals of 7 differ-
ent zoological orders and 45 species, a considerable dependency of the titers of
PNABs on the zoological (i.e., evolutionary) system was demonstrated - the
more distantly related the species, then the higher the titers [7].
Only primates seem to produce significant amounts of isohemagglutinins.
Closely related mammals within a zoological family have no PNABs against
other members of that family. Many environmental factors such as age, do-
mestication, biorhythm, disease, and radiation influence the production of
these PNABs. PNABs can be specific, as demonstrated by absorption experi-
ments. The close relationship between two related species (fox and dog) can
be proven by the fact that their erythrocytes absorb to a major extent the cross-
reacting specificity, leaving some fully specific antibodies behind. Absorption
of such antibodies decreases with the divergency of the species [8].
One example of differing liver metabolism between species is illustrated by
cholesterol. High values are found in man (±200 mg/IOO ml) and extremely
low values in pigs (36-58 mg/100 ml), with the dog, horse and cattle (approxi-
mately 100 mg/lOO ml) and sheep (64 mg/IOO ml) in the middle demonstrating
intermediate levels. Blood sugar levels in man (80-100 mg/lOO ml) are differ-
ent from sheep and cattle (30-60 mg/lOO ml), but similar to the pig (80-95
mg/lOOml).
Other immunological and genetic aspects of species difference have been
described elsewhere [8-11].
Biochemical Considerations
Biochemical data allow a more precise taxonomic classification of the species

than do morphological characteristics [8]. Molecular data have therefore been
used to develop a so-called molecular clock of evolution [12, 13]. Sequence dif-
ferences of homologous peptides and DNA-hybridization experiments make
it possible to estimate the zoological distance between species and thus allow
some judgment to be made regarding metabolic compatibility, a prerequisite
for the adequate function ofxenogeneic organs [14, 15].
Evolutionary Aspects of Enzymes of Importance in Xenotransplantation
All living organisms, especially highly developed species, possess a wide arse-
nal of proteolytic enzymes which have many physiological functions, begin-
ning with general protein digestion, ranging over blood coagulation and the
activation of complement, and extending to the release of hormones and phar-
macologically active pep tides and their precursor molecules. They are all un-
der specific and multivarious controls, because, if uncontrolled, they can de-
stroy the protein components of the cells and tissues - as occurs in hyperacute
rejection between widely divergent species [16].
Differences in amino acid sequence, three-dimensional structure, and even
enzymatic reactions confirm the existence of distinct families of proteins that
have developed during phylogeny and ontogeny.
In addition, these changes in molecular structure bring about similar varia-
tions in species-specific inhibitors. Proteases are supposed to have arisen in
the earliest phase of biologic evolution. In the course of evolutionary develop-
ment they have adapted from purely digestive functions to complex physiolog-
ical regulators.
Their heterogeneity (despite similar enzymatic specificities), their ontoge-
ny, and tissue differentiation are of wide biologic importance. Their tissue
specificity is highly expressed and well-known as isoenzymes. In adult verte-
brates, separate organs and tissues show a characteristic distribution and ac-
tivity of enzyme forms, and this individuality not only differs in organs, it also
changes during ontogeny. Fetal tissue can contain isoenzymes which barely
function in adults of the same species [17].
In cattle, for example, the embryonic form of lactate dehydrogenase
(LDH) develops in a unidirectional way into the adult form, whereas in rabbits
and pigs embryonic LDH remains well represented at all stages of ontogeny
[11]. Ovine embryos are less well oxygenated than rabbit or avian embryos.
This phenomenon is reflected in the embryonic isoenzyme situation and in the
A- and B-type contribution of the subunit of LDH, thus indicating an indirect
action of oxygen on the biosynthesis ofLDH [18].
Carboxylesterases exhibit the most extensive multiplicity of all the esterase
classes in vertebrate tissues and show considerable species variations with
respect to the electrophoretic properties of the isoenzymes, their tissue
distribution, and degree of multiplicity and, therefore, immunogemclty,

Carboxylesterase type 5, for example, exists in two forms in pig, sheep, horse,
ox and possum, with molecular weights of 80 000 and 160000, respectively, but
only as one isoenzyme of molecular weight of 80 000 in the guinea pig and rat
[19,20],
Catalase may be the most extensively investigated enzyme. It exists as five
major isoenzymes in such species as the horse, rat, and rabbit, whereas most
other mammalian species display only a single form of catalase. Its distribution
in the cell and subcellular units varies as markedly as its activity in different
species [21]. Another observation regarding catalase is its development during
ontogeny; it exists at a low level in the early stages of postneonatal develop-
ment, but increases during sexual maturation, and reaches a maximum in the
adult male.
Such multiplicity and heterogeneity regarding substrate specificity is prob-
ably common to most enzymes and takes the form of primary or secondary
modifications of protein structure and, therefore, of immunogenicity. Only
forms which are not involved in such differences are likely to function in an
undisturbed manner, and, therefore, not cause problems after xenogeneic
transplantation.
Differences in physiology may well reflect the degree of divergency be-
tween species, the pressure of selection in particular biologic environments,
and specific developmental events in vertebrate animals that might be suitable
for clinical xenotransplantation. It should be emphasized that activators and
inhibitors of enzymes probably show similar species differences. Their func-
tions, however, have not been explored, nor are they even fully understood.
Evolutionary Considerations Regarding Hormones
Steroid hormones function in mammals as sexual hormones or regulate carbo-

hydrate and mineral metabolism. The spectrum of all biologically active
steroid hormones seems to be similar in all mammals, but there are differences
in concentration. The synthesis of steroid hormones is similar in all verte-
brates, and so probably is their metabolism. A few species, however, possess
specific molecules, such as equilin and equilenin in horses.
Sexual hormone binding globulin (SHBG) is present in many species, but is
lacking in rodents and elephants [22]. Peptide hormones vary from tripeptides
to polypeptides of over 100 amino acids. Their function is regulated by inhib-
ines. Both the hormones and their inhibitors are mainly species-specific. One
example is the growth hormone, which differs in primates by only 2%, but be-
tween man and whale by 34%.
The function of growth hormones is not only to control body and organ
growth, but also the metabolism of protein, carbohydrates, and fat. First ex-
periments have demonstrated that transgenic mice carrying the bovine or
porcine growth hormone gene develop into giant mice. The products of the
gene are not recognized as foreign, due to neonatal tolerance, and, therefore,
no immunological reaction occurs. However, there is no physiological down-

regulation by inhibitors, leading to overproduction, with resulting marked
nephropathy, obsolescence of numerous glomeruli and massive cystic dilata-
tion of the tubules, together with uncontrolled liver hypertrophy. In contradis-
tinction, bovine growth hormones do not function in humans [23].
Blood Clotting
Blood clotting is a characteristic not only of vertebrates, but is also found in

such lowly forms of life as arthropods and molluscs. The clotting reaction is a
cascade of proteolytic processes. The exact interactions of the factors involved
in man have been well described.
Initiation of blood clotting can occur in two ways: (i) via the exogenous sys-
tem of tissue damage, and (ii) via the endogenous system, when blood and its
factors recognize foreign surfaces. The action of PNAB, forming antigen-anti-
body complexes, activates the endothelial cells into the state of procoagula-
tion [24]. Only potent anticoagulants, such as cobra venom factor and heparin,
are able to prevent thrombosis [25]. All factors show species-specific anti-
genicity.
Complement
Together with the blood clotting system, complement is activated during the
rejection process when xenografting is preformed between widely divergent
species. The complement system comprises 20 different proteins, mostly pro-
teolytic enzymes. Fixation of complement in immunoglobulin G (IgG) classes
and subclasses differs markedly in different species. In man, IgG j , IgG 2 , and
IgM are able to bind the C jq molecule, but in cattle only IgG j and in guinea pigs
only IgG 2 can bring about such binding.
In general, immunological similarity between species decreases as the zoo-
logical relationship becomes more disparate. For example, while man and ape
show similar amino acid sequences, cross-reactivity with factors from New
World monkeys is already lacking [26].
Adhesion Molecules
Recent investigations indicate that adhesion molecules such as endothelial

leukocyte adhesion molecules (ELAM), intercellular adhesion molecules
(ICAM), and vascular cell adhesion molecules (VCAM) also seem to playa
central role in hyperacute xenogeneic rejection. As adhesion molecules isolat-
ed from chicken embryos interact also with mammalian cells, good compati-
bility between species may be expected.
Respiratory Pigments
Mammals possess two of the four oxygen binding pigments (myoglobin and
hemoglobin). The hemoglobin content varies from 14.4 gil 00 ml in man to 10.6
g/100mlin the goat, with 13.3 g/100ml in the pig. Since 1 gofhemoglobin binds
1.34 ml of oxygen, the capacity for oxygen binding obviously varies between
the different species.
Comparisons between mammalian hemoglobins have shown that the alpha
and beta chains derive from one common ancestor, but with different degrees
of mutational change. Furthermore, the speed of mutation has changed [27]
(Fig. 27.1). Hemoglobins of closely related species are similar, if not identical
[28].
Comment
It is the influence of evolution on the animal kingdom that is responsible for

the extensive tissue differentiation that we see between the species. The above
synopsis gives some indication of the multiplicity of anatomical, physiologicaL
100
16.Human y
17.Lemur fl
90 18. Mouse fl
19. Rabbit fl
20. Tamarin a
80 21. Squirrel monkey a
3. Lamphrey- 22. Spider monkey a
23. Human a
'"
c Q)
.- .r:
globin
24 .Tamarin fl
Q) () 70 25. Spider monkey fl
() c
c ~ 26. Squirrel monkey fl
Q).o 27. Rhesus monkey fl
E"c 28. Gorilla fl
Q) Q) 60
> Q) Bovine (J; 29. Human Jl
iJ~ 30. Horse fl
Goat II (J;
~ll 50
Goat (J; 31. Pig Jl
0", 9. Sheep (J; 32. Bovine fetal Jl
"ia .~ 10. Pig 33. Bovine B fl
::i'"
:2 c0 11. Horse (J;" 34. Bovine A fl
40 12. Mouse 35. Sheep B Jl
;,g-o
o 0 13. Rhesus (J; " 36. Sheep A fl
()
14. Gorilla" 37. Goat A fl
15. Man
30 "
20
10
3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36
Fig. 27.1.Vertebrate globulin gene phylogeny (after Barnabas et al. [27])

References 437
and biochemical parameters that might be involved in the mechanisms of

adaptation to and rejection ofaxenotransplanted organ. Emphasis has been
directed towards the relevance of the ontogenic and taxonomic differences
that exist over a wide variety of biologic phenomena, particularly as they affect
important enzymatic and hormonal interactions.
The structural properties of the products of a perfectly functioning
xenograft, and their individual antigenicity, have to date not been investigated
because of our inability to achieve long-term function of grafts between wide-
ly divergent (i.e., discordant) species. However, recent information concern-
ing transgenic animals indicates new and unexpected problems due to incom-
patibility of biochemical systems and their regulatory features. Further
intensive studies are required if we are to understand the complexity of xeno-
transplantation as a whole.
References
1. Herre, Wo, Rohrs. M. Hallstiere zoologisch gesehen. Gustav Fischer Verlag, Stuttgart.
1973.
2. Eder, H. Blut, Lymphe und andere Kiirperfliissigkeiten. In: Lehrbuch der Vet.
Physiologie. Scheunert, A., Trautmann, A. (eds.) P. Parey; Berlin, Hamburg, 1976,
p.403.
3. Weinhold, C, Weingartner. J., Hammer, C. Gokel, 1.M., Peters, D., Reichart, B. In vivo
investigation of kangaroo aortic valve xenobioprostheses: an experimental animal mod-
el. In: Biologic and bioprosthetic valves. Bodnas, E., Yacoub, M. (eds.) Yorke Medical
Books, New York, 1986, p. 669.
4. Hammer. R.E., Pursel, V.Go, Rexroad. CE., Wall, RJo, Bolt. D.l.. Ebert, KM.,
Palmiter, R.D .. Brinster, R.L. Production of transgenic rabbits, sheep and pigs by mi-
croinjection. Nature. 315,680,1985.
5. Schilling, A., Land, W., Pielsticker, K Experimental xenografting in widely divergent
species; interaction of humoral factors in hyperacute xenograft rejection in the rat-dog
system. Res. Exp. Med. 156,79,1975.
6. Hammer, C, Chaussy, C. Brendel, W. Preformed natural antibodies in animals and
man. Ellr. Sllrg. Res. 5, 162, 1972.
7. Hammer. C Isohemagglutinins and preformed natural antibodies in xenogeneic organ
transplantation. Transplant. Proc. 19,443,1987.
8. Hammer, C Evolutionary considerations in xenotransplantation. In: Xenograft 25.
Hardy, M.D. (ed.), Elsevier; Amsterdam, New York, Oxford, 1989, p. liS.
9. Bach, F.H. Immunogenetic and cellular studies of xenogeneic responses. In: Xenograft
25. Hardy, M.A. (ed.), Elsevier; Amsterdam, New York, Oxford, 1989, p. 183.
10. Lindahl, KF., Bach, F. Human lymphocytes recognize mouse alloantigens. Nature. 254,
607,1975.
II. Lindahl, K.F., Bach, F. Generic and cellular aspects of xenogeneic mixed leukocyte cul-
turereaction.J. Exp. Med. 144,305,1976.
12. Kimura. M. Molecular evolutionary clock and the neutral theory. J. Mol. Evol. 26.24.
1987.
13. Sarich, Y.M., Wilson, A.C Rates of albumin evolution in primates. Proc. Natl. Acad. Sci.
USA. 58, 142. 1967.
14. Sarich, V.M., Wilson, A.C Immunological time scale for hominid evolution. Science.
158,1200,1967.
IS. Rake. A.C DNA reassociation kinetics of closely related species. Biochem. Genet. 11,
261. 1974.
16. Walsh, K.A. Methods in protein sequence analysis. In: Proteases and Biological Control.
E. Reich, D. Rifkin, E. Shaw (eds.), Cold Spring Harbor, New York, 1975, p. 1.
17. Markert, CL., Shaklce, J.B., White, G.S. Evolution of a gene. Multiple genes for LDH
isoenzymes provide a model of the evolution of gene structure, function and regulation.
Science. 189, 102, 1975.
18. Wilson, AC, Kaplan, N.O., Levine, L., Pesce, A, Reichlin, M., Allison, W.S. Evolution
of lactic dehydrogenase. Fed. Proc. 23, 1258, 1964.
19. Masters, CJ., Holmes, R.S. Peroxisomes - their roles in mammalian tissue. Trends.
Biochem. Sci. 4,233, 1979.
20. Holmes, RS., Masters, C.J. A comparative study of the multiplicity of mammalian es-
terases. Biochem. Biophys. Acta. 151,147,1968.
21. Deiseroth, A, Dounce, AL. Catalase: physical and chemical properties, mechanism of
catalysis and physiological role. Physiol. Rev. 50,319,1970.
22. Santa-Coloma, T.A, Muschietti, J.P., Carreau, E.H. Sex steroid binding protein from
Bufo arenarum: further characterization. Comp. Biochem. Physiol. 85A, 401,1986.
23. Wanke, R, Hermanns, W., Folger, S., Wolf, E., Brem, G. Accelerated growth and vis-
cerallesions in transgenic mice expressing foreign genes of the growth hormone family.
Pediatr. Nephrol. In press.
24. Bach, F. Endothelial cell activation by hyperacute rejection. The xenograft problem.
Eur. Surg. Res. 22, 183, 1990.
25. Snyder, G.B., Ballestereos, E., Zarco, RM., Linn, B.S. Prolongation of renal xenografts
by complement suppression. Surg. Forum. 17,478,1966.
26. Becherer, J.D., Alsenz, J., Lambris, J.D. Molecular aspects of C3 interaction and struc-
turallfunctional analysis ofC3 from different species. Curro Topics Microbiol. Immunol.
153,45,1989.
27. Barnabas, J., Goodman, M., Moore, G.W. Evolution of hemoglobin in primates and
other therian mammals. Comp. Biochem. Physiol. 39,455, 1970.
28. Cradock-Watson, J.E. Immunological similarity of horse, donkey and mule
hemoglobins. Nature. 215,630,1967.
Chapter 28
Nonhuman Primate Blood Group Serology:

Some Implications for Xenotransplantation
W.W. SOCHA AND J. MOOR-JANKOWSKI
Introduction
Nonhuman primates appear to be the natural choice among prospective

xenograft donors for man. Their taxonomic proximity to humans is expressed
in similar anatomy and, above all, in immunological likeness, the two basic cri-
teria evoked in selection of an ideal organ donor. For similar reasons, experi-
mental organ transplantations among individuals of closely related primate
species constitute animal models that most intimately approximate conditions
of primate-to-man transplants, and should, therefore, precede any serious at-
tempts to use nonhuman primate organs for human patients.
Blood groups are the immunological parameters of primate animals that
have been most thoroughly studied; their clinical importance in organ trans-
plantation has been assessed. Investigation of blood groups in primates fol-
lowed in the wake of the discoveries of the major human blood groups. Using
techniques developed for human blood grouping and, to some extent, the
same reagents, abundant information has been amassed on the blood group
genetics and serology of the ape and monkey species most commonly encoun-
tered in captivity [1-3].
Tables 28.1-28.3 give an overview of the blood groups of representative
primate species within three main taxa that constitute the suborder
Anthropoidea, namely, anthropoid apes (Hominoidea), Old World monkeys
(Cercopithecoidea), and New World monkeys (Ceboidea).
Much less is known on blood groups of prosimians, another suborder of pri-
mates mostly represented by numerous Malagasy families. Due to their rarity
in captivity and probably limited importance for xenotransplantation, discus-
sion of the latter group will not be included.
ABO System
All primate animals share with man the ABO groups, but, unlike the situation
in man, the A, B, and H antigens are found only occasionally on primate red
cells. They are, however, regularly present in tissues and, in a soluble form, in
various body fluids and secretions of almost all primate animals [4].
ABO red blood cell polymorphism is an exclusive attribute of man and an-
thropoid apes. A and/or Band H antigens of chimpanzee, orangutan, and gib-
Table 28.1. Blood group systems of apes: overview ....
....
o
ABO MN Rh Other
z
o
Chimpanzee Most group A, some group O. All M-positive Highly polymorphic; 8 unrelated simian-type hioOlI ::l
;:;
Reciprocally related N highly polymorphic; 7 Rh-negative ReEF group systems c
anti-A and/or anti-B as many as 17 sub- phenotypes; 3
always in serum; ::l
'"
types ofN (VABD 20 Rh-positive
"0
...,
All secretors of A and/or phenotypes) defined ReEF phenotypes
H group suhstances 3'
(0
'"
Gorilla All group B with anti-A N orMN only All Rh-positive 3 monomorphic simian-type CO
in serum 3 ReEF phenotypes specificities detected to date c
All secretors of Band H distinguished 6.
group substances Cl
...,
o
c::
Orangutan A, B, and AB types with Most M-positive Most-Rh-positive; 2 unrelated simian-type hlood V
suhgroups of A All N-negative Vl
rare Rh-negative group systems ()
...,
Reciprocally relatcd encountered Strong heterophile antihodies o
anti-A or anti-B in serum in serum against red blood C
(JQ
Most ABH secretors, but cells of various species, '<

rare nonsecretors also including man
found
Gihhon A, B, and AB types with M,MN,orN All Rh-like positive 3 unrelated simian-type hlood
subgroups of A group systems
Reciprocally related
anti-A or anti-B in serum
All ABH secretors
Table 28.2. Blood group systems of Old World monkeys: overview
ABO Simian-type groups on red cells
Baboon All secretors of H and A and/or B group substances, 14 unrelated simian-type blood group systems, some showing
(various also present on various tissues, but not on rbcs. homology with macaque blood groups.
species) Reciprocally related anti-A and/or anti-B always in serum. Naturally occurring antibodies against simian-type antigens
All four ABO types encountered, but distributions vary. often detected in serum.
Rhesus All secretors of group substances, also present 13 unrelated simian-type blood group systems, some antigens
on various tissues, but not on rbcs. showing homology with baboon blood groups.
Reciprocally related isoagglutinins regularly in serum. Naturally occurring antibodies against simian-type antigens
Most type B, but A, AB, and 0 also encountered. occasionally detected in serum.
Crab-eating All secretors of group substances, also present on 6 unrelated simian-type group systems, some showing homology
macaque various tissues, but not on rbcs. with rhesus monkey blood groups.
Reciprocally related isoagglutinins always in serum. Naturally occurring antibodies against simian-type antigens
A1l4 ABO types encountered, but distributions vary, occasionally detected in serum.
depending on origin; 0 type very rare.
Pig-tail All secretors of group substances, also present on Share with rhesus monkeys 13 unrelated simian-type blood
macaque various tissues, but not on rbcs. group systems.
Reciprocally related isoagglutinins present in serum. Naturally occurring antibodies against simian-type antigens
A1l4 ABO types encountered, group 0 being the most occasionally detected in serum.
frequent.
Bonnet All secretors of group substances, also present on 3 unrelated simian-type blood group systems defined to date.
macaque various tissues, but not on rbcs.
Reciprocally related isoagglutinins present in serum. ;p
to
A, B, and AB types encountered. ofJJ
Vervet All secretors of group substances, also present on Unknown. Available reagents inactive with rbcs '<
;a.
monkey various tissues, but not on rbcs. of this species. ~
Reciprocally related isoagglutinins present in serum. a

A, B, and AB types encountered.
~
~
rbcs, red blood cells I-'
Table 28.3. Blood group systems of New World monkeys: overview
~
ABO Comment
z
o
Spider All secretors of H and A and/or B group substances, B-like agglutinogen regularly present on red blood cells. ::I
monkey probably also present on various tissues. independent of ABO salivary type. ::r
c
(various Reciprocally related anti-A and/or anti-B often 3
III
::I
species) missing in serum.
0, A, and B types encountered. '"....3'
III
Squirrel All secretors of A and/or Band H group substances. B-like agglutinogen regularly present on red blood cells. 8
monkey Reciprocally related anti-A and/or anti-B independent of ABO salivary type. ttl
isoagglutinins often weak or missing. 0'
o
All 4 ABO types encountered. 0..
o....
Capuchin All secretors of A and/or Band H group substances. B-like agglutinogen regularly present on red blood cells. o
C
Reciprocally related anti-A and/or anti-B independent of ABO salivary type. "0
isoagglutinins often weak or missing. VJ
A, B, and 0 types encountered. '"o....

0'
Howler All secretors ofB and some also ofH group substances. B-like agglutinogen regularly present on red blood cells. ~
monkey Anti-A isoagglutinins regularly present in serum.
Marmoset All secretors of A blood group substance, H Strong B-like epitope detectable on red blood cells.
substance very weak or absent.
Anti-B isoagglutinins regularly present in serum.
MN System 443
bon red cells are immunologically and structurally very close to, if not identical
with, human A, B, and H. They also display heritable variations analogous to
human subgroups of A. As in man, the ABO blood groups of apes are defined
by hemagglutination tests using polyclonal and/or monoclonal sera as well as
some other reagents such as plant extracts (lectins), eel serum, snail extracts,
etc., originally developed for testing human blood, but also strongly and
specifically cross-reacting with ape red cells. Only red cells of gorillas, which
are all group B, react very weakly with average anti-B reagents, and the ABO
typing of these animals is usually carried out by saliva testing.
B- or A-like structures are also present on the erythrocytes of some Old
World and South American monkeys. They do not display, however, individu-
al intraspecies differences, and appear as parts of species-specific configura-
tions on the red cell membrane rather than expression of allotypic differentia-
tion.
Practically all apes and monkeys are secretors of blood group substances
(thus far only a few nonsecretor orangutans have been found) which are im-
munologically indistinguishable from A, B, and H substances present in hu-
man secretor saliva and other body fluids. Inhibition of anti-A, anti-B, and
anti-H reagents by A, B, or H substance-containing saliva is the basic tech-
nique employed for ABO typing of monkeys.
As in man, ape and monkey sera contain anti-A and/or anti-B isoagglutinins
which are reciprocally related to the A and/or B antigens expressed on the red
cells and in tissues and secretions. Titers and avidity of primate natural anti-A
and anti-B antibodies are comparable to those of human isoagglutinins.
A reciprocal relationship between antigens and isoagglutinins (Land-
steiner's law), which has important consequences in transfusion and trans-
plantation [4], does not always hold in South American monkeys or in pro simi-
ans. The frequent occurrence of irregular anti-A and/or anti-B antibodies in
the sera of these animals is linked to the presence on their red cells of B-like
and, rarely, also A-like structures.
MNSystem
Humans and gibbons display all three types of the MN system - M, MN, and N
- but only two types are detected in gorillas and orangutans (Table 28.1).
Among Old World monkeys, baboon, macaque and vervet monkey red cells
give uniformly strong reactions with most, but not all, rabbit antihuman M an-
tisera. The same is true of some monoclonal antibodies raised against human
M. By the same token, antimacaque or -baboon (or -chimpanzee) rabbit or
guinea pig sera, properly absorbed to remove heterophile antibodies, give
with human M and/or MN cells reactions indistinguishable from those pro-
duced by rabbit antihuman M. Only orangutan and some chimpanzee red cells
react with anti-N reagents (rabbit antisera or lectin). No N activity is displayed
by the red cells of Old and New World monkeys.
444 Nonhuman Primate Blood Group Serology
Linked genetically and serologically to the MN system is the chimpanzee

V ABD blood group system which is defined by panels of specific chimpanzee
isoimmune reagents. Elements of this very complex system are also prcsent on
the red cells of orangutans and gorillas. Unlike antigens M and N, some of the
antigens of the V ABD system are highly immunogenic and may become a
source of undue reactions in mismatched transfusions and incompatible preg-
nancies. Their role in organ transplantation is not known.
Rh-hr System
Tests with human antisera of various Rhesus (Rh) specificities reveal epitopes
on the red cells of chimpanzees, gorillas, orangutans, and gibbons that are
identical with or closely related to Rho (D) and/or hr'(c) of human blood.
Although such polyclonal reagents failed to react with the red cells of lower
monkeys, a number of monoclonal antibodies against human Rh antigens
were found to agglutinate red cells of various Old World monkey species, thus
indicating that some of the Rh epitopes are also present on the red cell mem-
brane of these primates [5]. The latter finding is of some significance in view of
the protracted discussion about the specificity of the original anti-Rh serum of
Landsteiner and Wiener and its relationship to human anti-Rho
The development of chimpanzee isoimmune reagents of the so-called R-C-
E-F specificities [6] substantially increased the number of Rh-related epitopes
thus far identified in the blood of apes. The chimpanzee RCEF blood group
system defined by these reagents reaches the complexity of the human Rh-hr
system, and the immunogenicity of its main component, the RC antigen, equals
that of the Rho (D) in man. All other ape species share with the chimpanzee
some of the specificities of the RCEF system.
Other Simian-Type Blood Groups
A number of other simian-type blood group systems, unrelated to any known

human groups, have been defined by means of iso- and cross-immune sera de-
veloped in chimpanzees, gibbons, orangutans, baboons, rhesus monkeys, and
crab-eating macaques. Sera produced in one species often cross-react, in a
type-specific manner, with the red cells of other, taxonomically related pri-
mate species. In some instances, entire complex blood group systems are
shared by several species. This is the case of the so-called Drh graded blood
group system of macaques [7], or the BP blood group system of baboons [8].
Spontaneous antibodies against epitopes of the two latter systems are often
detected in the sera of apparently nonimmunized monkeys. This is of practical
importance when choosing proper species for experimental xenotransplanta-
tion [9].
Heterophile Antibodies 445
Heterophile Antibodies
When the red cells of an animal are mixed with the normal serum of an individ-
ual of another species agglutination or hemolysis of the red cells may result
due to the presence in the serum of naturally occurring species-specific (het-
Table 28.4. Antichimpanzee antibodies in normal human sera
ABO group ABO group of human serum

of chimpanzee
red blood cells o (K.G.y o (A.S.) Al (F.B.) Al (N.M.)
Antibody titera
S AG ETC S AG ETC S AG ETC S AG ETC
o (Ch-168) 2 32 32 12 32 24
o (Ch-472) 2 64 8b 8 32 8
o (Ch-486) 1 64 32 8 32 8
o (Ch-503) 2 64 16 12 32 64
A (Ch-124) 8 16 24 4 16 24
A (Ch-335) 8 12 12 4 16 16
A (Ch-366) 8 8 16 1 4 12
A (Ch-505) 12 8 16 4 12 24
a Method used to measure antibody titer: S, saline; AG, antiglobulin; ETC, enzyme-treated
red blood cells. Titer is expressed in units as the reciprocal of the highest dilution of the
serum that gave a distinct (one plus) reaction.
b Hemolysed
C Initials of donors of serum
Table 28.5. Antihuman antibodies in normal chimpanzee sera
ABO group ABO group of chimpanzee serum

of human
red blood cells o (486)b 0(429) A (366) A (124)
Antibody titer a
O(KG.y 2 6 8 0 0 2
O(W.K) 2 4 8 0 0 2
o (V.N.) 2 6 8 0 0 1
o (A.S.) 2 4 8 0 0 4
Al (F. B.) 0 0 2 0 0 1
Al (L.c.) 0 0 2 0 0 0
Al (D.F.) 0 0 2 0 0 0
Al (B.K) 0 0 2 0 0 1
red blood cells
b Identification number of chimpanzee donor of serum
C Initials of donors of red blood cells
Table 28.6. Antimonkey antibodies in normal human sera
ABO group of monkey ABO group of human serum .p..

red blood cells .p..
0\
O,M,rh O,MN,Rh 2 rh A I, M,Rh Irh A 2 , M, Rhl rh B,M,rh B, M, Rh2rh A 2 B, M, Rhl Rh I
(W. K.)h (M.S.) (D. F.) (L.l.) (J.M.) (J.M.) (N.C)
z0
:l
Strength of antibody reaction" :r
":3
S AG ETC S AG ETC S AG ETC S AG ETC S AG ETC S AG ETC S AG ETC '":l
..,'"t:I
Rhesus monkey 3'
B (473) 5 5 5 K K 8 2 2 5 K K 10 0 0 5 5 2 8 8 2 8 '"
(;
B (477) 5 5 5 8 K 8 2 2 5 5 K 10 0 0 5 5 5 8 8 2 8 OJ
B (485) 5 5 5 K K 8 2 2 5 5 5 10 0 0 5 0 0 8 2 0 8 0
0
B (495) 5 5 5 K K 8 0 2 5 :; 5 10 0 0 5 5 2 8 5 0 5 P-
0(505) 5 5 5 K K 8 2 2 5 5 5 10 0 0 5 :; 5 8 0 0 2 0..,
0
Crab-eating macaque '0
"
AB (424) 5 5 5 K 8 8 5 2 5 5 5 10 0 0 0 0 0 5 0 0 () VJ
(')
K 2 5 10
..,
A (726) 5 5 5 K 8 5 5 5 0 0 0 0 0 2 0 0 0 0
AB (728) 5 5 5 K 8 8 5 2 5 2 5 10 0 0 0 0 0 0 0 0 0 0-
CJq
AB (424) 5 5 5 K 8 8 5 2 5 K 5 10 0 0 0 0 0 2 0 0 2 '<
AB (424) 5 5 5 8 K 8 5 2 5 2 5 10 0 0 0 0 0 5 0 0 2
Baboon
A (127) 5 5 5 8 8 8 0 0 2 5 5 10 0 0 0 0 () 0 () 0 2
B (948) 5 5 5 8 8 K 2 2 2 5 5 10 0 0 0 2 0 0 0 0 2
AB (514) 5 5 5 K 8 8 2 2 2 5 5 10 0 0 2 0 0 2 0 0 2
Marmoset
A (70033) K 8 10 10 10 10 8 8 10 8 10 12 10 10 10 10 K 10 K 8 10
A (70049) K 8 ]() 10 10 10 8 8 10 8 10 12 10 10 10 10 K 10 K 10 10
Squirrcl monkey
AB (49) K 8 10 10 10 10 8 K 10 8 10 12 10 10 10 8 K 10 K 8 10
AB (55) 8 8 10 10 10 10 8 8 10 8 10 12 10 10 10 8 K 10 K 8 10
a Method used to measure strength of antibody reaction: S, saline: AG, antiglobulin; ETC, enzyme-treated red blood cells. Strength of agglutination
expressed as score: 2=weak; 5= 1 plus; 10=3 plus; 12=4 plus.
h Initials of donors of serum
Heterophile Antibodies 447
erophile) antibodies. The intensity of species-specific reactions generally in-

creases with the taxonomic separation of the two species (Landsteiner's im-
munological perspective), but the presence of heterophile antibodies directed
against red cells of closely related species does not always follow that general
rule.
Relatively potent antichimpanzee antibodies are found in most, if not all,
normal human sera. (Table 28.4 shows representative titers of such antibodies
in tests with ABO-identical chimpanzee red cells.) However, the reverse is
Table 28.7. Representative titers of antimonkey antibodies in normal human sera
Red blood cells ABO group of human serum
0 AJ B AlB
Representative antibody titer a
Rhesus monkey 16 32 8 8 12 8 4 2 8 2 2 4
Crab-eating macaque 4 12 4 4 4 8 3 2 4 2 2 4
Baboon 2 12 4 32 6 2 2 2 4 1 2 2
Marmoset 24 16 32 24 12 64 16 12 8 8 4 8
Squirrel monkey 16 24 24 16 48 64 32 16 32 8 4 32
red blood cells
Table 28.8. Antihuman antibodies in normal baboon sera
ABO group Serum of group AB baboons

of human
red blood 108b 110 111 113
cells
Strength of reaction a
o (K. W.)" 0 2 0 2 0 2 0 0 5 0 8 5
o (T.1.) 0 0 0 0 0 2 0 0 5 0 8 5
o (W.R.) 0 5 0 0 0 2 0 5 5 0 8 5
AJ (B.F.) 0 0 0 0 0 0 0 0 5 0 8 5
AJ (C.L.) 0 0 0 0 0 0 0 0 5 0 8 5
Al (H.S.) 0 0 0 2 8 8 0 0 5 0 8 5
B (T. K.) 0 0 0 0 0 5 0 0 5 0 8 5
B (J.M.) 0 2 0 0 0 8 2 5 5 5 8 5
B (W.S.) 0 0 0 0 0 8 0 0 5 5 8 5
a Method used to measure strength of antibody reaction: S, saline; AG, antiglobulin; ETC,
enzyme-treated red blood cells. Strength of agglutination expressed as score: 2=weak; 5=1
plus; 8=2 plus; 10=3 plus; 12=4 plus
b Identification of baboon donors of serum
C Initials of donors of red blood cells
rarely true; normal chimpanzee sera are either devoid of antihuman antibod-
ies or show only very weak activity against ABO-identical human red cells
(Table 28.5). When normal human sera are cross-matched with the red cells of
Old World monkeys. which are taxonomically more distant from man. wide
individual differences may be observed (Table 28.6). On the other hand. hu-
man sera uniformly and strongly agglutinate red cells of the South American
monkeys. such as marmosets or squirrel monkeys. which occupy a lower posi-
tion on the taxonomic scale. The avidity of these species-specific reactions is
usually. but not always. paralleled by the titer of these reactions (Table 28.7).
The presence and strength of antihuman antibodies in normal sera of Old
World monkeys are often erratic. as exemplified by tests with representative
baboon sera (Table 28.8). Monkey antihuman antibodies are usually of the im-
munoglobulin G (IgG) class and their titers are low. not exceeding 2-4 units.
By definition, species-specific antibodies recognize characteristic struc-
tures present on the red cells of all members of a given species. However. as
the data in Tables 28.6 and 28.8 show, not infrequently the antibodies present
in normal primate sera selectively react with some, but not all, red cells of an-
other species. The specificity of such antibodies, as assessed by parallel tests
with batteries of isoimmune reagents of various specificities. is often related to
some highly immunogenic blood group antigens of baboons and macaques
[10].
The origin of some of the type-specific antibodies could be related to isoim-
munization in pregnancy; others may have developed in response to exoge-
nous immunogens [11].
Practical Implications
Primates as Donors for Man
Anatomy, immunological characteristics, as well as general availability are the

basic criteria of suitability of a nonhuman primate as a possible organ donor
for human recipients. The risk of transmission of certain infectious agents and
parasites habitually carried by various primates is another point that must be
taken into account when considering a primate xenograft for man (Chap. 29).
Among the apes, the chimpanzee seems to meet most of the anatomical and
immunological criteria of a suitable donor. In its naive state, the chimpanzee
normally does not carry infectious agents dangerous for man, with the ex-
ception of some common viruses such as herpes, Epstein-Barr, and cy-
tomegalovirus. From the point of view of blood group serology. the common
chimpanzee (Pan troglodytes) appears as the closest relative of man, sharing
with him all major red cell antigens [12]. There is also a striking homology
among a number of chimpanzee and human leukocyte antigens [13-15].
Since most chimpanzees are blood group A, and only some group O. their
usefulness as donors is limited to group A or, rarely, group 0 human recipi-
Practical Implications 449
ents. The major impediment, however, is the limited availability of these apes.
As an endangered species, chimpanzees cannot be imported from the coun-
tries of their origin and the numbers of those maintained in captivity are small
and will remain so for the foreseeable future. The total current USA chim-
panzee population probably does not exceed 1500; those available for
biomedical purposes can hardly meet the current and continuous require-
ments of various vaccine testings and of hepatitis (B and non-A, non-B) and
acquired immunodeficiency syndrome (AIDS) studies, etc. Breeding of cap-
tive chimpanzees cannot keep up with the growing needs of the biomedical
science establishment, and no immediate improvement in their birth rate can
be expected.
Other anthropoid apes - gorillas, orangutans, gibbons - are immunologi-
cally more remote from man, their numbers are small and steadily diminishing
in the wild, and those few maintained in captivity cannot be even contemplat-
ed for any use other than for public display in zoological gardens.
The Old World monkeys constitute another class of primate with some po-
tential as organ donors for man. Some of these monkeys are still quite abun-
dant in the wild as well as in captivity. They share with man ABO polymor-
phism, and the species-specific barrier between macaques, baboons, vervet
monkeys and man may not prove too difficult to overcome by modern meth-
ods of immunosuppression. Due to their size, however, their organs can be
considered only for transplantation - possibly only as temporary "bridges" -
in children. Last but not least, one has to bear in mind the risk of transferring
infectious and parasitic agents carried by these monkeys, some of which (e.g.,
Ebola or Marburg viruses) are deadly for man.
Primates as Experimental Models for Xenotransplantation
Notwithstanding the steady improvement in methods of immunosuppression

and a greater understanding of nonhuman primate immunology, there still re-
main a number of theoretical and technical problems to be resolved before
simians can become a common source of organs for human patients. It is,
therefore, of practical importance to develop animal models for experimental
xenotransplantation between closely related primates that would closely ap-
proximate primate-to-man transplantation. A few such primate models have
gained particular recognition due to anatomical and immunological charac-
teristics as well as to the availability of the species.
Crab-Eating Macaque-to-Baboon Model
Both crab-eating (cynomolgus) macaques (Macacafascicularis) and baboons

(Papio cynocephalus/anubis) have human-type A, B, and H antigens ex-
pressed on the tissues and in secretions, while their sera regularly contain re-
ciprocally related anti-A and/or anti-B isoagglutinins. The distribution of the
ABO groups is comparable in both species - approximately one-third of ba-

boons (yellow and olive) and crab-eating macaques are group B, one-third
group A, and one-third group AB. Group 0 is extremely rare in both species;
it has been found thus far only among Guinea baboons (Papio papio) [16],
and, recently, among crab-eating macaques imported from mainland China
(unpublished data). Although the frequencies of the ABO groups may vary
from batch to batch, matching the animals according to their ABO type is, by
and large, a relatively easy task.
Baboon and crab-eating macaque red cells share a number of simian-type
antigens (monkey blood groups defined by specific antibodies raised in pri-
mate animals) which points to immunological closeness of the two species.
This conclusion is also supported by the low level or absence of reciprocal het-
erophile antibodies in the sera of the two species. The presence of simian-type
epitopes on tissues other than red blood cells has thus far been only indirectly
confirmed [17].
The anatomy of both species is sufficiently similar and, last but not least, the
supply of baboons as well as of crab-eating macaques is still plentiful, both
from the wild and from captivity, and these animals are relatively easy to main-
tain and breed under laboratory conditions.
Pig-tail macaques (Macaca nemestrina) are an alternative choice as organ
donors for baboons. They are particularly interesting as they are the only mon-
keys that have all four ABO types, with group 0 being quite frequent [18].
They share with baboons, to the same extent as crab-eating macaques, a num-
ber of simian-type red cell antigens [19]. However, supplies of pig-tail
macaques from the wild are somewhat limited and there are only a very few
sources of captivity-born animals. The price of a pig-tail macaque is, accord-
ingly, higher than that of a crab-eating macaque.
Rhesus monkeys (Macaca mulatta) have many simian-type blood group
antigens in common with baboons and the species-specific barrier between
these two species is negligible. Unlike baboons, however, they are almost all of
human-type group B, which renders ABO donor-recipient matching difficult.
More importantly, their supply is very limited and their price high.
Vervet monkeys (Cercopithecus) are easily available, but their immunolog-
ical characteristics are not favorable. Their ABO blood group distribution
shows a very high frequency of type A, which, in many batches, particularly
those originating from East Africa, reaches 100% [20]. Groups Band AB are
rare and encountered mostly among animals from South Africa [21]. Their
simian-type blood groups have little in common with those of baboons and
macaques. However, species-specific reactions between vervet monkeys and
baboons are weak or erratic.
As evidenced by the number of pretransplantation tests requested by in-
vestigators and carried out in the last few years by the Primate Blood Group
Reference Laboratory (New York University Medical Center), crab-eating
macaques and baboons are considered the most appropriate animals for use in
experimental xenotransplantation and are of growing popularity among re-
search teams involved in this field.
Blood Group Compatibility Testing 451
Blood Group Compatibility Testing
Tests aimed at selecting blood group compatible and matched donor-recipient

pairs for experimental xenotransplantation are usually carried out at the deal-
er's compound, so that only selected animals are actually purchased and trans-
ferred to the researcher's animal holding facility. When the pool of available
animals is sufficiently large, mUltiple donors are chosen for a single recipient,
so that additional criteria such as size, sex, anatomy, and health condition can
be taken into consideration for the final selection of the best donor.
Usually. the pre transplantation tests include the following:
1. Tests for human-type ABO groups are carried out either by hemagglutina-
tion tests (in apes) or saliva inhibition tests (in lower monkeys) for the pres-
ence of A, B, and H group substances using poly- and monoclonal anti-A,
anti-B and anti-H reagents and human A], A 2, Band 0 red cells as test cells.
Red cell and saliva testing is supplemented by serum tests for the presence
of anti-A and/or anti-B isoagglutinins. Generally, only pairs of ABO identi-
cal animals are chosen.
2. Cross-matching tests between red cells and sera of animals are carried out in
order to detect incompatibilities resulting from the antibodies directed
against simian-type red cell epitopes that occur spontaneously in the sera of
various primate species, including baboons and macaques. The tests are car-
ried out, at a wide range of temperature, by saline, antiglobulin, and en-
zyme-treated red cell techniques. Whenever possible, only those animals
whose major cross-matches (recipient serum against donor red cells) are
negative are paired for transplantation. In some cases the results of minor
cross-matches (donor serum against recipient red cells) are also taken into
consideration.
3. Tests for simian-type blood groups are carried out by an indirect antiglobu-
lin hemagglutination technique using panels of rhesus and baboon isoim-
mune sera specific for epitopes shared by the red blood cells of baboon and
various macaque species [17]. Since perfect matching within all specificities
tested is rarely feasible, the recipients and donors are matched at least for
antigens considered to be highly immunogenic. such as antigens of the Drh
[7] or BP series [8].
In the 4-year period 1986-1989 prexenotransplantation tests were carried

out at our center on 496 baboons and 610 cynomolgus macaques (Table 28.9).
Altogether, 4216 random cross-matches were performed - on average, each
baboon was cross-matched with 8.5 macaques - of which 2950 (70%) were
among ABO-identical animals. It is noteworthy that the frequency of positive
cross-matches (17.7%) among ABO-matched animals was not significantly
different from that found in tests among all animals irrespective of their ABO
type (18.5%). This confirmed that the positive cross-matches were not ABO-
related.
Table 28.9. Monkey pretranspiantation compatibility testing and cross-matching (19K6-19K9) ~
Ul
tv
Experimental Numbers of animals
procedure z
o
(species) Total tested Major cross-matches Crossmatches among Positive crossmatches among :;
:::-
ABO-identical ABO-identical animals c
Total Positive animals :3
Total Strong reactions '":;
..,""0
S·
Xcnotransplantation Macaques 61 0 } 295() 235 (45.()')\) )
4216 7KO (I K5'Yo ) 522 (17.7%) e
(")
(macaq uc- to-baboon) Baboons 496
~
Allotransplantation Baboons 330 3310 430 (13.0%) 2150 272 (12.6%) 105 (3K.6%) o
(baboon-to-baboon) 8..
..,Cl
Allotranspla ntation Macaques 1Y5 2700 324(12.0%) 1870 210(IIN!.») K2 (37.K'X») o
c
(macaque-to-macaque) '0
Vl
(")
..,
2-
c
~~
Blood Group Compatibility Testing 453
Table 28.10. Pre- and post-transplantation antibody titers in xenograft recipient sera
Titer of serum against donor red blood cells
Saline a Antiglobulina Enzymc a
Recipient 1
Initial 0 0 0
Post-transfusion b 6 12 16
Post-transplant day 0 2 16 6
Recipient 2
Initial 0 4 2
Post-transfusion b 32 64 128
a Method uscd to measurc antibody titer.

b One week after a series of threc donor-specific blood transfusions givcn at weekly inter-
vals.
For comparison, Table 28.9 shows the results of cross-matches in preallo-

transplantation tests carried out during the same period in baboons and
macaques. Here again, the frequency of positive cross-matches among ran-
domly paired animals was not different from that among ABO-identical ani-
mals. However, the frequency of positive cross-matches among ABO-
matched macaque-baboon pairs was significantly higher than among simi-
larly matched baboon-baboon pairs (p<O.OOl) or macaque-macaque pairs
(p<O.Ol). The frequency of strong reactions (two plus or more) in macaque-
baboon cross-matches was also significantly higher than the frequencies of
such reactions in cross-matches between animals of the same species.
The increased frequency of positive macaque-baboon cross-matches does
not seem to result from the action of preformed species-specific antimacaque
antibodies. Such antibodies (that clump red cells of all macaques or all ba-
boons) are usually of low avidity and rare occurrence; they have been found in
less than 2% of baboon sera tested thus far. As indicated by comparative test-
ings and titrations, spontaneously occurring antibodies responsible for the
positive cross-matches are generally type-specific and directed against simian-
type red cell epitopes that are shared, with various frequencies, by baboons
and macaques. Cross-reacting type-specific antibody directed against a very
high frequency antigen may be difficult to distinguish from a true heterophile
antigen [11].
An assessment of the role of various serologic parameters in the survival of
primate xenografts was attempted in a retrospective analysis of the results of a
small series of macaque-to-baboon and baboon-to-baboon cardiac transplan-
tations [17]. Although no correlation could be found between a positive pre-
transplantation cross-match and graft survival, there were indications that
fewer simian-type mismatches between donor and recipient may have result-
ed in a longer cardiac xenograft survival. This indirect evidence, however, has

not been confirmed by the necessary serologic investigations. Due to a lack of
serial post-transplantation serum samples, systematic serologic follow-up of
the recipients could not be carried out. We obtained post-transplantation
serum samples of only two baboon recipients of macaque heterotopic cardiac
transplants and titrated them against donor red blood cells. Both recipients re-
ceived pretransplantation donor-specific transfusions in addition to a conven-
tional immunosuppressive regimen. As shown in Table 28.10, there was a
build-up of antibodies after the series of pretransplantation blood transfu-
sions. This was consistent with earlier observations in rhesus monkeys and ba-
boons that a high-titer antibody response regularly followed multiple transfu-
sions of simian-type mismatched blood [22]. A transient decrease of antibody
titer immediately after transplantation suggests that adsorption of antibodies
onto the graft occurred, due either to the presence of donor blood cells in the
transplant or to the presence of simian-type epitopes on the grafted tissues.
References
I. Erskine. A.G., Socha. W.W. The Principles and Practice of Blood GWl/ping. 2nd edition.
C.V. Mosby. St. Louis. 1978.
2. Socha. W.W .. Ruffie. J. Blood Groups of Primates: Theory. Practice, Fvoilltionary
Meaning. Alan Liss. New York. 1983.
3. Socha. W.W. Blood groups of non-human primates. In: Comparative Primate Biology,
vol. 1. Systematics. Evolution and Anatomy. (Edited by D.R. Swindler and J. Erwin)
Alan Liss. New York. 1986. pp. 299.
4. Socha. W.W .. Marboe. e.e.. Michler. R.E .. Rose. E.A .. Moor-Jankowski. J. Primate an-
imal model for the study of ABO incompatibility in organ transplantation. Transplant.
Proc. 19.4448. 1987.
5. Socha. W.W .. Ruffic. J. Monoelonal antibodies directed against human Rh antigens in
tests with the red cells of non-human primatcs. Rev. Fr. Tram:fus. Hemohiol. 33.39. 1990.
6. Socha. W.W .. Moor-Jankowski. J. Chimpanzee R-C-E-F blood group system: a counter-
part of the human Rh-hr blood groups. Folia Primatol. 33. 172. 1980.
7. Socha. W.W .. Wicner. A.S .. Moor-Jankowski. 1.. Valerio. D. The first isoimmunc blood
group system of rhesus monkeys (Macaca mulatta): the graded Drh system. Int. Arch.
Allergy Appl. Imm1lno/. 52.355. 1976.
8. Socha. W.W .. Moor-Jankowski. J .. Ruffic. J. The BP graded blood group system of the
baboon; its relationship with macaque red cell antigens. Folia Primatol. 40.205.1983.
9. Socha. W.W .. Moor-Jankowski. J. Primate animal model for xenotransplantation: sero-
logical criteria of donor-recipient selection. In: Xenograft 25. (Edited by M.A. Hardy)
Elsevier Science Publishers. New York. 1989. p. 87.
10. Socha. W.W .. Wiener, A.S., Moor-Jankowski. J., Schcffrahn, W .. Wolfson. S.K. Jr.
Spontaneously occurring agglutinins in primate sera. In!. Arch. Allergy App/. Iml1111nol.
51,656,1976.
11. Wiener. A.S., Socha. W.W. Spontaneously occurring agglutinins in primate sera. II.
Their classification and implications for the mechanism of antibody formation.
liaematologia. (Budapest) 10.464.1976.
12. Socha. W.W., Moor-Jankowski. J. Blood groups of anthropoid apes and their relation-
ship to human blood groups . .I. Hum. }~vol. 8.453. 1979.
13. Bainer. H., Van Vreeswijk, W., Dersjant. H .. d·Amaro. 1.. Van Leeuven. A .. Van Rood.
J.1. Lcukocyte antigens of chimpanzee (ChL-A). Transplanl. Proc. 4.43.1972.
References 455
14. Van Rood, J.J., Van Leeuven, A., Bainer, H. HL-A and ChL-A: similarities and differ-
ences. Transplant. Proc. 4,55, 1972.
15. Ward, F.E., Seigler, H.F., Metzgar, RS. Cytoxocity reactions of chimpanzee antisera
with human lymphocytic donors phenotyped or genotyped for HL-A. Transplant. Proc.
4,63,1972.
16. Wiener, A.S., Moor-Jankowski, J. The A-B-O blood groups of baboons. Am. 1. Phys.
Anthropol. 30, 117, 1969.
17. Michler, RE., Marboe, e.e., Socha, W.W., Moor-Jankowski, J., Reemtsma, K., Rose,
E.A. Simian-type blood group antigens in non-human primate cardiac xenotransplanta-
tion. Transplant. Proc. 19,4456,1987.
18. Socha, W.W., Moor-Jankowski, J., Sackett, G.P. Blood groups of pig-tailed macaques
(Macaca nemestrina). Am. 1. Phys. Anthropol. 48,321,1978.
19. Moor-Jankowski, J., Socha, W.W. Blood groups of macaques. A comparative study.
1. Med. Primatol. 7, 136, 1978.
20. Jolly, e.J., Turner, T.R, Socha, W.W., Wiener, A.S. Human-type A-B-O blood group
antigens in Ethiopian vervet monkeys (Cercopithecus aethiops). 1. Med. Primatol. 6,54,
1977.
21. MeDermin, E.M., Vos, G .H., Downing, H.J. Blood groups, red cell enzymes, and serum
proteins of baboons and vervets. Folia Primatol. 19,312,1973.
22. Socha, W.W., Rowe, A.W., Lenny, L.L., Lasano, S.G., Moor-Jankowski, J. Transfusion
of incompatible blood in rhesus monkeys and baboons. Lab. Anim. Sci. 32,48, 1982.
Chapter 29
The Nonhuman Primate as Potential Organ

Donor for Man: Virological Considerations
S.S.KALTER
Introduction
The shortage of human donor organs makes it desirable to find a secondary

source of these much needed organs. Historically, attempts have been made to
transplant organs from several different animal species. The most promising
donor candidate for xenotransplants is the nonhuman primate, and, although
it differs from the human primate with regard to tissue, other immunological
factors [1], and microbiologic flora [2], its phylogenetic relatedness to man
supports this suggestion. Results with nonhuman primates, particularly the
chimpanzee and the baboon, are promising, especially since xenografts may
be viewed as temporary solutions until a human donor organ or tissue be-
comes available. This chapter provides information on the microbiologic flora
of nonhuman primates that is of greatest concern in xenotransplants - the
viruses of nonhuman primates.
Viruses of Nonhuman Primates
Both human and nonhuman primates harbor not only their own viruses, but
may also be infected with a number of viruses that are not of primate origin.
These nonprimate viral infections are not of primary concern unless they have
the ability to become latent in a new host, which then results in a natural infec-
tion ofthe primate.
The finding by Sabin and Wright [3] of Herpesvirus simiae (B virus) in the
rhesus monkey (M acaca mulatta) was a cause of great anxiety, and remains so,
because of its virulence in human infection. It was subsequently shown by nu-
merous investigators that this finding was not unique, and it was eventually
realized that the nonhuman primate plays host to a large number of viruses [2,
4-10]. Many of these simian viruses are counterparts of human agents, but are
antigenically distinct.
The majority of simian viruses may be included in existing virus families,
such as Herpesviridae, Picornaviridae, and Adenoviridae, and some are capa-
ble of causing infection/disease in both human and nonhuman primate (Table
29.1). There are, however, a number of miscellaneous, as yet unclassified simi-
an viruses. The number of viruses isolated from nonhuman primates is exten-
458 The Nonhuman Primate as Potential Organ Donor for Man
Table 29.1. Viruses isolated from nonhuman primates and considered to be of simian origin
Adell()virtlses
Adenovirus S-I (SY I, M I) Adenovirus S-13 (SY 36. Mil)
Adenovirus S-2 (SY II. M S) Adenovirus S-14 (SY 37)
Adenovirus S-3 (SY IS, M4) Adenovirus S-IS (SY 38)
Adenovirus S-4 (SY 17, M 6) Adenovirus S-16 (SA 7)
Adenovirus S-S (SY 20, M 7) Adenovirus S-17 (SA 17)
Adenovirus S-6 (SY 23. M 2) Adenovirus S-18 (SA 18)
Adenovirus S-7 (SY 2S. M 8) Adenovirus S-19 (AA IS3)
Adenovirus S-8 (SY 30) Adenovirus S-20 (Y 340)
Adenovirus S-9 (S Y 31 ) Adenovirus S-21 (C -I)
Adenovirus S-IO (SY32, M3) Adenovirus S-22 (Pan S)
Adenovirus S-II (SY 33. M 10) Adenovirus S-23 (Pan 6)
Adenovirus S-12 (SY 34) Adenovirus S-24 (Pan 7)
Herpesviruses
Herpesvirus S-I (Herpesvirus simiae)
Herpesvirus S-2 (SA6)
Herpesvirus S-3 (SA8)
Herpesvirus S-4 (HerpesviruS'tamarinlls)
Herpesvirus S-S (Herpesvirus saimiri)
Herpesvirus S-6 (SMY)
Enrerovirllses
Enterovirus S-I (SY2) Enterovirus S-I 0 (SY 43)
Enterovirus S-2 (SY 6) Enterovirus S-II (SY 44)
Enterovirus S-3 (SY 16) Enterovirus S-12 (SY 4S)
Enterovirus S-4 (SY 18) Enterovirus S-13 (SY 46)
Enterovirus S-S (SY 19) Enterovirus S-14 (SY 47)
Enterovirus S-6(SY26) Enterovirus S-IS (SY 49)
Enterovirus S-7 (SY28) Enterovirus S-16 (SA4)
Enterovirus S-8 (SY3S) Enterovirus S-17 (SAS)
Enterovirus S-9 (SY 42) Enterovirus S-18 (A 13)
sive. For example, over 35 simian herpesviruses isolates are recognized, and
undoubtedly others have not been defined. In addition, there are distinct simi-
an poxviruses, and perhaps of greater interest are the numerous simian retro-
viruses that have been recently isolated. Many of the simian retroviruses have
been given a vernacular name to coincide with the human retroviruses.
Included among the retroviruses are the foamyviruses, some of which are also
distinctly of nonhuman primate origin. Other viruses exist, isolated from simi-
ans, but their original host is not known. Hopefully, all these will be appropri-
ately classified in the not too distant future. In addition to these distinctive
simian viruses, there are other viruses common to both the human and nonhu-
man primate which may be considered primate viruses (Table 29.2).
Nonhuman primate viruses may be (i) natural- those viruses indigenous to
a species and responsible for natural infection/disease primarily within that
group of animals, and those viruses which may be common not only to the non-
human primate but to its human counterparts and other animal species as well,
and (ii) experimental- viruses that are alien to a particular host species, but in-
troduced either directly or as genomic material as a result of laboratory stud-
Viruses of Nonhuman Primates 459
Table 29.2. Viruses common to both human and nonhuman primates
Herpesviruses a Adenoviruscs
HSV Most scrotypes
CMV
Retroviruses
EBV
V_Zh Endogcnous type C
Immunodeficiency viruses
Lymphotropic hcrpes
Foamyviruses
Enteroviruses
Miscellaneous
Poliovirus
Ebola-likc (7) and Marburg (Filoviruscs)
Coxsackievirus
Rubella
Echovirus
Kyasanur Forest
EMC
Yellow Fever
HAV
Dengue and other Arboviruses
Rhinoviruses
Poxviruses
Paraint1ucnza viruses LCM (Arenavirus)
Measles (rubcola) Reoviruses (rotaviruses)
Parainfluenza 1-5 (SV5) Rabies
RSV HBV
Chimpanzee coryza agent NANB
Mumps(?) Others (?)
Influenza
HSV, herpes simplex virus: CMV, cytomcgalovirus: EBV. Epstein-Barr virus: V-Z. vari-
cella-zoster virus: EMC. encephalomyocarditis virus: HA V. hepatitis A virus: LCM, lym-
phocytic choriomeningitis virus: HBV. hepatitis B virus: NANB. non-A. non-B hepatitis.
a The exact status of herpesvirus infection of human and nonhuman primates is not clear.
h V -Z and relatcd viruses.
ies. Any of these viruses, once introduced into a host animal may then be trans-
mitted either vertically or horizontally.
Sources ofInfection
Viral infection of primates may be exogenous or endogenous. Endogenous in-

fection implies transmission of a virus from mother to offspring as genomic
material. A herpesvirus infection occurring in utero is an exogenous infection;
a retrovirus transferred from mother to offspring may be an endogenous infec-
tion, depending upon the epidemiology of the mother's infection. Infections
may also be vertically or horizontally transmitted. Vertical transmission is pas-
sage from mother to offspring, whereas horizontal transmission is from one in-
dividual to another directly or indirectly via fomites.
Exogenous infections are most frequent and come from a source such as
another animal. Exogenous infections in nature are either the result of natural
phenomena, in which the primate becomes infected through contact with an-
other animal (humans, rodents, birds, insects) or by contact with another non-
human primate. These other nonhuman primates may be of another species or
of the same species but from different geographic areas.
Table 29.3. Viruses present in nonhuman primate tissues that may be activated when trans-
planted into the immunocompromiscd host
Herpesviruses" Papoyaviruses
SAil JCvirus
EBV BK virus
V_Zh SV40
CMV
Papillomaviruses
Lymphotropie herpes
HPV
Enteroviruses
Adenoviruses
Poliovirus
Most serotypes
Coxsackievirus
Echovirus Parvoviruses
EMC AAY
HAV
Retroviruses
Hepatitis viruses Endogenous type C
HAV Immunodeficiency viruses
HBV Foamyviruses ('I)
HCV(NANB)
EBYc Miscellaneous
CMYc Hemorrhagic fever viruses (?)
Rubella C!)
Parainfluenza viruses Monkeypox en
Measles (rubeola) LCM (Arenavirus)
RSV(?) Reo-Rotaviruses (?)
EBV, Epstein-Barr virus: Y-Z, varicella-zoster virus: CMV, cytomegalovirus: EMC

eneephalomyoearditis virus: HAY, hepatitis A virus: HBV, hepatitis B virus; HCV
(NANB); hepatitis C virus (non-A, non-B); RSY, respiratory syncytical virus: HPV, human
papillomavirus; AA V, adcno-associatcd viruses; LCM, lymphocytic choriomeningitis virus
" Herpes B (B virus) is not included as macaques should not be considered as a donor ani-
mal because of this virus, The exact status of herpesvirus infection of human and non-
human primates is not cleaL
b Y-Z and related viruses,
C Theses herpesviruses can cause clinical hepatitis,
In addition, and a major conccrn in xcnografting, are those agents that

cause latent or chronic infections that may persist by integrating within the
host nucleic acid. Routine detection methods often fail to detect the presence
of these agents. All these possible sources of infection must be considered be-
fore use of any animal for xenotransplantation.
Pathogenesis of Primate Viruses
Viruses associated with transplants are principally the same whether the
source is the human or nonhuman primate (Table 29.3). The susceptibility of
primates to viruses may vary from host to host, but in general the pathogenesis
s the same once infection occurs. The variability in susceptibility makes the
search for primate model systems for the study of human disease difficult.
However, when a virus crosses from one species to another, a clinical picture
very different from that seen in the host of origin may result. Often a virus
causing inapparent or mild disease in one species may cause a more severe,
and perhaps fatal, outcome in another species.
The nonhuman primate reacts to infection much as the human does. As in
the human, exogenous infection is dependent upon many predisposing fac-
tors. To be successful, an infecting organism must overcome natural nonspe-
cific barriers, such as the mucocutaneous covering, nonspecific host inflamma-
tory response, and generalized, nonspecific responses associated with blood
and blood products. Having overcome these defensive mechanisms, an infect-
ing agent then encounters other defensive modalities. Of these defenses, the
most significant are the various components of the immune system. Any im-
pairment of the T cells, B cells, phagocytes, and complement will result in an
immunological deficiency. A number of primate viruses have the ability to in-
terfere with the host's immune mechanism, which in turn may activate a latent
inf~ction.
Viral Infection of the Immunocompromised Host
As expressed above, host defenses are important in the type of clinical re-
sponse elicited, but in an immunocompromised host such as the transplant
patient, the defense mechanisms are impaired. Table 29.3 lists viral infections
associated with the immunocompromised patient. In xenotransplant, the
virus( es) may be of human or nonhuman primate origin, even though the tis-
sue source may be of a nonhuman primate. Overt disease is not always ob-
served in the donor animal, making it necessary to test the tissue or organ for
viruses. However, current laboratory procedures are not always adequate to
detect the presence or guarantee the absence of a virus: the animal may not be
shedding virus, viremia may not be present, and genomic material mayor may
not be detected, although serologic testing will generally indicate antibody to a
previous infection if the methodology employed is sufficiently sensitive.
Accordingly, the potential for the transfer of these agents is omnipresent. An
overview of the viruses that are found in nonhuman primates may be of value
for their detection and prevention.
Herpesviruses
The herpesviruses are of greatest concern and probably present the greatest
challenge in terms of xenotransplants. The simian herpesviruses are very
closely related to their human counterparts, and their pathogenesis in the nat-
ural host parallels that seen in the human. Persistent infection occurs, with
virus residing in the host ganglia for the lifetime of the animal. Reasons for
virus shedding are obscure and a recipient of tissue is always at risk.
An interesting generalization occurs with regard to the herpes simplex

(HSV)-B virus-SA 8 complex in primates. HSV types 1 and 2 are found natu-
rally only in humans. Nonhuman primate infection with HSV occurs as a result
of human contact. HSV infection of nonhuman primates may be fataL a situa-
tion analogous to human infection with B virus. According to Plummer [11],
man is the only known natural host to HSV. Detection of herpesvirus antibody
in a primate is often nonspecific in the sense that the antibody detected may be
indicative of cross-reaction with a herpesvirus from another animal source. B
virus is a natural infection of Macaca spp., and when present in another species
always indicates contact with a macaque. In African species, represented by
Cercopitheclls spp. and Papio spp., SA 8 is their natural herpesvirus. All three
herpesviruses are closely related in all biologic parameters including anti-
genicity. This antigenicity also overlaps with nonprimate animal herpes-
viruses.
New World monkeys are infected with a number of distinct, but related
herpesviruses. Of these, H. saimiri and H. tamarinus are natural infections in
the squirrel monkey (Saimiri sciureus). These herpesviruses, along with a
number of others, have been isolated from a variety of New World monkeys.
Included among these herpesviruses are several that are Iymphotropic, and
these are important because several are oncogenic in various animal hosts.
However, the small size of New World monkeys generally precludes their use
in a transplantation program. Lymphotropic herpesviruses have also been iso-
lated from various Old World simians. Although none of these lymphotropic
herpesviruses has shown any predilection for human infection, this capability
does need consideration.
Other herpesviruses of primates include cytomegaloviruses (CMV),
Epstein-Barr virus (EBV), and varicella-zoster (V-Z). Only minor antigenic
variations are found among the isolates, whether recovered from human or
nonhuman primates.
The CMV isolates show the greatest variation, but for clinical purposes are
sufficiently closely related to be considered as singular. CMV is universally
distributed among nonhuman primates. In most instances, infection occurs
without overt disease. However, fatalities have been reported in chimpanzees
as well as latent infection with localization of virus in submaxillary glands,
blood, leukocytes, and other tissues [12, 13]. Muchmore [14] suggests that
chimpanzee CMV has the potential to cause human infection. It is well estab-
lished that the incidence of CMV infection following transplants with human
organs is very high [13, 15). Although there are no data to substantiate CMV
infection following transplants from simian to human, it must be assumed that
the potential for such would be very high, especially among immunosup-
pressed individuals.
EBV is another herpesvirus with widespread distribution in both the hu-
man and nonhuman primate popUlation. Its prevalence and association with
several clinical disease entities - infectious mononucleosis, Burkitt's lym-
phoma, nasopharyngeal carcinoma - and the ability to be oncogenic in several
species of nonhuman primates places an importance on this virus unexceeded
by many others within the context of transplantation. Adding to this concern is

the ability of EBV to be B cell tropic and capable of immortalizing primate B
cells in culture. EBV-like herpesviruses have been isolated from Old World
simians and all these isolates share some homology with EB V -DNA. With the
possible exception of the baboon EBV-like virus (H. papio) [16], the other iso-
lates from various monkeys and apes are not known to cause disease in their
natural hosts nor when experimentally inoculated into other primates. The
epidemiology of EBV infection in nonhuman primates resembles that seen in
human infection. Viral transmission is horizontal by means of oral secretions
and the incidence of natural infection is high, occurring at an early age. EBV
becomes latent, and the virus persists in leukocytes for the life of the animal
[17].
Of some concern is the widespread distribution of lymphomas seen in the
Sukhumi baboon colony - non-Hodgkin's lymphoma of the lymphoid type,
lymphosarcomas, pro lymphocytic lymphosarcomas, reticulosarcomas, lym-
phoplasmacytic and immunoblastic lymphomas, and lymphogranulomatosis.
These animals are hyporesponsive and frequently demonstrate immunosup-
pression in the course of their disease [18]. A number of lymphoproliferative
herpesviruses (Gammaherpesviruses) have been recovered from monkeys
and apes [19]. Any agent that becomes integrated into host nucleic acid and re-
mains undetected is of major concern when considering a source of donor tis-
sue.
V -Z is not a common infection of nonhuman primates; however, it does oc-
cur and causes a chickenpox-like, sometimes fatal, disease. In addition to V-Z,
several other antigenic ally related viruses have been recovered from nonhu-
man primates - Liverpool vervet virus (LVV), human papillomavirus (HPV),
Pat as monkey herpesvirus (PMH), Delta herpesviruses 1 and 2 (DHV),
macaque vesicular disease virus (Medical Lake Macaque virus, MLMV), and
chimpanzee herpesvirus (CZHV). A gorilla isolate is also recognized. All
these isolates share the same typical V-Z characteristics: cell association,
growth with difficulty in culture, and defective extracellular virus. The simian
isolates appear to be more closely related antigenically to each other than to
their human counterpart. The CZHV isolate is more closely related to human
V -Z than the other isolates. It is not clear whether the human V -Z virus infects
nonhuman primates and produces disease; however, as no other source has
been found, it is likely that CZHV may be of human origin. Zoster has not
been reported in simians, and dorsal root ganglia lesions have not been ob-
served. As zoster does not appear in humans until many years after primary
varicella infection, the failure to observe zoster in nonhuman primates may be
a matter of their short lifespan. Padovan and Cantrell [20] provide an excellent
review of varicella-like herpesvirus infections of nonhuman primates.
A herpesvirus originally isolated from the African green monkey as well as
from baboons, which is antigenically closely related to B virus and HSV, is SA
8. No confirmed report of human infection with this virus is known, but its fre-
quent presence in the baboon, an animal frequently considered as a potential
organ source, suggests consideration be given to this virus. Widespread infec-
tion in baboon colonies has been reported involving hundreds of animals. Few
deaths have occurred as a result of primary infection; oral and genital lesions
do occur, and fatalities result from secondary bacterial infection [21-23].
Some 37 herpesviruses of primates have been reported by McCarthy and
Tosolini [24], and others have since been recognized. The antigenic related-
ness of all these herpesviruses is not clear, but it is apparent that such a vast
number implies an infectivity requiring serious consideration in terms of
zoonoses and transplantation. Herpesviruses also exist in other animals in-
cluding amphibia, and the possible role these may play in antibody stimulation
or immunosuppression is unknown.
Enteroviruses
The enteroviruses are a large group of viruses frequently found in nonhuman

primates. A series of distinctive simian, as well as human, enteroviruses are
seen in nonhuman primates [2]. In general, although the human enteroviruses
are notorious for causing disease - poliomyelitis, meningitis, respiratory dis-
ease - , a few of their simian counterparts have been recognizably pathogenic.
They need be considered, however, because of the presence of antibody in
nonhuman primates to many of the enteroviruses as well as their widespread
distribution in nature. One enterovirus, in particular, is an exception.
Encephalomyocarditis virus (EMCV) has been responsible for periodic out-
breaks of disease in both the human and nonhuman primate population. With
the predilection of EMCV for heart tissue, the unwitting transfer of this virus
could have serious consequences. Recently an outbreak ofEMCV occurred in
a baboon colony with the loss of a large number of animals. Deaths occurred
with little prodromal symptomatology. Pathology was associated principally
with the respiratory tract and the heart [25].
The rhinoviruses, which are in the same family as the enteroviruses
(Picornaviridae) and known to cause infection in primates, do not appear to be
agents of concern in transplants. Enterovirus 72, or hepatitis virus A, is dis-
cussed with the other hepatitis viruses below.
Hepatitis Viruses
Human hepatitis A (HA V) infection has been reported as a consequence of

contact with infected chimpanzees [26]. The precise epidemiology of human
infection resulting from contact with chimpanzees is not clear. Other nonhu-
man primates have been shown to have antibody to HA V and a number of
species are known to be susceptible to HA V infection. Several nonhuman pri-
mate species are also known to have antibody to human hepatitis B virus
(HBV), and some have HBV antigen [27,28]. In addition to a clinical picture
similar to that seen in the human, HA V and HBV liver pathology also resem-
bles that seen in the human [29]. Few data are available on non-A non-B hep-
atitis (NANB) infection of simians, but it must be assumed that certain nonhu-
man primates become infected [30].
Adenoviruses
Some 25 serotypes of adenovirus are frequently isolated from nonhuman pri-

mates in kidney cultures, and in the respiratory and intestinal tracts. Disease
has been associated with these isolates, but more often, adenovirus isolation
has been from "normal" animals. Serologic surveys indicate that adenovirus
infection is common to all primates and antibody to human adenoviruses has
been found in nonhuman primates. Cross-reaction between the human and
nonhuman primate adenoviruses also occurs. An unknown aspect of adeno-
virus infection is the status of non cultivatable adenoviruses in nonhuman pri-
mates. Since these strains occur in humans, they probably are also present in
monkeys and apes. All adenoviruses appear to cause some pathology in the
immunocompromised host - gastroenteritis, cystitis, and renal failure.
A number of human and nonhuman adenoviruses are known to be onco-
genic and produce tumors in rodents. Mukai et al. [31] were able to induce
retinoblastomas in baboons following inoculation with human adenovirus 12.
Adenoviruses are one of the commonest causes of conjunctivitis in humans,
and human adenovirus infection is recognized in pediatric transplant recipi-
ents [32].
Influenza Viruses
There are no known simian influenza virus strains. Influenza does occur in the
nonhuman primate, with overt disease and significant increases in antibody
titer to the current strain. Serologic data suggest that the nonhuman primate
does not serve as a reservoir for influenza viruses, but apparently responds
clinically much as the human does.
Parainfluenza Viruses
Three genera of parainfluenza viruses (Paramyxovirus, Morbillivirus, and

Pneumovirus) are recognized, all with the capacity to infect primates.
Originally, two simian parainfluenza types (SV 5 and SV 41) were described.
SV 5 is frequently encountered in macaque kidney cell preparations, but does
not appear to be pathogenic and may not be a simian virus. This virus is fre-
quently isolated from humans and dogs. SV 41 has been seen only infrequent-
ly since its original isolation. Intracerebral inoculation of young monkeys with
SV 41 can produce a severe lymphocytic choriomeningitis.
The other parainfluenza viruses infect both humans and nonhumans and so
may be considered primate viruses. Parainfluenza viruses 1-5 (SV 5 is often in-
cluded with parainfluenza virus 5), mumps, Newcastle disease, and several
other animal and avian viruses comprise the Paramyxovirus genus; in the
Morbillivirus genus are the measles-rinderpest-distemper viruses; and the
genus Pnellnlovirus consists of the respiratory syncytial virus (RSV) group.
All parainfluenza viruses cause infection and disease along with persistent
infections in primates. The clinical pattern usually is one of an acute infection
in both human and nonhuman primates. Infection with measles virus may pre-
sent a different problem.
Although human infection with measles virus (rubella) has long been rec-
ognized as a major childhood disease, its recent link with disease in adults, par-
ticularly involvement of the central nervous system as a result of latent infec-
tion, makes this virus important in transplantation. Measles infection of
nonhuman primates is fairly common, but there have been no reports of cen-
tral nervous system complications occurring in the nonhuman primate. It is
also not known if chronic disease occurs in these species. Limited data suggest
that measles latency may occur in nonhuman primates [33] as it does in the hu-
man following measles infection [34]. Measles is also associated with immuno-
suppression and other immunological functions. In addition, a wide variety of
central nervous system complications also occur after the acute phase of
measles infection. In the human, measles has been associated with subacute
sclerosing panencephalitis (SSPE), as a result of infection and following vacci-
nation. Experimental attempts to produce SSPE in monkeys have thus far
been equivocal [35, 36]. Other diseases have also been linked to measles virus
infections - systemic lupus erythematosus, rheumatoid arthritis, polymositis,
scleroderma, Goodpasture's syndrome, Sjogren's syndrome, and Reiter's syn-
drome [37]. It would, therefore, appear that any measles seropositive animal
would be a poor choice as a donor animal for xenotransplantation.
RSV infection of chimpanzees results in lower respiratory disease and can
be fatal (P. Alford, personal communication). Other nonhuman primates be-
come infected (seropositive) with RSV, but apparently do not become ill. It is
not known if RSV becomes latent following infection, but as this virus is a
paramyxovirus the possibility exists.
Papovaviruses and Papilloma viruses
SV 40, one of the earliest of the recognized simian viruses [38], is not consid-
ered infectious for humans and multiplies poorly in human cells. This virus has
been isolated from patients with progressive multifocalleukoencephalopathy
(PML) [39]. Widespread human exposure to SV 40 resulted inadvertently
from its presence in early poliovirus vaccines. Antibody to SV 40 may be found
in both poliovirus vaccinated and unvaccinated populations. Antibody to SV
40 is also present in nonhuman primate popUlations, but it is not clear which of
the nonhuman primates, if any, is the natural host for this virus. SV 40 has been
isolated from immunologically suppressed macaques with spontaneous PML
[40].
The precise role pap ova viruses play in human infection/disease is not clear.
Several papovaviruses (JC virus, JCV, and BK virus, BKV), as well as the
closely related papillomaviruses, have been recovered from humans under
normal and clinical conditions. SA 12, an African monkey papovavirus, is
widespread among nonhuman primates as evidenced by the presence of anti-
body [41]. Antibodies to a B celllymphotropic papovavirus have been report-
ed to be prevalent among human and nonhuman primates [42]. Of concern in
the use of papovavirus-positive primates as tissue donors would be the ability
of these viruses to produce tumors, transform cells, and to become latent. JCV
has been shown experimentally to cause brain tumors in intracerebrally inocu-
lated owl monkeys. It was also noted that the JCV genome was integrated into
the cellular DNA in all tumors examined [43].
Papillomaviruses have been reported in nonhuman primates (including
chimpanzees) and produce cutaneous papillomas. Histologically these papil-
lomas resemble those seen in humans. It has been demonstrated that papillo-
mavirus-associated venereal lesions occur in the nonhuman primate [44].
Association of human genital warts (condyloma acuminata) with HPV raises
the question of possible transfer during transplantation from one primate to
another. Over 60 HPV types are recognized in the human. Of these, seven are
prevalent (HPV 6, HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, and HPV 35).
Those prevalent in nonhuman primates have not been reported.
Parvoviruses
Although long recognized in veterinary practice in association with a number

of animal diseases, it was only recently and by chance that a human parvovirus
was found [45]. Originally isolated from asymptomatic blood donors and
thought to be of minor clinical relevance, it is now clear that human par-
voviruses, as represented by strain B 19, cause erythema infectiosum and the
aplastic crisis that complicates chronic hemolytic anemia. B 19 virus is proba-
bly responsible for a wide range of clinical effects [46].
The wide spectrum of animal diseases associated with various parvoviruses,
particularly fetal infection, raises the possibility of transfer from animal to hu-
man. The only recognized simian parvoviruses are the adeno-associated viruses
(AA V) which are found in preparations of simian adenoviruses. Adenoviruses
are one of the most common of simian viruses. Antibody to AA V -1 has been
found in monkey and human sera [47] suggesting that infection of humans with
simian AA V does occur. The significance of this finding is unknown.
Retroviruses
This family of viruses, apparently found in all animals including humans, is one
of the more important virus groups in terms of xenotransplantation. The retro-
viruses are one of the more complex viral families with regard to their biologic
and clinical capabilities. Classified according to morphology into a number of

different subfamilies, these viruses vary in origin, epidemiology. pathogenesis.
genomic structure, and clinical manifestations. Genomic material of endoge-
nous retroviruses probably resides within all living animals. Expression of
these genes varies from cell to cell, and such genes are difficult to detect al-
though molecular probes may eventually be of value for their detection.
Careful and diligent search has demonstrated the presence of C-type retro-
virus particles in most vertebrate placental tissues examined [48].
Within this grouping are both endogenous and exogenous retroviruses
which may be vertically or horizontally transmitted. As a group, the retro-
viruses have gained prominence because a number arc oncogenic
(Oncovimses) and more recently as the causative agents of human acquired
immunodeficiency virus (AIDS) and simian AIDS, i.e., human immunodefi-
ciency virus (HIV) and simian immunodeficiency virus (SIV), respectively,
and a number of human and nonhuman primate cancers and other diseases
(e.g., human T celllymphotropic virus. HTL V. and simian T celllymphotropic
virus, STLV). The AIDS/SAIDS viruses are classified as either lcntiviruses or
type D oncoviruses. SAIDS in nonhuman primates may also be caused by sev-
eral type D oncoviruses (SR 1-5). The HTLV/STLV viruses are type C on-
coviruses. Among other retroviruses are the slow viruses (M aedilVisna) which
are included among the lentiviruses. The foamyviruses (Spumavimses). pre-
sent in human and nonhuman primate tissues, are generally considered labo-
ratory nuisances. No disease has been attributed to these foamyviruses.
Type C oncoviruses were first described in birds by Rous in 1910 (Rous sar-
coma) [49]. The presence of type C viruses in mice is also well known. An ex-
ogenous type C virus was reported by Theilen et al. [50] following its isolation
from a woolly monkey (Lagothrix sp.) with a fibrosarcoma. This isolate (SSV-
1) was recovered only in the one cited instance. However, the animal from
which it was recovered had been in contact with gibbon apes (Hylobates lar).
Several type C viruses have been isolated from gibbons with lymphosarcomas
and leukemias [51]. Because there is an immunological relationship between
the woolly monkey isolate and the gibbon isolates. the status of the woolly
monkey isolate is not clear. The gibbon ape leukemia virus (GALV) is excret-
ed into the environment and leads to infection of antibody-negative gibbons.
Infection in utero also occurs with virus transmission from mother to fetus.
The origin of GALV is uncertain, but it appears that gibbon infection results
from contact with feral mice (Mus caroli) as this animal carries an endogenous
virus closely related to GAL V and SSV -1.
Endogenous retroviruses have been observed in nonhuman primates by a
number of investigators. First observed in baboon and human placental tissue
[52,53]. their presence in various other primates and vertebrates was quickly
confirmed [54-57]. Of interest was the inability to isolate these agents from
most animals, but both types C and D were observed by electron micros-
copy [58]. Isolates were obtained from baboons (Papia cynocephalus.
P. hamadryas, P. papio, P. anubis, Theropithecus gelada), stumptail macaques
(M. arctoides) , rhesus (M. mlllalta) , colobus (c. polykomus), and owl monkeys
(Aotus trivigatus) [59-65]. Type D isolates were recovered from the langur
(Presby tis obscurus) [66] and squirrel monkeys (S. sciureus) [67]. Little is
known about the pathogenesis of these isolates with the exception of those
from the baboon. Less is known about those agents that were seen, but never
isolated.
A baboon isolate (BaEV) containing the Kirsten murine sarcoma genome
(KMSV[BaEV]) produced a brain tumor with metastasis to the lung in an in-
tracerebrally inoculated baboon [68]. Tumors were also observed in other
species, but these generally regressed. A newborn chimpanzee inoculated
with this pseudotype developed metastatic disease and was euthanized. The
results of these studies indicated that host susceptibility depended upon the
immunological response of the animal to the expression of antigens develop-
ing in the placenta and other embryonic cells. According to Ahmed et al. [69],
the interaction between the baboon endogenous virus and host cell may be
more than immunologically dependent. The baboon virus (BV) may interact
with human cells carrying the RSV genome, producing syncytia which in turn
may help spread oncogenic virus infection to noninfected cells. BV syncytium-
infected cells, upon passage, lose their ability to form syncytia, but establish a
chronic BV infection.
Recovery of the type D retrovirus from a rhesus monkey mammary tumor
was the forerunner of the current AIDS/SA IDS retrovirus studies [70]. This
Mason-Pfizer monkey virus (MPMV) was not originally recognized as a
SAIDS virus and not considered to be tumorogenic. In retrospect, the clinical
picture produced by MPMV is now recognized as AIDS/SAIDS (SRV 3)
[71-75]. Interest in MPMV and other endogenous retroviruses lagged, princi-
pally because of the inability to associated tumors unequivocally in experi-
mental animals. The recognized relationship between MPMV and the newly
isolated type D retroviruses (SRV 1-5) would indicated that the significance
of the other endogenous retroviruses should be reconsidered.
Since the early 1970s, veterinarians have observed periodic outbreaks of
opportunistic infections along with neoplasias and immunosuppression in
macaque colonies. The similarity of the disease in monkeys to the emerging
human AIDS quickly became apparent. It is now quite clear that a parallel
clinical situation exists in the human and nonhuman primate populations.
Accordingly, the viruses responsible for the human and simian diseases share
a parallel nomenclature - HIV and SIV (lentiviruses) for the human and non-
human agents, respectively. These viruses also share cross-reactive viral pro-
teins. A number of distinct, but related, SIV isolates have been found from dif-
ferent nonhuman primate species. These isolates were derived from
immunodeficient macaques at several primate centers. In addition to SAIDS,
many of the animals also displayed another clinical condition - retroperi-
toneal fibromatosis (RF). This latter group of animals yielded the type D
retroviruses, SRV 1-5 (see above).
Other simian retroviruses, counterparts of human agents, also exist. These
viruses, in keeping with current nomenclature, have been designated STLVas
they are closely related to the human HTLV viruses. These are type C retro-
viruses and in humans are responsible for adult T cell leukemia (ATL). tropi-
cal spastic paraparesis (TSP). B cell lymphoma. myelopathy. immunodeficien-
cy. hairy celilcukemia. and mycosis fungoides (HTL V-I. II. V). In macaques.
STLV-I causes malignant lymphomas [76.77]. STLV-I has been recovered
from Old World monkeys. and antibody is particularly prevalent in the
African green monkey (c. aethiops). Antibody to HTL V-I has also been ob-
served in possible donor candidates such as the chimpanzee and the baboon.
Serologic surveys indicate that HTL V -1 antibody is prevalent in a wide variety
of monkeys and apes [78-83 j. Evidently there is a wide disparity in HTL V-I
infection among the apes in particular.
Regardless of the lack of recognized infection in apes with HTLV -1. use of
these species as donor animals must be approached cautiously. As retrovirus-
es are transmitted vertically from mother to offspring. the unknown potential
for the transfer of genomic material is reason enough for concern.
Miscellaneous Viruses
When using nonhuman primates. as with any group of animals. the unexpect-
ed must be anticipated. In addition to infection/disease with recognized virus-
es. ""new" or unreported viral outbreaks occur. A series of virus diseases. prin-
cipally hemorrhagic diseases. due to previously unrecognized agents or
obscure viruses, has been recorded. The simian species most affected has been
macaques. Kyasanur Forest disease in langurs (Presby tis entellus) and bonnet
(M. radiata) monkeys. as well as in humans, is typical of a localized zoonotic
condition that is apparently restricted to only one area [84]. The singular oc-
currence of African green monkey-related Marburg disease in humans and
monkeys is well documented [85]. Other occurrences of human Marburg dis-
ease unrelated to monkeys have since been recognized.
After a lapse of approximately 20 years, simian hemorrhagic fever (SHF)
has again appeared in monkeys. First observed in the Soviet Union, England,
and the United States in the late 1960s. it is a highly fatal (100%) hemorrhagic
disease. Recently. an outbreak of disease due to this same agent occurred in
macaque colonies. In the original outbreaks. transmission of virus was by con-
taminated syringe needles and tattooing needles. These were not changed be-
tween animals. The epidemiology of the current SHF outbreak, as best deter-
mined, was either by way of rodents. aerosols. or an insect vector. but not
contaminated needles. As in the first SHF outbreak. the 1989 outbreak that
occurred in the United States and Germanywas 100% fatal. Humans are unaf-
fected by SHF virus. SHF. although highly fatal for the macaque, is evidently
limited to this genus. The natural host for SHF is the patas (Erythrocebus
patas) and African green monkey (and possibly the baboon). These species do
not show any evidence of clinical disease with this virus. but antibody may be
demonstrated (D.M. Renquist. personal communication).
An Ebola-like virus. in association with an SHF outbreak. has also recently
been found (1990). Ebola virus infection. which in macaques is also highly fa-
Practical Considerations 471
tal and clinically similar to SHF, is highly fatal for humans. Although there
were more than 100 human contacts with these Ebola-like virus infected ani-
mals, no human cases have been recognized. This is the first recognized occur-
rence of Ebola virus in nonhuman primates. The epidemiology of this out-
break is not clear. The original Ebola virus cases occurred in Africa. The
monkeys (M. fascicularis) carrying this Ebola-like virus most recently were
from the Philippines [86].
Occurrence of Ebola virus in primates implies consideration be given to a
number of other diseases (principally hemorrhagic) that occur in the geo-
graphic areas from which nonhuman primates are derived. Included among
these diseases are Lassa fever, Machupo virus (Bolivian hemorrhagic fever),
dengue, Rift Valley fever, and others (arbovirus diseases). Clinically, these
diseases are all similar. making differential diagnosis difficult.
Practical Considerations
The concept of xenotransplantation has intrigued the medical community for

many years. A number of different animal species have been used as donor an-
imals in spite of the knowledge that rejection of the transplanted tissue or or-
gan was likely. After the turn of the century, several investigators made use of
primates as organ donors with questionable results [87,88]. Modern primate
xenotransplantation research owes its existence to the efforts of several
groups of investigators using the baboon and chimpanzee [89-91]. In all in-
stances, although patient survival varied, the tissue was rejected in spite of in-
tensive immunosuppressive regimens.
Much has been accomplished during the past years in terms of developing
more appropriate approaches to the immunosuppressive therapy. Once again,
principally because of the scarcity of human organs, serious consideration is
being given to the use of primate organs, particularly in neonates, as an interim
measure prior to availability of human organs. Although the chimpanzee has
always been considered the most desirable of primates for xenotransplanta-
tions, limited availability plus the almost universal outcry by various conserva-
tion and "animal rights" groups restricts the choice of the chimpanzee.
Colony-bred baboons (P. ursin us) and the African green monkey have been
recommended as potential organ donors [92-94].
In making these recommendations, these investigators express a concern
for the presence of infectious agents, particularly viruses in the donor animal.
Recognition that infectious agents may playa major role in transplantation is
not new. Viral infections are known to increase following immunosuppression
as a consequence of organ transplants from human to human [15]. This same
enhanced infectivity should be expected following xenotransplants inasmuch
as the nonhuman viral flora parallels that of the human [2]. It is not surprising
that any suggestion to use simian tissues would immediately raise the question
of the likelihood of transmitting potentially pathogenic viruses (as well as oth-
er organisms). Accordingly, using nonhuman primate organs for transplants

would in effect double the risk of infection in the organ recipient. Not only is
the organ recipient at risk from activation of his or her latent infection because
of immunosuppression, but, being immunologically suppressed, their suscep-
tibility to any agent that may be in the transplanted organ is also enhanced.
Gold [95] expressed concern regarding infections associated with immuno-
deficiency. In addition to various bacterial, parasitic, and fungal diseases that
result from immunosuppression, a number of viral diseases are of concern -
polioviruses, herpesvirus (HSV, CMV, V-Z), measles, and vaccinia. Gold em-
phasizes that, although no live vaccines should be used, attempts should be
made whenever possible to protect immunocompromised individuals with
killed vaccines even though their immune response is not normal.
Ho [15] reviews the status of virus infections of humans after transplanta-
tion and concludes that, following major organ or tissue transplantation, the
most common viral infections are latent infections which reactivate.
Herpesviruses are the most frequently activated virus group, with CMV ap-
proaching 100%, followed by HSV, V-Z and EBY. Reactivation of BKV is
probably more than 50%. lCV, which has been associated with PML is also ac-
tivated during immunosuppression. Other unknown or unrecognized latent
infections probably occur, but are undetected. According to Ho, primary in-
fections also occur resulting from viruses in the donor tissue (CMV, hepatitis
B). Although most viral infections are inapparent, there is significant morbidi-
ty and mortality resulting from herpesvirus infections.
Ho [15] concludes that the factors that account for increased infections are:
(i) suppression of cell-mediated immunity by cytotoxic immunosuppressants
and antilymphocytic serum, (ii) graft-versus-host and probably host-versus-
graft reactions, (iii) exposure to a source of virus, and (iv) underlying disease.
Suppression of antibody formation does not appear to be an important factor.
However, a number of viruses (herpesviruses, retroviruses, others) are known
to suppress antibody formation by their action on antibody-forming cells. One
may speculate that exposure to these organisms could precipitate a chain reac-
tion, setting off an enhanced susceptibility or severity of reaction following
this exposure. Such reactions have been reported as a result of herpesvirus in-
fections in AIDS patients [96]. Heise [97], in a review of diseases connected
with immunosuppression, emphasizes that any impairment of major compo-
nents of the immune system (T cells, B cells, phagocytes, or complement) may
result in clinical immunodeficiency. This impairment is expressed as a marked
susceptibility to opportunistic and pathogenic organisms which are difficult to
control.
Although there are few data on this subject concerning nonhuman viruses,
the close relationship between human and nonhuman primate viruses sug-
gests that a similar reaction could occur following xenotransplantation.
Recognizing the possibility of such a predicament, van der Riet et al. [92]
screened promising baboon and African green monkey donors for antibody to
a number of major viruses. This study also attempted to define the influence of
captivity on the presence of the agents. The results indicated that infection is
Comment 473
commonplace in wild-caught animals, which in essence would preclude their

use for xenografts. Similar findings were previously reported in an extensive
review of the virological flora of wild and captive nonhuman primates [98].
Captive breeding does tend to lower the incidence of viral infections, suggest-
ing that such a mechanism may produce desirable donor animals. Captive
breeding requires a carefully planned regimen of monitoring animals as well
as husbandry protocols [99].
Other considerations have generally overridden the aspect of infection in
the transplant process. The major concern has been to provide an organ that
will function. However, it is evident that organ and tissue transplantation,
whether from human or nonhuman sources, does present the same basic prob-
lem - the introduction of potentially pathogenic organisms into an immuno-
logically deficient individual. Meyers et al. [100] observed that infections oc-
curred in 28% of transplantation patients within 18 months post operation.
Use of nonhuman tissues would probably result in the same incidence of infec-
tion, if not higher. The simian retroviruses, which are present in several species
under consideration as organ donors, need investigation. Cross-infectivity by
these agents is recognized, although apparently human infection has not oc-
curred [78-80, 83].
Recently a baboon heart was used in a baboon-to-human orthotopic trans-
plant in a neonate with hypoplastic left heart syndrome. The baboon, a young
female free of microfilaria, hepatitis antigen, CMV, and parasites, was the
donor animal. The infant survived for 20 days with no evidence of systemic in-
fection [101], suggesting optimism for future investigations. Information on
long-term effects is obviously unavailable.
If the use of nonhuman primates as a source of donor organs becomes a vi-
able option, considerations other than rejection and infection must be exam-
ined. Many social, legal, ethical, and even political considerations require at-
tention. From the pragmatic aspect, the simian of choice for xenografts would
be the chimpanzee. This species is simply not available and, even assuming
that breeding programs were instituted, sufficient numbers would probably
never be available. The baboon might be a reasonable alternative. Sufficient
numbers are available to initiate breeding colonies, and the baboon breeds
well in captivity. The virological flora of these animals has been well studied.
In spite of recent publicity with regard to the use of animals in research, if
transplants are to be successful in the human, continued research in live ani-
mals is a must. Substitute model systems simply do not exist, nor do prostheses
function satisfactorily. A choice will have to be made.
Comment
Lacking sufficient human sources for donated tissues and organs, it is highly
likely that there may be a need for xenografts. The nonhuman primate would
undoubtedly serve as the most viable alternative. It is probable that immuno-
logical advances will overcome difficulties related to rejection. What has heen
only superficially addressed. and possihly more difficult to resolve. is the pres-
ence of infectious agents in the donor organ. In addition to their infectivity is
their potential for integrating into the host nucleic acid. For practical purposes
this host relationship might appear to eliminate the use of animals as a source
of tissue. However. advances in chemotherapy. like those seen in immunolo-
gy. suggest that infections will also he overcome.
The development of a colony of animal donors free of potentially danger-
ous viruses would he a monumentaL hut not impossible. task. The ability to
rapidly screen nonhuman primates at the time of capture is now under study
[99 j. Recently. several studies addressing this concern suggest colony breeding
as a method for reducing the prohlem of host viruses [92. 94 J. In these studies
long-term expression of viruses was not considered. If the use of nonhuman
tissues were to be only a stop-gap measure until a suitahle human organ he-
came available. long-term incubation as a prelude to infection would not be a
major issue. Good colony management. including the monitoring of both the
animals and attending human personneL should do much to provide a suitable
animal organ until an appropriate human organ becomes available [102-1051.
If the animal organ were to he transplanted with a view to permanency. how-
ever. then the development of late viral infection would need to be seriously
considered and excluded.
Acknowledgements. Much of the data reported herein were the result of a long
collaboration with Dr. R.L. Heherling. His efforts are very much appreciated.
Numerous staff memhcrs have also been involved and their efforts are ac-
knowledged. Support for our studies of viral diseases of nonhuman primates
principally has been from grants from the National Institutes of Health and
the World Health Organization. More recently. support for development of
diagnostic procedures applicable for use in the field or nonlaboratory setting
has been through the Small Business Innovative Research Program.
References
1. BaIner, H. Choice of animal species for modcrn transplantation rcsearch. Tral1spfa11l.

Pmc. 6. 19 1974.
2. Kalter. S.S .. Ablashi. 0 .. Espana. C .. Heberling, F.L.. Hull, R.N., Lennette. E.H ..
Malherbe. H.H .. McConnell, S.. Yohn. D.S. Simian virus nomenclature. 1980.
Jl1tervirofogr. 13.17.1980.
3. Sabin, A.B .. Wright. A.M. Acute ascending myelitis following a monkey bite. with the
isolation of a virus capable of reproducing the disease..!. f:'xp. Mf'rf. 59. 115, 1934.
4. Hull, R.N .. Minner. J.R.. Smith.J.W. New viral agents recovered from tissue cultures of
monkey cells. 1. Origin and properties of cytopathogenic agents. SVI. SV2. SV4. SV5.
SV6. SVI1. SVI2. and SVI5. Alii..!. Hvg. 63.204.1956.
5. Malherbe. H .. Harwin, R. Scven viruses isolated form the vervet monkey. Brit. .!. Exp.
Pa/hol. 38.539. 1957.
6. Hsiung, G.D .. Melnick. J.L. Orphan viruses of man and animals. AI1I1. N. Y. Amd. Sci.
70.342. 1958.
References 475
7. Heberling, R.L., Cheever, F.S. Enteric viruses of monkeys. Ann. N. Y. Acad. Sci. 85,
942,1960.
8. Shah, K., Southwick, CM. Prevalence of antibodies to certain viruses in sera of free-liv-
ing rhesus and captive monkeys. Int. J. Med. Res. 53,488,1965.
9. Bhatt, P.N., Goverdhan, N.K., Shaffer, M.J., Bandt, CD., Fox, J.P. Viral infections in
monkeys in their natural habitat in Southern India. Am. J. Trop. Med. Hyg. 15, 551,
1966.
10. Soave, O. Viral infections common to human and non-human primates. Jama. 179,
1385,1986.
11. Plummer, G. Comparative virology of the herpes group. Prog. M ed. Virol. 9,302, 1967.
12. Vogel, F.S., Pinkerton, H. Spontaneous salivary gland virus disease in chimpanzees.
Arch. Pathol. 60,281,1955.
13. Jordan, M.C Latent infection and the elusive cytomegalovirus. Rev. Infect. Dis. 5,205,
1983.
14. Muchmore, E. Possible cytomegalovirus infection in man following chimpanzee bite.
Lab. Anim. Sci. 21,1080, 1971.
15. Ho, M. Virus infections after transplantation in man. Arch. Virol. 55,1,1977.
16. Lapin, B.A., Ayrba, VX., Yakovleva, L.A., Sangulia, l.A, Timanovskaja, V.V.,
Chuvirov, G.N., Kokosha, L.V. The establishment of suspension lymphoblastoid cell
cultures containing herpes-like virus from haematopoietic organs of hamadryas ba-
boons with hematosarcoma. Dokladi. Akademii. Nauk. Sssr. 222, 244, 1975 (in
Russian).
17. Rabin, H. In vitro studies of Epstein-Barr virus and other lymphotropic herpesviruses
of primates. In: The Herpesviruses. Volume 4, Roizman, B. and Lopez, C (eds.)
Plenum, New York, London, 1985,p.167.
18. Neubauer, RH., Rabin, H., Strnad, B.C, Lapin, B.A, Yakovleva, L.A, Indzie, E.
Antibody responses to Herpesvirus papio antigens in baboons with lymphoma. Int. J.
Cancer. 23, 186, 1979.
19. Kalter, S.S. Herpesvirus infection in Old and New World monkeys. In: Virus Diseases in
Laboratory and Captive Animals. Oarai, G. (ed.) Martinus Nijhoff Publishers, Boston,
1988, p. 101.
20. Padovan, D., Cantrell, CA. Varicella-like herpesvirus infections of non-human pri-
mates. Lab Anim. Sci. 36,7,1986.
21. Eichberg, J.W., McCullough, B., Kalter, S.S., Thor, D.E., Rodriguez, AR. Clinical, vi-
rological, and pathological features of herpesvirus SA8 infection in conventional and
gnotobiotic infant baboons (Papio cynocephalus). Arch. Virol. 50,255,1976.
22. Kalter, S.S., Weiss, S.A, Heberling, RL., Guajardo, J.E., Smith, CG., III. The isolation
of herpesvirus from trigeminal ganglia of normal baboons. (Papio cynocephalus). Lab.
Anim. Sci. 28,705,1978.
23. Levin, J.L., Hilliard, J.K., Lipper, S.L., Butler, T.M., Goodwin, W.J. A naturally occur-
ring epizootic of simian agent 8 in the baboon. Lab. Anim. Sci. 38,394, 1988.
24. McCarthy, K., Tosolini, F.A A review of primate herpes viruses. Proc. Roy. Soc. Med.
68,145,1975.
25. Hubbard, G.B., Carey, K.O., Butler, T.M., Levin, J.L. An epizootic of encephalomyo-
carditis virus in baboons. Personal communication, 1989.
26. Hillis, W.D. Etiology of viral hepatitis. John Hopkins Med. J. 120,176,1967.
27. Deinhardt, F. Hepatitis in primates. Adv. Virus Res. 20, 113, 1976.
28. Eichberg, J.W., Kalter, S.S. Hepatitis A and B: serologic survey of human and non-hu-
man primate sera. Lab Anim. Sci. 30,541, 1980.
29. Deinstag, J.L.. Popper, H., Purcell, RH. The pathology of viral hepatitis A and B in
chimpanzees: a comparison. Am. J. Pathol. 85, 131, 1976.
30. Bradley, D.W., Krawczynski, K., Cook, E.H., Mccaustland, K.A, Robertson. B.H.,
Humphrey, CD., Kane, M.A, Spelbring, J., Weisfuse, l., Andjaparidze, A., Balayan.
M., Stettler, H., Velazquez, O. Enterically transmitted non-A, non-B hepatitis: etiology
of the disease and laboratory studies in non-human primates. In: Viral Hepatitis and
Liver Disease. Zuckerman, A.J. (ed.) New York, Alan R Liss, 1980, p.138.
31. Mukai, N .. Kalter, S.S., Cummins, L.B., Matthews, V.A.. Nishida. 1'.. Nakajima.
T. Retinal tumor induccd in thc hahoon by human adenovirus 12. Science. 210, 1023,
19KO.
32. Koneru. B .. Jaffc. R .. Esquivel. CO., Kunz, R., Todo, S., Iwatsuki, S., Starzl, TE.
Adenoviral infections in pediatric liver transplant recipients . .Iailla. 25R. 4K9. 19K7.
33. Albrecht. P .. Shabo. A.L.. Burns, G.R., Tauraso, N.M. Experimental measles en-
cephalitis in normal and cyclophosphamide-treated rhesus monkeys . .I. Infeci. Dis!.
126.154,1972.
34. Wild, TF. Measles virus and chronic infcetions. Palh. Bio/. 29.429, 19KI.
35. Schumacher, H.P., Alhrecht, P .. Clark, R., Kirschtein. R.L.. Tauraso. N.M. Intra-cere-
hral inoculation of rhesus monkeys with a strain of measles virus isolated from a case of
subacute sclerosing panenccphalitis.ln/i'ct. 11Il1II1l1l. 4,419, 1971.
36. Ueda. S., Otsuka, 1'., Okuno, Y. Experimental suhacute sclerosing panencephalitis
(SSPE) in a monkey by subcutaneous inoculation with a defective SSPE virus. Bikell. I
IK, 179, 1975.
37. Morgan, E.M .. Rapp. F. Measles virus and its associated diseases. Baclerio/. Rev. 41,
636.2977.
38. Rustigian, R., Johnston, P.B., Reichart, H. Infection of monkey tissue with virus like
agents. Pmc. Soc. Exp. Bio/. Med. R8, 8, 1955.
39. Weiner, L.P., Herndon, R.M., Narayan, O.N., Johnson, R.T, Shah, K., Rubenstein,
L.L Presiosi, T.L Conley, F.K. Isolation of a virus related to SV40 from patients with
progressive multi focal leukoencephalopathy. N. Eng/. I Med. 286, 3K5, 1972.
40. Holmherg, CA., Grihble, D.H., Takemoto. K.K .. Howley. P.M .. Espana, C, Oshurn,
B.1. Isolation of simian virus 40 from rhesus monkeys (Macaca 11111/atta) with sponta-
neous progressive multifocalleukoencephalopathy . .I. In/ Dis. 136,593,1977.
41. Braun. L., Kalter, S.S., Yakovleva, L.A., Kaschula, V.R., Shah, K.V. Neutralizing anti-
hodies to simian papovavirus SA 12 in Old World primates in laboratory colonies: high
prevalence in bahoons. I Med. Prinwtoi. 9,240, 19RO.
42. Takemoto, K.K., Furuno, A., Kato, K., Yoashiia, K. Biological and biochemical studies
of African green monkey Iymphotropic papovavirus . .I. Vim!. 42,502, 1982.
43. Miller, N.R., Mckeever, P.E., London, W., Podget, B.L.. Walker. D.L.. Wallen. W.C
Brain tumors of owl monkeys inoculated with JC virus contain JC virus genome.
I Viro!. 49,848,1984.
44. O'Banion, M.K .. Sundberg, J.P., Shima, A.L., Reichmann, M.E. Venereal papilloma
and papillomavirus in a colohus monkey (C%hllS gllere;:a). Inlervir%gy. 2R. 232,
1987.
45. Cossart, Y.E., Cant, B., Field, A.M., Widdows, D. Parvovirus-like particles in human
sera. Lanc!!!. 1,72.1967.
46. Anderson, M.J. Human parvovirus infections..!. Vim/. Meth. 17,175, 19R7.
47. Rapoza, N.P .. Atchinson, R.W. Association of AAV-I with simian adenoviruses.
Nature. 215, 1186, 1967.
48. Kalter, S.S., Heberling, R.L., Hellman, A., Todoro, G.J., PanigeL M. C-type particles in
hahoon placenta. Pmc. Roy. Soc. Med. 68, 135, 1975.
49. Rous, P.A. A transmissihle avian neoplasm (sarcoma of the common fowl). I Exp.
Med. 12,686,1910.
50. Theilen, G.H., Gould, D., Fowler, M.E., Dungworth, D.L. C-type virus in tumor tissue
of a woolly monkey (Langolhrix sp.) with fibrosarcoma. I Nat!. Cancer Insl. 47, RRI,
1971.
51. Kawakami, TG .. HufL S.G., Buckeley, P.M., Dungworth, D.L., Snyder, S.P .. Gilden,
R.V. C-type virus associated with gibhon lymphosarcoma. Nallire. 235. 170, 1972.
52. Kalter. S.S .. Helmke. R.L Heherling, R.L.. PanigeL M., Felsburg. P.J .. Axelrod, L.R.
Observations of apparent C-type particles in bahoon (Papio cynocephalus) placentas.
Science. 179.1332.1973.
53. Kalter, S.S., Helmke, R.J., Heherling, R.L., Panigel. M., Fowler, A.K., Strickland. J.E.,
Hellman, A. C-type particles in normal human placentas. I Nat!. Cancer Ins!. 50, 10RI.
1973.
References 477
54. Schidlovsky, G., Ahmed, M. C-type virus particles in placentas and fetal tissues of rhe-
sus monkeys.i. Natl. Cancer Inst. 51,225,1973.
55. Vernon, M.L., McMahon, J .M., Hackett, J.J. Brief communication: additional evidence
oftype-C particles in human placentas.i. Natl. Cancer Inst. 52,987,1974.
56. Seman, G., Levy, B.M., Panigel, M., Damochowski, L. Brief communication: type-C
virus particles in placenta of cottontop marmoset (Saguinus oedipus). i. Natl. Cancer
Inst. 54,251, 1975.
57. Feldman, D. An electron microscopic study of virus particles in rhesus monkey placen-
ta. Proc. Natl. Acad. Sci. USA. 72, 118, 1975.
58. Dalton, AJ., Hellman, A, Kalter, S.S., Helmke, RJ. Brief communication: ultrastruc-
tural comparison of placental virus with several type-C oncogenic viruses. i. Natl.
Cancer Inst. 52, 1379,1974.
59. Kalter, S.S., Heberling, RL. Isolation of C-type viruses from baboon (Papio cyno-
cephalus) placenta tissue. Presented to the Annual Meeting of the American Society of
Microbiology. 1974, p. 233 (VI94) (Abstract).
60. Benveniste, RE., Lieber, M.M., Livingston, D.M., Sherr, CL Todaro, GJ .. Kalter, S.S.
Infectious C-type virus isolated from a baboon placenta. Nature. 248, 17, 1974.
61. Todaro, G.J., Sherr, CJ., Benveniste, RE. Baboons and their close relatives are unusu-
al among primates in their ability to release non-defective endogenous type-C viruses.
Virology. 72,278, 1976.
62. Todaro, G.J., Sherr, CJ, Sen, A, King, N., Daniel, M.D., Fleckenstein, B. Endogenous
New World primate type-C viruses isolated from owl monkey (Aotus trivirgatus) kid-
ney cell line. Proc. Natl. Acad. Sci. USA. 75,1004, 1978.
63. Todaro, G.J., Benveniste, RE., Sherwin, S.A, Sherr, CJ. MAC-I, a new genetically
transmitted type-C virus of primates: "low frequency" activation from stumptail mon-
key cell culture. Cell. 13,775,1978.
64. Sherwin, S.A., Todaro, G.J. A new endogenous primate type-C virus isolated from the
Old World monkey Colobus polykonos. Proc. Natl. Acad. Sci. USA. 76,5041, 1979.
65. Rabin, H., Benton, CV., Tansky, M.A, Rice, N., Gilden, RV. Isolation and characteri-
zation of an endogenous type-C virus in rhesus monkeys. Science. 204,841,1979.
66. Benveniste, RE., Todaro, G.J. Evolution of primate oncornaviruses: an endogenous
virus from langurs (Presby tis sp.with related virogene sequences in other Old World
monkeys. Proc. Natl. Acad. Sci. USA. 74,4557,1977.
67. Heberling, RL., Barker, S.T., Kalter, S.S., Smith, G.C, Helmke, RJ. Oncornavirus:
isolation from a squirrel monkey (Saimiri scuireus) lung culture. Science. 195,289,1977.
68. Heberling, F.L., Kalter, S.S., Eichberg, J.W., McCullough, B. Fibrosarcomas in dogs
and non-human primates induced by a baboon type-C virus-murine sarcoma pseudo-
type. Presented at the RNA Tumor Virus Meeting. Cold Spring Harbor Laboratory,
NewYork,1976,p.21.
69. Ahmed, M., Korol, W., Larson, D.L., Harewood, K.R, Mayyasi, S.A Interactions be-
tween endogenous baboon type-C virus and oncogenic viruses. I. Syncytium induction
and development of infectivity assay. Int. i. Cancer. 16,747, 1975.
70. Chopra, H.C, Mason, M.M. A new virus in a spontaneous mammary tumor of a rhesus
monkey. Cancer Res. 30, 2081, 1970.
71. Fine, D.L., Landon, J.C, Pienta, RJ., Kubicek, M.T., Valerio, M.G., Loeb, W.F.,
Chopra, H.C Responses of infant rhesus monkeys to inoculations with Mason-Pfizer
monkey virus materials. i. Natl. Canter Inst. 54,651, 1975.
72. Stromberg, K., Benveniste, RE., Arthur, L.O., Rabin, H., Giddens, W.E., Ochs, H.D.,
Morton, W.R, Tsai, CC Characterization of exogenous type-D retrovirus from a fi-
broma of a macaque with simian Aids and fibromatosis. Science. 224,289,1984.
73. Marx, P.A., Bryant, M.L., Osborn, K.G., Maul, D.H., Lerche, N.N., Lowenstine, L.F.,
Kluge, J.D., Zaiss, CP., Henrickson, RV., Shiigi, S.M., Wilson, B.J., Malley, A, Olson,
L.C., Mcnulty, W.P., Arthur, L.O., Gilden, RV., Barker, CS., Hunter, E., Munn, RJ.,
Heidecker, G., Gardner, M.B. Isolation of a new serotype of simian acquired immune
deficiency type-D retrovirus from Celebes black macaque (Macaca nigra) with im-
mune deficiency and retroperitoneal fibromatosis.i. Viral. 56,571.1985.
74. Letvin. N.L.. Eaton. K.A .. Aldrich. W.R .. Sehgal. P.K .. Blake. B.1 .. Schlossman. S.F ..
King. N.W .. Hunt. R.D. Acquired immunodeficiency syndrome in a colony of macaque
monkeys. Pmc. Natl. Aunl. Sci. USA. 80.2718. 1983.
75. Giddens. W.E.. Bielitski. J.T.. Morton. W.R .. Ochs. H.D .. Myers. M.S .. Blakely. G.A ..
Boyce. J.T Idiopathic retroperitoneal fibromatosis: an enzootic disease in the pigtail
monkey Macaw nemestrina. Lab. Invest. 40.294. 1979 (Abstract).
76. Miyoshi. l.. Yoshimoto. S .. Fujishita. M .. Taguchi. H .. Kubonishi. I.. Niiya. K .•
Minezawa. M. Natural adult T-cell leukemia virus infection in Japanese monkeys.
Lancet. 2.658. 1982.
77. Homma. T. Kanki. P.1 .. King. N.W. Jr.. Hunt. R.D .. O'Connell. M.1 .. Letvin. N.L..
Daniel. M.D .. Desrosiers. Re.. Yang. C.S .. Essex. M. Lymphoma in macaques: associ-
ation with virus of human T Iymphotropic family. ScicnC!'. 225.716. 1984.
78. Hunsman. G .. Schneider. J .. Schmitt. J .. Yamamoto. N. Detection of serum antibodies
to adult T-cell leukemia virus in non-human primates and people from Africa. Int. 1.
Cancer. 32.329. 1983.
79. Hayami. M .. Komuro. A .. Nozawa. K .. Shotake. T. Ishikawa. K .. Yamamoto. K ..
Ishida. T. Honjo. S .. Hinuma. Y. Prevalence of antibody to adult T-cell leukemia virus-
associated antigens (Atla) in Japanese monkeys and other non-human primates. Int. 1.
Cancer. 3. 179. 1984.
80. Becker. W.B .. Beckcr. M.L.B .. Homma. T. Bredc. H.D .. Kurth. R. Serum antibodies to
human T-cellicukemia virus type I in different ethnic groups and in non-human pri-
mates in South Africa. S. Ali-. Med. 1. 67.445.1985.
81. Ishida. T. Yamamoto. K .. Shotake. T. Nozawa. K .. Hayami. M .. Hinuma. Y. A field
study of infection with human T-cell leukemia virus among African primates.
MicruiJioi. 1I7Im lin 01. 30.315.1986.
82. Botha. M.C. Jones. M .. De K1erk. W.A.. Yamamoto. N. Spread and distribution of IlU-
man T-cell leukemia virus type I reactive antibody among baboons and monkcys in the
Northern and Eastern Transvaal. S. Afr. Med.1. 67.665.1985.
83. Lowenstine. L.J.. Pederson. N.C. Higgins. J .• Pallis. K.C.. Uyeda. A .. Marx. P .. Lcrchc.
N.W .. Munn. F.L Gerdner. M.B. Seroepidemiologic survey of captive Old World pri-
mates for antibodies to human and simian rctroviruses. and isolation of a lentivirus
from sooty mangabeys (CcrcoceiJlls atys). Int. 1. Cancer. 38.563. 1986.
84. Work. TH .. Trapido. H. Kyasanur Forest disease. A new virus diseasc in India.
Summary of preliminary report of invcstigations of the Virus Rcsearch Ccnter on an
epidcmic diseasc affecting forest villagers and wild monkeys of Shimoga District.
Mysore. Ind. l. Med. Sci. 11.340. 1957.
85. Martini. G.A .. Siegert. R MariJllrg Virus Disease. Springer-Verlag. New York. 1971.
86. Center for Disease Control. Morbidity and Mortality Weekly Report. 39.22.1990.
87. Unger. E. Nierentransplantationcn. Berlin Klill. Wschr. 47.573.1910.
88. Schonstadt cited by Moral & Paris. Les applications physiologiques et chirurgicales des
transplantations renales. Bioi. Aled. 10.397.1913.
89. Hitchcock. CR. Kiser. J.C. Telander. R.L.. Scljoskog. E.L. Baboon renal grafts. lama.
189.934.1964.
90. Reemtsma. K .. McCracken. B.H .• Schlegel. J.R .. Pearl. M.A.. Pearce. CW .. DeWitt.
CW .. Smith. P.E .. Hewitt. RL.. Flinner. RL.. Creech. O. Jr. Renal heterotransplanta-
tion in man. Ann. Sllrg. 16D. 384.1964.
91. Starzl. T.E .. Marchioro. T.L.. Ifeters. G.N .. Kirkpatrick. CH .. Wilson. W.E.C. Porter,
K.A .. Rifkind. D .. Ogden, D.A., Hitchcock, CR, Naddell, W.R. Renal heterotrans-
plantation from baboon to man: experiences with six cases. Transplantation. 2. 752,
1964.
92. Van der Riel. F. de St., 1.. Human. P.A., Cooper. D.K.C. Reichart. B .. Fincham. J.E..
Kalter. S.S .. Kanki. P.J.. Essex. M .. Madden. D.L.. Lai-Tung. M.T .. Chalton. D .. Sever.
J.L. Virological implications of the use of primates in xenotransplantation. Transplant.
Proc. 19.4068.1987.
93. Human. P.A.. Van der Riet, F. de St.. J., Cooper. D.K.C., Kalter, S.S., Fincham, J.E.,
Smuts. H.E. Reichenspurner, H .. Madden. O.L., Sever. J.L., Reichart. B. The virologi-
References 479
cal cvaluation of non-human primates for xenotransplantation. Transplantation. 19,
146,1987.
94. Human, P., van der Riet, F .. Cooper, D.K.C, Fincham. 1.. Kalter. S.S .• Reichart. B.
Aids-Forschung (Aifo). 4. 188.1989.
95. Gold, E. Infections associated with immunologic deficiency diseases. Med. Clin. N.
Amer. 58.649. 1974.
96. Fauci, A.S .. Macher, A.M., Longo. A.L. Acquired immunodeficiency syndrome: epi-
demiologic, clinical, immunologic. and therapeutic considerations. Ann. Int. Med. 100,
92.1984.
97. Heise, E.R. Diseases associated with immunosuppression. Environ. Hlth. Perspec. 43,
9,1982.
98. Kalter. S.S .. Heberling, RL. Comparative virology of primates. Bacteriol. Rev. 35,310,
1971.
99. Kalter. S.S .. Heberling, RL. Viral battery testing in non-human primate colony man-
agement. Lab. Anim. Sci. 40.21, 1990.
100. Meyers, A.M .. Kallenbach, J., Milne, F.J .. Zwi, S .. Rabkin, R., Myburgh, J.M. Post-
transplant pulmonary complications. S. Afr. l. Surg. 11. 124.1973.
101. Bailey. L.L.. Nehlsen-Cannarella. S.L.. Concepcion, W., Jolley, W.B. Baboon-to-hu-
man cardiac xenotransplantation in a neonate. lama. 254.3321,1985.
102. Held. 1.R Requirements for laboratory animals in health programs. Bull. WHO. 59,
513.1981.
103. Ott-Joslin, J.E. Viral diseases in non-human primates. In: Zoo and Wild Animal
Medicine. 2nd Edition, Fowler, M.E. (ed.), W.B. Saunders, Co., Philadelphia. 1986,
p.624.
104. Renquist. D.M .. Whitney. RA. Jr.. Zoonoses acquired from pet primates. Vet. Clinics
of North America: Small Animal Practice. 17,219,1987.
105. Johnson-Delancy, CA. The pet monkey: human health biohazards and protective
measures. ESVC Proc. Montreal, 275, 1990.
Chapter 30
The Pig as Potential Organ Donor for Man

D.K.C. COOPER, Y.YE,L.L. ROLF JR., AND N.ZUHDI
Introduction
If the immunological problems could be overcome, the ideal animal organ

donor for man would be an animal that is readily available throughout the
world in large numbers and grows to an adequate size. This would immediate-
ly exclude the nonhuman primates; the larger species are endangered and
those that are not reach only a limited size which make them unsuitable as
donors of some organs for adult humans.
The risk of controversy with regard to the use of animals as organ donors
for man would be greatly minimized if the animal were already being killed in
large numbers on a daily basis to provide food for human consumption.
Animals of adequate size in this group would include the pig, the sheep, and
possibly the calf, although this latter animal would rapidly grow to a size that
would make the organs unsuitable for transplantation purposes. It would
clearly be preferable for the animal not to be generally known as a pet, thus ex-
cluding the dog, which would in any case be too small in most cases.
In 1989, the U.S. Department of Agriculture's National Agricultural
Statistical Service reported that more than 89 million pigs were reared and
slaughtered in the USA to provide meat and other food products. Ethical ar-
guments against the use of a few thousand pigs each year for the purposes of
transplantation will be greatly weakened in the light of the vast number al-
ready killed annually for human use.
The advantages of the pig (Table 30.1) over the sheep and calf include: (i)
the pig requires less space to breed and be raised; (ii) it is easy to feed; (iii) the
costs of breeding and maintaining pigs are probably rather less than those for
Table 30.1. Classification of the pig
Phylum Chordata
Class Mammalia
Order Artiodactyla
Suborder Suiformes
Infraorder Suina
Family Suidae
Genus Sus
Species Scrota
482 The Pig as Potential Organ Donor for Man
sheep and certainly less than those for calves; (iv) the pig produces a large litter
size; (v) the pig rapidly grows to a size where it would be suitable as an organ
donor for even the largest of adult humans; conversely, during infancy, the pig
is of a size to be a suitable donor for infants and children.
For purposes of organ transplantation in man it would seem advisable, if
not mandatory, to use a gnotobiotic (germ-free) animal. if one were available,
to ensure that no infectious organism was transferred to the human organ
recipient [1]. Gnotobiotic pigs are readily available [2], and there are several
advantages of raising germ-free pigs rather than sheep or calves. These will be
discussed later.
Furthermore, there are considerable similarities between certain pig organs
and physiological systems with those of man, e.g., the heart and cardiovascular
system [3,4]. Additionally, much is known of pig physiology from its use as a
research animal. particularly with regard to the cardiovascular system [4-8].
Kirkman [9], in reviewing aspects of the pig as a potential organ donor for
man, has drawn attention to other similarities between pig and man relating to
(i) size, (ii) dietary habits - both omnivorous, (iii) digestive physiology, (iv)
kidney structure and function, (v) respiratory rate and tidal volume, (vi) coro-
nary artery distribution, (vii) hemodynamics, (vii) propensity to obesity. (viii)
susceptibility to disease, and (ix) social behavior.
Immunological Considerations
All of these advantages of the pig are dependent, of course. upon the develop-
ment of a successful method of overcoming the major immune response that
would be mounted by man against a grafted pig organ. It is well-documented
that rejection following transplantation between widely divergent (discordant
[10]) species (e.g .. pig-to-dog [11-14]. pig-to-baboon [15. 16]. pig-to-man [17],
sheep-to-man [18]) is followed by rapid vascular (hyperacute, humoral) rejec-
tion [19], which is much less common when transplantation is performed be-
tween closely related (concordant [10]) species (e.g., chimpanzee-to-man
[18-26]. baboon-to-man [24. 27, 28], vervet monkey-to-baboon [29, 30].
cynomolgus monkey-to-baboon [31, 32], or goat-to-sheep [33]).
Nevertheless, evidence is slowly increasing to indicate that even transplan-
tation between closely related species is complicated in many cases by a vascu-
lar or humoral form of rejection which may prove to be a more difficult prob-
lem to overcome than the cellular rejection that occurs. Evidence from the
baboon-to-human heart transplant carried out at Loma Linda in 1984 [28] and
from studies ofvervet monkey to baboon heart grafts where histopathological
features of vascular rejection were seen in 80% of cases [29], would suggest
that vascular rejection may playa significant role in graft failure in a large
number of concordant cases.
In a pig-to-man transplant it seems clear that a method must be developed
of removing the antipig antibodies from the human serum before transplanta-
Immunological Considerations 483
tion, though there is some evidence that complement activation takes place in-
dependently of an antigen-antibody reaction via the alternative pathway [34].
Furthermore, drugs or other therapy must be employed to prevent the produc-
tion of new anti pig antibodies. Finally, cellular rejection must also be prevent-
ed.
Though the presence in man of natural preformed antibodies directed
against pig tissues and the initiation of the complement cascade that results
from such an antigen-antibody reaction form a major barrier to the success of
discordant xenotransplantation, these very same factors may ultimately prove
of value in the management of patients undergoing transplantation with ani-
mal organs. Measurement of the titer of antibody and of the complement com-
ponents may provide some measure of the rejection status of the recipient, and
may indicate a need for an increase or decrease in immunosuppressive thera-
py. The search for a blood testes) that indicates the rejection status of the re-
cipient following allografting has been disappointingly unsuccessful. If a
means of successfully reducing the antibody titer and/or complement compo-
nents becomes available, then the plasma levels of these various factors might
prove a valuable method of monitoring the immune status of the recipient and,
therefore, also of the graft.
To overcome the first problem, namely the presence of preformed circulat-
ing antipig antibodies in human serum, experimental work is advancing in the
field of removal of such antibodies. Various forms of plasma exchange and ex-
tracorporeal immunoadsorption are being investigated (Chap. 6), and it
would seem likely that one of these techniques will prove successful.
The second problem, that of preventing the further production of such anti-
bodies, has to date proved insoluble, but with the development of new drugs
(e.g., 15-deoxyspergualin [30,35] and FK-506 [35], possibly in combination
with each other and/or antithymocyte globulin [30]), and of other techniques
(e.g., total lymphoid irradiation [36,37]), then a solution to this problem may
be found in the foreseeable future.
When xenotransplantation is performed between closely related species
the need to remove preformed circulating anti donor species antibodies from
the recipient serum may not be required, as such antibodies may not be
demonstrable. The experimental and clinical evidence, however, suggests that
such antibodies may develop after transplantation and may playa significant
role in the rejection of the graft [28,29]. It would therefore appear that a solu-
tion to the problem of the production of antispecies antibodies may be re-
quired when transplantation is performed between closely related species as
well as between widely disparate species.
The third problem, that of the prevention of cellular rejection, should in all
likelihood be overcome by the presently available drugs used successfully fol-
lowing allografting. There is some evidence to suggest that cellular rejection
may be no more difficult to prevent, and may even be less so, when transplan-
tation is performed between widely disparate species [38,39].
There is, however, little information available on the cellular and humoral
immune responses to pig antigens in man. Studies on this topic are included
elsewhere in this volume. One further recent study performed by Citterio et al.
[39]. looked at mixed lymphocyte cultures using lymphocytes isolated from
the peripheral blood of healthy human subjects and from pig splecn. Human
lymphocytes reacted to irradiated pig lymphocytes. increasing the H3-thymi-
dine incorporation by 53 (±35) times (range of the Stimulation Index 18-104).
This activation was less than that obtained with phytohemagglutinin (10
ng/ml). but significantly higher than the proliferation induced by human anti-
gens in mixed lymphocyte culture. The addition of 100 ng/ml cyclosporine to
the mixed lymphocyte culture inhibited the lymphocytic response to phyto-
hemagglutinin by 69% (±27%). to human antigens by 87% (±13%). and to pig
antigens by 97% (±3%).
Different concentrations of preformed antipig antibody were found in dif-
ferent human subjects [39]. Moreover. sera taken after the introduction of im-
munosuppressive therapy following transplantation in human patients
showed no significant decrease in the antibody titer (against pig antigens)
when compared with pretherapy levels.
These data suggest that cellular responses in man to pig antigens may be
easily controlled by cyclosporine. and that the major problem remains that of
pre-existing antibodies and continuing antibody production.
Observations in six patients undergoing heart allografting at our own cen-
ter would confirm that immunosuppression with combined cyclosporine. aza-
thioprine. methylprednisolone and anti thymocyte globulin does not signifi-
cantly reduce the antipig lymphocytotoxic or hemagglutinating antibody
levels (unpublished data). Attempts to identify antigens on pig lymphocytes
using known human HLA A. B, and DR antisera have been unsuccessful (F.
Neethling. unpublished data). Reactions were nonspecific and no definite
antigens could be identified. Similarly. attempts at cross-matching using pig
cells and human sera were not successful.
Blood Group Compatibility
When transplantation is performed between widely disparate species, the an-

tispecies antibody appears to override any problems that might arise from
blood group antibodies [11, 15]. With regard to transplantation between close-
ly related species the evidence is that blood group compatibility is of impor-
tance, and incompatibility increases the risk of early vascular rejection [28,29].
There are few data in the literature, however, with regard to the effect of
blood group compatibility or incompatibility in discordant transplantation.
Most evidence suggests that all pigs are of blood group A or 0 [40], though 0
blood is able to provoke antibody formation if injected into a suitable organism
[41]. There are also pigs (type 1) that demonstrate an absence of A and 0 sub-
stance on the red cells [42]. The general finding is that the ABO system (and the
large number of other blood group systems that have been reported in this ani-
mal) does not affect the outcome of organ allografting in pigs and therefore can
be ignored (R. Hickman and R. Binns. personal communications).
Nonimmunological Considerations 485
Perper and Najarian [11] studied organ transplantation between the pig
and the dog. In the selection of donor and recipient, each pig was saline cross-
matched preoperatively with at least 12 dogs in an effort to keep red cell in-
compatibility to a minimum. The animals used for transplantation were those
with the lowest titers of red cell incompatibility, as evidenced by the degree of
agglutination or hemolysis. In all cross-matching experiments, fresh serum
was used within 15 min after it was obtained from the animal in order to pre-
serve its complement activity. No combination of pig and dog red cell types in-
fluenced the fate of the xenografted organ.
In the pig-to-baboon cardiac transplant model there appeared to be no in-
fluence of major blood group incompatibility on outcome [15]. In a total of
eight experiments the blood group of the recipient baboon was AB in five cas-
es and B in three cases. Therefore. if all pigs are A. O. or 1. in only three cases
was it likely that blood group incompatibility might have played a significant
role. Donor heart function was for a mean of 161 min in these three baboons
compared with 106 min in the remaining five where ABO compatibility might
have been expected to be present. Though the data are not conclusive. there
appeared to be no significant difference in the rate of pig kidney rejection
when the organ was perfused with human AB or 0 blood [43].
Nonimmunological Considerations
If such immune problems could be overcome, the remaining potential compli-

cations with regard to the use of any animal as a donor would include: (i) dis-
ease processes that affect the animal's organs that might lead to dysfunction of
the transplanted organ after transplantation in man; (ii) the presence of infec-
tious agents that might be transferred with the organ to man; and (iii) malig-
nant tumors or other neoplastic conditions that could be transferred with the
organ to man. These problems are common to any potential animal donor, not
just the pig. For example, the baboon may harbor viruses that could prove a se-
rious problem if transferred to man [44.45] (Chap. 29). These various prob-
lems with regard to the use of the pig as a potential donor will be briefly ex-
plored.
Disease Processes Affecting the Major Organs of Pigs
The subject of disease processes affecting the major organs of pigs will be
briefly explored by consideration of the heart as the donor organ. The poten-
tial cardiovascular diseases from which the pig may suffer have been clearly re-
viewed by Van Vleet and Ferrans [46], and the following outline summarizes
their review. The reader is directed to the original paper for further informa-
tion and references.
Congenital Anomalies
A wide variety of congenital abnormalities has been described in pigs [47].
These include nearly all of the anomalies commonly seen in man, together with
one or two others. Fortunately they appear to be rare, occurring in only approx-
imately 0.16% of pigs in one series in which over 300 000 pigs were necropsied
and in 0.49% in another series [48]. Hsu and Du [47]. however, found an inci-
dence of 4.35% in necropsies of I906pigs during an II-month period. Incidence
of cardiac malformation was highest in pigs examined between 29 and 110 days
of age. One hundred and twenty-two anomalies were found in a total of 83 pigs
in this series. The most common abnormalities were dysplasia of the tricuspid
valve in 42, atrial septal defect in 31, and subaortic stenosis in 22 (Table 30.2). A
fibrous sub aortic ring leading to stenosis has been the most common disorder in
other series, occurring in between 0.22% and 3.1 % of pigs.
A heritable ventricular septal defect (VSD) has been described in a strain
of Yucatan miniature swine [49]. This has been demonstrated to be a high
membranous VSD analogous to the most common form seen in humans.
Pigs with significant congenital cardiac anomalies are poorly developed
with severe dyspnea, lethargy, and anorexia. Sudden death is not uncommon
[47]. The high incidence of congenital heart disease seen in pigs within the first
3-4 months of life [47] might indicate that pigs so afflicted commonly die with-
in this age period. It should be possible, therefore, to exclude them from use as
organ donors by identification of symptoms and signs of cardiac defect.
Furthermore, the majority of the defects described would be clearly visible on
examination of the heart after excision. Only in the case of a small VSD might
it prove difficult to detect the lesion by direct inspection before insertion of the
heart into the recipient.
As there is a genetic foundation for many such congenital defects, it should
be possible to reduce the low incidence even further by selection of stock and
selective breeding.
Table 30.2. Congenital cardiac malformation in 83 pigsa
Malformation Number of
abnormalities Percentage
Dysplasia ofthe tricuspid valve 42 34.0

Atrial septal defect 31 25.0
Sub aortic stenosis 22 18.0
Ventricular septal defect 9 7.0
Persistent common atrioventricular canal 8 7.0
Malformation of moderator band 7 6.0
Persistent cranial vena cava 1 1.0
Persistent truncus arteriosus 1 1.0
Pulmonary stenosis 1 1.0
Total 122
a A total of 1906 pigs were examined (from Hsu and Du [47]).

Pericarditis
Pericarditis can occur in pigs with several infectious diseases, especially those
related to hemophilus and mycoplasma organisms.
Bacterial Valvular Endocarditis

Bacterial valvular endocarditis is an important cardiac disease in swine, most
frequently caused by streptococci.
Other Endocardial Lesions

Other endocardial lesions, such as localized areas of thickening and/or calcifi-
cation, and cysts containing blood or clear fluid, can occur either murally or as
valvular lesions, but are rare in pigs less than 6-12 months of age.
Hypertrophic and Congestive Cardiomyopathy

Hypertrophic and congestive cardiomyopathy are both extremely rare in pigs.
Myocarditis
Myocarditis may result from encephalomyocarditis virus and foot-and-mouth
disease virus. It may also result from bacterial infections, toxoplasmosis, cys-
ticercosis, and pseudorabies (a herpesvirus infection).
Cardiomyopathy of Selenium-Vitamin E Deficiency

Myocardial necrosis with vascular lesions ("mulberry heart disease") and
skeletal muscle necrosis ("white muscle disease") are characteristic lesions of
selenium-vitamin E deficiency in the pig. They can be prevented by attention
to dietary requirements.
Myocardial Necrosis
Myocardial necrosis may occur in porcine stress syndrome, malignant hyper-
thermia, and herztod, as well as in pigs subjected to restraint stress. A high de-
gree of heritability has been shown for porcine stress syndrome in several
breeds. The basic metabolic defect involves abnormal calcium movement in
cardiac and skeletal muscle. The clinical syndrome may be precipitated in sus-
ceptible pigs by administration of halothane and/or succinylcholine, or by var-
ious emotional and physical stresses such as transportation, high ambient tem-
perature, or high humidity.
Myocardial Alteration Induced by Chemical Toxins

Pigs are susceptible to poisoning by gossypoL which is found in cottonseed
meaL a protein supplement used in swine rations. Affected pigs develop con-
gestive heart failure. with prominent ventricular dilatation and pulmonary
edema. Myocardial lesions also occur in pigs fed diets containing long-chain
monoenoic fatty acids such as erucic. which is found in rapeseed oil.
Myocardial calcification has occurred in pigs fed a calcinogenic plant or large
amounts of vitamin D.
Rhabdomyomatosis
Though rhabdomyomatosis (congenital rhabdomyoma. nodular glycogenic
tumor. nodular glycogenic infiltration) is common in young pigs. usually in-
volving the ventricular myocardium. it is not a malignant tumor and. there-
fore, would not be an increased problem in the immunocompromised recipi-
ent. However. it would clearly lead to dysfunction of the transplanted heart.
The lesions appear as multiple. poorly circumscribed. pale areas scattered in
the myocardium. Histologically. the neoplastic cells are composed of large
myoblastic cells, the cytoplasm of which contains an abundant amount of
glycogen. Since these neoplasms are found mostly in newborn and young
piglets they are believed to be of congenital origin [50 J.
Atherosclerosis
Numerous studies of spontaneously occurring and of experimentally induced
atherosclerosis have been reported for swine. The distribution and morpholo-
gy of atherosclerotic lesions in swine are in many respects similar to those in
man. The spontaneous disease is seen usually only in aged swine.
A review of the above naturally occurring conditions indicates that the ma-
jority of them can be prevented if the pig is kept free of infection. and. there-
fore, will not be seen in the gnotobiotic pig. Those that cannot be so excluded
include the rare congenital deformities, those related to dietary variances
(which can be prevented by attention to diet), porcine stress syndrome (which
can be minimized by careful selection of strain), rhabdomyomatosis, and
atherosclerosis (which is rare in young pigs). It would seem. therefore. that the
only problems that it might not prove possible to exclude totally are the occa-
sional congenital anomaly or rhabdomyoma. It might be possible to exclude
rhabdomyomatosis by performing echocardiographic studies, and by careful
visual examination and palpation of the donor organ at the time of transplan-
tation.
There are. however. several other artificially induced conditions that can
affect the pig heart, such as those induced by chemical agents (generally intro-
duced in the diet or from drug therapy for other conditions). These include
cobalt and monensin cardiotoxicity. They would not occur under normal con-
ditions if diet were well controlled in the gnotobiotic pig.
Several nematodes may infect pigs maintained under normal conditions,
and the larvae of some of these migrate through the body and may injure the
organs to be utilized for transplantation purposes (Table 30.3). Some of these
provide a further hazard in that they could cause disease if transferred to man
(Table 30.4). All could be excluded by using pigs reared under germ-free con-
ditions.
Infection in the Pig That Could Be Transferred to Man
The pig can harbor bacterial, viral, fungal, protozoal, and/or helminth organ-
isms (Table 30.4) [51,52]. Some of these organisms could lead to infection in
man if transferred with the transplanted organ. The questions must therefore
be asked: how easy is it to make the diagnosis of significant transferrable infec-
tion in the selected pig, and perhaps more important, how easy is it to exclude
significant infection in the selected pig?
After much consideration and discussion with veterinarians, it would seem
that the only reliable method of ensuring the pig is free from all infectious
agents is to breed and raise pigs in a germ-free environment - gnotobiotic pigs
[2]. This is clearly a time-consuming and expensive exercise, but if this policy is
not followed it would seem impossible to be certain that the transfer of signifi-
cant infectious agents to the recipient would not occur.
If gnotobiotic pigs were not used, then numerous questions arise, many of
which would be difficult to answer with complete certainty. Which diseases
could the pig carry without symptoms or signs being present? Is a test available
to confirm that the disease is or is not carried by the selected pig, and is this test
100% reliable? Could the infection be eradicated in the pig by therapy before
organ excision? If not, could the (covert) infection be transferred to man?
What specific organisms might be transferred with (i) the heart, (ii) the liver,
(iii) the kidney, etc? Would the transferred organism be equally harmful in
man - in other words, is man at equal or greater risk from the same strain of or-
ganism as is the pig? Could the infection be readily diagnosed at an early stage
in man? Is a test available to confirm whether or not the organism has been
transferred to man?
Table 30.3. Non-zoonotic helminths found in swine
Trichuris SlIis (whip worm)

Oesophagostomum sp. (nodular worms)
Hyostrongylus rubidus (red stomach worm)
Macracanthorhynchus hirudinaceus (thornheaded worm)
Metastrongylus sp. (lung worm)
Ascarops strongylina (stomach worm)
Physocephalus sexalatus
Stephanurus dentatus (kidney worm)
Strongyloides ransomi (threadworm)
Table 30.4. Swine zoonoses"
Bacterial
Brucellosis (Brtlcella SlIis) (B)
Leptospirosis (Leptospira interogenesc) (B)
Listeriosis (Listeria II/onocytogencs) (B)
Anthrax (Baeilllls allthracis) (B)
Erysipeloid (Erysipelothrix insidiosa)
Viral
Vesicular stomatitis (Herpes) (B)
Influenza, all strains (B)
Lymphocytic choriomeningitis (B)
Equinc encephalitis (eastern and wcstern) (B)
Fllngal
Coccidioidomycosis (Coccidioides imlllitis) (B )/(H)
Dermatophytosis (Microsporum canis) (S) )
(Microsporum gypseum) (S)
ringworms
(7i-ichophyton mentagrophvtes) (S)
(Trichophwon VCITllcosllm) (S)
Protozoal
Coccidiosis (Eimeria spp and iso,lpora spp) (B )/(Br)/(M)
Toxoplasmosis (Toxoplasma gondii) (B )/(Br)/(M)
Trypanosomiasis (Trypanosoma crtlzi) (B)
Balantidiasis (Balantidillm coli) (B)
Helmilltlz
Ascariasis (Ascaris Sllum) (B)
Trichinosis or Triehincllosis (Trichinella spiralisj (B )/(M)
Paragonimiasis (Paragonimus westerman i) (B/Lung)
Capillariasis (Capillaria hepatica) (B/Liver)
(Capillaria phillipillensis) (B/Intestines)
Schistosomiasis (SchistosomajaponiCllm) (B)
Ectoparasites
Sarcoptes scabiei (scabies) (S)
Ilaematopinlls suis (louse) (S)
Demodex phylloides (S)
(B), blood; (H), heart: (S), skin: (Br), brain: (M), skeletal muscle
a Letters in parentheses indicate where organisms may be found.
If transferred to man, could the organism be eradicated by therapy Of,

preferably, could the human recipient be pretreated to prevent the develop-
ment of any disease from any transferred organism? In other words, could the
recipient be immunized against specific infectious diseases of swine'? In gener-
al, no immunization is possible against helminth, fungal, or protozoal infecting
agents,
Finally. which diseases might be greatly disabling or fatal in man even if di-
agnosed and treated correctly? For example. the ascaris helminth may well be
impossible to eradicate from the recipient human. and could well lead to seri-
ous infestation and possibly death.
Gnotobiotic Pigs
All of these doubts and concerns can be dismissed by the use of germ-free pigs,
with which there is already considerable experience. Miniats [2] has reviewed
developments in the rearing of germ-free farm animals, and the reader is re-
ferred to this source for further information.
The pioneer work in gnotobiosis was done by Kuster in 1917 who raised
gnotobiotic goats. In 1960, Landey and Sondberg initiated work with germ-
free pigs, and Whitehair and his colleagues began rearing germ-free pigs and
ruminants. The subject has been more fully reviewed by Dufty [53]. Many sig-
nificant contributions to the understanding of basic immunological phenome-
na have been made by the use of the gnotobiotic pig [2].
Because of the large size and rapid growth of farm animals, their mainte-
nance in isolators presents technical difficulties. They cannot be maintained as
gnotobiotes to maturity, and none are known to have reproduced in isolators.
Each individual animal or litter, therefore, has to be derived surgically and fed
on sterilized diets which resemble the colostrum or milk of the species con-
cerned. Experimentation has thus been limited to young animals only.
The success in the rearing of neonatal gnotobiotic animals depends on the
timing of surgical delivery, satisfactory anesthetic and surgical techniques, a
suitable isolator, and proper management and diet for the newborn animals.
On the positive side, being well-developed at birth, farm animals learn readily
to feed themselves and, apart from problems related to confinement and re-
straint, they are relatively easy to rear.
The pig is the farm animal reared most commonly as a gnotobiote, and has
been preferred over other species as it has a relatively uniform duration of
pregnancy (Table 30.5), large litter size (Table 30.5), and is easy to rear under
isolated conditions (Table 30.6).
After surgical delivery under aseptic conditions, either by hysterectomy or
hysterotomy, the piglet is maintained in an isolator, either in a germ-free state
or as a gnotobiote exposed to a limited harmless amount of microbial flora.
Pigs have been reared easily under such conditions until the age of 3 or 4
months, after which age difficulties develop regarding the size of the isolator
required and the amount of waste material that has to be cleared each day.
Table 30.5. Gestation period and usual litter size in various farm animal species a
Gestation period in days N umber of fetuses
Species Mean Variability Usual Variability
Sow 114 (98-124) 10 1-20

Ewe 148 (144-152) lor2 1- 4
Goat (nanny) 151 (144-156) lor2 1- 4
Cow 283 (278-288) 1 1- 2
Mare 340 (310-370) 1 1- 2
a Compiled from Emsinger [71]; quoted by Miniats [2].

Table 30.6. Factors influencing successful rearing of gnotobiotic pigs"
I. Construction of a suitable isolator is facilitated as the pig has short legs

2. If delivered close to term. gnotobiotic piglets are well-developed and. if maintained in an
environment of35°C, are active and begin feeding themselves from a trough within 6 h
3. They must be kept separately
4. They require good support and traction for their feet
5. There is considerable experience with suitable diets for such pigs
" From Miniats [2].
With larger pigs, there is an increased risk of isolator damage and the provi-
sion offeed and water consumption becomes excessive. For purposes of trans-
plantation. it would be necessary to rear the pig in a germ-free environment
for at least 6 months. At this age the conventionally reared pig weighs approx-
imately 100 kg, but the gnotobiotic pig would weigh significantly less than this.
Piglets reared under gnotobiotic conditions gain weight less quickly than if
reared under conventional conditions. Gnotobiotic pigs weigh 4.8 kg at 4
weeks of age and 9.5 kg at 8 weeks, compared with corresponding average
weights of conventionally reared pigs of 8.2 kg and 19.6 kg [54]. Early weight
gain is therefore only approximately half of that of the piglet suckling the sow.
However. the pig would appear to be the farm animal most suited to being
reared under gnotobiotic conditions. For example. the following disadvan-
tages have been encountered in the production of gnotobiotic sheep as com-
pared with pigs. Sheep have a limited breeding season, small litter size (Table
30.5). and there are few reliable signs indicating the closeness of parturition,
and a relatively high mortality in the newly delivered neonate. Lambs are
more likely to require bottle feeding for the first few days after delivery.
Furthermore, problems associated with the digestion of solid diets by small
ruminants that do not have the normal gut micro flora remain to be solved.
Neoplastic Diseases in the Pig That Could Be Transferred to Man
The incidence of neoplasms in swine in the USA is approximately 0.004 % [55].

This estimate was made following ante mortem and post mortem inspection of
over 83 million pigs by the US Department of Agriculture in 1972. The actual
incidence would almost certainly be higher if swine were allowed to survive
into middle life. Over 90% of pigs are slaughtered between 4.5 and 6 months of
age. Neoplasms, however, would appear to be significantly less common in
swine than in cattle, where the incidence is approximately 0.35%.
Malignant Lymphoma
Malignant lymphoma is possibly (with embryonal nephroma) the most com-
mon tumor found in swine, contributing 46% of all tumors in one survey [56]
(Table 30.7). There is no known breed or sex predisposition. Malignant lym-
Table 30.7. Neoplasms in pigs
Organ System Type of neoplasm
Skin and appendages Melanoma"

Papilloma
Squamous cell carcinoma
Musculoskeletal system Osteo( sarco )mas
Chondro( sarco )mas
Hemic and lymphatic system Malignant lymphoma a
Mastocytoma
Respiratory system Nasal mucosa
Cardiovascular system Rhabdomyoma (heart)"
Digestive systcm Hepatic neoplasms
Carcinoma (stomach)
Mesothelioma
Urinary system Embryonal nephroma (kidney)a
Genital system Testicular neoplasms
Granulosa cell tumor (ovary)
Adenocarcinoma and
cystadenoma (ovary)
Teratoma
Mammary gland tumor
Endocrine system Adrenocortical tumor
(adenomas and carcinomas)
Pheochromocytoma
Thyroid neoplasms
Nervous system Primary neoplasms
a Most common
phoma (also known as lymphosarcoma or leukosis) has been estimated to

have an incidence between 0.001 and 0.3% [57].
The diagnosis can be suspected in any animal showing loss of weight and
strength, by which time the disease is usually well advanced. The most com-
mon specific manifestation and presenting sign, however, is the pronounced
enlargement of a number of lymph nodes. Several internal organs may also be
involved. Pigs are difficult to examine clinically, and, therefore, signs of the
disease may be difficult to detect.
A few cases (approximately 5%) have been reported where lesions are
found in viscera without any detectable lymph node involvement. The heart is
rarely affected, but, if so, it is almost always the base of the heart and the walls
of the atria that are involved, occasionally with later extension to the ventri-
cles. The spleen is commonly enlarged, sometimes to great dimensions. Some
animals afflicted with the condition demonstrate exceedingly high white blood
cell counts with a predominance of lymphocytes. If the diagnosis has not been
made earlier, it should be possible to make it, or at least strongly suspect it, by
careful examination of the major organs and lymph nodes at the time of organ
excision for the purposes of xenotransplantation.
A chronic granulomatous disease in pigs with histological features similar
to those of Hodgkin's disease in man has been described [58].
Embryonal Nephroma
Embryonal nephroma is relatively common with an incidence of approxi-
mately 0.02%. It is most often seen in pigs under 1 year of age. Metastatic le-
sions are seldom observed.
Melanoma
Multiple melanoma have been described in Sinclair and Hormel miniature

swine [59-61] and conventional swine [62-65]. Lesions, believed to be
metastatic from skin primaries, have been found in many internal organs in-
cluding the heart. In pigs, melanomas are found most often in the jowl (cheek)
and flank regions, and thus can be differentiated from melanosis. The latter
condition is a very common pigmentary entity, but discreet nodular masses are
not present.
Nonmalignant Cardiac Rhabdomyoma

The nonmalignant cardiac rhabdomyoma has been discussed above.
As with potential transferrable infectious diseases, the possibility of the pres-

ence of these tumors leads us to ask a number of questions. Can we exclude
melanoma by careful external examination of the pig, including the mouth?
Can we adequately exclude malignant lymphoma? Can we exclude renal tu-
mors by careful external examination and palpation of the organ at the time of
excision, or previously by renal scans and/or arteriography?
The answers to these questions remain uncertain, but with meticulous
physical examination and the use of echo cardiographic techniques, it should
prove possible to exclude most significant tumors before the organ is trans-
planted. Possibly the use of echocardiography or arteriography at the time of
organ excision may also prove of value.
Metabolic Compatibility Between Pig and Man
With regard to xenotransplantation, CaIne was one of the first to draw atten-
tion to what he termed the "milieu interieur" of the recipient, and whether the
environmental conditions of the recipient would be satisfactory for good func-
tion of the grafted organ [10]. This is possibly more important with regard to
organs such as the liver and kidney than the heart. Would all the necessary
metabolites be present to allow normal function? Minor differences in, for ex-
ample, pH or serum hormone levels could conceivably have profound and un-
favorable effects on graft function. The cells in the blood might have difficulty
in traversing the capillaries of the graft and, by interfering with the circulatory
pattern, might impair the graft's function. Because no discordant organ grafts
have functioned for long periods, such questions, raised by CaIne in 1970, still
cannot be answered.
This topic was discussed again more recently by Auchincloss [66]. From his
review of the literature, his findings were that, with regard to primate organs,
there has not yet been a clear example of the inability of an organ to survive or
function except for considerations that would apply equally to allografts.
Though experience with organs from widely disparate species is extremely
limited, Auchincloss drew attention to the fact that pig livers used in cross-cir-
culation systems have been shown to perform at least some of the functions
necessary to support human life [67,68]. Auchincloss's view, however, was
that it is almost inconceivable that some examples of organ dysfunction will
not be found under such circumstances, for it would seem unlikely that all of
the enzymes and hormones that showed species variation will function with
equal efficiency in xenogeneic recipients. There is, as yet, no evidence to re-
fute this viewpoint.
It is hoped, however, that particularly with regard to metabolically "sim-
ple" organs such as the heart (as opposed to an organ such as the liver which
has a large number of different biochemical transactions to perform) this will
not prove a barrier. The experience of pig hearts functioning in baboons for
periods of3 or4 days [16] and of pig kidneys for, on one occasion, over 20 days
Table 30.8. Approximate range of hemodynamic parameters in man and swine
Human" Pigb
Heart rate (beats/min) 111-123

Cardiac output index 2.6--4.2 (llmin per m2) 170-210 mllmin per kg
Pressures (mm Hg)
Right atrium 0-8 1-9
Right ventricle 15-30/0-8 24-30/2-5
Pulmonary artery 15-30/3-12 11-24 (mean)
Pulmonary capillary wedge
and left atrium 1-10 2-12
Left ventricle 100-140/3-12 116W
Aorta and systemic arteries 70-105 (mean) 114-126 (mean)
Systemic vascular resistance 700-1600 dyn s/cm-5 719 torr/I per min/kg
Pulmonary vascular resistance 20-130 dyn s/cm-5 69 torr/I per min/kg
" Range of normal resting hemodynamic values for man from [72].
b Approximate values in resting swine from [5,8,73,74].
C Systolic pressure may be low (±60 mm Hg) in some minipig and micropig species [5].
Table 30.9. Normal hematologic and blood chemistry parameters in the pig and man
Swine" Human"
Hematology
Hemoglobin (g/dl) 10-16 12-18 (M); 11-15 (F)
PCV(%) 32-50 32-52 (M); 34-45 (F)
RBC (xlO/lll) 5.0-8.0 4.3-5.8 (M); 3.9-5.1 (F)
MCV(ll m') 50-68 82-99
MCH (pg) 17-21 28-34 (M); 37-34 (F)
MCHC(g/dl) 30-34 33-35
Platelets (/111) 320000-520000 130000-400000
Total white blood count (/111) 11000-22000 3800-10900
Neutrophils (mature) (1111) 3080-10450 2000-7150
Lymphocytes (/111) 4290-13640 1100-3000
Monocytes (1111) 200-2200 60-750
Eosinophils (1111) 55-2420 25-350
Basophils (1111) 0-440 10-160
Chemistry
Sodium (mEq/L) 140-150 136-145
Potassium (mEq/L) 4.7-7.1 3.5-5.0
Chloride (mEq/L) 100-105 104-111
HCO, (mEq/L) 18-27 24-30
Calcium (mg/dl) 11.0-11.3 8.7-11.0
Inorganic phosphorus (mg/dl) 4.0-11.0 2.2-4.7
Magnesium (mg/dl) 1.9-3.9 1.8-2.4
Iron (Ilg/dl) 73-140 50-160 (M); 60-135 (F)
BUN (mg/dl) 8-28 5-25
Creatinine (mg/dl) 1.0-2.7 0.5-1.4
Glucose (mg/dl) 65-95 65-105
Cholesterol (mg/dl) 117-119 140-220
Total bilirubin (mg/dl) 0-0.2 0.1-1.0
Alkaline phosphatase (mU/ml) 60-269 40-130
SGOT(mU/ml) 25-87 7-27
CPK(mU/ml) 65 30-220
SGPT (mU/ml) II (Karman units) 0-40
Total plasma protein (g/dl) 3.5-6.0 6.0-8.0
Albumin (g/dl) 1.9-2.4 3.2-4.8
Globulin (g/dl) 1.0-1.3
Uric acid (mg/dl) 0.35-1.95 3.5-8.5 (M); 2.5-7.0 (F)
Triglycerides (mg/dl) 0-145 40-160 (M); 35-135 (F)
LDH(mU/ml) 211 80-200
Fibrinogen (mg/dl) 100-500 200-400
M, male; F, female; PCV, packed cell volume; RBC, red blood cells; MCV, mean corpus-
cular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin
concentration; BUN, blood urea nitrogen; SGOT, serum glutamic oxaloacetic transami-
nase; CPK, creatine phosphokinase; SGPT, serum glutamic pyruvic transaminase; LDH,
lactate dchydrogenase
" Based on [51,75). Other biochemical valucs for swine can be found in Tumbleson [3).
" Based on Miale [76).
References 497
[69], lends encouragement to the view that long-term function of pig organs in
man will be possible. An orthotopically transplanted pig liver was also ob-
served to support life satisfactorily in a baboon for a period of 3 days [70].
Data regarding the hemodynamic performance of the pig heart are well
documented [4-8], and would appear to indicate that the pig heart would func-
tion satisfactorily in man, both at rest (Table 30.8) and during exercise [8]. The
resting heart rate and the mean systemic pressure are both rather higher in the
pig than in a human of corresponding size.
Speculation over the ability of other pig organs - kidney, pancreas and liver
- to function adequately in man has been documented recently by Kirkman
[9]. He concludes that there is no evidence to suggest that the pig kidney will
not function adequately in man as metabolic and regulatory parameters, and
size and anatomical structure, are all similar. A porcine pancreas would proba-
bly function successfully as porcine insulin can clearly control human glucose
levels, and the regulation of insulin secretion in the pig is very similar to that in
man. Because of the complexity of the metabolic functions of the liver, it is less
certain that a pig liver would successfully support life in man. Kirkman, like
Auchincloss [66], suggests that while many biochemical pathways may be the
same, it seems unlikely that every enzyme and every factor produced will have
the same structure. A comparison of some of the major hematologic and bio-
chemical parameters in swine and man is presented in Table 30.9.
Comment
It would seem, therefore, that, if the immunological problems can be over-

come, the pig would in many ways prove a satisfactory organ donor for man.
Gnotobiotic techniques would probably need to be used until the pig reached
a size enabling the organs to be used in adult humans. An unlimited number of
donor organs of all types would then be available for use in man.
Transplantation could then be offered to every suitable recipient.
References
1. Coates, M.E., Gustafsson, B.E. (eds) The Germ-Free Animal in Biomedical Research.
Laboratory Animals Ltd., London, 1984.
2. Miniats, O.P. Production of germ-free animals. Part II. Farm Animals. In The Germ-
Free Animal in Biomedical Research. (edited by Coates, M.E. and Gustafsson, B.E.)
Laboratory Animals, Ltd., London, 1984, p. 49.
3. Tumbleson, M.E. (ed) Swine in Biomedical Research. Volume 3. Plenum Press, New
York,1985.
4. Stanton, H.C., Mersmann, H.J. (eds) Swine in Cardiovascular Research. Volumes I-III.
CRC Press, Inc., Boca Raton, 1986.
5. Smith, A.c., Spinale, F.G., Swindle, M.M. Cardiac function and morphology of Hanford
miniature swine and Yucatan miniature and microswine. Lab. Anim. Sci. 40,47,1990.
6. Hughes, H.C. Swine in cardiovascular research. Lab. Anim. Sci. 36,348, 1986.
7. Kirkman. RL. Of swine and men: organ physiology in different species. In Hardy. M.A.
(cd). Xenogra{125. Elsevier: Amsterdam. New York. Oxford 19R9. p. 125.
R. Hannon. J.P. Hemodynamic characteristics of the conscious resting pig. In Tumbleson.
M.E. (cd). Swine in Biomedical Research. Plenum. New York and London. 1985. p. 13.+1.
9. Erickson. H.H .. Faraci. F.M .. Olsen. S.C Effect of exercise on cardiopulmonary func-
tion in domestic pigs. In Tumbleson. M.E. (cd). Swine in Biomedical Research Plenum.
New York and London. 1985. p. 709.
10. CaIne. RY. Organ transplantation between widely disparate species. Transplanl. Pmc.
2.550. 1970.
II. Perper. R.1.. Najarian. J .S. Experimental renal heterotransplantation: l. In widely diver-
gent species. Transplallwliol1. 4.376.1966.
12. Giles. G.R. Boehmig. J.H .. Lilley. 1.. Amemiya. H .. Takagi. H .. Coburg. A.1 ..
Hathaway. W.E.. Wilson. CB .. Dixon. F.J .. StarzL TE. Mechanism and modification of
rejection of heterografts between divergent species. Transplant. Pmc. 2.522. 1970.
13. Merkel. F.K .. Bier. M .. Beavers. CD .• Merriman. W.G .. Wilson. C. StarzL TE.
Modification of xenograft response by selective plasmapheresis. Transplant. Froc. 3.
534.1971.
14. Mozes. M.F .. Gewurz. H .. Gunnarson. A .. Moberg. W .. Westbery. N.G .. Jetzer. T.
Najarian. J .S. Xenograft rejection by dog and man: Isolated kidney perfusion with blood
and plasma. Transplant. Pmc. 3.531.1971.
15. Lexer. G .. Cooper. D.K.C. Rose. ;\.G .. Wicomb. W.N .. Rees.J .. Keraan. M .. Du Toit. E.
Hypcracute rejection in a discordant (pig to hahoon) cardiac xenograft model.
1. Ileart Tramplant. 5.411.19R6.
16. Cooper. D.K.e.. Human. P.A .. Lexer. G .. Rose. A.G., Rees, J., Keraan, M., Du Toit, E.
The effects of cyc1osporine and antihody adsorption on pig cardiac xenograft survival in
the haboon. 1. Heart Transplant. 7. 23R. 198K
17. Ross. D.N. In Experience With liuman lieart Transplantatiol1. (editcd by H. Shapiro)
Bulterworths. Durban. 1969. p. 227.
18. Cooley. D.A .. Hallman. G.L.. BloodwelL RD .. Nora. J.1 .. Leachman, RD. Human
heart transplantation: cxperience with 12 cases. Am . .!. Cardiol. 22. R04. 1968.
19. Rose. A.G .. Cooper. D.K.e.. Human. P.A., Reichenspurner, H .. Reichart. B. Histo-
pathology of hyperacute rejection ofthe heart: experimental and clinical observations in
allografts and xcnografts.'!' Hearl Transplant. In prcss.
20. Rcemtsma. K.. McCracken. B.H .. Schlegel, J.Y .. Pearl. M. Heterotransplantation of the
kidney: two clinical experiences. Science. 143.700.1964.
21. Reemtsma, K .. McCracken. B.H .. Schlegel. J.Y .. Pearl. M.A .. Pearce. CW .. Dewitt.
CW .. Smith. P.E.. Hewitt, R.L., Flinner. R.L.. Creech. O. Jr. Renal heterotransplanta-
tion in man. Anl1. Sllrg. 160.3R4. 1964.
22. Reemtsma. K .. McCracken. B.H., Schlegel, J.Y .. Pearl. M.A .. Dewitt, CW .. Creech. O.
Jr. Reversal of early graft rejection aftcr renal transplantation in man . .!. Am. med. Ass.
187.691,1964.
23. Reemtsma, K. Hcterotransplantation. Transplant. Proc. 1.251. 1969.
24. Barnard. CN .. Wolpowitz. A., Losman. J.G. Heterotopic cardiac transplantation with a
xenograft for assistance of thc left heart in cardiogenic shock after cardiopulmonary by-
pass. S. Afr. Med. 1. 52, 1035, 1977.
25. Giles. G.R .. Boehmig. H.1 .. Amemiya. H .. Halgrimson. e.G .. StarzL TE. Clinical
hetcrotransplantation of the liver. Transplant. Proc. 2.506.1970.
26. StarzL TE .. Ishikawa. M .. Putnam. CW., Porter. K.A., Picache. R, Husberg, B.S .•
Halgrimson. CG., Schroter. G. Progress in and deterrents to orthotopic liver transplan-
tation, with special refercnce to survival, resistance to hyperacute rejection. and biliary
duct reconstruction. Transplant. Proc. 6. 129. 1974.
27. StarzL TE .. Marchioro. TL.. Peters. G.N .. Kirkpatrick. CH., Wilson, W.E.C, Porter,
K. A .. Rifkind. D., Ogden. D.A .. Hitchcock, CR, Waddell. W.R Renal heterotrans-
plantation from baboon to man: experience with six cascs. Transplantatiol1. 2.752,1964.
28. Bailey. L.L.. Nehlsen-Cannarella. S.L.. Concepcion. W .. Jolley. W.B. Baboon-to-human
cardiac xenotransplantation in a neonate . .I. Am. Med. Ass. 254.3321.1985.
29. Cooper. D.K.e.. Human. P.A .. Rose. A.G .. Rees. 1.. Keraan. M .. Reichart. B., Du Toit.
References 499
baboon cardiac xenograft model.J. Thorac. Cardiovasc. Surg. 97,447,1989.
30. Reichenspurner, H., Human, P.A., Boehm, D.M., Rose, A.G., May, R, Cooper, D.K.C,
transplantation in primates.J. Heart Transplant. 8,200,1989.
K., Rose, E.A Cardiac xenotransplantation in primates. J. Thorac. Cardiovasc. Surg. 93,
809,1987.
32. Reemtsma, K., Pierson, RN., Marboe, CC, Michler, RE., Smith, CR, Rose, E.A,
Fenoglio, 1.1., lr. Will atherosclerosis limit clinical xenografting? Transplant. Proc. 19,
108,1987.
33. Bailey, L., lang, J., Johnson, W., Jolley, W.B. Orthotopic cardiac xenografting in the
newborn goat. J. Thorac. Cardiovasc. Surg. 89242,1985.
Matsumoto, M., Uenaka, A., Kitamura, H. The mechanism of discordant xenograft re-
jection. Transplantation. 46,825, 1988.
35. Thomas, F.T., Demasi, R, Aranada, D., Marchman, W., Alqaisi, M., Larkin, E.W.,
Condie, RM., Carobbi, A, Thomas, J .M. Comparative efficacy of immunosuppressive
drugs in xenografting. Transplant. Proc. 22, 1083, 1990.
36. Knechtle, S.J., Halperin, E.C, Bollinger, RR Cardiac xenograft survival using total
lymphoid irradiation and cyclosporine. Heart Transplant. 4,605,1985.
37. Cooper, D.K.C, Human, P.A., Reichart, B. Prolongation of cardiac xenograft (vervet
munosuppressive drug therapy. Transplant. Proc. 19,4441, 1987.
38. Auchincloss, H., Jr., Pierson, RN., III, Russell, P.S., Winn, H.J. The cell-mediated re-
sponse to xenogeneic antigens is similar to that to minor allogeneic histocompatibility
antigens. In Hardy, M.A (ed). Xenograft 25. Elsevier; Amsterdam, New York, Oxford,
1989, p. 207.
39. Citterio, F., Pozzetto, U., Serino, F., Fioravanti, G., Castagneto, M. Cyclosporine effects
on xenogenic lymphocyte activation: cellular immune reactions to pig antigens in man.
40. Szymanowski, Z., Stetkiewicz, S., Wacheler, B. Les groupes serologiques dans Ie sang du
pore et leur relation avec les groupes du sang humain. Compt. Rend. Soc. Bioi. 94, 204,
1926.
41. Szymanowski, Z., Wacheler, B. Contribution a I'etude des immuno-iso-agglutines du
sang du pore. Compt. Rend. Soc. Bioi. 95,932,1926.
42. Andresen, E. Blood groups in pigs. Ann. NY Acad. Sci. 97,205, 1962.
43. Otte, K.E., Andersen, N., 10rgensen, K.A, Kristensen, T., Barfort, P., Stark lint, H.,
Larsen, S., Kemp, E. Xenoperfusion of pig kidney with human AB or 0 whole blood.
44. Van der Riet, F.de St. J., Human, P.A, Cooper, D.K.C, Reichart, B., Fincham, I.E.,
Kalter, S.S., Kanki, P.J., Essex, M., Madden, D.L., Lai-Tung, M.T., Chalton, D., Sever,
1.L. Virological implications of the use of primates in xenotransplantation. Transplant.
Proc.19,4068,1987.
45. Kalter, S.S. Overview of Simian viruses and recognized virus diseases and laboratory
support for the diagnosis of viral infections. In: Benirschke, K. (ed). Primates - The Road
to Self-Sustaining Populations. Springer-Verlag, New York, 1986, p. 681.
46. Van Vleet, 1.F., Ferrans, V.J. Cardiovascular diseases of swine. In Swine in
Cardiovascular Research. Volume I (edited by Stanton, H.C and Mersmann, H.I.). CRC
Press, Boca Raton, Florida, 1986, p.121.
47. Hsu, F.S., Du, SJ. Congenital heart disease in swine. Vet. Pathol. 19,676, 1982.
48. Wang, F.I. Pathological study on cardiac disease in swine. MS thesis. National Taiwan
University, Taipai, Taiwan, 1978, (quoted by Hsu and Du (47».
49. Swindle, M.M., Thompson, RP., Carabello, B.A, Smith, AC, Hepburn, B.J .S., Bodison,
D.R, Corin, W., Fazel, A, Biederman, W.W.R, Spinale, F.G., Gillette, P.C Heritable
ventricular septal defect in Yucatan miniature swine. Lab. Anim. Sci. 40, 155, 1990.
50. Migaki. G. Naturally occurring neoplastic diseases (VIII Cow, horse, pig, sheep, and
goat.) In Hamlhook of La/Joratorv Animal Science Vol. Il/ Melhy. E.C. lr. and Altman.
N.H. (cds). CRC Press. Inc .. Cleveland, Ohio, p. 2K9, 1974.
51. Blood. D.C.. Radostits, O.M .. Henderson, 1.A. Veterinary Medicine: A iext/Jook of the
Diseases of Caltle, Sheep, Pigs, Coats and /Ior.lt's. Bailliere Tindall. Philadelphia. 19K3.
52. Benenson. A.S. (cd). COllIrol of COlllllllllzica/Jle Diseases ill Mall. Twelfth Edition,
American Public Health Association, 1979.
53. Dufty, 1.H. Specific pathogen free and gnotobiotic farm animals. Allstralian Veterinarr
1. 52,355, 1970.
54. Miniats, O.P .. Valli, V.E. The gastrointestinal tract of gnotobiotic pigs. In Cerlllfiec
Research. Biologiml Effect of Gnoto/Jiotic Environments. (edited by Heneghan. 1.B.)
Academic Press. N cw Yark. London, 1973, p. 575.
55. US Department of Agriculture, Animal and Plant Health Inspection Service. Federal
Meat and Poultry Inspection Statistical Summary for 1972,1973.
56. Plummer. P.l.G. A survey of six hundred and thirty-six tumors from domesticated ani-
mals. Canad. 1. Compo Mer!. Vet. Sci. 20,239,1956.
57. Smith, H.A. Malignant lymphoma in animals. Amer. .I. Ciin. Path. 38,75,1962.
5K. Forbus, W.D .. Davis, c.L. A chronic granulomatous disease in swine with striking re-
semblance to Hodgkin's disease. Am . .I. Pathol. 22,35,1940.
59. Flatt, RE., Nelson, L.R., Middleton, e.e. Melanotic lesions in the internal organs of
miniature swine. Arch. Path. 93,71,1972.
60. Strafus, A.C., Dommer!. A.R, Tumhleson, M.E., Middleton. c.c. Cutaneous mela-
noma in miniature swine. Lab Anim. Sci. lK, 165, 190K.
61. Flatt. R.E .. Middleton. c.e., Tumbleson, M.E .. Perez-Mesa, e. Pathogenesis of benign
cutaneous melanomas in miniature swine. 1. Amer. Vet. Merl. Assoe. 153.936. 1968.
02. Nordby, 1.E. Congenital melanotic skin tumors in swine . .I. Hered. 24.301,1933.
63. Caylor. H.D .. Schlotthauer. c.F. Melano-epitheliomas of swine: transplantation and cul-
tural experiments. Arch. Path. Lab. Med. 2,343,1920.
64. Case, M.T. Malignant melanoma in a pig.J. Allier. Vet. Merl. Assoe. 144,254.1904.
65. Hjerpe. C.A .. Theilen. G.H. Malignant melanoma in porcine littermates. 1. Amer. Vet.
Med. Assoc. 144,1129,1904.
00. Auchineloss. H. lr. Xenogeneic transplantation. Transplantation. 40,1. 198K.
67. Eiseman. B. Cross-circulaton in heterologous systems. Colstun Papers. 279, 1967.
08. Norman, l.e., Saravis, C.A., Brown, M.E .. McDermott, W.V. lr. Immunochemical oh-
servations in clinical heterologous (xenogeneic) liver perfusions. BII/I. Soc. /nt. Chir. 25,
071. 1966.
09. Alexandre, G.P.1., Gianello, P., Latinne, D .. Carlier, M., Dewaele. A.. Van Ohhergh. L..
Moriau. M .. Marhaix. E .. Lambotte, 1.L., Lamhotte, L., Squifflet. 1.P. Plasmapheresis
and splenectomy in experimental renal xenotransplantation. In Hardy, M.A. (cd).
Xenograft 25. Elsevier; Amsterdam, New York, Oxford, 19K9, p. 259.
70. CaIne, R.Y .. White. H.1.0 .. Herhertson, B.M .. Millard, P.R, Davis, D.R, Salaman, 1.R ..
SamueL 1.R. Pig to bahoon liver xenografts. Lancet. 1. 1176, 196K.
71. Emsinger, M.E. Animal Science. Seventh Edition. The Interstate Printers and
Puhlishers. Dennville. Illinois. 1977, p. 311.
72. Grossman, W .. Barry. W.H. Cardiac catheterization. In Heart Disease. Chapter 9.
(E. Braunwald, cd.) W.B. Saunders Co .. Philadelphia, 191\8, p. 242.
73. WisekopL R.B .. Bogetz. M.S .. Reid, I.. Roizen, M.L Keil, L.c. Cardiovascular, en-
docrine and metabolic responses of conscious swine to hemorrhage. In Swine in
Biomedical Research. Volume 3, (Tumbleson, M.E., cd.) Plenum, New York and
London. 1980, p. 1405.
74. Hannon, 1.P., Bossone, C.A., Wade, e.E. Normal physiological values for conscious pigs
used in hiomedical research. /,ab. Anim. Sci. 40,293,1990.
75. Benirschke. K .. Garncr. F. M. (eds). Patlzologr of Laboratory Animals, Springer-Verlag,
Berlin, Heidelherg, New York, 1974.
76. Miale, 1. B., Lahoratory Medicine; Hematology. C. V. Moshy, St. Louis, 191\2. p. 070.
Chapter 31
Human Antibodies to Pig Determinants and

Their Association with Hyperacute Rejection
of Xenografts
K.I. WELSH, D.H. TAUBE, M. THICK, A. PALMER, N. STEVENS, AND R. BINNS
Introduction
In 1985, our group began a series of literature searches, in vitro tcsting, and ex-
tracorporeal experiments on antibody removal, which, coupled with deduc-
tions made from this work, led us to connect a pig kidney to the arteriovenous
shunt of a patient awaiting renal transplantation. It is the series of events that
led up to this experimcnt and its outcome which form the subject of this chap-
ter.
The potential problems of using a pig as an organ donor for man as we saw
them in 1985 were:
1. Preformed human antibody responses against pig.
2. Primary antibody responses to pig post-transplant (i.e., new immunoglobu-
lin (IgM) responses not included in point 1).
3. Secondary antibody responses to pig (i.e., IgG responses following IgM re-
sponses defined in points 1 or 3).
4. Primed T cells to pig (assuming point 1 was partially T cell-dependent).
5. T cell responses to pig post-transplant.
6. Complement problems (human complement is well known to be a poor
effector molecules for the lysis of human cells but would, by analogy with
other systems, be effective against pig cells).
7. Non-T and non-B cell immunity, e.g., natural killer (NK) cells.
8. Inefficiency of hormones, etc. produced by pig organs to function with hu-
man receptors and vice versa.
Our interests in dissecting the antibody responses thoroughly (points 1 and
2 above) were to give us clues as to which were likely to be directed against car-
bohydrate (i.e., those which persisted as IgM) and to provide some insight into
the overall magnitude of the problems likely to be faced.
Pig Histocompatibility Systems
Our first literature review revealed that three pig histocompatibility systems-
swine leukocyte antigen systems A, B, and C (SLA, SLB, and SLC) - and 15
blood group molecules had been identified, and which we considered major
potential targets for the antibodies outlined in points 1-4 above.
502 Human Antibodies to Pig Determinants and Their Association
The SLA is the equivalent of human leukocyte antigens (HLA) class I and
II. Three polymorphic chains (A B, and C) have been identified [1]. Two loci
are defined in the SLA-D region [2-4]. SLB is a separate histocompatibility
that has been identified, and is closely linked to the L blood group locus. Two
alleles are known [5]. SLC locus is not linked to SLA SLB, or blood groups A
E and N. Two alleles are known (SLC1 and SLC2), expressed in frequencies of
0.1 and 0.9 [6].
Pig Blood Groups
Fifteen pig blood groups have been identified and two are known to behave as
minor histocompatibility antigens - groups A and E. Two allelic forms of A
(A and 0) and 14 alleles ofE are known [7].
Aims of Study
Based on this information, we set about screening the sera from 100 humans,
drawn from groups anticipated to have different ranges of humoral responses
to pig. These included vegetarians, vegans, Moslems, Jews, significant eaters
of pork, renal transplant patients known to be heavily sensitized against HLA
and human red cell antigens, and individuals known to have worked with or
been bitten by pigs.
The aims of this initial work were twofold:
1. To identify, if possible, a human antibody which reacted with pig lympho-

cytes but not with kidney structural elements, i.e., we hoped to identify the
equivalent of a CD45 antibody.
2. To define the extent and magnitude of the problem of preformed natural
antibodies in man against pig tissues.
Methods
Pig Lymphocyte Preparation
Although ethylenediamine tetra-acetic acid (EDT A), citrated, or heparinized

blood gave adequate yields of pig cells after the standard human density gradi-
ent separation methods (1.077 g/cl), monocyte contamination in the lympho-
cyte fraction led to significant background killing in the cytotoxic tests.
Defibrination of pig blood with glass beads prior to separation avoided such
problems and allowed lymphocytes to be used up to 4 days after preparation in
cytotoxic tests without appreciable rise in background. To achieve storage for
Methods 503
3-4 days 0.5% pig serum in phosphate-buffered saline (PBS), McCoys, or
RPMI 1640 all proved satisfactory.
Rabbit Complement-Mediated Cytotoxicity
A standard National Institutes of Health (NIH) human-type cytotoxic test

protocol of complement-mediated cytotoxicity in Terasaki plates proved not
adequate for pig lymphocytes, but could be easily modified by a fivefold re-
duction in the amount of complement used. Some preparations of pig lympho-
cytes were sufficiently lysed with shorter incubation times than the NIH proto-
col. Routinely, we used a 30-min preincubation period and assessed, by the use
of inverted phase contrast microscopy, when optimal cell death was occurring.
Usually 30-60 min gave good results on pig lymphocytes prepared from defib-
rinated blood. Two commercial and one in-house source of complement gave
indistinguishable results. One commercial complement source gave consis-
tently higher titers.
The method consisted of the following steps:
1. Dilution of 50 ~l human serum through 50 ~l PBS containing 0.5% pig serum

(0.5% -1.5% of pig serum is essential for cell viability).
2. Transfer of 2 ~l of each serum dilution by the use of a Hamilton syringe to
the wells of an oiled Terasaki plate. A 50-~1 Hamilton was utilized and,
starting transfer at the highest dilution, 2 ~l aliquots were added to the wells
of five separate plates. After discharge of the remaining serum the next dilu-
tion was taken and the process repeated. This procedure, although wasteful
of serum, allowed rapid transfer of material without a specific wash step be-
tween dilutions; indeed it proved more reproducible than tests with a wash
step with PBS being used between dilutions.
3. Addition ofl ~l of pig cells (2 million per mlin PBS/1 % pig serum).
4. Incubation for 30 min at 22°C.
5. Addition of rabbit complement (1 ~l).
6. Incubation for 60 min at 22°C (but see above for necessary variation).
7. Visualization of cell death by the use of eosin or trypan blue.
Hnman Complement-Mediated Cytotoxicity
When using human complement, the method was exactly as above, except:
1. No rabbit complement was added; thus each individual serum acted as its
own complement source. This method suffered the disadvantage that com-
plement activity diluted out much more rapidly than the IgM antipig anti-
body.
2. Exactly as above, but I ~l pig-adsorbed human serum was used in place of
rabbit complement. Serum from the individual being titered was adsorbed
as described below, and the adsorbed sera used as complement in each well
of diluted sera from each individual. Adsorbed serum was tested as negative
in the rabbit complement-mediated test prior to its use as complement.
Adsorption of Human Antibodies on Pig Cells
One million pig spleen or peripheral white blood cells were used to adsorb 1 ml
serum titer 1. Thus, to adsorb 1 ml human serum, cytotoxic titer 11300, 300 mil-
lion pig leukocytes were divided into three aliquots and sequential adsorp-
tions carried out for 2 h, 2 h, and overnight, with occasional mixing. A single
2-h adsorption with 300 million cells removed around 80% of the titer.
Agglutination
Agglutination was assessed by direct visualization of red blood cell clumping

in the wells of Terasaki plates set up in parallel to the human complement cy-
totoxic tests but where the test sera used had been complement-inactivated by
heat treatment at 56°C for 20 min.
Flow Cytometry
Flow cytometry was achieved using a Becton Dickinson Facstar machine and
utilized specific anti-IgM and -IgG second reagents. Difficulty was observed
with the pig/human discriminatory capacity of some secondary anti-IgM
reagents.
Histology
After 3 months of problems with second reagent specificity (see "Flow

Cytometry") we were able to utilize standard pathology laboratory techniques
to visualize human antibodies on pig kidney sections. Endothelial cells were
the main targets of the standard human antipig IgM antibody which we had de-
fined by lymphocytotoxicity. This result showed that this reagent would not
function as an antipassenger cell reagent and that antibody removal would be
necessary before transplantation. It also showed that the standard cytotoxic
test against pig lymphocytes could be used as a cross-match test for pig-to-hu-
man transplantation of kidneys.
Results of Initial Antibody Studies 505
Results ofInitial Antibody Studies
The results of the lymphocytotoxicity tests are shown in Table 31.1. We also
obtained the following additional data on incomplete subsets. For cytotoxicity
with human complement, the range of variability was considerable and did not
correlate with the C4 allotypes of the individuals. In agglutination tests, some
human sera were particularly good at agglutination, whilst some sera did not
agglutinate.
We concluded that the rabbit complement test is the most reproducible and
cosistent between humans. The other tests are more complicated to perform,
interfere with one another (lysis hides agglutination), and are less repro-
ducible, and sequential samples from the same individuals differ widely.
In spite of the range of subjects tested, all humans appeared to have as their
main xenoantibody an IgM which reacted with pig red cells, lymphoid cells,
and kidney epithelium and endothelium. Adsorption with anyone of these
targets removed almost all IgM activity from most humans (cf. 1969) [8].
Western blot analysis and Ouchterlony analysis suggested that all humans had
IgM antibody to the same pig determinant and that in each human the re-
sponse to this determinant was the major natural antibody. The different hu-
man groupings had different titers of IgM, and there was some correlation of
response with human A and 0 blood groups. However, no human had an IgM
cytotoxic titer against lymphocytes of less than 11128 or greater than 11512
(1132-11128 with weaker complement).
Using flow cytometry and adsorption techniques we were able to identify a
low level of IgG antibody in humans which was not against the same determi-
nant but which was to a common determinant. Additional IgM and IgG anti-
Table 31.1. IgM and IgG titers of anti-pig Iymphocytotoxic activity in the sera of the human
groups studied
Study group Number Titerrangc
Prc-DTT Post-DTT
IgMand IgG IgG
Normal omnivores 28 256-512 0-2

Renal patients 24 256-1028 0-8
Normal vegetarians 28 128-512 0-2
Normal vegans 14 128-512 o
Moslems 6 256-512 0-2
DTT, dithiothreitol
For cytotoxicity with human complement, the range of variability was considerable and did
not correlate with the C4 allotypes of the individuals. In agglutination tests, some human
sera were particularly good at agglutination whilst some sera did not agglutinate.
We concluded that the rabbit complement test is the most reproducible and consistent be-
tween humans. The other tests are more complicated to perform, interfere with one another
(lysis hides agglutination), and are less reproducible, and sequential samples from the same
individuals differ widely.
bodies were occasionally observed. These were determined more by the indi-
vidual pig used as target than by interhuman variation. There was consider-
able variation in the ability of human sera to lyse or agglutinate pig cells in the
absence of rabbit complement. A complete analysis of these data has not been
made.
Experimental Study
Armed with the above evidence, in the summer of 1986 we defined the follow-
ing strategy:
1. All future experiments would be carried out using an inbred pig line which
was of blood group O.
2. The blood group 0 nature would be checked on each tissue because of the
known human variation in A and possibly B substance quantitation in dif-
ferent tissues.
3. Only antibodies reacting to the inbred pig kidney would be considered in
this phase of the work.
4. T cell responses to pig were unlikely to be greater than T cell responses to
alloantigens. (This was purely a guess in 1986, and was based on the fact that
the cellular immune system is designed to react maximally to minute differ-
ences.) T cell responses would therefore be ignored for this phase of the
work.
The inbred pig line produced by Dr. Richard Binns and owned by the
AFRC, Cambridge, typed with human typing reagents as blood group O.
Samples of various tissues were tested biochemically for the presence of A and
B substance and were found to be negative (Prof. B. Samuelson, Gothenberg,
Sweden).
Experimentally, we chose two connected approaches:
1. The assessment of the antipig responses in individuals who were having anti-
HLA antibodies removed for clinical reasons.
2. The use of blood from these individuals and others to perfuse pig kidneys
with samples from which the IgM and or IgG had been removed, and to as-
sess damage by a range of criteria. Human blood donors would be chosen
whose serum naturally agglutinatedllysed pig cells since these represented
the worst possible cases.
3. If points 1 and 2 proved feasibility, we would connect a pig kidney to a hu-
man via a dialysis shunt.
Results of Experimental Study 507
Results of Experimental Study
Removal of Antipig Antibodies
We studied the removal of these antibodies in five patients undergoing anti-

HLA antibody removal by plasma exchange or immunoadsorption (PEllA)
[9] who were immunosuppressed with prednisolone and cyclophosphamide to
prevent antibody resynthesis. We found that PEllA effectively removed hu-
man antipig IgM. However, despite immunosuppression, these antibodies
were resynthesized with a return to pre PEllA titers within 6-8 weeks, as
shown in Table 31.2.
Perfusion Experiments
Pig kidneys were attached to a dialysis pump and human whole blood (250 ml
in heparin) circulated at a flow rate of approximately 170 mllmin. Within a few
minutes all such kidneys went black and flow ceased. Biopsies showed charac-
teristic features of hyperacute rejection.
Table 31.2. Total IgM levels and antipig antibody titers pre- and post-plasma exchange/im-
munoadsorption
Patient Pre-PEllA Post-PEllA
IgM level (gil) Pig titer IgM level (gIl) Pig titer
1 0.59 >1/500 0.06 1/32

2 0.85 >1/500 0.26 1/64
3 1.0 >1/500 0.07 1/8
4 0.7 >1/500 0.2 1/128
5 2.5 >1/500 0.1 1/8
PEllA, plasma exchange/immunoadsorption
Table 31.3. Simplified summary of perfusion experiment results
Patient Hyperacute rejection IgM deposition
Pre-PEllA Post-PEllA Pre-PEllA Post-PEllA
1 +++ +++
2 + + ++ ++
3 + ++
4 + + ++ +
5 +++ +++
PEllA, Plasma exchange/immunoadsorption. These data suggested that in the shortterm,

removal of IgM to levels <0.1 gIl and reduction of antipig titers to 1/8 or below were associ at -
ed with the abolition of hyperacute rejection and no IgM binding.
In a second series, human serum was used in place of whole blood. Here the
major measurable rapid event was excessive IgM and fibrin deposition observ-
able on renal biopsy at 10 min. Sera from the same five patients defined in
Table 31.2 were used. These experiments were performed to test whether the
removal of antipig IgM from these patients resulted in significant reduction in
binding of IgM to pig kidneys. The results are summarized in Table 3l.3. The
data suggested that in the short term, removal of IgM to levels <0.1 gil and re-
duction of antipig titers to 118 or below were associated with the abolition of
hyperacute rejection and no IgM binding.
Clinical Trial
The encouraging results from this series of experiments carried out over a pe-
riod of over I year led us to define the following clinical trial. The design was:
1. To prove by cytotoxicity and flow cytometry that the human antibody to pig
had been reduced to a minimal titer before transplant.
2. To maintain circulation through the kidney for a period up to 6 h with a
pump utilizing full flow measurements.
3. To maintain the pig kidney in a sterile bag with a facility for collecting urine.
4. To maintain the donor pig alive in order to repeat the same experiment with
the second kidney 3 weeks later.
5. To collect blood samples from the human subject during and after the peri-
od of kidney perfusion in order to assay for T cell and antibody responses.
Ethical permission was applied for and granted on the condition that con-
ventional transplant immunosuppressive therapy was administered to cover
the "experimental period". The first hour after the connection of the organ is
remembered second by second by the immunologist on the team because of
fear of hyperacute rejection. The last few hours are remembered second by
second by the nephrologist since full clinical responsibility switched to him at
this time.
In detail, this experiment was performed immediately after an IA session
when the patient's totallgM level was <1.0 gil and his antipig titer was <112 by
flow cytometry; in addition, there was a negative cytotoxic cross-match against
target lymphocytes from the pig kidney donor. Prostacyclin (2.5 nglkg per
minute) was infused into the arterial limb of the shunt to prevent clotting in the
extracorporeal circuit. The pig kidney began to pass urine immediately and
continued to do so for 6 h until the experiment was terminated. Urine output
varied between 0.5 and 1.0 IIh. and the patient's plasma creatinine fell by ISO
/-lmolll during the experiment. Other than hypovolemia (which was corrected
with intravenous fluids), the patient experienced no side effects.
Histological examination of the kidney at the end of the experiment
showed no evidence of hyperacute rejection or IgM deposition. Very slight
Clinical Trial 509
fibrin deposition was observed. Following this experiment, the patient's anti-
pig IgM titers gradually rose at a similar rate to that seen in the other patients
who had been similarly immunoadsorbed but had not been connected to a pig
kidney. No new antipig IgG has been detected in this patient since the proce-
dure.
Problems were encountered with the follow-up experiment intended to be
carried out 3 weeks later. Firstly, it was delayed 1 week to allow further prepa-
ration and testing. Secondly, after preparing the patient, and with 1 h hour to
the time of organ perfusion, the donor nephrectomy was performed. The pig
had grown a considerable amount in the 4 week period since the first study and
its remaining kidney had grown even more. Although this could have been
predicted, it was not. As a result, the kidney would not fit into the sterile bag
specially designed for the experiment. The attempt to squeeze it in caused a
split in the bag which, although repaired, became a problem immediately after
connection of the kidney to the human and the pump. The experiment was,
therefore, terminated after 1.5 h of kidney perfusion.
Immunologically, the experiment was sound with no hyperacute rejection
and no histological abnormalities or evidence of fibrin deposition. Although
immunosuppressive therapy was discontinued, no excessive humoral respon-
ses were observed during the 6 months monitoring period after the experi-
ment. The IgM response never returned to normal levels, but the IgG re-
sponse was measurable by cytotoxicity (at neat) whereas originally it had been
detectable only by flow cytometry.
We were concerned that the pig kidney might induce responses that inter-
fered with the HLA antibody removal therapy. This proved totally unfound-
ed, and the patient was able to undergo transplantation with a human cadaver
kidney.
Although intended to be the first of five such experiments, no further work
has been carried out. This is because worldwide press coverage led to several
threats being made by animal rights campaigners against the hospital and the
members of the medical team. Members of our group have, however, de-
scribed these experiments at scientific meetings because we believe they con-
tribute towards achieving the goal of successful xenotransplantation.
In scientific terms we have shown that of the eight potential problems of
xenotransplantation defined at the commencement of our study, problems 1,
2,3, and 4 are soluble. For example, each human does not have antibodies to a
wide range of pig determinants, and our volunteer did not produce massive
secondary responses after perfusion of the first kidney. Furthermore, when
the second kidney was attached, additional antibodies were neither present at
the time nor formed later, suggesting that the T helper response was well con-
trolled by the standard immunosuppressive regimen used.
We have stored in liquid nitrogen T cells covering the period of the experi-
ment and the following 6 months, but we have not yet investigated problem 5.
Nor do we have any information regarding problems 7 or 8. Unfortunately,
our adsorption of Ig might well have depleted complement components and so
we can make no statement on problem 6. However, we did observe variations
between individuals in their ability to lyse pig lymphocytes in the presence of

their own complement. We suspect. therefore, that problem 6 may need a con-
siderable amount of further investigation.
References
I. Chardon. P .. Vaiman. M .. Renard. c.. Arnaux. B. Pig histocompatibility antigens and B2

microglobulin. Transplantation. 26, 107,1978.
2. Lunney, 1.K., Sachs, D.H. Transplantation in miniature swine II. In vitro parameters of
histocompatibility in MLSA homozygous minipigs. Transplant Proc. 23.271, 1979.
3. Pennington. L.R.. Lunney. 1.K .. Sachs, D.H. Transplantation in miniature swine VII:
recombination within the major histocompatibility complex of miniature swine.
4. Chardon, P., Vaiman, M., Arnaux, B. Characterization of class II major histocompatibili-
ty antigens in pigs. Anim. Blood Groups Biochem. Genet. 12,59,1981.
5. Hruban, V., Hradecky, 1.. Pazdera, M., Simon, L.. Veselsky. 1. SLB - a new alloantigenic
system of the pig.J.lmmunogenet. 5.173.1978.
6. Hruban, V .. Hradecky. 1., Veselsky. 1. Non-MHC allo-antigenic system in pigs (SLC) de-
tected by leuco-agglutination. Anim. Blood Groups Biochem. Genet. 14.299. 1983.
7. Leight, G.S., Sachs, D.H., Rosenverg, S.A. Transplantation in miniature swine III. Effects
of MSLA and A-O blood group matching on skin allograft survival. Tissue Antigens. 12.
65.1978.
8. McKenzie, I.F.C.. Stocker. 1., Ting. A., Morris, P.l. Human lymphocytotoxic and hemag-
glutinating activity against sheep and pig cells. Lancet. 2,386, 1968.
9. Palmer, A., Taube. D., Welsh. K., Bewick, M., Gjorstrup, P., Thick, M. Removal of anti-
Lancet. 1.10.1989.
Chapter 32
An Ethical Framework for Considering

The Development of Xenotransplantation in Man
R.A. WRIGHT
Introduction
As we look to the future, re-examination and furthcr consideration of the eth-

ical issues inherent in the development and utilization of xenotransplantation
is of great importance. This chapter will address those ethical issues in two
ways: first, by general indication of the areas in which broad ethical concerns
arise; and second, by raising some very specific questions which need to be ad-
dressed during the process of the scientific development of xenograft technol-
ogy, before that technology is applied.
Since the transplantation of Baby Fae by the Lorna Linda group in 1985,
there have been no further whole organ xenografts done; however, the prob-
lem has not been set to rest. Although the initial concern with the Baby Fae
case focused on the primary ethical issues of consent, subsequent discussion
has focused on concern about the use of animals in medical experimentation
[1-8]. The ethical point in such transplantation programs is, however, far more
complex than a simple question of consent or of animal utilization. Rather, it
covers the entire spectrum of ethical concerns with health care in general and
organ transplantation in particular. All of that is then compounded by the fact
that clinical xenograft procedures have been and, for some time in the future,
will continue to be experimental therapy. Insofar as xenografts are used for
children or those debilitated by terminal illness, its experimental nature raises
additional questions of ethics [9, 10].
Rather than rehearse the litany of various key articles [7, 11-19], this chap-
ter will try to give an overview of the issues from a different perspective, one
that might allow us to understand the problem in more detail and gain insight
into how these issues may be resolved. This in turn will allow us to frame our
thinking as we look toward the future development of this aspect of medicine.
Ethical Framework
An examination of the history of ethics shows that, regardless of their specific

beliefs on individual topics, people generally account for three important
components of ethical decision-making [20]: (i) determining possible courses
512 An Ethical Framework for Considering The Development
of action and assessing the moral worth of each action in terms of its potential
production of harm and/or benefit; (i,i) recognizing that humans as moral
agents have duties to one another, and assessing possible actions according to
how they exemplify the fulfillment ofthose duties; and (iii) recognizing that in-
dividuals have rights which affect their interactions with others, especially in-
sofar as one person's right imposes duties on other persons, and assessing how
possible actions affect those rights.
Our understanding of the ethical dimensions of xenotransplantation re-
quires recognizing how various concerns raised by that process fit within these
categories of consideration: (i) consequences, (ii) duties, and (iii) rights.
Conseqnences
One of the primary arguments in favor of pursuing xenotransplantation is that

there are insufficient transplantable organs available to save the lives of those
persons whose lives could otherwise be saved. This has been highlighted par-
ticularly by the increasing need for transplantation in children with congenital
anomalies and dysfunctions. The urgency for these considerations stems in
large measure to one single consequence - these children will die without a
transplant.
There is considerable dispute about the actual number of organs available
and how that problem can be solved [11,21-26]. Nonetheless, it is a fact that
there are simply not enough transplantable organs to meet the growing need.
This may be particularly true with infants and children, given the relatively
lower number of traumatic deaths in these age groups and the possible propor-
tionate increased reluctance to donate organs of infants, given the psychologi-
cal trauma caused the parents by the death of their child. To prevent the deaths
of those needing transplant (a harmful consequence), the argument goes, we
must look to xenotransplantation as the only possible means for obtaining the
organs to save lives (a good consequence).
More important is the assumption that medicine must do all that it can to
produce a very specific outcome (consequence), namely, the preservation of
life. Xenotransplantation is in that respect no different than any other medical
procedure; it "is only a departure in degree, not in kind, from the assumptions
and techniques that drive medicine in general" [18]. The question, of course, is
whether or not the medical imperative requires that the prolongation of life
can be accomplished irrespective of costs (where costs are broadly construed
as social, economic, and moral) [27-29].
If we assume that the moral imperative of medicine is to make every effort
to sustain life, and if we accept as the fundamental justifying element of
medicine the principal of beneficence (to produce the maximum of good) [30],
it is still not clear how far we should proceed with xenograft programs. The
reason is that the consequences of our action must take into account more than
just the specific results to the individual affected, i.e., the particular life saved
by the procedure [11].
Ethical Framework 513
What this means is that our actions have a wider scope than the simple
physiological effect on the individual who receives the transplant. For exam-
ple, the family and support system of that individual is significantly affected
[31]. In the case of high-tech medical applications, the professionals who uti-
lize the technology have devoted their lives and intellectual resources to the
advancement of this activity. For the social environment in which the trans-
plant occurs, there are the monetary costs for the procedure. In addition are
the physical and emotional costs to those who may be deprived of their health
care needs because the resources necessary to care for them have been divert-
ed to the xenograft project. There are also psychosocial consequences to the
survivors of the transplant, who may see themselves differently or may be
somehow discriminated against because of their unique transplant. Finally,
there is the consequence of utilizing animal resources amid concerns over ani-
mal rights and animal experimentation which are becoming increasingly prob-
lematic for the advancement of medical science.
Medicine has progressed tremendously since several clinical xenografts
were attempted in the early 1960s. The advancement, however, has been par-
alleled by a greatly increased public attention to the activities of the medical
community. As a result, scrutiny of the medical community by outsiders has
made necessary considerations of far greater complexity than might be other-
wise required. Taken together, the spectrum of consequences outlined is far
broader than that usually imagined to affect any specific medical treatment, or
the process of treatment development. The problems of xenotransplantation
are significant enough, however, that we must recognize each item in this spec-
trum of consequences as needing consideration before deciding whether to
utilize the procedure and, if so, to what extent.
Duties
Since the beginning of medicine's recorded history, it has been clear that the
physician is unlike his or her social contemporaries because the profession of
medicine imposes upon the practitioner a set of duties which is found in no
other area of human endeavor [32, 33]. Although these duties have been set
out across the years in many different ways and in many different forms, the
duties may be seen to revolve around four basic moral responsibilities: (i)
beneficence; (ii) nonmaleficence, (iii) autonomy, and (iv) justice [34,35]. The
physician has a duty to exercise beneficence (promote the good of the patient)
while also adhering to the principle of nonmaleficence (preventing or doing as
little harm as possible), and recognizing the autonomy (right to self-determi-
nation) of the patient in a system which adheres as closely as possible to the
equality, fairness, and openness required by justice.
The primary manifestation of this set of physicians' duties in xenotrans-
plantation is the overarching concern to provide a means of preserving pa-
tients' lives. The preservation of life is seen as a duty of beneficence, because
life itself is taken as a good; as nonmaleficence because unnecessary death is
understood as a harm which must be avoided; as upholding the patients' au-

tonomy, because autonomy presupposes life itself; and, as an exercise in jus-
tice insofar as people have equal and fair access to the procedure if they need
it. Xenotransplantation, as any other high technology life-sustaining care, can
thus be understood to be a moral duty insofar as it serves to preserve life.
A problem arises, however, if this duty to preserve life is not an absolute
duty; for then conflicting duties can easily arise, wherein one treatment
modality might have to be discontinued in order to facilitate utilization of an-
other. At the economic level, for example, justice requires fairness among
persons. Yet if the funds necessary to underwrite xenotransplantation pro-
jects significantly deplete the funds available for other projects, it could be ar-
gued that the physician's duty to xenotransplants is less clear. This raises as
well the question of whether or not the physician's duty is to preserve life it-
self, or preserve that life which has the best potential quality and longest term
net value. A question can then be raised whether the physician's duty to pur-
sue xenograft development is, in fact, outweighed by the duty to supply other
medical care which enhances the length and quality of many more individual
lives [15, 36, 37].
Put differently, medicine is usually understood as having duties to specific
individuals on the basis of the physician-patient relationship. Consideration
must also be given, however, to the question of whether or not physicians have
responsibility to those who are not their individual patients, i.e. society as a
whole. In this regard important ethical questions must be asked of those pur-
suing xenograft development - what specific duties does a physician have, and
do those duties in fact justify proceeding with xenografts in an environment
where other medical care must be forgone?
At the same time, since society is the source of support for medical activi-
ties, there are questions of duty which also arise for society as a whole. These
are analogous to the questions raised for physicians, but broader in the sense
that a physician's concern is clearly and unequivocally his or her patient, while
society's concern is clearly and unequivocally the well-being of society as a
whole. Insofar as the well-being of an individual is in conflict with the well-
being of society as a whole, the social question becomes - what duties does
society have and to whom? This is especially important in our current climate
of medicine wherein crisis care and rescue seem to be our primary activities
[38-40]). For surely society might legitimately ask whether or not its duty in
these circumstances is to continue to rescue individuals who have question-
able survival potential, and about whom nothing is known for their potential
lives even if they do survive. The social question of duty then becomes whether
or not society has a duty to prevent or prohibit medical activities which are
deemed detrimental to the perceived social welfare.
The problem of individual versus social good becomes acute when we rec-
ognize that the welfare of society is linked to the well-being of its individual cit-
izens, but is not at the same time a simple conjunction of that well-being. For
while it is true that a society is well when each of its members is well, it does not
follow that the means to social well-being is the health of any particular citizen.
Ethical Framework 515
Consequently, a social obligation to overall wellness could entail an obligation

to deny care to some portion of society on behalf of the remainder.
On the other hand, if the social obligation is understood to necessitate at-
tention to the well-being of each member, then there is entailed a necessity to
fund and support the activities required. The decision process thus involves, at
some point, a need to force the issue of social duties. Otherwise, we might pro-
ceed on one understanding only to have the social viewpoint force the other.
It should be noted that this social duty, in conjunction with the medical
duty, is not relevant solely to the question of xenotransplantation, but cuts
across the entire health care spectrum and should be considered for any appli-
cation of health care technology.
Rights
The final concern at this level is the notion that individuals have rights. The
problem, of course, is the extent to which the rights of an individual control the
situations in which that individual might be found [41--43]. In addition, there is
the significant question of how to handle competing rights. For example, if we
accept the position that an individual has a right to life, which entails a correla-
tive right to access any and all health care technology which might sustain that
life, we must ask how we then accommodate other compelling rights. One pos-
sible approach is to decide that the only relevant right is the right to life and
that this right must be protected first and above all else, with all other rights
being secondary. For the sake of argument, we could accept this position, how-
ever, and still ask whether or not an individual has a right to a specific resource,
and, if so, on what basis. This issue becomes particularly crucial when that re-
source is available primarily (or exclusively) at public expense.
Our society clearly has not reached consensus on this absolute notion of the
right to life, as witnessed by ongoing discussion and court cases involving re-
fusal of life-sustaining medical treatment. Moreover, we socially condone
abortion, we are ambivalent to child abuse which frequently ends in death, we
are increasingly vocal in our support for the freedom of individuals to end their
lives as they see fit, and we increasingly exercise the social prerogative of capi-
tal punishment. Even within the medical community there is a growing con-
sensus that the absolute preservation of life itself is a vitalistic endeavor which
is not appropriate to health care. Rather, the emerging consensus is that an im-
portant difference exists between sustaining life and prolonging dying, and
medicine should be involved in the former but not in the latter [44--49].
The issue this raises for xenotransplantation is multiply complex. First, do
individuals have some sort of fundamental right according to which they legit-
imately should have access to xenotransplantation and, accordingly, research
and development of the science is appropriate? Or is it legitimate to disconti-
nue certain approaches in medicine in order to focus on others, thus perhaps
discontinuing all xenotransplantation? Second, if there is a right, is it such that
there is a social duty generated according to which xenografts and other life-
sustaining treatment must continue to be developed? Third, if there is an indi-

vidual right, is it absolute or contingent upon, for example, the ability to pay,
or a state's determination of suitability (as occurred recently in Oregon in the
USA).
Animal Utilization
To understand more fully the concern with consequences, duties, and rights, it
is instructive to briefly consider the controversy surrounding the use of ani-
mals in medicine. Moreover, given the current climate of protests and violence
against those utilizing animals, a better understanding of the issues which arise
from these three concerns is of paramount importance to xenograft develop-
ment.
Initially, the current debate over whether or not animals have rights and, if
so, whether their rights are sufficient to preclude their use in medical work,
makes it appear as if the sole question to be resolved is a determination of
rights. That is a gross oversimplification, however, because in addition to con-
cerns with consequences and duties, there are concerns with the metaphysical
presuppositions that raise the question in the first place. For example, there is
a significant body of literature arguing that there is no metaphysical difference
between humans and animals [50-52]; that the continuum of life is such that to
draw lines between species is arbitrary and capricious [53]. On this basis, to
treat animals differently from humans is "speciesism", which is just as bad
morally as racism or sexism [19, 54]. Accordingly, if a human has a right not to
be killed solely to save the life of another human, an animal has the same right,
especially given its vulnerability [10, 18].
It might be argued that animal rights are not an issue, either because ani-
mals do not have such rights, or because whatever rights they might have are
clearly overridden by the rights of humans. Notice, however, that both of these
responses presuppose resolving numerous questions about animals, the na-
ture of their existence, and the relationship which is morally appropriate be-
tween humans and animals because of that nature.
However, even if animals do not have a wide range of rights, or any rights
at all, there is still a question about what duties humans have toward animals
[54]. After all, we do punish as criminals people who mistreat animals even
where that mistreatment does not entail death. Yet the very process of
xenografting is known ahead of time to necessitate the death of the animal; as
a result, we must also question whether we have in fact exhausted all other
alternatives to the use of animals [19, 55]. These notions must thus be re-
conciled and understood if we are to be able to make sense of our claim to
both a human right to treatment which preserves life, and a human right to
utilize animals in such treatment, when that utilization intentionally results in
death.
Regardless of the question of rights and duties, there are also questions of
consequences. For example, the "trade-off' of an animal life for a human life
Factors for Consideration 517
has consequences for the animal and the human [17]. For the animal, it means
an end to its existence, an existence which, at least to the animal, must be un-
derstood to have some level of significance. In short, as Frey has noted, "until
we decide the comparative value of animal and human life, the majority of
these practices will never be finally resolved" [55].
There is also broader significance, as in the potential for eliminating specif-
ic primate species in order to satisfy the human demand for organs (as, for ex-
ample, with the current shortage of baboons and chimpanzees) [54]. Beyond
that, the economic consequences become important because intraspecies in-
compatibility and immunology problems will require enormous resources to
resolve.
The debate between so-called animal rights proponents and those in the
medical community is most clearly seen, however, as a disagreement over
whether consequences or rights are most important. For on the one side, the
most cogent argument for using xenografts, despite the sacrifice of the animal,
is that the consequence to humans of not using such organs, namely death, is so
much worse than the same consequence to the animal; despite any notion of
animal rights, the duty to preserve human life justifies the xenograft. On the
other side, the most cogent argument for not using xenografts is that there is no
justification for the human claim to an absolute right to life, taking as evidence
the fact that we simply do not accept such a claim in any other circumstance.
Since the right to life claim is the basis for justifying any nonhuman sacrifice,
xenografting is not morally acceptable because is inappropriately elevates hu-
man existence to an unjustified level within the entirely of the natural order,
solely for the selfish purpose of preserving human life.
Finally, there is the concern with human interference in the "natural" order
of things. If we do, in fact, have legitimate duties toward nature and the envi-
ronment, then our responsibilities to animals, especially those with the higher
levels of intelligence, must be taken seriously. Xenografting, which requires
the death of an animal, it is argued, is thus a violation of our duty to the protec-
tion and "stewardship" we have over the environment. This particular con-
cern is especially troublesome when the xenotransplant is necessitated by the
human's self-destructive behavior.
Factors for Consideration
Having raised these issues concerning consequences, duties, and rights as

components of decision-making in xenotransplantation, it is important to sug-
gest a direction for our thinking about ethics as we determine the ultimate uti-
lization of this technology. However, instead of focusing on a particular ideol-
ogy or narrow ethical viewpoint, e.g. upholding animal rights, or never doing
transplants, it seems more productive to think about the specific areas of con-
cern which must be reviewed in our decision-making process. To this end we
will utilize the decision factor construct described elsewhere [20, 56]. This will
involve recognizing and dealing with the five factors of decision-making: (i)
data, (ii) values, (iii) action constraints, (iv) decision theory, and (v] ethical
framework.
Data
Although we tend to focus on science, and the data that it gives to us, it is im-
portant to recognize that in resolving xenotransplantation issues there is a
wider range of data which are important. These data include, in addition to
the scientific information, information about the types of individuals, institu-
tions, and systems within which xenotransplantation will occur. For example,
we need more information on family systems theory in an effort to develop a
better understanding of the family support systems necessary for success.
This will of necessity require information about the psychosocial dimensions
of a person's life and the impact of the outcome of the transplantation proce-
dure. Although at this point we largely attempt to use only scientific criteria
for determining acceptability for transplants, given the problems peculiar to
xenotransplantation, a much better understanding of the psychosocial di-
mensions of transplantation itself, as well as the use of xenografts, will be cru-
cial.
It will be important to develop an understanding of the social systems with-
in which the transplant recipient must function, so that appropriate support
services will be available. This is particularly important with regard to this
form of therapy for infants and children. These patients will have a longer life,
with the longest potential time span both for life improvement and for prob-
lems to arise. Knowledge and development of the appropriate support sys-
tems may thus be more crucial to the success of the transplantation than the
medical science itself.
Finally it is necessary to continue our work to understand the reasons for
the low rate of human organ donation so that education and encouragement
may be appropriately applied. This is a fundamental problem because allo-
grafting seems more obviously appropriate than xenografting if for no other
reason than the genetic similarities. As a result, one crucial component of our
data development as we proceed with xenotransplantation must be whether or
not we are justified in using animals as a resource for humans without a greater
effort to obtain human donors as a resource for humans [55]- the sequelae of
the Baby Fae case amplified this concern [4,5, 19,55-59]. The importance of
these data lies in the fact that while the so-called animal rights movement can-
not be totally eliminated, it can be diffused by careful, thoughtful, well-round-
ed justification for the use of animals. We thus must develop much more fully
data on the availability of allograft materials and much greater sophistication
in our utilization of those materials. Further, by continuing to focus our efforts
on developing educational approaches which will improve understanding and
lead to a higher rate of available allograft materials, the addition of xenografts
will then greatly enhance the overall transplantation program.
It is not necessary that each of these additional data components, or related

studies to which they might lead, be completed before we proceed with any
further xenotransplantation work. However, these areas should be explored
in conjunction with the ongoing scientific development so that there will be
some congruence of the data at the point when a decision is made concerning
the ultimate utilization of xenotransplantation.
Values
All of medicine has an inherent reliance upon both explicit and implicit values.
Each individual who works in the field brings to his or her work that set of per-
sonal values upon which he or she operates. The professions and institutions of
which they are a part also impose a set of values on both the individuals and the
health care system. It is crucial to understand these values and how they func-
tion in the xenotransplantation situation because otherwise a major compo-
nent of success (or failure) will be overlooked. Equally important, it must be
recognized that the transplant recipient similarly functions from basic sets of
values as does the society in which all those involved must interact. Perhaps
one of the most serious mistakes that could be made in our continuing pursuit
of xenotransplantation development is to ignore, or fail to recognize, the ef-
fect of values and value judgments in all aspects of the decision process
[60-64].
While recent work in transplantation has begun to focus on these issues
[65], there has yet to be a systematic evaluation of two value factors; namely,
(i) what are the values which have a pivotal influence on decision making
about an application of transplant technology, and (ii) how may we effectively
deal with differences of value, especially as those differences affect decision-
making regarding transplantation. Admittedly, the value questions are ex-
tremely sensitive for those involved in transplantation, particularly in light of
the extreme criticism leveled at early attempts to categorize patient suitability
to receive transplants. The current ranking systems used by UNOS have
evolved out of our intent to avoid at all costs a subjective imposition of person-
al values in determining human worth, and subsequent determination of
transplant suitability.
At the same time, we must recognize that this is not entirely an honest en-
deavor because it is simply impossible to separate human values and notions
of worth from individual decision-making. Even the "scientific" criteria are
based upon value judgments as to the significance of specific factors.
Moreover, since the scientific criteria are not absolute, there is lots of room for
value imposition in the decision process. What is crucial for further study in
support of xenotransplantation is a re-examination of value appropriateness
in the decision process. This would be accomplished in conjunction with com-
ponents of decision theory to be discussed below, but would focus heavily on
the early rejected notions of social worth, quality of life, and perhaps of most
importance, the values of the individual receiving care. It would also recognize
the proper role of value judgments in determining application criteria for

xenotransplantation.
The approach to these value issues should be twofold. First, we must sepa-
rate out the values which lead to self-destructive behaviors that result in the
need for transplantation. This is crucial because of the growing concern that
the tremendous expenditures of technology and finance should not be made
for those whose need results from their direct self-abuse, e.g., the alcoholic cir-
rhosis of the liver necessitating transplantation, or the smoking-induced em-
physema necessitating a lung transplant. Second, we must delineate appropri-
ate sets of social, economic, religious. legal. and moral values which can in
some sense of consensus be seen as appropriate consideration in this process.
Although this value inquiry is clearly sensitive and certainly problematic,
our failure to undertake its examination means not that we are being value
neutral (since that is impossible); rather, it means that we are utilizing values in
our decision process which are not openly recognized as playing that role, thus
creating in our decision process, if not a hidden agenda, certainly a hidden set
of presuppositions.
Action Constraints
Action constraints - those elements of our practical life which seem either to
prevent us from acting as we wish, or force us to act in specific ways - may be
theoretically unimportant, but are a practical necessity. In fact, it makes no
sense to consider the practical application of anything wherein the action con-
straints that may be imposed on us are not taken into account. There are two
general directions for this consideration of action constraints in xenotrans-
plantation.
First. we must look at the question of whether or not we should even perfect
the technology and science necessary to carry out xenotransplantation, by
looking at whether the social system will permit us to do so. Second, we must
consider whether, even with social approval, there might be reasons, e.g., eco-
nomic, resource allocation, potential disruption from animal activists, etc.,
which are sufficient to negate whatever progress we might make in develop-
ment. These two major considerations are important because negative results
in either might be a sufficient justification for not proceeding with the scientif-
ic development in the first place. Most important to this consideration is the
notion that it makes no sense to spend years developing something which is in-
tended for practical application that will never be realized.
In addition to those two major considerations, there is the value-related ac-
tion constraint problem of determining how, if at all, the use ofxenotransplan-
tation might have both positive and negative effects on those who receive such
transplants. This consideration is important because throughout the history of
transplantation we have generally focused on the good. i.e .. the positive re-
sponses, and tried to ignore or displace the negative. While this makes sense in
terms of psychologically justifying our pursuits and endeavors, it does not make
sense at the level of practical application. For while we might want to paint the
rosiest picture possible, our ethical responsibility of beneficence would pre-
clude proceeding where the objective results are overwhelmingly negative.
Throughout these considerations it must be recognized that the potential
constraints of action may come at numerous levels. For example, a general so-
cial consensus allowing xenotransplantation may nonetheless be locally disap-
proved, so that reason would dictate not utilizing the technology. Veatch for
instance has noted religious objections to the use ofxenografts [14]. Locations
such as the "bible belt" of the United States, despite a general social consensus
at the national or international level, might thus be inappropriate sites for
xenotransplantation because of those objections. Normally such objections
might be thought unreasonable by those who do not share the basic viewpoint;
however, the greater the percentage of society that shares a viewpoint, the
greater the proportion of risk in contravening the viewpoint. This is particular-
ly the case where public support of the institution is crucial to the continued
existence of that institution. The offering of xenotransplantation in such cir-
cumstances has little to do with the technology itself but is constrained by the
social and political environment in which the technology is to be offered.
In a similar vein, if opposition to the utilization of animals is more prevalent
in one location than in another, significant social objection to the utilization of
animals might, in the same way as religious objections, constrain the offering
of xenotransplantation in that location. The point, of course, is that we need to
develop our understanding of these potential constraints as we correlatively
develop our understanding of xenotransplantation itself.
The problem with considering such action constraints is that persons who
could benefit from xenotransplantation might for these reasons be precluded
from access. While the principle of justice, and the principle of autonomy, in
their abstract forms make this objection theoretically irrelevant, the practical
fact is that it is not irrelevant. A major consideration then for understanding
action constraints in xenotransplantation is to understand the legitimate depth
to which these constraints must affect the overall project.
Decision Theory
Decision theory - commonly understood in medicine as clinical decision anal-

yses - has a quantitative appearance [56,66,67]. While that is important for
our considerations here, there are a number of dimensions of decision-making
as a process that must also be considered. Put differently, the decision theory
component of developing xenotransplantation is not simply the perfection of
quantified utility. Rather, it entails in addition an understanding of several key
components in decision-making. For example, as with all other areas of
medicine, there is a serious question about who is or should be the appropriate
decision maker.
Since some significant applications of xenotransplantation would be to
children, the problem of decision-making is compounded because of the po-
tential disagreements between parents and physicians over what is in the med-
ical best interests of the child. Thus. for example. if Baby Fae's parents had ob-
jected to the transplant on the grounds of religious beliefs concerning the in-
termingling of species. could we envision the decision situation in which the
appropriate avenue of resolution would be a guardianship court order? Since
we regularly do that in other situations to overcome parental objection based
on religious beliefs (e.g .. Jehovah's Witness or Christian Science families). it
makes sense to foresee this as a significant decision problem for xenotrans-
plantation as well.
Equally important. and related to the data, values. and action constraint
components already discussed. is the theoretical question about the criteria
and process for the decision itself. We must examine in detail the relationship
between decisions based on empirical information. decisions based on values.
and decisions which combine the two. We must also better understand the re-
lationships between other persons in the decision process and their effective
impact on each other. For example. in most medical situations the physician is
the primary decision maker and the patient. or patient's surrogate. is a collab-
orating decision maker. The key question is at what point the transition occurs
from physician to patient. or patient surrogate. in the "go-no go" determina-
tion. Accordingly, at what level do the physician's values and data become sec-
ondary to the data and values of others in the decision process?
Finally. we must give considerable attention to the decision content. One of
the major criticisms of the consent process in the Baby Fae case was that the
parents may have been put in situations where they were overwhelmed by in-
formation, or in which they felt pressured to reach a certain conclusion [1, 3,
68, 69J. Insofar as these elements were present, legitimate questions were
raised about the validity of the consent process. Part ofthe problem is to devel-
op a decision process which incorporates a context for the decision that miti-
gates as much as possible these contravening interests. Since the utilization of
xenotransplantation would only rarely be at a point of immediate crisis. there
would be sufficient lead time to discuss and deal with these decision problems
and develop a consent process independent of pressure-filled adversarial or
confrontational interaction. In this sense, anticipatory decision-making bc-
comes a key component of the overall decision process.
Ethical Framework
The ethical framework of a decision is directly related to the considerations of

consequences, duties, and rights discussed earlier. We note historically that
few decisions are such that the three can be equally weighed. As a result, deci-
sion makers focus on one of the three as the primary consideration in decision
making [20, 56J and conflict resolution [70,71]. As we look at different deci-
sions reflectively. we see that such a focus may mean not only consideration of
different decision factors. but may also frame the decision in such a way that
radically different outcomes may be possible. The crucial factor for develop-
ment of xenotransplantation is going to be a determination of whether the po-
tential consequences, emphasizing beneficence and nonmaleficence, should
be the primary concern or whether the rights and duties considerations should
take precedence.
The consequences analysis would frame the problem in such a way that
xenotransplantation could be precluded were we unable to show its benefit in
terms of life-years, or that the cost ratio for that benefit was within some
agreed bounds of acceptability. This framework initially might seem to be the
most attractive, but runs two significant risks. First, there is the risk that the
life-years adjusted benefit will, in fact, not turn out to be what we had hoped,
making the procedure inappropriate; or second, that the cost analysis might be
other than what we would wish. For example, the cost ratio might be much
higher than anticipated (as with the end-stage renal disease program). In ei-
ther case, the analysis will hinge upon three factors: (i) what criteria are being
used to weigh and balance the life-adjusted benefit to the risks and costs, (ii)
what scheme of decision-making is being used to accomplish those balances,
and (iii) what ratio of cost and benefit is the life of acceptability beyond which
we should not go.
On the other hand, the rights and duties framework might initially seem
more appropriate because an emphasis on autonomy and justice would weigh
the decision in favor of preserving life, thus reinforcing the physician's duty to
pursue xenotransplantation regardless of the cost/benefit analysis. As for the
consequence base, however, there are potential problems here as well. To be-
gin, the life-year adjusted benefit and cost analysis might produce duties which
conflict with a basic duty to preserve life, or a duty to uphold and respect pa-
tient autonomy. For example, why should we expend such tremendous re-
sources to save the lives of a few, especially when their quality of life may be
permanently diminished. Or, why should we claim as a duty the application of
this high technology at a great expense when low technology-low expense
needs, such as prenatal care, well-baby care, universal immunization, and
good nutrition, are not supplied. In this regard the rights and duties frame-
work requires argument for the basic rights and/or duties which are alleged to
take precedence in that system.
This is not intended to argue that we are hopelessly mired in a philosophical
dispute which has no obvious outcome. Rather it is to suggest that part of the
determination for proceeding with xenografting necessitates open discussion
of these problems and a reaching of consensus on which of the two general ap-
proaches we will take, and how we will understand that approach once it is tak-
en. For it is absolutely clear that we cannot continue to pursue this project
meaningfully in the absence of such consensus, given that there is inherent
conflict between the two positions which cannot otherwise be resolved.
Comment
It has been the attempt of this chapter to raise a number of important ethical
concerns which are inherent to the overall development of xenotransplanta-
tion, as well as its utilization in the medical community. The attempt has not
been to present a specific ethical viewpoint, but to present both a range of is-
sues and a framework for approaching them.
Roger Evans has pointed out quite properly that there are concerns about
the role of ethics in these decision considerations and the role of specific ethi-
cal expertise in their outcome [72]. I do not share his pessimism about the role
of ethics, nor do I share his pessimism about the possibility of achieving con-
sensus among ethicists or physicians regarding the concerns raised in this pa-
per. Rather, the attempt has been to put forward a set of considerations which
are intended to show how crucial it is to understand the importance of the eth-
ical debate. It is also intended to show that the importance of the ethical de-
bate lies much more in its process than in its participants' "expertise". In this
regard then the resolution of the ethical issues in xenotransplantation, as with
the resolution of the technical issues, may not be properly seen as a peripheral
exercise in cocktail party futility, but rather as an integral component of the
overall project.
Medical ethics is not limited as Evans would have us believe, but is, in fact,
one of the key components in conflict resolution as well as development of our
overall framework for understanding and utilizing any specific medical tech-
nology. The entire community of those participating - not just physicians, or
just ethicists, or just lawmakers, or just judges, but society as a whole - has a re-
sponsibility in assuring that outcome which results from open honest delibera-
tion of all relevant concerns, not just the scientific.
References
1. Knoll, E., Lundberg, G.D. Informed consent and Baby Fae. lAMA. 254,3359,1985.
2. Levine, C. NIH approves the consent for Baby Fae. or does it? Hastings Center Report.
15,1985.
3. Capron, A.M., Regan, T., Reemtsma, K., Sheldon, R., McCormick, R.A., Gore, A. The
subject is Baby Fae. Hastings Center Report. 15,8-13,1985.
4. Breo, D.L. Is "Baby Fae" transplant worth it? Am. Med. News. 27,1,41,1984.
5. Preston, T.A. Baby Fae: the ethics of medical adventurism. Washington Post. Nov. 14,
07,1984.
6. Provonsha, J.W. Ethics of experimental surgery: the Baby Fae case - an interview and
comment [interview by Jack E. Bynum]. Pharos of Alpha Omega Alpha Honor Medical
Society. 49,23,1986.
7. Annas, G.J. Baby Fae: the "anything goes" school of human experimentation. Hastings
Center Report. 15,15,1985.
8. Katz, D.M. Baby Fae and the ethics of benefits. National Underwriter. 88,2,1984.
9. Murray, T.H., Caplan. A.L. Which Babies Shall Live? Humanistic Dimensions of the
Care of Imperiled Newborns. Clifton, NJ: Humana Press. 1985.
References 525
10. Caplan, AL. Ethical issues raised by research involving xenografts. JAMA. 254,3339,
1985.
11. Drugan, A, Evans, W.J., Evans, M.l. Fetal organ and xenograft transplantation. Am. J.
Obstet. Gynecol. 160,289,1989.
12. Francione, G.L. Xenografts and animal rights. Transplant. Proc. 22, 1044, 1990.
13. Reemtsma, K. Ethical aspects of xenotransplantation. Transplant. Proc. 22, 1042,
1990.
14. Veatch, RM. The ethics ofxenografts. Transplant. Proc. 18,93, 1986.
15. Leeder, S.R. All for one or one for all? The ethics of resource allocation for health care.
Med. J. Australia. 147,68,1987.
16. Tucker, W.R Xenografts and scientific evaluation. IRB: A Review of Human Subjects
Research. 8, 10, 1986.
17. Werner, R The rights and wrongs of animal use for human organ transplant. In:
Biomedical Ethics Reviews 1987. Humber, J.M. and Almeder, RF (eds.), Clifton, NJ:
Humana Press, 1988, p. 65.
18. Nelson, J.L. Xenograft: ethics and the use of animals as organ donors. Rio Law. 2, S, 71,
1987.
19. Kushner, T., Belliotti, R Baby Fae: a beastly business. J. Med. Ethics. 11,178,1985.
20. Wright, RA Human Values in Health Care: the Practice of Ethics. New York: McGraw-
Hill,1987.
21. Robertson, J.A Supply and distribution of hearts for transplantation: legal, ethical, and
policy issues. Circulation. 75,77,1987.
22. Fletcher, J.e., Robertson, J.A., Harrison, M.R. Primates and anencephalics as sources
for pediatric organ transplants. Medical, legal and ethical issues. [Review]. Fetal
Therapy. 1,150,1986.
23. Caplan, AL. Equity in the selection of recipients for cardiac transplants. Hastings Center
Report. 75, 1, 10, 1987.
24. Caplan. AL. Ethical and policy issues in the procurement of cadaver organs for trans-
plantation. N. Eng/. J. Med. 311,981,1984.
25. Caplan. AL. Requests, gifts, and obligations: the ethics of organ procurement.
26. Caplan. AL. Obtaining and allocating organs for transplantation. In: Human Organ
Transplantation: Societal, Medical-Legal, Regulatory and Reimbursement Issues.
Cowan, D.H., Kantorowitz, J.A, Moskowitz, J. and Rheinstein, P.H. (eds.) Ann Arbor:
Health Adminstration Press, 1987, p. 5.
27. Churchill, L.R Rationing Health Care in America: Perceptions and Principles ofJustice.
Notre Dame, Ind.: University of Notre Dame Press, 1987.
28. Callahan, D. Setting Limits: Medical Goals in an Aging Society. New York: Simon and
Schuster, 1987.
29. Callahan, D. Rationing health care: will it be necessary? Can it be done without age or
disability discrimination? Issues in Law and Medicine. 5,353, 1989.
30. Pelligrino, E.D., Thomasma, D.e. For the patient's good: the restoration of beneficence
in health care. In: For the Patient's Good: The Restoration of Beneficence in Health Care.
E.D. Pelligrino (ed.) New York: Oxford University Press, 1988, p. 3.
31. Wright, e. Concerns of the family. Nursing Times. 1,53,1987.
32. Graber, G.e. Physician-patient relationships. In: Ethical Analysis of Clinical Medicine.
Graber, G.e., Beasley, A.D. and Eaddy, J.A (eds.) Baltimore: Urban and
Schwarzenberg, 1985, p.1.
33. Bayles, M.D. Professional Ethics. Bellmont, CA: Wadsworth, 1989.
34. Beauchamp, T.L., Childress, J.F. Principles of Biomedical Ethics. New York: Oxford
University Press, 1989.
35. Harron, F., Burnside, J., Beauchamp, T. Health and Human Values; a Guide to Making
Your Own Decisions. New Haven: Yale University Press, 1983.
36. Reimer, L.G. The power of the individual's story. Second Opinion: Health, Faith and
Ethics. 12,40,1989.
37. Lee, J.M. Ethics and resource allocation.J. Family Practice. 28,140,1989.
3il. Jonsen. A.R. Ethical issues in organ transplantation. In: Medical Ethics. Veath. R.M.
(cd.) Boston: Jones and Bartlett. 19i19. p. 22lJ.
3lJ. Daniels. W. Why saying no to patients in the United States is so hard. Cost containment.
justice. and provider autonomy. N. Engl. 1. Med. 314. 13ilO. 1986.
40. Jonsen. A.R. Bentham in a box: technology assessment and health care allocation. Law,
Medicine and Health Care. 14.172. 19i16.
41. Curran. W,]. Thc constitutional right to health care. N. Eng/..J. Med. 320112. 7il8. 19i1lJ.
42. Banks. T. The right to medical treatment. In: Aids and the Law. Dalton. H.L. and Burris.
S. (cds.) New Haven. CT: Yale University Press. 19i17. p. 175.
43. White. A.R. Rights. Oxford: Clarendon Press. 19i14.
44. Rachels. 1.. Beauchamp, T.L. Killing and letting die. In: Ethical Issues in Death and
Dying. Beauchamp. T.L. and Perlin. S. (eds.) Englewood Cliffs: Prentice-Hall. I97il.
p.240.
45. Tolstoy, L.. Camus. A.. Taylor. R., Hare. R.M" Nagel. T" Margolis. J. The significance of
life and death. In: Ethical Ismes in Death and D,ring. Beauchamp, T.L. and Perlin S.
(eds.) Englewood Cliffs: Prentice-Hall. IlJ7il, p. 313.
46. Gula, R.M. Moral principles shaping public policy on euthanasia. Second Opinion:
Health, Faith and Ethics. 14.72, 19lJO.
47. Veatch, R.M. Choosing not to prolong dying. Medical Dimensions. Deccmber. 1972.
48. Dottes. A.L. Death with dignity: an alternative to chronic dialysis. Dialysis and
Transplantatiol1. 9.732. 1980.
49. Veatch. R.M. Death, Dying, and the Biological Revolution: Our Las! Ques! fi)r
Responsibilitl,. New Haven: Yale University Press, 1989.
50. Singer. P. Animal Liberatiol1. NY: New York Review Book. 1975.
51. Singer. P., Frey, R.G. Animal rights and animal research. In: Ethics and Public Policy.
Beauchamp. T.L. and Pinkard, T.P. (cds.) Englewood Cliffs: Prentice-Hall. 1983, p. 3il7.
52. Singer, P. Animals and the value of life. In: Matters of Life and Death; New Illtroductory
Essays in Moral Philosophy. Regan. T. (ed.) NY: Random House, 19i1O. p. 218.
53. Regan, T. The other victim. Hastings Center Report. 15.9.1985.
54. Nelson. J.L. Animals as a source of human transplant organs. In: Biomedical Ethics
Reviews. Humber,J.M. and Almeder, R.F. (eds.) Clifton. NJ: Humana Press. 19i17.
55. Frey, RG. Animal parts, human wholes: on the use of animals as a source of organs for
human transplants. In: Biomedical Ethics Reviews. Humber, J.M. and Almeder. R.F.
(cds.) Clifton. NJ: Humana Press. 1988. p. il9.
56. Wright. R.A. Clinical judgment and bioethics: the decision making link . ./r. Med. Phil. 16,
71.1990.
57. Vinocur, B. Less dramatic remedy for Baby Fae cases. New York Times. Nov. 9. A30.
1984.
5il. Russell. C. Rensberger, B. Baby Fae case leaves tremors: heart specialists find fault with
transplants. Washington POS!. Nov. 17. AI. 1984.
59. Capron. A.M. When well-meaning sciencc goes too far. Hastings Center Report. 15, il,
1985.
60. Fry. S.T. Moral values and ethical decisions in a constrained economic environment.
Nursing Economics. 4,160,1986.
61. Engelhardt, H.T. Jr. Fashioning an ethic for life and death in a post-modcrn society.
Hastings Center Report. Jan/Feb, 1989.
62. Mahon, K.A.. Fowler. M.D. Moral development and clinical decision-making. Nursing
Clinics of North America. 14.3.1979.
63. Lynn. J. Conflicts of interest in medical decision-making. 1. Am. Geriatr. Soc. 36, 945,
1988.
64. Ornery. A. Values, moral reasoning, and ethics. Nursing Clinics of North America. 24,
499,1989.
65. Martyn, S .. Wright. R .. Clark. L. Required requcst for organ donation: moral. clinical.
and lcgal problems. Hastings Cellter Report. 18,27,1988.
66. Forrow. L.. Wartman. S.A" Brock. D.W. Science. ethics and the making of clinical deci-
sions: implications for risk factor intervention . ./AMA. 259,3161, 1988.
References 527
67. Kassirer, J.P. Adding insult to injury: usurping patients' prerogatives. N. Engl. 1. Med.
308,898, 1983.
68. Russell, C. Lack of scrutiny fueled Baby Fae controversy. Washington Post. Nov. 18,
AI5,1984.
69. National Institutes OF Health. NIH report of its review of the Baby Fae case. IRB: A
Review of Human Subjects Research. 8, 1,1986.
70. Benjamin, M. Splitting the Difference; Compromise and Integrity in Ethics and Politics.
Lawrence, KS: University Press of Kansas, 1990.
71. Benjamin, M. Moral agency and negative acts in medicine. In: Medical Responsibility;
Paternalism, Informed Consent, and Euthanasia. Robinson, W.L. and Pritchard, M.S.
(eds.) Clifton, NJ: Humana Press, 1979, p. 170.
72. Evans, R.W. Some ethical and logistic issues in transplantation. In: The Transplantation
and Replacement of Thoracic Organs. Cooper, D.K.C. and Novitzky, D. (eds.) Kluwer;
Dordrecht, Boston, London, 1990, p. 515.
Section VI
Clinical Experience
Chapter 33
Experience with Clinical Kidney

Xenotransplantation
K. REEMTSMA AND A.I. BENVENISTY
Introduction
Attempts at renal xenotransplantation predating 1963 appear only as sporadic

reports [1-4] and were uniformly unsuccessful (Chap. 2). These early attempts
involved both nonprimate and nonhuman primate donor-to-human combina-
tions. Technical imperfection and no understanding of, or means to control,
immunological host reactivity led to a waning in interest in this surgical field.
However, in the early 1960s interest in xenografting was rekindled after
clinical studies demonstrated that renal allotransplantation could be success-
fully performed under the cover of adequate immunosuppression either with
total body irradiation [5-8], immunosuppression with 6-mercaptopurine [9,
10], or other drug therapy [11-13].
Several factors led to reinvestigation of the viability of xenotransplantation
as a therapeutic option for patients with advanced renal failure. Firstly,
hemodialysis was still an imperfect option for the treatment of terminal ure-
mia, with hemoaccess still a difficult and cumbersome problem and dialysis fa-
cilities a limited resource. Peritoneal dialysis was still not refined as a long-
term solution to the problem of renal failure. Secondly, human allograft
donors were in short supply and significant logistic and technical problems
with procurement and organ preservation still existed.
Initial attempts at human renal xenotransplantation were performed with
monkey [14] or baboon [15] donor organs. Although they were unsuccessful,
interest in this area persisted. The first major series ofxenotransplants was re-
ported by Reemtsma from Tulane University utilizing chimpanzees as donors
[16]. Starzl, at the University of Colorado, reported a series of baboon-to-hu-
man renal xenografts. These two series remain the only significant experience
in human renal xenotransplantation.
Selection and Preparation of the Recipient
Patients selected for a xenotransplantation at Tulane were terminal uremics,

maintained on dialysis, for whom a renal allograft could not be found from a
"volunteer" donor and after a search for a cadaveric organ had been unsuc-
cessful. The patients were then counseled on the experimental nature of the
532 Experience with Clinical Kidney Xenotransplantation
xenograft protocol and were entered into the study [16]. In some cases, bilater-
al nephrectomy/splenectomy was carried out. Pretreatment was with azathio-
prine. corticosteroids, and actinomycin C.
Selection and Procurement of the Nonhuman Primate Donor
Chimpanzee
At Tulane, the chimpanzee (Pan troglodytes) was selected as the source of the
donor organ for several reasons. The chimpanzee is classified in the category
of great apes, and displays several characteristics similar to man, including
size. Several studies have suggested that the renal function of chimpanzees
and man is quite similar [18, 19]. Chimpanzees express only blood types 0 or
A, with no identifiable B or AB groups [20]. Although the tempting possibility
of a "universal donor" was attractive, only 18% of chimpanzees express blood
group 0 [21], making donor availability (in a species already in limited supply)
a problem. This shortage actually led to the trial of a cross-blood group trans-
plant which will be discussed below. The animals used were surplus to the
needs of the aerospace program or were obtained from circuses.
Baboon
Starzl and his colleagues at the University of Colorado selected the East
African baboon (Papio doguera) for their clinical studies with renal xeno-
transplants. Previous work had demonstrated that at least an early diuresis
from the baboon kidney could be obtained after implantation in a human ure-
mic patient [15]. In addition, the cost of the donor baboon was much less when
compared with the chimpanzee. An important factor in the decision to use the
baboon was the fact that it was about 500 times as numerous as the chim-
panzee, making it a more likely source for grafts. Furthermore, experimental
comparisons could be made with the New Orleans experience.
Although the baboon exhibits an AB blood group system (in contrast to the
ABO system in humans), expression of the antigen(s) is not strong on the ery-
throcyte. Blood typing needed to be done using baboon saliva [20].
The Operative Procedure
The conduct of the donor and recipient operations was as follows. After deter-
mination of creatinine clearance, the donor animal was anesthetized and intu-
bated, and core-cooled to 30°C. The recipient patient was simultaneously pre-
pared. The donor kidneys, ureters, aorta, and vena cava were removed en
The Operative Procedure 533
iow me! :
Vlt. dex:rd:'.
(lO·C) w:.h
o.5QM
?I'OC3L - ~. ,
5lnQ n
Fig. 33.1. Diagrammatic representation of the donor operation. (After Starzl et al. (17])
bloc. In the Starzl experience, cold (lO-lYC) low molecular weight dextran in
normal saline, containing heparin and procaine chloride, was used for en bloc
perfusion. The entire complex was excluded from the circulation using appro-
priate placement of clamps and venting of perfusate through the divided up-
per inferior vena cava [17] (Fig. 33.1).
Fig. 33.2. The renal complex from the donor nonhuman primate is implanted into the ex-
traperitoneal space in the human recipient. The ends of the donor aorta and vena cava are
anastomosed to the sides of the recipient external iliac artery and vein, respectively. The
ureters are implanted into the bladder through submucosal tunnels. (After Starzl et al. [17])
The graft was then brought into the recipient operating room, and anasto-
mosed with an end-to-side technique to either the recipient aorta/vena cava or
the external iliac vessels. The ureters were implanted into the bladder via sub-
mucosal tunnels (Fig. 33.2).
Results of Clinical Renal Xenografting 535
Postoperative Management
Representative postoperative immunosuppressive protocols are displayed in

Figs. 2.6 (see page 15) and 33.3 for the Tulane and Colorado experiences, re-
spectively. The immunosuppressive protocols were similar with azathioprine,
steroids, and actinomycin C being given postoperatively, with graft irradiation
and adjustment of medications used during episodes of rejection [22]. Careful
records were kept of the usual clinical parameters and daily creatinine clear-
ances.
Results of Clinical Renal Xenografting
Twelve chimpanzee and six baboon xenograft procedures were performed in

the mid-1960s at Tulane and Denver, respectively. Following chimpanzee
xenografting, excellent early renal function with marked polyuria was seen,
PT. M.M
, 7YO ~
URINE No 411_ 64 . 1110'"
(mE q/L)
URINE
UREA
CONCOlT'RATION
(mom-I.)
URINE
VOLUME
WBe
AZATHIOPRINE
(mQm/doy)
PREDNISONE
(mQm/doy)
TIME IN o..YS .
Fig. 33.3. Chart of Starzl's third case illustrating certain aspects of the clinical course and
drug treatment. The human recipient was of blood group AB + and the donor baboon was of
blood group B. The xenograft was replaced by an allograft after 2 months. (After Starzl et al.
117])
536 Experience with Clinical Kidney Xcnotransplantation
with 24-h urine volumes greater than 61 not uncommon. Hume performed one
chimpanzee xenograft which produced 541 of urine in the early postoperative
period, leading to severe electrolyte disturbances, and resulting in the death of
the recipient [23]. In general, the diagnosis of xenograft rejection was made ac-
cording to patterns seen with allograft rejection (fever, decreased urine out-
put, and tenderness over the graft).
In the Denver series, similar early postoperative function and diuresis was
observed. Rejection was a serious problem in five of the six cases. The diagno-
sis of xenograft rejection was again made according to patterns seen in allo-
graft rejection. Occasionally, isotope renograms were used adjunctly. Usually
there were abrupt episodes of dysfunction, but slow, insidious episodes were
also noted. Although episodes could be managed with actinomycin D and lo-
cal radiotherapy, the interval between rejection episodes was short, and in-
tense immunosuppression could not be reduced, which resulted in extremely
poor clinical outcomes. Two patients had the baboon grafts replaced by a hu-
man allograft, and septic complications intervened secondary to intense initial
immunosuppression. Ultimately, all six patients died within 10-60 days, with
mean xenograft function being 36 days.
.
150
z~
::lE
aJ~
608~~\
AlOO ./ \
200' \ /'" ....... : ."
o °
0 ••••••••• ""
Fig. 33.4. Courses of two patients, one fol-
;~~'\'
..
38 ::..
~
lowing a living related (sibling] allograft,
the other following a primate-to-man
xenograft. Note the similarity between the
!;~ two courses, including the development
2 4 6 8 10 12 14 16 18 and subsequent reversal of rejection
Days fallowing transplantation episodes. (After Reemtsma et al. [16])
Immunological Studies in Clinical Renal Xenotransplantation 537
In the first six Tulane cases, the primary problems were infection and
wound sepsis, particularly with Aerobacter aerogenes infections. For the most
part, grafts functioned immediately and were able to maintain function. They
responded to antirejection therapy, and, in fact, some behaved in a curiously
similar manner to concurrently performed allotransplants (Fig. 33.4). Graft
function in certain cases was greater than 6 months, but there were no long-
term successes. In the last six cases, rejection was a more serious problem, with
early and irreversible rejections seen in all [24].
Pathological Changes in Renal Xenografts
Baboon-to-Human
Pathological changes in cross-species grafting have been reviewed by Porter

[25] (see also Chaps. 11, 12). Upon removal, all grafts were edematous with pe-
techial hemorrhages in parenchyma, calyces, and pelves, with patent arteries
and veins [17]. On microscopic section, cortical and interstitial cellular infil-
trates were observed with edema and patchy hemorrhage and patchy infarc-
tion; total infarction had occurred in one graft. Fibrin and platelet deposits in
interlobular arteries were seen in all of the grafts, causing narrowing of a vari-
able number of vessels with some luminal blockage by superimposed throm-
bus.
Chimpanzee-to-Human
Findings in the chimpanzee-to-human group were more varied than those in

the baboon-to-human combination, but in general were felt to be less severe
[25]. The first chimpanzee-to-man allograft examined 9 weeks after implanta-
tion showed interstitial edema, and tubular necrosis and repair, but no other
cellular infiltrate and no glomerular or vascular changes. Sections of chim-
panzee kidneys that functioned for 9 months showed no cellular infiltrate but
marked intimal thickening of the interlobular arteries. The last six chim-
panzee-to-human grafts all demonstrated the classical signs of rejection with
edema, cellular infiltrates, and vascular lesions [24].
Immunological Studies in Clinical Renal Xenotransplantation
Unfortunately, the immunological studies carried out were performed prior to

the modern era of understanding of transplantation immunobiology. Most of
the studies related to measurement of antiblood group antigen antibody levels
which, at the time, were thought to be important in transplantation immunobi-
538 Experienee with Clinical Kidney Xenotransplantation
ology. The role of cytotoxic antibodies in humoral rejection had then not been
clearly elucidated [26J.
Baboon-to-Human
In the baboon grafts, attention was concentrated on the baboon AB system,

which was, as mentioned above, difficult to ascertain because of poor antigen
expression on the erythrocytes, necessitating the use of saliva for typing.
Heteroagglutinins and isoagglutinins to baboon erythrocytes were measured.
The preoperative sera of all six recipients of baboon kidneys contained het-
eroagglutinins to baboon erythrocytes. After transplantation, the titers fell,
implying that antigenic determinants specific for this serum antibody were
present on the xenograft. Furthermore, in five of six cases, titers rose after
each rejection episode [17].
When the antiblood group A and B isoagglutinins were examined, a fall in
titers of preformed isoagglutinins was found when transplants were per-
formed across blood group barriers, again implying adsorption of antibody on
to the xenograft. Titers would rise again with the final rejection episodes
(Fig. 33.5).
Chimpanzee-to-Human
In the chimpanzee experience, similar findings were noted. A human anti-

chimpanzee hemagglutinin existed in all of the recipients. However, since the
antibody did not adsorb onto kidney (unlike the baboon), the antigen seemed
PT.5
c .. ~
CD 0
~~-----------
AnUria
51"
-'0 Antl- B
iJ
0-- - --0
o----------<>Antl- A
"0. - Heteroagglutinins
II: " 10
I
/
I
/
10 12 14 16 18
Post operative days
Fig. 33.5. Serial measurements of anti-A and anti-B isoagglutinins and agglutinins after
xenotransplantalion from a hlood group AB donor hahoon inlo a hlood group 0 human re-
cipient. Initial reductions in antihody tilers were followed hy rises. (After Slarzl et al. [17])
References 539
to exist only on the erythrocyte and not on the kidney. Nevertheless, when ex-
amination for cytotoxic antibodies was performed in recipients of chimpanzee
xenografts, they were found in all but one patient as immunoglobulin M (IgM)
and IgG fractions [24]. High titer serum did not reveal any specificity other
than xenogeneic despite extensive cross adsorption [27]. Most importantly,
the administration of immunosuppression did not seem to prevent or reduce
the appearance of these antibodies.
Comment
These few cases of clinical renal xenografting a quarter of a century ago are no-
table in that they represent almost the world's total experience in this field.
They demonstrated the important findings that a xenograft from a nonhuman
primate could, in fact, even with rudimentary forms of immunosuppression,
function to sustain a patient previously in terminal renal failure. Although the
immunological studies were unsophisticated by current standards, the clinical
and pathological examinations that were carefully performed and document-
ed demonstrated that the behavior of these grafts was similar to concurrent al-
lografts (at that time termed homografts). These pioneering efforts have sub-
sequently stimulated a great deal of experimental work in this field of
xenografting.
References
1. Princeteau, M. Greffe renale. f. Med. Bordeaux. 26,549,1905.

2. J aboulay, M. Greffe de reins au pli de coude par soudures arterielles et veineuses. Lyon
Med.107,575,1906.
3. Unger, E. Nierentransplantationen. Klin. Wschr. 47,573,1910.
4. Neuhof, H. The Transplantation o/Tissues. Appleton and Co., New York, 1923, p. 260.
5. Hume, D.M., Miller, J.P., Miller, B.F., Thorn, G.W. Experiences with renal homotrans-
plantation in humans: report of9 cases.f. Clin. Invest. 34,327,1955.
6. Murray, J.E., Merrill, J.P., Dammin, G.J., Dealy, J.B. Jr., Walter, CW., Brooke, M.S.,
Wilson, RE. Study on transplantation immunity after total body irradiation. Surgery. 48,
272,1960.
7. Hamburger, J.J., Vaysee, J., Crosnier, J., Auvert, J., Lalanne, CM., Hopper, J. Jr. Renal
homotransplantation in man after irradiation of the recipient: experience with 6 cases
since 1959. Amer. f. Med. 32,854,1962.
8. Kuss, R, Teinturier, J., Milliez, P Quelques essais de greffes de hein chez l'homme.
Mem. Acad. Chir. 77,775,1951.
9. Schwartz, R, Dameshek, W. Drug induced immunologic tolerance. Nature. 183,1682,
1959.
10. Schwartz, R, Dameshek, W. The effects of 6-mercaptopurine on homograft reactions.
f. Clin. Invest. 39,952,1960.
11. Starzl, T.E., Marchioro, T.L., Waddell, W.R. Reversal of rejection in human renal homo-
grafts with subsequent development of homograft tolerance. Surg. Gynec. Obstet. 117,
385.1963.
12. Hume. D.M .. Magee. J.H .. Kauffman. H.M. Jr. Renal homotransplantation in man and
modified recipients. Ann. Slirg 15X. liOK 1963.
13. Murray. J.E .. Merrill. J.P .. Harrison. J.H .. Wilson. RE .. Dammin. G.J. Prolonged sur-
vival of human kidney homografts by immunosuppressive drug therapy. iV. Enfi/. 1. Mell.
268. !3l5. 191i3.
14. Reemtsma. K .. McCracken. B.H .. SchlegeL J. U .. Pearl. M. Heterotransplantation of the
kidney: two clinical experiences. Science. 143.700. 19M.
IS. Hitchcock. CR .. Kiser. J.C. Tclander. RL.. Seljeskob. E.L. Baboon renal grafts.
lAMA. 189.934. 19M.
16. Reemtsma. K .. McCracken. B.H .. SchlegeL J.U .. PearL M.A .. Pearce. CW .. DeWitt.
CW .. Smith. P.E .. Hewitt. RL.. Flinner. RL.. Creech. O. Renal heterotransplantation
in man. Ann. Slirg. 1liO. 3X4. 1964.
17. StarzL T.E.. Marchioro. T.L.. Peters. G.N .. Kirkpatrick. CH .. Wilson. W.E.C. Porter.
K.A.. Rifkind. D .. Ogden, D.A., Hitchcock. CR, WaddelL W.R. Renal heterotrans-
plantation from baboon to man: experience with Ii cases. Transplal1lation. 2.752, 1964.
18. Gagnon, J.A., Clarke. RW. Renal functions in the chimpanzee. Amer. 1. Phrsiol. 190.
117.1957.
19. Smith. H.W., Clarke. RW. The excretion of inulin and creatinine by the anthropoid apc
and othcr infra-human species. Amer. l. Physiol. 122,132, 193X.
20. Wiener, A.s.. Moor-Jankowski. J. Blood groups in anthropoid apes and baboons.
Science. 142.67.1963.
21. Goldsmith, E.A. Personal communication to K Reemtsma.
22. Rcemtsma. K .. McCracken. B.H., SchlegeL J.U .. Pearl, M.A.. Dewitt, CW .. Crcech, O.
Reversal of early graft rejection after renal hetcrotransplantation in man . .lAMA. 1X7.
691. 19M.
23. Hume. D.M. (as discussant in reference 16].
24. Reemtsma, K. Renal hetcrotransplantation from non-human primatcs to man. Anl1.
NY. Acad. Sci. 162.412. 19li9.
25. Porter. KA. Pathologic changes in transplanted kidneys. In: Experience in Rellal
Transplantation. T.E. Starzl (cd.) W.B. Saunders, Philadelphia. 1964.
26. Starzl, T.E. Baboon renal and chimpanzee liver heterotransplantation. In: Xenofiraft 25.
M.A. Hardy (ed.) Elsevier. New York. 1989, p. 17
27. Dewitt, CW .. Reemtsma, K, Oliver. CB., Ahlschier, A. Production of anti-chimpanzee
cytotoxin in human subjects. In: Advances in Transplantation. Hamburger, 1 .. Malke. G.
(cds.) Williams and Wilkins. Baltimore. 196X. p. 409.
28. Reemtsma. K Xenotransplantation: a personal history. In: Xenofiraft 25. M.A. Hardy
(cd.) Elsevier. New York. 1989. p. 7.
Chapter 34
Experience with Clinical Heart

Xenotransplantation
D.K.C. COOPER AND Y.YE
Introduction
To our knowledge, there have been eight documented attempts at clinical

heart xenotransplantation, the first being in 1964 and the most recent being in
1984. Five of the eight have used nonhuman primates as donors, and three
have used widely divergent species, namely the sheep and the pig. The longest
surviving primate heart has been 20 days, and the nonprimate hearts func-
tioned either not at all or for only a few minutes (Table 34.1).
First Attempt
The first cardiac xenotransplant, and indeed the first cardiac transplant of any
type in man, was performed by James Hardy (Fig. 34.1) and his colleagues at
the University of Mississippi Medical Center on January 23, 1964 [1]. As heart
transplantation had not been performed previously, this surgical group had to
contend with many problems that would not be faced today.
It was their intention to use a recipient who was absolutely terminal, i.e.,
where cessation of heart function was imminent, and also a donor in whom the
same situation was occurring. The concepts of brain death and the removal of
a beating heart from a patient whose respiratory function was supported by a
ventilator had not yet been accepted. The team was clearly aware that the like-
lihood of heart function ceasing simultaneously in a potential recipient and a
potential donor was extremely low, but felt that this situation should be
sought. If a suitable recipient presented, however, with no suitable human
donor, they had a back-up plan to use a chimpanzee heart. This plan had
evolved following the encouraging results of kidney transplantation using
chimpanzees as donors obtained by Reemtsma and his colleagues in the previ-
ous few months [2-5].
Hardy's group had performed extensive experimental work in dogs and
calves and had developed a technique of short-term preservation of the donor
heart by retrograde coronary perfusion using cold oxygenated blood adminis-
tered by gravity flow [6, 7]. Furthermore, they had recently attempted the
world's first lung transplant in man [8].
Table 34.1. World experience in clinical heart xenotransplantation lJl
~
N
Case Year Surgeon Institution Donor Type of Outcome Reference

Transplant source m
x
"0
r;
'"i
1964 Hardy University of Mississippi, Chimpanzee OHT Functioned 2 h II] (S'
Jackson, Mississippi, USA Heart too small to ::l
(")
r;
support circulation
§.
2 196X Cooley Texas Heart Institute, Sheep OHT Immediate cessation [14] ;:
Houston, Texas, USA of function ('I vascular Q
rejection) S·
o·
3 196X Ross National Heart HospitaL Pig HHT Cessation of fune- -::r:'"
London. UK tion within 4 min ()
('1 vascular rejection) [21]. (personal '"

:::.
communication) ><
r;
4 196X Ross National Heart HospitaL Pig Perfused Immediate cessation ::l
London. UK with human of function ('? 2-
'"i
blood but vascular rejection) OJ
::l
C/O
not trans- "0
planted 5:
::l
5 1969 Marion Lyon. France Chimpanzee ?OHT Rapid failure? raised [25J ~.
pulmonary vascular g
resistance
6 1977 Barnard University of Cape Town. Baboon HHT Functioned 5 h [22J
Cape Town. South Africa Heart too small to
support circulation
7 1977 Barnard University of Cape Town. Chimpanzee HHT Functioned 4 days [22]
Cape Town. South Africa Failed from probable
vascular rejection
8 1984 Bailey Lorna Linda University. Baboon OHT Functioned 20 days [29]
Lorna Linda. California. USA Failed from vascular
rejection
OHT. orthotopic heart transplantation; HHT. hcterotopic heart transplantation

First Attempt 543
Fig. 34.1. James Hardy, who, in 1964, led

thc surgical team that performed the
world's first heart transplant, using a chim-
panzce as donor. In the previous year,
Hardy and his group had carried out the
world's first singlc lung allotransplant
I .'4' f; ~ I ... I
,I , ~_ f,o- ~ to I
Si red Hal' '0.-: I,.),."....+.~_.....~_~~. . .'"')~.--;-____
f. •
Fig. 34.2. Consent permit for operation signed by a relative of the first patient to undergo
heart transplantation in 1964. (Courtesy of Professor J.D. Hardy.) Compared with what
would be required today, it is surprisingly brief and less than comprehensive.
544 Experience with Clinical Heart Xenotransplantation
With regard to a human donor, their original plan was to insert catheters
into the femoral vessels and begin total body perfusion, the instant death (i.e.,
cessation of cardiac and respiratory function) was determined by a physician
not associated with the transplant team. They would then continue to store the
heart by means of retrograde coronary sinus perfusion.
The recipient was a 68-year-old man who had had hypertensive cardiovas-
cular disease for many years for which he had been taking digitalis and diuret-
ics. He had been admitted in a semicomatose state in cardiogenic shock and
had required vasopressor drugs to maintain a systolic blood pressure of 100
mmHg. His impaired mental state was considered to be secondary to cardio-
genic shock with hypoxia imposed upon an already atherosclerotic cerebral
vascular bed, or possibly to an intracranial vascular accident. By today's stan-
dards, he would clearly be a less than ideal candidate for heart transplantation
in view of his doubtful cerebral state, the presence of widespread atherosclero-
sis, and his age. Furthermore, he had gangrene of the lower portion of the left
leg which required amputation a few days before his heart transplant was per-
formed (Fig. 34.2). He was clearly terminal and his respirations were irregular
and inadequate without mechanical assistance.
When it became clear that he was about to die from total cardiac failure , he
was transferred to the operating room as there was a potential donor in the
hospital at that time (Figs. 34.3, 34.4). Cardiopulmonary bypass was initiated
and the patient's own effective heart action ceased. The prospective human
donor lingered in the recovery ward and was clearly not going to undergo car-
diorespiratory arrest in the immediate future. A decision was therefore made
to utilize a chimpanzee heart, and this animal was anesthetized in an adjacent
operating room. The chimpanzee weighed 96 lb (44 kg), which was consider-
ably less than the potential recipient, but the chimpanzee's cardiac output had
been measured at 4.25 IImin while under anesthesia on a previous occasion. It
Fig. 34.3. The operating room team at th e University of Mississippi during the performance
of the world 's first heart xenotransplant in J 964. (Courtesy of Professor J.D. Hardy)
First Attempt 545
Fig. 34.4. The operating room team at the University of Mississippi during the performance
of the world's first heart xenotransplant in 1964. (Courtesy of Professor J.D. Hardy)
was hoped that this would be adequate for the patient, particularly if the chim-
panzee heart could be paced to increase cardiac output.
The donor heart was excised and a coronary sinus catheter inserted imme-
diately. The heart was briefly perfused with a cold heparinized Ringer's lactate
solution through which oxygen had been bubbled to increase the P0 2 . After
the primate blood had been washed out, the perfusion fluid was changed to
cold oxygenated human blood, administered by gravity flow, with the heart re-
maining submerged in cold Ringer's lactate solution. A slow and satisfactory
ventricular fibrillation developed.
The recipient's heart was then excised and the donor organ sutured in place
using a standard technique which had been developed over a number of years
in the experimental animal [9] (Fig. 34.5). The aorta was undamped approxi-
mately 45 min after the beginning of the actual insertion of the donor organ.
The blood from the pump oxygenator quickly rewarmed the transplanted
heart, and a strong ventricular fibrillation was developed. A single shock with
a pulse defribillator produced complete cardiac arrest, which was followed by
a regular and forceful beat at a rate of approximately 80 per minute.
It was soon apparent, however, that the smaller chimpanzee heart would
not be able to handle a large venous return unless its rate were increased. The
heart was, therefore, paced at 100 beats/min. All perfusion catheters were re-
moved and protamine sulfate was administered. The paced primate heart was
able to maintain a systolic blood pressure ranging from 60 to 90 mmHg.
Unfortunately, as time passed, the heart became increasingly unable to handle
Fig. 34.5. Photograph of the chimpanzee heart after implantation in the first patient to
undergo heart transplantation in January 1964. Pacing wires can be seen entering the peri -
cardial cavity to the right of the heart (left). (Courtesy of Professor J.~. Hardy)
Fig. 34.6. Histopathological appearances of myocardium of chimpanzee heart at necropsy

following the first attempted heart transplant. The myocytes are poorly preserved and there
is some vascular congestion, but no specific features suggestive of hyperacute rejection or
myocyte necrosis can be seen. The lack of nuclear staining in the myocytes may be due to
autolysis. The appearances suggest that vascular (humoral) rejection had not been a faclor
in the failure of the heart to support the circulation. (Courtesy of Professor J.D. Hardy, with
review of the histopathology by Professor A.G. Rose)
the large venous return, Some 2 h after the heart had been defibrillated and 1 h
after removal of the cardiopulmonary bypass cathcters, the heart was judged
incapable of accepting the large venous return without intermittent decom-
pression by manual cardiac massage. Further support was abandoned.
Case Two 547
It is not certain from the report of this case whether it was purely a question
of inadequate size of the chimpanzee heart or whether early vascular (hu-
moral) rejection was also affecting cardiac function adversely. As there is no
mention that the heart became swollen or edematous or took on the
blue-black color of a heart which is undergoing rapid hyperacute rejection, it
seems most likely from the description that it was purely a matter of inade-
quate size. This assumption is supported by a review of the histological ap-
pearances of the myocardium at necropsy (Fig. 34.6). There are no definite
features of significant vascular rejection (A.G. Rose, personal communica-
tion).
The report of this operation was greeted by considerable controversy in the
lay press and even by some members of the medical profession [10]. It was
deemed justified by the transplant team in view of recent attempts at liver
transplantation l11, 12] and the use of a cardiac mechanical assist device [13]
and, as previously mentioned, by encouraging results utilizing primate kidneys
for kidney transplantation.
Case Two
The second recorded attempt was made by Denton Cooley and his associates
in Houston, Texas, on June 12, 1968 [14, 15]. This group had performed four
previous heart transplants using human donor organs, and, therefore, many of
the problems facing Hardy had already been clarified. Details of this case are
few. The patient was a 48-year-old man weighing 165 lb (75 kg) with ischemic
heart disease, and his circulatory status was deteriorating several hours after a
cardiac arrest. As no human donor was available, the heart from a 125lb (57
kg) sheep was inserted. It seems likely that hyperacute rejection occurred im-
mediately on the operating table.
About 10 min after the operation had begun, whilst the surgical team was
still suturing the right atrium, the sheep heart began to develop "spasm", and
before reperfusion of the heart was initiated the spasm virtually obliterated
the left ventricular cavity. When full coronary flow through the ascending aor-
ta from the pump oxygenator was restored, only the atria contracted; the ven-
tricles did not contract. The atrial contractions persisted for a short time, but
then diminished. The heart was described as taking on the appearance of a
"uterus" [15].
No histopathological specimens are now available, but the appearances
were described by Dr. Cooley in 1968 [15]. The myocardium "showed a mini-
mum of round cell infiltrates around the vessels, and in the interstices".
Opinion at the time was that these appearances were "not unequivocal evi-
dence of rejection".
At this time (1968), there was little published experimental work regarding
the outcome of transplantation in widely disparate species. What evidence
there was [16] and subsequent work [17-20] has clearly shown that such an at-
tempt would be doomed to early failure. At the time, therefore, there were no
experimental data to suggest that the outcome would be favorable. Even to-
day, techniques have not evolved which would lead to success in such a case,
even though the use of plasmapheresis or extracorporeal adsorption of anti-
species antibodies might lead to prolongation of adequate cardiac function for
a few hours or even a few days.
Cases Three and Four
Although a pig heart was actually incorporated into the patient's circulation in
only one of these cases - the second was only perfused with blood from the
pump oxygenator - both experiences will be included here. These cases have
never been reported fully in the literature, but basic details are available from
the proceedings of a symposium held in Cape Town in 1968 [21]. The exact
date of these two operations, performed (on the same day) by Donald Ross
and his colleagues in London in the United Kingdom, remains obscure, but al-
most certainly took place in the first 6 months of 1968, possibly before Dr.
Cooley's operation (case 2). Ross's group was faced with the unusual circum-
stance of having two patients at the same time in adjacent operating rooms
who could not be weaned from cardiopulmonary bypass support following
open heart procedures.
In the first patient, a pig heart was anastomosed in parallel as a heterotopic
heart transplant in the hope that this heart would be able to maintain the circu-
lation until either the patient's own heart recovered or a human donor heart
became available to allow orthotopic transplantation. Anastomoses were
made from donor to recipient at the left and right atrial level (using Dacron
conduits) and between the two aortae and pulmonary arteries (D.N. Ross,
personal communication). Both right and left ventricles were therefore sup-
ported by the pig heart. Within 4 min of reperfusion, however, the pig heart
went absolutely "'rigid" and started oozing edema fluid.
Following this experience, in the second patient the heart was not anasto-
mosed, but a preliminary test was carried out by inserting the coronary perfu-
sion lines from the pump oxygenator into the pig's coronary arteries to see
whether the same response would occur. The reaction was identical, and so
this heart was not transplanted. Information on any histopathological changes
in the myocardium of either heart is not available.
To Donald Ross and his colleagues, therefore, must be credited the concept
of using an animal heart as a "bridging" device towards transplantation with a
human heart, an idea that was taken up subsequently by Barnard [22] (see be-
low), and has gained further support and interest in recent years [23,24].
Cases Six and Seven 549
Case Five
Case five is only briefly documented by Professor Pierre Marion in the French
medical literature in October, 1969 [25]. The exact date ofthe procedure is not
recorded, but presumably was late 1968 or early 1969.
The patient was a young woman with mitral and tricuspid valve disease.
After mitral valve surgery, left ventricular support was necessary but, by the
4th postoperative day, her condition was terminal. As no human donor was
available, a chimpanzee heart was transplanted and initially functioned well.
Rapid deterioration occurred, however, believed to be due to a high pul-
monary vascular resistance that proved irreversible. No further details were
given.
Cases Six and Seven
The Cape Town group, headed by Christiaan Barnard, had introduced the op-
eration of heterotopic heart transplantation clinically in 1974 [26], and their
initial results were good. As first attempted by Ross (above), one of the indica-
tions for the use of the heterotopic cardiac transplant was considered to be
temporary support of a failing heart in the anticipation of its recovery when all
other measures of support had been unsuccessful. In 1977, the Cape Town
group made two attempts to support failing human hearts with primate hearts
placed in the heterotopic position [22].
The first of these two patients was a 25-year-old woman who had previous-
ly undergone aortic valve replacement with a size 17 Bjork-Shiley aortic pros-
thesis. Extensive intravascular hemolysis resulted in persistent anemia which
could not be controlled by any form of medical therapy, including blood trans-
fusions. After 6 weeks, the hemolysis was still present, and it was decided to
perform another operation in an attempt to insert a bigger aortic valve. On
June 20, 1977, an aortoventriculoplasty was performed and a 23-mm Bjork-
Shiley prosthesis inserted.
After the heart had been rewarmed, difficulty was initially encountered in
defibrillating the ventricles. Eventually this was achieved, with the heart in si-
nus rhythm, but with intermittent episodes of complete heart block. Pacing
was commenced. On attempting to discontinue extracorporeal circulation, ad-
vanced left ventricular dysfunction was evident. Conventional medical thera-
py failed to improve the condition. As it was thought that the cardiac pump
failure was due to ischemia resulting from a small right coronary ostium, a
saphenous vein bypass graft was performed between the ascending aorta and
the proximal right coronary artery. Withdrawal of extracorporeal support re-
mained unsuccessful, however, even after the insertion of an intra-aortic bal-
loon pump.
Since a human donor was not available, the heart of a 30-kg male Chacma
baboon with the same blood group as the patient was removed and transplant-
ed heterotopically using the technique previously described by this group [261.

Although it is not stated, it is presumed that only the left ventricle was support-
ed by this technique. Once the baboon heart had been defibrillated and shared
the circulation of the patient, it was possible to discontinue all mechanical sup-
port. However, the patient's own heart showed severe ventricular irritability
and several attacks of ventricular fibrillation had to be electrically defibrillat-
ed. During the periods of ventricular fibrillation, it was noted that the baboon
heart alone could not carry the full circulatory load, the mean arterial pressure
dropping from 60 to 30 mmHg and the venous pressure rising from 14 to 30 cm
H 20 .
The operation was completed, however, and the patient returned to the in-
tensive care unit. At this stage she maintained a good circulation with moder-
ate inotropic support. She regained consciousness, started warming up periph-
erally, and passed copious amounts of urine. With the two hearts functioning,
she maintained a mean arterial pressure of70 mmHg. At 2 h after her return to
the intensive care unit, and 5.5 h after the completion of the transplant, her
heart again developed repeated attacks of ventricular fibrillation. Initially
these were successfully treated by cardioversion, but eventually all attempts to
establish a coordinated ventricular beat failed. The baboon heart continued to
maintain a moderate circulation for another 30 min, but then also went into
ventricular fibrillation and all attempts to resuscitate it were unsuccessful.
At autopsy, the donor heart appeared macroscopically normal (Fig. 34.7).
On histological examination, the heart appeared normal in many areas, with
small scattered foci of contraction bands and polymorphs, and mild interstitial
edema in some areas. The capillaries were not occluded by platelet thrombi.
The epicardial and intramyocardial arteries appeared normal. [n the initial re-
port of the autopsy findings, there was thought to be no evidence of classical
hyperacute rejection . A recent review of the specimens by Rose at al. [27],
Fig. 34.7. Macroscopic appearances at autopsy or the native human heart (to the right) and
the heterotopically placed baboon heart (10 the left) in case six. (Courtesy of Proressor A.G.
Rose)
Cases Six and Seven 551
however, suggests the possibility of early hyperacute rejection, though elec-

tron microscopic studies revealed no significant abnormalities [28].
This group performed a second such heterotopic cardiac xenotransplant
utilizing a chimpanzee as the donor on October 13, 1977. The patient was a 60-
year-old man operated on for severe calcific aortic stenosis. His preoperative
left ventricular ejection fraction was only 19%, and he had diffuse coronary
artery disease, but no critical lesion which needed revascularization. The aor-
tic valve was replaced without complication but on attempting to discontinue
cardiopulmonary bypass support severe left ventricular dysfunction was evi-
dent. Conventional medical therapy failed and irreversible ventricular fibrilla-
tion ensued. Extracorporeal circulatory support was restarted, and cardiac
function was assisted by the heterotopic transplant of a chimpanzee heart. It
was then possible to discontinue all mechanical support. The patient returned
to the intensive care unit with a good circulation and no evidence of irritability
of the native heart.
High doses of immunosuppressive drugs were used to try to prevent early
rejection of the heterotopic transplant. It must be remembered that cyclo-
sporine was not then available. In the 4 days following surgery steady deterio-
ration in the function of the transplanted heart as well as of the patient's own
heart was noticed. On the 4th postoperative day, the recipient's own heart
stopped and could not be restarted, and the markedly reduced function of the
transplanted heart was unable to support the circulation.
At autopsy, there was reported extensive subendocardial infarction of the
recipient heart and severe acute rejection of the donor heart (Fig. 34.8). A re-
cent review of the specimen by Rose et al. [27] revealed widespread capillary
destruction and interstitial hemorrhage (Fig. 34.9); scanty thrombi occluded a
few capillaries and some arterioles showed intimal cell detachment with ery-
throcyte entrapment within the intima. These changes were similar in nature
Fig. 34.8. Macroscopic appearanccs at autopsy of the native human heart (to the right) and
the heterotopically placed chimpanzee heart (to the left) in case seven. (Courtesy of
Professor A.G. Rose)
552 Experience with Clinica l Heart Xenotransplantation
Fig. 34.9. Histopathological appearances of the chimpanzee heart in case seven. Interstitial
hemorrhage due to destruction of the capillaries is a promine nt feature (hematoxylin and
eosin. x600). (Courtesy of Professor A.G. Rose)
to those seen in hyperacutely rejecting experimental xenografts. but appeared

mild in nature [21]. Ultrastructural appearances confirmed microvascular
damage with free erythrocytes in the interstitium [28].
With the relatively primitive nature of the intra-aortic balloon device in
1977 and encouraged by the reported results of the use of xenografts in the ear-
ly days of kidney transplantation [2-5]. the Cape Town group felt that. in the
absence of a human donor. heterotopic heart xenotransplantation might be
the best means of providing circulatory support when it was anticipated that
the patient's own heart would subsequently recover. The available evidence
was that the increased immunological onslaught on a primate xenograft (as
opposed to a pig or sheep heart) could be controlled until the patient's own
heart had recovered or until the primate heart could be replaced by a human
donor heart.
In their first case. however. it appeared that even the heart of an adult male
baboon was too small to maintain an adequate cardiac output when the pa-
tient's own heart stopped beating. From the experience of the second case. it
appeared that a heterotopic transplant using a chimpanzee heart could sup-
port the patient's circulation for 48- 72 h only, and should , therefore. be em-
ployed only where there was evidence that the patient's own heart function
might recover rapidly.
Case Eight 553
Case Eight
The experience of Leonard Bailey and the Loma Linda group in California is
by far the most detailed study of xenotransplantation in the human subject to
date [29]. It is also the most successful to date, in part because cyclosporine was
available to this group, while it was not in all previous cases. The patient was a
4.8-1b (2.2-kg) female neonate born prematurely with hypoplastic left heart
syndrome. She required mechanical ventilation, inotropic support, and con-
tinuous infusion of prostaglandin E j • In the absence of a human donor it was
decided to use the heart of a baboon.
A detailed immunological assessment was made of both the neonate and
six size-matched potential baboon donors. This involved human leukocyte
antigen (HLA) typing, microlymphocytotoxic cross-matching, and mixed
lymphocyte culture (MLC) reactions. HLA typing and serum cross-match
failed to distinguish a "preferable" donor. Results of the MLC appeared more
helpful. The baboon chosen was the one that elicited the weakest MLC re-
sponse from the patient. This was only about 7%-13% as great as the response
elicited against the other baboons.
Unfortunately, the patient was of blood type 0, a blood group that has al-
most never been recorded in any baboon species. An ABO mismatch between
donor and recipient was, therefore, unavoidable. The baboon chosen was of
blood group A.
U nits of human donor blood to be used during and after the operation were
free of lymphocytotoxic antibaboon antibody. Washed erythrocytes were the
only blood component utilized, except for 25% human serum albumin.
The baboon was carefully screened to ensure that it was free of microfilaria,
hepatitis antigen, cytomegalovirus, and skin, blood, and fecal parasites. Other
screening was also performed, including a tuberculin test.
Cyclosporine was administered intravenously to the potential recipient
for 38 h before the transplant procedure in a dose of 0.5-1.0 mg/h.
Xenotransplantation was performed on October 26,1984. The operative tech-
nique involved near total thymectomy, hemodilution, hypothermia and circu-
latory arrest. Reconstruction of the aortic arch well beyond the large patent
ductus arteriosus was also accomplished. Xenograft function returned sponta-
neously following orthotopic implantation. and regular sinus rhythm was ob-
served. No inotropic support was required.
The initial plan was to replace the xenografted heart with a human donor
heart as soon as possible, using the xenograft only as a bridging device. No
allograft was procured, however, during the lifetime of the patient.
Tracheal extubation was possible after 3 days, and a switch to oral cyclo-
sporine was possible a further week later. It was also possible to discontinue
supplemental oxygen. At about this time (postoperative day 11). however. the
infant had decreased activity and appetite and mild tachypnea. A single intra-
venous dose of methylprednisolone (100 mg/kg) was administered and this
achieved immediate reversal of these signs.
Functional impairment of the cardiac graft, however, was clearly docu-

mented 3 days later when again the patient demonstrated diminished appetite
and activity. Echocardiography revealed a progressive reduction in the car-
diac fiber shortening fraction. Elevation of creatine kinase isoenzyme and
serum myoglobin levels paralleled echocardiographic findings and supported
a diagnosis of myocyte necrosis. The reappearance of tachypnea, hypoxemia,
and gradually progressing dyspnea prompted reintubation and ventilator as-
sistance. A reduced cardiac output was documented. This presumed acute re-
jection episode was treated with anti thymocyte globulin, azathioprine, and
methylprednisolone.
Features of cardiac failure increased, however, and the patient eventually
became anuric. Metabolic acidosis developed and complete heart block oc-
curred 2 h before death on November 15, 1984, some 20 days after the trans-
plant operation.
Examination of the xenografted heart showed that it was only slightly en-
larged, but the myocardium, while soft and compliant, was hemorrhagic.
Microscopic sections revealed absence of normal interstitial spaces between
myocardial fibers and marked vacuolization of myocyte nuclei. Muscle cells
making up the walls of arterioles were swollen, some to near occlusion. Deeply
situated ventricular myocardial sections showed regions of coagulation necro-
sis, interstitial hemorrhage, and myofibrillar contraction bands characteristic
of anoxia. There was an infiltrate of mononuclear cells in several regions of
ventricular subepicardium.
Electron microscopy demonstrated swollen, partially disrupted mitochon-
dria occupying spaces between separate myofibrillar bundles. Fat vacuoles ap-
peared to have formed from swollen mitochondria. Modest amounts of finely
granular deposits of immunoglobulin G (IgG), IgM, IgA, and C3 were demon-
strated. Scattered areas of immunofluorescence involved no more than 15%
of ventricular myocardial specimens.
The infant cellular response to the cardiac xenograft was, therefore, weak
and played virtually no role in graft compromise. Almost certainly, cyclo-
sporine played a significant role in minimizing the degree of cellular rejection
that occurred. The histological findings appeared to be antibody and/or com-
plement-mediated despite immunosuppression.
It was clear that the patient died of progressive graft necrosis, complicated
by acute renal and pulmonary insufficiency. Lymphocytotoxic antidonor anti-
bodies had been absent preoperatively, but anti-A and anti-B isoagglutinins to
human erythrocytes and low titered heteroagglutinins to baboon erythrocytes
were present in the patient's circulation before transplantation, though they
disappeared afterwards. Anti-A and anti-B isoagglutinins were notably ab-
sent terminally. Traces of heteroagglutinins were still detectable in terminal
serum samples.
The suspected sequence of events was as follows. The ABO antibodies
and/or antibaboon antibodies were gradually adsorbed by the graft, producing
injury to the largest endothelial bed, namely the microcirculatory vessels. This
phenomenon resulted in widespread microvascular luminal narrowing.
Comment 555
Circulatory sludging, thrombosis, cellular hypoxia, and myocyte injury and/or
necrosis followed. Edematous and porous microvascular endothelium permit-
ted interstitial hemorrhage and lipid accumulation.
The fact that graft destruction was related more to an antibody phe-
nomenon than to cellular rejection seems to have been well documented. The
relative roles of ABO antibodies and/or antibaboon antibodies are less cer-
tain. Subsequent experimental work suggests that the antispecies antibody
may playa more major role in gradual graft destruction than the anti-ABO an-
tibody, though the presence of ABO incompatibility between donor and re-
cipient is associated with a higher incidence of early hyperacute rejection [30].
It would seem from this clinical experience, together with further experi-
mental work [30, 31], that even transplantation between closely related pri-
mate species is complicated by humoral rejection. Though this is not so rapidly
destructive as when transplantation is performed between widely disparate
species, nevertheless it plays a significant and major role in graft destruction.
Further experimental work, therefore, will need to be directed at overcoming
the humoral response.
Comment
At the present time, therefore, it would appear that the use of nonhuman pri-
mate hearts coupled with standard present day immunosuppressive therapy
(cyclosporine, azathioprine and steroids) might result in good cardiac function
for a short period of time of days or a few weeks. The experimental evidence is
that the addition of antithymocyte globulin to the immunosuppressive regi-
men, particularly if used in association with one of the newer pharmacologic
agents, such as 15-deoxyspergualin, FK-506, rapamycin, or RS-61443 might
lead to extended graft survival of weeks or even months [31,32].
Even with the use of new agents such a 15-deoxyspergualin, which has a sig-
nificant suppressive effect on B cell activity with reduction of antibody pro-
duction, existing antispecies antibodies, though of low titer in man against oth-
er primate species, may need to be removed from the plasma by a technique
such as plasma exchange or extracorporeal immunoadsorption.
The larger nonhuman primate species, such as the chimpanzee, are already
endangered species and will not be available as organ donors for man. The
ethics of their use in this way today are also highly controversial. Nonhuman
primate hearts will only be available from the smaller primate species such as
the baboon, vervet (African green) monkey, or cynomolgus monkey, and,
therefore, will be most suited for use in infants and young children, possibly
primarily as "bridges" until a human donor of suitable size and blood group
becomes available. Current evidence is that the use of a primate organ, with
the possible development of increased levels of antibodies directed against the
species, would not jeopardize the success of the subsequent use of a human
donor organ (E.A. Rose, personal communication). A high level of antiba-
boon antibodies, for example, would not lead to an increased incidence of lym-
phocytotoxic antibodies against human lymphocytes. There would remain.
however. significant ethical questions and controversy to deal with. particular-
ly with animal rights groups who would be opposed to the use of such animals
for this purpose.
The use of pigs or sheep as donors for man, though logistically ideal and
ethically far less controversial, remains a more distant reality. The presence of
strong preformed antispecies antibodies in man to these animals will preclude
their use as donors until a reliable method has been developed to remove these
antibodies and prevent their further production. A combination of extracor-
poreal immunoadsorption and some of the new immunosuppressive agents
holds out hope in this regard in the foreseeable future. The development of
transgenic animals, e.g., pigs carrying human transplantation antigens, is a dis-
tant goal that may hopefully revolutionize the field of xenotransplantation
[23].
References
1. Hardy. J.D., Kurrus, F.E., Chavez, CM., Neely, W.A., Webb, W.R., Eraslan, S., Turner.
M.D., Fabian, L.W., Labecki, J.D. Heart transplantation in man: developmental studies
and report of a case. f. Am. Med. Ass. 188,1132,1964.
2. Reemtsma, K., McCracken, B.H., Schlegel, J .V., Pearl, M. Heterotransplantation of the
kidney: two clinical experiences. Science. 143,700, 1964.
3. Reemtsma, K., McCracken, B.H., Schlegel, J.V., Pearl, M.A., Pearce, CW., Dewitt,
CW., Smith, P.E., Hewitt, R.L., Flinner, R.L., Creech, O. JR. Renal heterotransplanta-
tion in man. Ann. Surg. 160,384, 1964.
4. Reemtsma, K., McCracken, B.H., Schlegel, J.V., Pearl, M.A., Dewitt, CW., Crecch, O.
JR. Reversal of early graft rejection after renal transplantation in man. f. Am. Med. Ass.
187,691, 1964.
5. Reemtsma, K. Heterotransplantation. Transplant. Proc. 1,251,1969.
6. Hardy, J.D., Kurrus, F.D., Chavez, CM., Webb, W.R. Heart transplantation in infant
calves: evaluation of coronary perfusion to preserve organ during transfer. Ann. NY
Acad. Sci. 120,786, 1964.
7. Kurrus, F., Hardy, J.D., Chavez, CM., Elliott, R.L. Heart transplantation. Fed. Proc. 23,
201,1964.
8. Hardy, J.D., Webb, W.R., Dalton, M.L. Jr., Walker, G.R. Jr. Lung homotransplantation
in man: report of initial case. f. Am. Med. Ass. 186, 1065, 1963.
9. Cooper, D.K.C Experimental development of cardiac transplantation. Br. Med. f. 4,
171,1968.
10. Hardy, J.D. The World a/Surgery, 1945-1985: Memoirs a/One Participant. University of
Pennsylvania Press, Philadelphia, 1986.
11. Starzl, T.E., Marchioro, T.L., Von Kaulla, K.N., Hermann, G., Brittain, R.S., Waddell,
W.R. Homotransplantation of the liver in humans. Surg. Gynec. Obstet. 117,659,1963.
12. Moore, F.D. Quoted by Hardy, et al. [1].
13. Debakey, M.E. Cited info Am. Med. Ass. 187,299,1964. Quoted by Hardy, et al. [11.
14. Cooley, D.A., Hallman, G.L., Bloodwell, R.D., Nora, J.J., Leachman, R.D. Human
heart transplantation: experience with 12 cases. Am. f. Cardiol. 22,804,1968.
15. Cooley, D.A. In: Experience With Human Heart Transplantation. (edited by H. Shapiro)
Buttcrworths, Durban, 1969, p. 228 and p. 203.
References 557
16. Perper, RJ., Najarian, J .S. Experimental renal heterotransplantation. I. In widely diver-
17. Giles. G.R, Boehmig, HJ., Lilley, J., Amemiya, H., Takagi, H., Coburg, AJ., Hatha-
way, W.E., Wilson, C.B., Dixon, F.J., Starzl, T.E. Mechanism and modification of rejec-
tion of heterografts between divergent species. Transplant. Proc. 2,522,1970.
IS. Merkel, F.K., Bier, M., Beavers, CD., Merriman, W.G., Wilson, c., Starzl, T.E.
Modification of xenograft response by selective plasmapheresis. Transplant. Proc. 3,
534,1971.
19. Mozes, M.F., Gewurz, H., Gunnarson, A., Moberg, W., Westbery, N.G., Jetzer, T.,
Najarian, J .S. Xenograft rejection by dog and man: Isolated kidney perfusion with blood
and plasma. Transplant. Proc. 3,531, 1971.
20. Cooper, D.K.C., Human, P.A, Lexer, G., Rose, AG., Rees, J., Keraan, M., Du Toit, E.
Effects of eyclosporine and antibody adsorption on pig cardiac xenograft survival in the
baboon. I. Heart Transplant. 7,238,1988.
21. Ross, D.N. In: Experience With Human Heart Transplantation. (edited by H. Shapiro)
Butterworths, Durban, 1969, p. 227.
22. Barnard, C.N., Wolpowitz, A., Losman, J.G. Heterotopic cardiac transplantation with a
pass. S. Afr. Med. I. 52, 1035, 1977.
23. Cooper, D.K.C. Cardiac xenotransplantation - overview. In: The Transplantation and
Replacement of Thoracic Organs. Edited by Cooper, D.K.C. and Novitzky, D. Kluwer
Academic, Dordrecht, Boston, London, 1990.
24. Cooper, D.K.C. Present status of cardiac xenotransplantation. Cardiac Surgery: State of
the Art Reviews. 3, 661,1989.
25. Marion, P. In: Les transplantations cardiaques et les transplantations hepatiques. Lyon
Med. 222,585,1969.
26. Barnard, C.N .. Losman, J.G. Left ventricular bypass. S. Afr. Med. I. 49,303,1975.
27. Rose, AG., Cooper, D.K.C., Human, P.A, Reichenspurner, H., Reichart, B. Histo-
pathology of hyperacute rejection of the heart: experimental and clinical observations in
allografts and xenografts. 1. Heart Transplant. In press.
28. Rose, A.G., Cooper, D.K.C. Ultrastructural appearances of hyperacutely rejected car-
diac xenotransplants. (Abstract) I. Heart Transplant. 9,72,1990.
29. Bailey, L.L., Nehlsen-Cannarella, S.L., Concepcion, W., Jolley, W.B. Baboon-to-human
cardiac xenotransplantation in a neonate. I. Am. Med. Ass. 254,3321,1985.
30. Cooper, D.K.C., Human, P.A., Rose, AG., Rees, J., Keraan, M., Reichart, B., du Toit,
baboon cardiac xenograft model. I. Thorae. Cardiovase. Surg. 97,447, 19S9.
31. Reichenspurner, H., Human, P.A., Boehm, D.M., Rose, A.G., May, R, Cooper, D.K.C.,
transplantation in primates. I. Heart Transplant. 8,200,1989.
32. Thomas, F.T., DeMasi, RJ., Araneda, D., Marchman, W., Alqaisi, M., Larkin, E.W.,
Condie, RM., Carobbi, A., Thomas, J .M. Comparative efficacy of immunosuppressive
drugs in xenografting. Transplant. Proc. 22, 1083,1990.
Chapter 35
Liver Xenotransplantation:
Clinical Experience and Future Considerations
D.V. CRAMER, L. SUER, AND L. MAKOWKA
Introduction
The use of xenografts in liver transplantation offers several distinct advan-

tages for the treatment of patients with end-stage liver disease and fulminant
liver failure. The first is the availability of a predictable and ready supply of
donor organs. At the present time the most critical element restricting the
widespread application of liver transplantation as a therapeutic modality is the
shortage of human donor organs. It has been estimated that approximately
20%-25% of patients awaiting liver transplantation die due to lack of an ap-
propriate donor organ. The limited supply of donor organs also has the effect
of limiting the size of the potential pool of recipients that may benefit from the
procedure. An increased supply of suitable organs will result in a large in-
crease in the number of transplants performed.
The availability of donor organs would provide the opportunity to practise
liver transplantation as an elective procedure. The timing of the transplant is
essential in achieving a good outcome for the patient; that is, the ability to de-
crease the waiting period for patients with signs of deterioration is an impor-
tant factor in improving graft and patient survival.
A third important advantage of xenografting would be the opportunity to
match the size of the graft for small adults and children. Despite the develop-
ment of procedures that allow for the transplantation of an isolated hepatic
lobe or segment, there remains a critical shortage of appropriately sized or-
gans for the pediatric population.
Finally, one major area in which xenografting will have immediate and
widespread impact will be in the treatment of fulminant hepatic failure. In
these patients the onset of disease is sudden and the time available to locate
and procure an appropriate organ is limited. At the present time, identifica-
tion of a suitable donor organ is frequently not possible before acute liver fail-
ure leads to a poor neurological outcome and/or death of the patient.
The mechanisms responsible for mediating the xenograft reaction, and the
response of individual organs to that reaction, are important to define if we are
to understand the biologic barriers that must be overcome before a clinically
successful outcome can be achieved. At the present time, progress in the appli-
cation of liver xenografting is primarily limited by our lack of understanding of
the reaction of human recipients to xenografted organs, and the importance of
the rejection reaction on the ability ofthe liver to function appropriately.
560 Liver Xenotransplantation: Clinical Experience and Future Considerations
The liver is generally more resistant than other organs to allograft rejec-
tion, particularly antibody-mediated hyperacute reactions, and long-term sur-
vival following immunosuppression is morc easily achieved. It is possible that
a similar resistance to hyperacute or discordant xenograft reactions may also
exist. Hyperacute xenograft reactions consists of an immediate, diffuse in-
travascular coagulopathy, followed by an aggressive cellular rejection reac-
tion. The hyperacute inflammatory coagulation reaction results from either (i)
the binding of naturally occurring xenoantibodies of the recipient to tissues of
the donor, followed by activation of the classical complement pathway, or (ii)
direct activation of the alternative complement pathway by antigens ex-
pressed in the donor graft. This type of reaction results in rapid loss of the graft
due to an obstructive coagulopathy of the microvasculature. Species that are
more closely related (concordant) exhibit a rejection reaction that does not
depend upon a hyperacute humoral phase, but rather is characterized by an
aggressive, acute cellular rejection.
In this chapter we intend to review the clinical and experimental experi-
ence with liver xenografting. Our objective is to provide a summary of infor-
mation that may relate to (i) the selection of potential liver xenograft donors
for man, and (ii) the importance that the type of xenograft reaction may have
on the ability of the liver to function in that environment.
Clinical Experience with Liver Xenografting
The clinical reports that are relevant for understanding the potential of liver
xenografting are very limited. They consist primarily of (i) the use of liver
xenografts in the ex vivo support of patients with acute hepatic failure, (ii) a
small number of attempts to use auxiliary heterotopic xenografts, and (iii)
three cases of orthotopic transplantation.
In general, these were attempts to provide temporary support of patients
with acute liver failure, and were made early in the development of organ
transplantation as a surgical discipline. Any retrospective evaluation of the re-
sults of these experiences, therefore, must be considered in the light of the
poor success rates for liver allografting being achieved during that period.
Recent improvements in surgical techniques and the use of more powerful im-
munosuppressive drugs have dramatically improved graft survival rates, and it
is reasonable to expect a similar improvement in the results that would be ob-
tained following xenografting if it were carried out today.
Ex Vivo Liver Perfusion
There have been several attempts to maintain the lives of patients with fulmi-
nant hepatic failure by using ex vivo isolated liver xenograft perfusion for the
temporary support and metabolic detoxification of the patient (for summary
Clinical Experience with Liver Xenografting 561
see [1,2]). Approximately 100 or more patients have been treated in this man-
ner, but the short perfusion times in most cases and technical complications
have limited the amount of useful information that has been obtained.
The perfusions usually lasted less than 8 h, preventing an evaluation of the
effect of the immunological response of the patient to the liver, and limiting
accurate determination of the therapeutic efficacy of these methods. Some of
the more detailed reports, however, have demonstrated that the liver
xenograft is metabolically active and functions to clear lactate and ammonia,
produces bile, secretes host serum proteins in bile, and produces donor-type
serum proteins [3,4]. The livers used for these perfusions were most frequent-
ly derived from pigs, although other species used as donors included subhu-
man primates (macaques, baboons), cattle, and occasionally man.
One of the more interesting examples of the use of ex vivo liver perfusion is
the report by Abouna et al. [5] that describes the use of 16 livers (ten pig, three
baboon, and one each from calf, monkey, and man) to support a single patient
in hepatorenal failure for 11 weeks. Perfusion of the patient's blood through
the livers was associated with a reversal of hepatic coma eight times, and the
perfused livers were shown to be capable of producing bile and synthesizing a
variety of clotting factors.
Perfusion with the pig livers was associated with the appearance of anti-
body to pig serum proteins and an increase in the titers of preformed human
antipig antibodies. Pig proteins synthesized by the donor liver appeared in the
patient's serum and persisted for approximately 23 days. At least one episode
of an anaphylactic response by the patient to the pig liver perfusion was seen.
To avoid this complication, livers from other species were used, and over a pe-
riod of 6 weeks the levels of antipig antibody decreased; subsequent perfu-
sions with pig livers, combined with steroid therapy, were successfully per-
formed.
Heterotopic Liver Transplantation
There have been a small number of reports (summarized in [1]) describing the
use of liver xenografts as heterotopic grafts to provide short-term support for
patients in hepatic failure. The first of these grafts was performed by Starzl et
al. [96] and consisted of a chimpanzee liver attached to the femoral vessels of a
young child in hepatic coma. The graft functioned for 24 h, during which peri-
od the patient's coma was partially resolved and clearance of bilirubin and al-
kaline phosphatase could be demonstrated. The loss of the graft was associat-
ed with the formation of thrombi in the hepatic artery and several small
vessels.
This result is typical for heterotopic xenografts, where occasional grafts
have been shown to function satisfactorily, improving neurological and coagu-
lation functions. Most of these grafts, however, failed within a few hours, ei-
ther due to rejection or technical complications accompanying the surgery.
Orthotopic Liver Transplantation
Orthotopic liver xenografting has been attempted in three patients by Starzl

(Fig. 35.1) and his colleagues [7-10].
The first case was a 28-month-old child with biliary atresia who was given
an orthotopic chimpanzee liver because of a lack of a human cadaveric donor
[7, 10]. Immediately following surgery there was evidence that the liver
xenograft was functioning; bilirubin levels dropped sharply and remained low
for the remainder of the time the child survived. The immunosuppressive ther-
apy used was a combination of azathioprine, prednisone, and antilymphocyte
globulin (ALG). The authors reported that, in retrospect , this therapy was in-
adequate, with insufficient levels of azathioprine and prednisone. In addition,
too little attention was paid to the thrombocytopenia that developed, which
was probably a result of the ALG.
Nine days after surgery the patient died from sepsis. The pathological
changes reported in the liver were similar to those seen for an allograft of simi-
lar duration [10]. There was heavy lymphocyte infiltration of the portal triad,
without evidence of the vascular lesions (fibrinoid necrosis) generally associ-
ated with hyperacute rejection, and centrilobular cholestasis.
The second case involved a 7-month-old child with extrahepatic biliary
atresia who was transplanted with a chimpanzee liver [8]. An attempt was
made to match the donor and recipient for antigens of the major histocompat-
ibility complex (MHC). Mixed lymphocyte reactions (MLR) were carried out,
and two potential donor animals that did not stimulate MLR responses by the
patient were identified. The patient received multiple transfusions, and subse-
Fig. 35.1. Thomas Starzl. one of the major

surgical pioneers or organ transplantation.
who led the teams that carried out the first
attempts at clinical liver xenografting using
chimpanzees as donors
The Choice of the Potential Liver Xenograft Donor for Man 563
quently developed a positive cross-match with the donor chimpanzee. In an at-
tempt to avoid an antibody-mediated hyperacute rejection, the donor kidneys
were perfused ex vivo with the patient's blood to absorb the cross-reacting an-
tibodies. Both kidneys were rejected hyperacutely, the first within 5 min and
the second at approximately 3 h. This absorption procedure was successful,
with elimination of the pre-existing antidonor antibodies. Once the liver graft
had been placed, bile was produced, but the patient developed bleeding com-
plications, including a precipitous drop in complement levels, and died 26 h
later. The pathological examination of the liver did not demonstrate any hepa-
tocellular damage.
The third case also involved a small child (23 months) with biliary atresia
who was transplanted with a chimpanzee liver [9]. This was a second liver
transplant for this child and was performed to replace a failed allograft that
had been implanted 10 days earlier. The graft functioned for 14 days, and the
cause of death was sepsis. Once again, the pathological lesions in the graft
were minimal [9]. There was no evidence of cellular infiltration of the portal
triad, no evidence of vascular fibrinoid rejection, and a primary finding of cen-
trilobular cholestasis.
There was evidence of postoperative liver function in at least two of these
three children (with graft survival times of 9 and 14 days). Of particular inter-
est was the observation that these grafts exhibited only limited histopathologi-
cal evidence of immune-mediated rejection damage [10]. In general, the type
and severity of the host antidonor reactions were qualitatively and quantita-
tively similar to those observed for allografts. The recipients were treated with
azathioprine, prednisone, and ALG to prevent rejection, and the histological
damage was limited, suggesting that the therapy was at least partially success-
ful or that the xenograft reaction was less severe than expected.
Although the attempts described above to use xenografts as complete liver
replacement have been very limited, it is clear that (i) the liver from another
species is capable offunctioning in a foreign host environment, (ii) there is lit-
tle evidence from this limited clinical (and experimental) data that liver
xenografts are subject to violent, hyperacute rejection, even in cases where an
organ from a species as widely divergent as the pig has been used, and (iii)
there is a suggestion that the xenograft reaction to a concordant primate
species may be amenable to effective immunosuppression. In view of recent
improvements in surgical techniques, infection control, and immunosup-
pressive therapy, these results are encouraging for the future use of liver
xenografts.
The Choice ofthe Potential Liver Xenograft Donor for Man
The choice of a species to act as a xenograft donor involves complex scientif-

ic, ethical, and practical considerations, many of which have not received seri-
ous attention. The choice of a species on scientific merit will be based on a
564 Livcr Xcnotransplantation: Clinical Experience and Future Considerations
solid informational data base that demonstrates that the donor organs are
physiologically and anatomically compatible, and that the genetic disparity
between the donor and recipient would be of such intensity that a reasonable
potential of modulating or completely preventing the rejection reaction
would exist.
The relationship between genetic distance (between donor species and
man) and xenograft rejection process has not been examined carefully enough
to allow for an absolute prediction as to the type of reaction that would be
seen. The commonly accepted assumption is that the greater the genetic dis-
parity, then the more likely is the xenograft reaction to be one of hyperacute
rejection mediated by natural antibody. There are, however, several questions
that will have to be addressed before a particular liver xenograft donor could
be considered for use in a clinical setting.
There are good examples of xenografting between closely related species
where the xenograft rejection patterns are markedly different from those pre-
dicted on the basis of phylogenetic relationships. Rats, for example, reject
guinea pig hearts within minutes (a hyperacute or "discordant" xenograft re-
action), while mouse or hamster hearts are rejected in 3-4 days ("concordant"
reaction) [11]. All three species are rodents, and the disparity in the reaction
patterns suggests that it may be difficult to predict the severity of the reaction
by human recipients to different xenograft donors, even if these were closely
related species.
The hyperacute reaction seen between "discordant" species may also con-
sist of different types of rejection reactions. There are clearly those that are
mediated by natural antibody, particularly those across ABO-like disparities,
and in other cases (guinea pig-to-rat) by the activation of alternative comple-
ment pathways in the absence of demonstrable natural antibody [12]. It is pos-
sible, for example, that differences between species could be the expression of
an antigen that has the ability to activate the alternative pathway. This type of
difference would make attempts to prevent xenograft rejection by depletion
of natural antibody ineffective.
Additionally, the liver has been shown to be more resistant to rejection re-
sulting from either (i) the traditional cell-mediated allograft response, or (ii)
hyperacute rejection mediated by preformed antibody (ABO mismatches) or
antibody that has been stimulated by previous sensitization of the recipient to
donor-type histocompatibility antigens [13]. It is not clear whether these dif-
ferences in the liver's sensitivity to rejection are the result of (i) the large mass
of this organ, which makes it capable of inducing high-dose tolerance, or (ii)
differences in the type and/or level of the tissue antigens that are the target of
the rejection process.
Liver xenografts exchanged between hamsters (as donors) and rats (as re-
cipients) survive for approximately 7 days [14]. This is longer than survival of
heart grafts exchanged in the same hamster-to-rat combination (3--4 days),
and yet shorter than that of rat heart allografts exchanged between a strong al-
lograft combination (10 days) [11]. This relative resistance to rejection may
enhance the effects of immunosuppressive drugs on liver xenografts, and sug-
gests that the liver might be one of the more attractive and first organs to con-
sider for xenotransplantation.
The choice of the appropriate donor species for human xenografting will
also depend upon ethical considerations. There is growing concern about the
use of animals for research, particularly for those with anthropomorphic simi-
larities to man, and it is to be expected that these concerns will apply equally to
the use of animals as xenograft donors.
Because of a variety of considerations (size, reproductive capacity, and ge-
netic and/or physiological similarities) there are three groups of animals that
represent the most likely candidates as xenograft donors. They include pri-
mates, swine, and small ruminants.
Primates
The use of nonhuman primates as xenograft donors for human recipients of-
fers the clear advantage of physiological and genetic similarities with man. The
genetic similarities should reduce the severity of the rejection response, which
(based upon a relative lack of preformed human antibody that reacts with
nonhuman primate donor tissues) is generally considered to be of a concor-
dant nature [15]. In general, humans display relatively low levels of noncyto-
toxic antibodies to chimpanzees and baboons, and this observation has led to
the use of these animals as donors for 27 hearts, kidneys, and livers for human
transplantation [2].
The results of these early clinical experiments can be viewed as both en-
couraging and discouraging. The primate kidney grafts were not rejected in a
hyperacute fashion, several of the grafts surviving for days or weeks
(Chap. 33). At least one graft functioned for 9 months, and no pathological
changes of rejection were reported in the graft at the time of the death of the
patient [16]. These results were similar to those obtained with chimpanzee liv-
er xenografts by Starzl and colleagues (see above), two ofthe three grafts func-
tioning and, at autopsy, showing evidence of rejection comparable to that in an
allograft.
In each case, xenogeneic organs functioned appropriately in the human
host, and did not exhibit the rapid, explosive hyperacute rejection seen be-
tween discordant species. None of these grafts, however, survived for greatly
prolonged periods of time, and rejection appeared to be more intense and
rapid than that seen with allografts, though the xenograft reaction did appear
to respond to conventional immunotherapy. The therapy available at that
time was much less effective than that available today, suggesting that the use
of modern immunosuppressive drugs may be associated with better control of
While the use of subhuman primates offers the potential of long-term graft
survival with current methods of immunosuppression, several medical and
ethical issues effectively eliminate the practical use of these species as
xenograft donors. The most immediate concern is the availability of sufficient
numbers of animals to act as organ donors. All primates exhibit limited repro-
ductive capacity, and nonhuman primates represent special problems for
breeding in captivity.
Ten regional primate centers were established in the United States in the
1960s to examine the potential of providing breeding facilities for primates for
purposes of experimentation. To date these facilities have not provided suffi-
cient numbers of animals for current research needs, let alone the number of
donors that would be necessary to meet the organ shortage associated with
transplantation. Many nonhuman primate species are also considered endan-
gered species and subject to limitations of capture and/or importation restric-
tions.
Medically, nonhuman primates also present an important health risk to hu-
man recipients. The close genetic relationships among primates include the
sharing of several serious and potentially fatal diseases. These include, for ex-
ample, simian B virus, tuberculosis, malaria and, most recently, the potential
that some nonhuman primates carry acquired immunodeficiency syndrome
(AIDS)-like viruses capable of infecting humans. Finally, the use of intelligent
species with many social similarities to humans is ethically unacceptable to an
increasing number of people.
Given these limitations, it is not reasonable to assume that the solution to
the source of xenogeneic grafts for human recipients will include the use of
substantial numbers of nonhuman primates.
Pigs
The pig is a species that anatomically, physiologically, and ethically may repre-
sent the best choice as a xenograft donor in humans (Chap. 30). As described
above, the pig liver has been used as a temporary ex vivo support for patients
with acute hepatic failure [3, 17, 18]. These early experiments demonstrated
the ability of the pig liver to function in humans and provide metabolic support
during acute hepatic failure.
The use of the pig as a xenograft donor is considered to be less desirable
than subhuman primates because of the genetic distance between pigs and hu-
mans. Humans and pigs are sufficiently distant genetically that, given the con-
siderations for species matching discussed above, the expectation is that pig-
to-human grafts would be rejected in a hyperacute fashion. The evidence for
such a discordant rejection reaction is, however, incomplete.
The use of pig livers to support patients with acute hepatic failure repeated-
ly for periods of several hours without evidence of significant pathological
changes in those livers clearly suggests that this donor/recipient liver
xenograft combination may not be susceptible to the classical hyperacute and
violent "discordant" reaction. There is evidence, however, that suggests that
this apparent lack of a hyperacute rejection may be organ specific (see below).
Natural antibodies that react with pig lymphocytes exist preformed in nor-
mal human serum, and consist primarily of IgM immunoglobulins [19, 20].
When tested against pig endothelial cells, these antibodies, in the presence of
complement, cause the cleavage and release of heparan sulfate proteoglycans
from the cell surface, suggesting that they may be capable of precipitating a
hyperacute rejection of grafts expressing these molecules on normal vascular
endothelium [21].
Ex vivo perfusion of pig kidneys with human blood is associated with in-
creased vascular resistance, platelet aggregation, and the deposition of human
IgG and complement components in the renal tubules and glomeruli [22]. We
have compared similar perfusion of the pig liver, using human whole blood
and plasma, and demonstrated that the deposition of antibody and comple-
ment components is quantitatively reduced (when compared with the kidney).
Changes in vascular resistance were not observed, and our results do not pro-
vide evidence for the rapid onset of hyperacute discordant reactions as ob-
served in the heart and kidneys in the same species combination.
Experimental testing of pig-to-human liver xenografts is clearly restricted
to in vitro studies, though much relevant information can be obtained from
pig-to-subhuman primate experiments. There have been no reports of at-
tempts to use a pig as a donor for human liver replacement, even though there
is, as suggested above, little evidence that such a combination would lead to an
uncontrollable hyperacute rejection.
As outlined previously, isolated perfusion of pig livers with human blood
has been attempted, with inconclusive results [4]. The livers were perfused for
up to 6 h; evidence for hepatic parenchymal damage consisted of a slight in-
crease in serum potassium level and a clear increase in the mean level of serum
aspartate transaminase. There was little evidence of a change in vascular resis-
tance, and the ability of the liver to clear sodium lactate and ammonium citrate
was not impaired. When combined with the data derived from the use of the
pig for temporary support of patients with acute hepatic failure, the results
suggest that the pig-to-human liver xenograft may be rejected at a rate that is
slow enough to be controlled with current (albeit experimental) immunosup-
pressive methods.
The most relevant evidence from experimental animals concerning pig-to-
human liver xenografting would be provided by the transplantation of a pig
liver into a nonhuman primate recipient. Two series of such experiments have
been conducted (Chap. 24).
In the initial studies, seven pig-to-baboon liver grafts were attempted with
limited success [23]. The grafted livers survived for short periods of time, usu-
ally less than 24 h, although one animal was treated with immunosuppression
and died from pneumonia at 3 days. In most animals death was attributed to
centrilobular necrosis and poor liver function. The liver of the 3 day survivor
exhibited the cellular inflammatory lesions seen in baboon-to-baboon allo-
grafts. The absence of pathological lesions consistent with hyperacute rejec-
tion suggests that failure of the remaining grafts may have been associated
with surgical technical problems.
The second group of experiments included pig-to-rhesus (three experi-
ments) and pig-to-chimpanzee (one experiment) liver transplantation [24].
None of the animals survived for more than 12 h, and there was histological
evidence of widespread intravascular coagulation in at least one of the pig-to-
rhesus grafts.
The xenografted liver, therefore, appears relatively resistant to hyperacute
rejection, and perhaps this is associated with its arterial blood supply for main-
taining graft function. The vascular supply of the liver does not have the same
end-organ distribution as that of the heart or kidney, and the disseminated in-
travascular events of hyperacute rejection may have less effect on organ func-
tion when distributed through the vascular sinusoids of the liver [25]. Liver
allografts are also less susceptible to hyperacute rejection mediated by anti-
body, including circulating preformed lymphocytotoxic antibody, and anti-
body produced by intentional immunization in rats [26-28]. With sufficient
sensitization, however, hyperacute rejection of the liver can be induced in the
rat, and the lesions associated with this accelerated rejection are similar to
those seen in other organs [29].
Despite the clinical and experimental evidence that the liver may be re-
sistent to hyperacute rejection mediated by antibody, there is a growing body
of evidence that suggests that the liver undergoes an accelerated rejection in
the presence of preformed or induced antidonor antibodies [13]. In humans,
ABO incompatibility is associated with an increased incidence of early liver
loss due to antibody-mediated damage [30]. After transplantation, the donor
organ functions well, but evidence of progressive liver damage can become in-
creasingly apparent during the first few days after graft placement. This liver
damage may be associated with the deposition of immunoglobulins and com-
plement components in the sinusoids and vessels, the onset of a disseminated
coagulopathy, and with hemorrhage and scattered foci of hepatic cell necrosis.
In the pig, immunization by repeated skin grafting can induce hyperacute
rejection of a subsequent liver allograft [31]. Three skin grafts at 2-week inter-
vals result in sensitization of the recipient animals, and subsequent allogeneic
liver grafts are lost within 4 h. This graft loss is associated with pathological ev-
idence of hyperacute rejection, including endothelial damage to the central
veins, parenchymal hemorrhage, and neutrophil infiltration.
Rumiuauts - Sheep aud Goats
There have been isolated attempts to use tissues from small ruminants (sheep
and goats) in humans. Sheep and goat kidneys have been transplanted to hu-
mans prior to the development of immunosuppression, and these grafts sur-
vived for 3 and 9 days, respectively (see [1] and Chap. 2). Experience with the
use of a liver from a small ruminant in subhuman primates or humans is very
limited. A calf liver was used in one of the support perfusions described by
Abouna et a1. [5], and resulted in a drop in bilirubin levels and a return to con-
sciousness for the patient.
The use of organs from small ruminants suffers from the same disadvan-
tages of genetic disparity as those seen with swine. The data available on the
Future Directions 569
intensity and type of xenograft reaction in humans are limited, but there is no
reason to expect that the reaction would be less severe than that expected be-
tween humans and pigs. In addition, there are greater physiological differ-
ences that may complicate the postsurgical function of the donor organ.
Humans have erythrocytes that are significantly larger than those of sheep and
goats, and these size differences may be important at the level of the capillary
bed. One other difference that potentially may be important is the depen-
dence of the ruminant liver on short chain fatty acids for energy metabolism;
there may be difficulties associated with a conversion to glucose metabolism
shortly after transplantation.
Future Directions
Improved Immunosuppression
In recent years the major improvement in the survival of allografts is due to the
discovery and use of new immunosuppressive drugs. The introduction of cy-
closporine resulted in a dramatic increase in the survival rate following liver
transplantation, and major improvements in the quality of life for the recipi-
ents of these grafts. More recently a new drug, FK-506, may offer additional
improvements in controlling the rejection of liver allografts in some individu-
als, and may exhibit better patient compliance for treatment. These two drugs
share similar mechanisms of action, primarily acting to prevent the production
and subsequent activity of interleukin-2 (IL-2) and other early lymphocyte-
activating cytokines.
Neither of these drugs, however, has been shown to be active in preventing
the rejection of concordant or discordant xenografts. Although the long-term
solution to xenografting may evolve from genetic manipulations, such as those
described below, which may not require additional new immunosuppressive
therapy, the potential application of new drugs should continue to be ex-
plored.
The focus of future experimental work will be directed simultaneously to-
wards prevention of the humorally mediated hyperacute reaction seen with
discordant grafts, combined with attempts to control the aggressive cellular
rejection seen in concordant (and possibly, though not certainly, discordant)
combinations. The hyperacute reaction potentially can be disrupted (i) at the
level of the target antigen, (ii) by interfering with the production and/or func-
tion of natural antibody or, (iii) by blocking the activation of the complement
cascade and its influence on coagulative and acute inflammatory reactions.
Recent experiments have shown that disruption of individual components
of this process can prolong xenograft survival and suggest that a polytherapeu-
tic approach may be possible with current immunosuppressive agents. A vari-
ety of studies have demonstrated that removal of natural antibody prolongs
liver xenograft survival [13], that the use of antagonists of acute inflammatory
mediators can interfere with antibody-mediated discordant or sensitized allo-

graft reactions [11,32], and that immunosuppressive agents that prevent the
production of antibody play a role in dclaying the rejection of cardiac
xenografts [33]. Combinations of these therapies have proven to be very effec-
tive in rodent xenograft models, most particularly the long-term survival of
hamster hearts transplanted to rats following treatment with irradiation and
cyclosporine [34].
It will be necessary to develop a multiple drug regimen that includes agents
that prevent any immediate humoral reaction to the graft (possibly combined
with methods of antibody depletion) and antagonists of the acute inflammato-
ry and coagulative events associated with complement activation [32], fol-
lowed by immunosuppressive agents that prevent the activation and function-
al responses of inflammatory cells and lymphocytes involved in the xenograft
reaction. This type of poly therapeutic approach to xenografts is currently pos-
sible, and combinations of new immunosuppressive agents now in develop-
ment may be effective when included in this type of regimen.
Modification ofthe Host Immune Response to Prevent Recognition

and Response to Foreign Antigen Targets
One area of investigation that has the potential of contributing towards per-
manent survival of xenografts is modification of the recipient immune re-
sponse to prevent the recognition and response to foreign donor antigens. The
transplantation of solid organs and bone marrow between genetically differ-
ent individuals is limited by our ability to control the immunological recogni-
tion and rejection of the graft by the recipient. The induction of specific toler-
ance to a foreign graft, without compromising the ability of the host to
generate an otherwise effective immune response, has been the goal of a wide
variety of experimental studies.
Two types of experimental models with potential for clinical application
have been described recently. Both of these models depend upon the creation
of chimeric recipients through bone marrow transplantation, and the subse-
quent induction of tolerance to tissues from the bone marrow donor (Chap. 8).
One model involves whole body irradiation of the recipient followed by bone
marrow transplantation. Partial shielding of the long bones allows for recon-
stitution with both donor and recipient bone marrow cells [35]. The second
model is similar, consisting of lethal irradiation followed by bone marrow
transplantation with a mixture of both donor and recipient cells [36].
The achievement of mixed bone marrow chimerism results in the induction
of tolerance to donor tissues and maintenance of immune competence
through the presence of syngeneic lymphocytes that can be processed normal-
ly by the host thymus. In both systems, the chimeric recipients have been re-
ported to be tolerant to skin grafts of the donor while remaining capable of re-
jecting third-party skin grafts with normal vigor. These experiments have been
successfully extended to include mouse-to-rat skin xenografts. To date these
References 571
procedures have not been fully tested in larger species, but the potential to in-
duce bone marrow chimerism as a means of inducing tolerance to xenografts is
an area of active experimental interest and pursuit.
Modification of the Donor Graft Target Antigens to Escape Recipient

Recognition
The long-term solution to the problem of donor/recipient incompatibilitie~

may depend upon the identification of the antigens that serve as targets for the
xenograft reaction, followed by the permanent alteration of these targets to
prevent their recognition by the recipient. Identification of the xenograft tar-
get antigens may allow for the development of techniques that prevent their
expression in the graft, thereby avoiding the host immune response.
It has recently been demonstrated, for example, that transgenic mice ho-
mozygous for a ~rmicroglobulin gene disruption do not express class I histo-
compatibility antigens on the surfaces of cells [37]. This antigen is a major tar-
get for the allograft reaction, and failure to express the antigen can be
expected to eliminate an allograft reaction directed against this cell surface-as-
sociated protein. One of the most surprising aspects of these experiments was
the observation that the lack of expression of class I did not have an effect on
the embryological development of the transgenic offspring.
In the future, the isolation and identification of the antigens that are the tar-
gets for the xenograft response will allow for modification of specific donor or-
gans or for the creation of animals that do not express any of the relevant tar-
get antigens. It is conceivable, for example, that specific retroviruses could be
developed that would target specific organs for the insertion of host DNA,
antisense RNA, or modified (nonfunctional) donor genes. Alternatively, the
development of transgenic pigs with similar genetic alterations is possible,
providing the opportunity to breed stocks of animals with selected histocom-
patibility alterations. In both cases, the availability of xenograft organs could
eliminate the absolute need for human donor organs and allow for the imple-
mentation of organ transplantation as a routine scheduled surgery rather than
as an unpredictable emergency.
References
1. Dubernard, J.M., Bonneau, M., Latour, M. Heterografts in Primates. Simep Editions,

Villeurbanne, France, 1974.
2. Auchincloss, H. Jr. Xenogeneic transplantation. A review. Transplantation. 46, 1,1988.
3. Norman, J.e., Saravis, e.A., Brown, M.E., McDermott, W.V. Jr. Immunochemical ob-
servations in clinical heterologous (xenogeneic) liver perfusions. Surgery. 60, 179, 1966.
4. Hickman, R., Saunders, S.J., Goodwin, N.E., Terblanche, J. Perfusion ofthe isolated pig
liver with human blood.i. Surg. Res. 11,519,1971.
5. Abouna, G.M., Serrou, B., Boehmig, H.G., Amemiya, H., Marineau, G. Long-term sup-
port by intermittent mUlti-species liver perfusions. Lancet. 2,391, 1970.
6. Starzl, T.E., Marehioro. T.L.. Faris, T.D., McCardle. M.1 .. Iwasaki, Y. Avenues of future
research in homotransplantation of the liver. Amer. 1. Surg. 112,391,1966.
7. Starz!' T.E. Experience in Hepatic Tramplantation. Saunders, Philadelphia, 1969, p. 4011.
8. Giles, G.R, Boehmig, H.l., Amemiya. H., Halgrimson, CG., Starz!' T.E. Clinical het-
erotransplantation of the liver. Transplant. Proc. 2.506. 1970.
9. Starz!. T.E., Ishikawa. M .. Putman. CW .. Porter. K.A.. Picache. R. Husberg. B.S ..
Halgrimson, CG., Schroter, G. Progress in and deterrents to orthotopic liver transplan-
tation, with special reference to survival, resistance to hyperacute rejection, and biliary
duct reconstruction. Transplant. Proc. 6, 129. 1974.
10. Porter, K.A. Pathology of the orthotopic homograft and heterograft. In: Experience in
Hepatic Transplantation. T.E. Starzl (ed.), W.B. Saunders. Philadelphia. 1969. p. 422.
11. Makowka. L.. Chapman, F.A.. Cramer, D.V .. Quian. S.G .. Sun, H., Starzl, T.E. Platelct-
activating factor and hyperacute rejection. The effect of a platelet-activating factor an-
tagonist, SRI 63-441. on rejection of xenografts and allografts in sensitized hosts.
12. Miyagawa, S., Hirose, H., Shirakura, R, Naka, y', Nakata, S .. Kawashima, V .. Seya, T ..
Matsumoto, M .. Venaka. A.. Kitamura, H. The mechanism of discordant xenograft re-
jection. Transplantation. 46,825,1988.
13. Starzl. T.E .. Demetris, A.l. Liver Transplantation: a 3I-year Perspective. Year Book
Medical Publ., Inc., Chicago, 1990.
14. Monden, M .. Valdivia, L.A., Gotoh, M., Hasuike. Y., Kubota, N .. Kanai, T., Okamura,
1., Mori, T. Hamster-to-rat orthotopic liver xenografts. Transplantatiun. 43.745.1987.
15. CaIne, RY. Organ transplantation between widely disparate species. Transplant. Proc.
2.550, 1970.
16. Reemtsma, K. Renal heterotransplantation from non-human primates to man. Ann.
N. y. Acad. Sci. 162,412, 1969.
17. Eiseman, B., Lam, D.S., Raffucci, F. Heterologous liver perfusion in treatment of he pat-
icfailure. Ann. Surg. 162,329, 1965.
18. Ham, 1.M., Pirola, RC. Davidson, RM .. Ayrrow, S .. Elmsliz. RG. Pig liver perfusion
for the treatment of acute hepatic coma. Surg. Gynecol. Obstet. 127,453, 1968.
19. Shons, A.R, Najarian, 1.S. Xenograft rejection mechanisms in man. Trans. Am. Soc.
Artif Intern. Organs. 20,562,1973.
20. Edwards, N., Ott, G., Berger, C, He, X., Teppler, I., Copey, L., Smith, C, Reemtsma, K.,
Rose, E. Incidence of preformed antibodies against potential xeno-donors in human
serum. Transplantation. 49, 1022, 1990.
21. Platt, 1.L., Vercellotti, G.M .. Lindman, B.l., Oegema, T.R lr., Bach, F.H., Dalmasso,
A.P. Release of heparan sulfate from endothelial cells. Implications for pathogenesis of
hyperacute rejection. 1. Exp. Med. 171, 1363, 1990.
22. Otte, K.E., Andersen, N., lorgensen. K.A., Kristensen, T., Barfort, P., Starklint, H.,
Larsen, S., Kemp, E. Xenoperfusion of pig kidney with human AB or 0 blood.
23. CaIne, RY., White, H.l.0., Herbertson, B.M., Millard, P.R, Davis, D.R, Salaman,l.R.
Samuel, 1.R Pig-to-baboon liver xenografts. Lancet. 1. 1176, 1968.
24. CaIne, RY., Davis, D.R., Pena, 1.R., BaIner, H., De Vries, M., Herbertson, B.M ..
Millard, P.R, loysey, V.C, Seaman, M.l., Samuel. 1.R, Stibbe, 1., Westbroek, D.L.
Hepatic allografts and xenografts in primates. Lancet. 1, 103, 1970.
25. Knoop, M., Steffen, R, Orlof, M.S., Cramer, D.V. Overview: The importance of hepatic
artery reconstitution in orthotopic liver transplantation. Submitted for publication.
26. Gordon, RD., Iwatsuki. S., Esquivel, CO., Tzakis, A., Todo, S., Starzl, T.E. Liver trans-
plantation across ABO blood groups. Surgery. 100,342. 1986.
27. Gordon, RD .. Fung, 1.1., Markus, B., Fox, I.. Iwatsuki. S., Esquivel. CO., Tzakis, A.,
Todo, S .. Starzl. T.E. The antibody crossmatch in liver transplantation. Surgery. 100,705,
1986.
28. Kamada, N .. Davies, H., Roser, B. Reversal of transplantation immunity by liver graft-
ing. Nature. 292,840, 1981.
29. Knechtle. S.l., Kolbeck, P.C, Tsuchimoto, S.. Coundouriotis, A., Sanfilippo, F.,
Referenccs 573
Bollingcr, RR. Hepatic transplantation into sensitized recipients. Transplantation. 43, H,
1987.
30. Demetris, A.J., Jaffe, R, Tzakis, A., Ramsey, G., Todo, S., Belle, S .. Esquivel, c.,
Shapiro, R. Markus, B., Mroczek, E. Antibody mediated rejection of human orthotopic
liver allografts: a study of liver transplantation across ABO blood group barriers. Am. 1.
Pathol. 132,489, 198K
31. Merion. RM., Colletti, L.M. Hyperacute rejection in porcine liver transplantation. I.
Clinical characteristics, histopathology, and disappearancc of donor-specific lympho-
cytotoxic antibody from serum. Transplantation. 49,861,1990.
32. Makowka, L.M., Chapman, F., Cramer, D .. Sher, L., Podesta, L.. Howard, T.. Starzl, T.E.
The role of inflammatory reactions in xenotransplantation. In: Xenograft 25. Hardy.
M.A. (cd.), Elsevier; Amsterdam, New York, Oxford, 1989, p. 159.
33. Valdivia. L.A., Monden. M., Gotoh, M., Nakano, Y., Tono, T., Mori. T. Evidence that
deoxyspergualin prevents sensitization and first-set cardiac xenograft rejection in rats by
suppression of antibody formation. Transplantation. 50. 132, 1990.
34. Knechtle, S.J., Halperin. E.C., Saad, T., Bollinger, R.R. Prolonged heart xenograft sur-
vival using combined total lymphoid irradiation and eyclosporine . .1. Heart Transplant. 5.
254. 19H6.
35. Slavin, S., Strober. S .. Fuks, Z., Kaplan, H.S. Induction of specific transplantation toler-
ance using fractionated total lymphoid irradiation in adult mice: long-term survival of
allogeneic bone marrow and skin grafts. 1. Exp. M ed. 146,34, 1977.
36. Ilstad, S.M., Sachs, D.H. Reconstitution with syngeneic plus allogencic or xenogeneic
bone marrow leads to specific acceptance of skin allografts or xenografts. Nature. 307,
168,19H4.
37. Zijlstra, M., Bix, M., Simister, N.E., Loring, J.M., Raulet, D.H., Jaenisch. R ~2-micro
globulin deficient mice lack CD4· K+ cytolytic Tcells. Nature. 34,743,1990.
Subject Index
ABO blood groups (See "Blood groups") cynomolgus-to-rhesus 372

Accommodation (See also "Adaptation" fox-to-dog 350,353
and "Anergy") 70,81,82,84,150,392 guinea pig-to-rat 57,61,149,211,314,
Acetyl glycerol ether phosphorylcholine 315,316,381,564
226 hamster-to-mouse 314,319
Acquired immunodeficiency syndrome hamster-to-rat 140,149,153,165,314,
(AIDS) 306 318,323,333,564,570
Acquired immunodeficicncy syndrome- lamb-to-newborn goat 356
like viruses, in non-human primates 566 macaque-to-baboon 453
Actinomycin C 12,14,532,535 mouse-to-rat 314,564
Actinomycin D 536 pig-to-baboon 55,233,234,239,243,
Action constraints, in medical ethics 520 244,390,392
Adaptation (See also "Accommodation" pig-to-dog 243,244
and "Anergy") 392 pig-to-rat 55
Adenoviruses 465 pig-to-rhesus 73,83
Adhesion molecules, different species 435 rabbit-to-newborn pig 172
Adrenal medulla 305 rat-to-mouse 29,314,319,335
Adsorption, of antibodies (See" Antibody vervet monkey-to-baboon 234,238,
adsorption ") 368,371
Afferent arm, of xenograft immune re- kidney 314
sponse 144 cat-to-dog 345,347,355,358
Agarose microcapsules 301 cat-to-rabbit 226
Agglutination 504,505 dingo-to-dog 341,351,353,360
ALG (Sce "Antilymphocyte globulin") dog-to-wolf 355
Alginate-polylysine capsules 278,298, fox-to-dog 341,345,350,351,359
300,302,304 guinea pig-to-rat 314
Allogeneic, definition 181 hare-to-rabbit 164,183
Allograft 3 lion-to-dog 345
ALS (See "Antilymphocyte serum") mousc-to-rat 314
Alzheimer's disease 306 pig-to-baboon 83,390,391
AMS (See "Antimacrophage serum") pig-to-dog 54,92,345
Anergy (See also "Accommodation" and pig-to-goat 355
"Adaptation") 85,392 pig-to-rabbit 184,208
Anesthesia, in rodent xenografting 142 rabbit-to-cat 184,377
Animal models ofxenotransplantation rabbit-to-pig 184
hamster-to-rat 165,331,324 sheep-to-dog 345
hare-to-rabbit 164 sheep-to-goat 356
macaque-to-baboon 449 tiger-to-dog 345
pig-to-dog 91 wolf-to-dog 163,341,345,351,354,
pig-to-rhesus 71 360
sheep-to-dog 91 liver
heart 113 baboon-to-pig 264
calf-to-goat 356 cynomolgus-to-rhesus 264
cynomolgus-to-baboon 370 dog-to-pig 264
576 Subject Index
Animal liver xenogeneic 56,398,400,406,420

guinea pig-to-rat 265,266,268,314 xenohemagglutinating 348,349
hamster-to-rat 149,266,267,314,319, xenoreactive 26,29
564 Antibody (see also "Antibodies")
pig-to-baboon 264,389,390,392,567 adsorption 82,89,93,507
pig-to-chimpanzee 264,390,567 assay 70,90
pig-to-dog 264 binding 73,84
pig-to-rhesus 264,390 depletion 171,316
lung elimination 25, 170
cat-to-dog 356 rebound 171
pancreatic islets sorbents 89,90,170,171,239,342,391,
fish-to-rat 272,292 483,556,563
hamster-to-mouse 301 Antibody-dependent cell-mediated cyto-
pig-to-rat 290 toxicity (ADCC) 30,52,60,104,134,
skin 144,151,283,331
calf-to-goat 356 Antibody-mediated graft injury 48,54
dingo-to-dog 354,360 Antigen
fox-to-dog 353 ABH, of nonhuman primates 439
hamster-to-rat 140,149,323 ABO blood group 70,82
mouse-to-rat 48,570 canine erythrocyte (CEA) 340,341,342
rat-to-mouse 29,53,141 cross-reactive 102
sheep-to-goat 356 dog leukocyte (D LA) 340,341
wolf-to-dog 354,355,360 donor, recognition of 570
Animal rights 290,513,516,556 endothelial cell 71,82,84
Animal rights groups 7,471,508,520 Forssman 58
Animal utilization 516 fox leukocyte (FLA) 341
Antibodies (see also "Antibody") Hanganutziu-Deicher 58
allo 24 heterophile 85
anti-ABO 57,70,82,93,133,555 in baboon 367
antidonor 139,149,344,563 injectable in antibody "neutralization" 93
antiendothelial cell 54,132 minor histocompatibility 127
anti-HLA 6,70,83,88 Paul-Bunnell 58
anti-idiotypic 90 recognition and alteration 571
antipig 501,507 sharing, species 56
anti species 555,556 synthetic 93
auto 71 xeno 32,58,103110,111,570,571
complement-fixing 60,349 Antigen-antibody (immune) complex 93,
cross-reacting 385,563 233
cross-species 56 Antigen-antibody reaction in discordant
cytotoxic 129,371,539 xenografting 385
dog leukocyte 340 Antigen-bearing cells, depletion of 280
Forssman 54,56,58,60 Antigen presenting cells (APCs) 102,103,
Hanganutziu-Deicher 56 106,110,122
heterophile 56,445 Antilymphocyte globulin (ALG) 95,281,
in chronic rejection 53 346,349,350,351,355,562,563
induced 26,29 Antilymphocyte serum (ALS) 276,279,
lymphocytotoxic 127,132,556 390,391
natural preformed 4,5,24,25,26,56,57, Antimacrophage serum (AMS) 346,350,351
60,69,70,71,72,81,84,86,91,131, Antisera
132,133,134,225,314,339,344,347, cat 346
385,395,420,432,448,483,507,556, dog 346
560,561,564,565,566 Forssman 54
non-complement fixing 52 horse 56
Paul-Bunnell 56 Paul-Bunnell 58
polyreactive 71 sheep 56
species-specific 385 wolf 346
Subject Index 577
Antithymocyte globulin (ATG) 29,139, isoenzymes 343
238,282,319,346,348,351,371,483,554, leukocyte antigens (DLA) 340,341
555 MHC 340
Arthus-like reaction 53 preformed natural antibodies 344
Artificial membranes 297 physiological markers 340
Atherosclerosis 5,54 serum proteins 340,343
Autogeneic, definition 181 Carbohydrate determinants 225
Azathioprine 12,14,238,305,391,532,535, Carboxylesterases, different species 433
554,555,562,563 Cardiac xenotransplantation, in man 541
in infants and children 555
B cells 60,70,88,95,555 Carnivores 340
BN 52063 267 "Carotid syndrome" 58
Baboon (See also "Nonhuman primates" Cat (See "Animal models")
and "Animal models") 7,162,239,367, Catalase, different species 434
531,532,549,550,553,554,555 Cell-mediated Iympholysis (CML) 102,
"Baby Fae", heart transplant 24,26,29,511 104,105,
Bailey, Leonard 553 Children as recipients 7,555,562
Barnard, Christiaan 549 Chimera 32, 122, 207,570
Benefits, xenotransplantation 6,559 Chimerism 122,125,130,131,132
Bioartificial Chimpanzee (See also "Nonhuman pri-
devices 297 mates" and "Animal models") 7,10,12,
pancreas 303 59,161,208,444,531,532,544,549,551,
parathyroid 307 555,561,562,563
Bioencapsulation (See "Encapsulation") Chiron 207
Blood groups Chitosan - alginate microcapsule mem-
ABO 6,12,27,367,439,451,532 branes 300
compatibility testing 451 Cholesterol, different species 432
different animals 431,432 Cholinergic neurons 306
incompatibility 70,82,88,93,238,256, Chromosomal analysis, canines 107,108,
257,367,485,538,553,554,555,568 343
MN system 443 Coagulation 51,81,131,173,384,395,435,
in nonhuman primates 12,367,439,443, 560
444,451,532 Cobra venom factor 53,61,85,166,318,
of pigs 484,502 384
Rh-hr system 444 Collagenase 276
Simian-type 368,444 Colloid osmotic deregulation 50
Blood group substances 391 Columns, staphylococcal protein 83,89
Bone marrow cells 28,32, 122 Competing rights 515
Bone marrow transplantation 121,123, Complement 49,73,316,317,348,503,554,
133,570 563
Brain, xenograft site 277 activation 49,51,60,61,69,73,75,81,
Brain death, concept of 541 84,131,211,225,316,323,384,395,400
"Bridging" by cardiac xenotransplant 7, alternative pathway 49,61,74,85,211,
548,552,553,555 316,560,564
Brockman bodies 290 cascade 52,167,483
classical pathway 49,60,73,316,560
C-peptide 282 deficiency of sixth component, rabbit
C3 deposition 21l 85,232
C6 deficiency, in rabbits 85,232 depletion 25,61
Caine, Sir Roy 69,313 deposition 255,267,406
Canine (See also "Animal models") 339 different species 435
as xenograft model 163,339,340 effect of cobra venom factor 85
chromosomal analysis 343 inhibition 83,85,86,173,384
erythrocyte antigens (CEA) 340,341, system 60
342 Complement-fixing antibodies 60,346,
immunogenetic markers 340,345 349
578 Subject Index
Complement-mediated cytotoxicity 127, discordant 5

134,503 disparate 4
Concordant xcnotransp1antation 4,5,25, evolutionary aspects 433
47,162,166,181,182,183,194,238,263, genetic engineering 86
267,313,318,331,339,560 modification of tissues 174
Congenital cardiac malformations in the nonhuman primates as donors for man
pig 486 12,448
Consent for xenotransplant operation organ function 6
511,543 organs, shortage of 3,24,512,559
Consequences in medical ethics 512 physiological considerations 430
Cooley, Denton 547 pig as donor for man 481
Corticosteroids 12,14,238,350,371, 391, selection of 532,565
532,535,553,554,555,562,563 Drug toxicity in rodent xenografting 143
Cross-reactivity, serologic 59 Duties in medical ethics 51
Cyclophosphamide 88,316,332
Cyclosporine 238,282,305,316,319,323, Economic consequences of xenotransplan-
331,332,350,352,391,484,553,554,555, tation 517
569 Efferent arm of xenograft immune re-
Cynomolgus monkey (Sec "Nonhuman sponse 49,144
primates" and "Animal models") Electronmicroscopy (See
Cytokines 54 "Ultrastructure ")
Cytolysis 51 Embryo, immunologically incompetent 47
Cytotoxic T lymphocytes (CTL) (See also Encapsulation 278,288,297,304,307
"Tcells") 104,107,108,110, II L 151, Endothelial cell
298 activation 51,69,74,81,84
Cytotoxicity 127, 151 antigen expression 91
antigen recognition 71
Daedalus 9 desensitization 85
Darwin 429 hyperplasia 185
Data in medical ethics 518 injury 53,212
Decay accelerating factor 85 integrity 225
Decision-making in medical ethics 518 loss of integrity 225
Decision theory in medical ethics 521 memhrane associated proteins 85
15-deoxyspergualin 29,30,95,139,238, membranes 86
282,319,352,371,372,483,555 pig 504,567
Diabetes 275,302 surface antigens 84
Diffusion chamber 297 vascular 144
Dingo (See also "Animal models") 340, vesiculation 75
353 Endothelial-leukocyte adhesion molecules
Discordant xenotransplantation 4,5,25, (ELAM) 74
47,55,81, 170, 174, 181, 182, 184,197, Enteroviruses 464
207,239,263,265,275,290,313,314,318, Enzyme replacement therapy 304
339,406 Enzymes, different species 395,433
Dithiol-reducing agents 94 Eseulapius 207
Dithiothreitol 94 Ethical framework regarding xenotrans-
DNA technological methods 204 plantation 518
Dog (See "Canine" and "Animal models") Ethical issues in xenotransplantation 511,
Domestication of animals 430 522
Donor Ethics of using nonhuman primates as
anatomical characteristics 430 donors for man 566
antigen recognition and alteration 571 Euglycemia (See "Normoglycemia")
biochemical considerations 433 Extracorporeal immunoadsorption of anti-
blood parameters 431 bodies (See "Antibody adsorption ")
closely related 4 Ex vivo perfusion of an organ (Sec also
concordant 5 "Organ perfusion" and "Xenoperfu-
defining a suitahle donor for man 429 sion") 33,57, 185,507,508,560,561, 568
Subject Index 579
F( ab)2 fragment 53,57 clinical attempts at xenotransplantation
Fc fragment 49,89 162,531,541,559
Fc receptor 52 heart, baboon-to-man 24,243,247,
Fibrin 81,245 365,549,553
Fibrinolytic system activation 384 heart, chimpanzee-to-man 20,243,
Fibrotic response 300 247,365,541,549,551
FK 506 5,95,139,282,372,483,555,569 heart, pig-to-man 548
Flow cytometry 334,504 heart, sheep-to-man 547
Fox (See also "Animal models") 340 kidney, baboon-to-man 22,365,531
Fox leukocyte antigens (FLA) 341 kidney, chimpanzee-to-man 14,20,
Freund's adjuvant 346 365,531,535
Fulminant hepatic failure 304 kidney, monkey-to-man 531
Function in a xenogeneic environment 6, liver, chimpanzee-to-man 264,265,
33,34,433,494 561
defining a suitable donor 429,531
Genetic distance between donor species early attempts at kidney transplantation
and man 564 9,10,568
Glycoproteins 60,84,225 immunological studies following renal
Gnotobiotic (germ-free) animals 6,482, xenotransplantation 537
491 nonhuman primates as donors for man
Goat (see also "Animal models") 568 448
Graft-versus-host disease (GVHD) 121, recipients of renal xenotransplants, se-
123 lection and preparation 531
Growth factors, species-specific 131 Hume, David 20
Guinea-pig (See "Rodent" and "Animal Hyperglycemia 282,302
models") Hyperparathyroidism 307
Hypersensitivity reactions 30
Hamster (See "Rodent" and" Animal H ypogammaglobulinemia following anti-
models") body adsorption 89
Hardy,James 20,541,543 Hypoglycemia 282
Heart (See also "Animal models" and Hypoimmunogenicity 289
"Human") 541,542
orthotopic transplantation 369,553 Icarus 9
heterotopic transplantation 243,369, Immune (antigen-antibody) complex 93,
548,549,551 233
surgical technique in rodents 324 Immune reactivity 279
xenograft model 113,314,369 Immune response
Hemagglutinin 538 afferent phase 144
Hemophilia 306 cellular 103,105,110
Heparan sulfate proteoglycan 51,76,225, destructive phase 298
567 effector phase 102, 104, 144, 298
Hepatitis viruses 464 induction phase 105,112
Hepatocyte 304 modification 288,570
Herpesviruses 461 to allografts 101
Heteroagglutinins 538 to xenografts 101,148
Heterograft 3 Immune sera, preparation 346
Histopathology (See "Morphology") Immunity 30,110,129
Homograft 3 Immunoadsorption of antibodies (See
Homologous restriction 85 "Antibody adsorption")
Hormone deficiency states 307 Immunoalteration 278,288
Hormone replacement therapy 307 Immunobiology of xenografts 139, 148
Hormones, different species 434 Immunocompromised patient, viral infec-
High-performance liquid chromatography tion 461
(HPLC) 72,282 Immunofluorescence 59,182,189,200,
Human 202,207,209,396,406,554
anti pig antibodies 501 Immunogenetic markers 345
580 Subject Indcx
Immunogenicity 279,280 Kidney homogenate, pig 94

Immunoglobulins 26,57,61,89,334,539, Killercells 30,104.130.146.150.151,283.
568 298.331
IgA 49,209,212,223,226,554 Kupffercells 314
IgE 49
IgG 49,56,60,6 I, 83, 84,iN, 91, 209, 212, Lactate dehydrogenase (LDH), different
223,226,255,334,398,406,539,554 species 433
IgM 49,57,60,61,83,84,89,91,94,132, Lamarque 429
211,223,226,316,334,398,406,507, Lion 340.345
539,554,566 Liver (See also "Animal models" and
Immunohistochemistry 168,204,209 "Human") 4.55,253,314.559
lmmunoincompetence 122 allograft rejection 253,560
Immunoisolation 277,288,297 auxiliary heterotopic xenotransplanta-
Immunological monitoring in discordant tion 560
xenografting 483 choice of donor for man 563
lmmunomodulation 289,370,570 chronic disease 306
Immunoprotection, humoral 298 ex vivo perfusion 33,560
Immunosuppression 569 function following xenografting 268
of concordant xenografts 161, 162,238 heterotopic transplantation 561
of discordant xenografts 170,174,239 in sensitized recipient 255
pharmacologic 5,29,95, 139, 141, 155, metabolism in different species 432
238,239,318,324,350,370,391.531. orthotopic transplantation 562
551.562,569 relative resistance to antibody-mediated
Immunosuppressive modalities 155,166 rejection 27,56,258,568
Inflammatorycascade 316.318 relative weights 430
Inflammatory mediators, inhibition 173,569 role of its large mass in inducing toler-
Influenza viruses 465 ance 564
Insulin 286,298 transplantation for hemophilia 306
Intercellular adhesion molecule (ICAM) transplantation in children 562
31, 107 transplantation in man 559
Interferon-gamma (IFN - y) 298 xenoagglutination in man 560
Interleukin-1 (IL-l) 106,298,300 Lung 356
Interleukin-2 (IL-2) 102,106 Lymphocyte function associated antigen
Intrathecal space 277 (LFA) 31. 107
Intrathymic space 277 Lymphokine activated killer (LAK) cells
Irradiation (See "Killer cells")
in combination with cyclosporine 570
in production of chimerism 570 Macrophages 84, 104
of graft 14,535,536 Major histocompatibility complex (MHC)
total body 32, 121,331.324.531 31,103,106,108,109,110,122,161,184,
total lymphoid (TLI) 139.156,268,280, 340
282,319.331,332,335,371,483 Man (See "Human")
ultraviolet (UY) 288 Marion, Pierre 549
Islets (See "Pancreatic islets") Mechanisms of graft injury 48.49
Isoagglutinins 538.554 Medawar, Sir Peter 3
Isoenzymes in canines 343 Membrane associated proteins 85
Isogeneic. definition 181 Membrane attack complex (MAC) 50,74,
Isohemagglutinins 432 85,316
Isolation-perfusion chamber 276 2-Mercaptoethano] 94
6-Mercaptopurine 531
Jaboulay 9,11 Metabolic compatibility of organ and host
6,33,34,433,494
Kallikrein-kinin system, activation 384 Methylprednisolone 238,350,553,554
Kangaroo as donor for man 430 MHC (See "Major Histocompatibility
Kidney (See also "Animal models," Complex")
"Human" and "Renal") 531 Mice (See "Mouse")
Subject Index 581
Microcapsule 300,301 Organ perfusion (See "Ex vivo perfusion"
Microencapsulation (See "Encapsula- and "Xenoperfusion")
tion") Organs, artificial 3
Microscopy, electron (See
"Ultrastructure ") Pancreas 55
Microscopy, light (See "Morphology") Pancreatic islets 28,32,55, 113, 144,275,
Minor histocompatibility antigens 127 299,300,302,303
Mixed lymphocyte reaction (MLR) 31, Papillomaviruses 466
102,105,342,484,553,562 Papovaviruses 466
Models, animal (See "Animal models") Parainfluenza viruses 465
Monkey (See "Nonhuman primates") Parathyroid cells 307
Monoclonal antibody (MAB) 123,128, Parkinson's disease 305
280,281,282,302,303,331,332,371, Parvoviruses 467
387 Penicillamine 94
Morphology, Perfusion of an organ (See "Ex vivo perfu-
following ex vivo perfusion of rabbit kid- sion" and "Xenoperfusion")
neys with human blood 185 "Phenotypic-modulation" 54
of cardiac rejection 168,231,232,233, Photochemotherapy 371
314,335 Phylogenetic disparity 7,69,232,356
of cardiac xenografts in man 546, 550, Phylogeny 564
551,552,554 Pig 70,71,72,73,90,91,94, 102, 106,254,
of kidney rejection 168,181,183,184, 255,481,504,566
185,197,207,209,348,406,504 as organ donorfor man 4,6,9,71,278,
ofliver rejection 184,253,257,261,264, 430,481,482,485,494,495, 501,541,
265,267,389,390 548,556,561,566
ofliver xenografts in man 562,563 blood groups 484,502
of renal xenografts in man 504,537 congenital cardiac anomalies 486
of xenograft rejection 59,154,165 diseases affecting major organs 485,
preparation of tissues 182, 191,209 489,490,492
Mouse (See also "Rodent") 102,103,139, gnotobiotic (germ-free) 482,488,489,
146 491
chimerism 32, 125 histocompatibility systems 501
specific pathogen free (SPF) 133 infection transferrable to man 489
Mycophenolic acid 5,372 neoplastic diseases 492
Mythology 9,207 zoonoses 489,490
Plasma exchange 83,86,88,313,316,483,
Natural killer (NK) cells (See "Killer 507,555
cells") Plasmapheresis 25,82,83,171,391
Neonates, immunologically incompetent Platelet activating factor (PAF) 52,74,
47 173,386,401
Neoplastic disease in the pig 492 Platelet-derived growth factor (PDGF)
Neovascularization of skin graft 27 54,300
Neuhof, Harold 9,13 Platelets 51,52,62,91
Neural tissue (fetal) 305 aggregation 196,197,381
Nerve growth factor (NGF) 304,306 antigen expression 91
Nonhuman primates 6,7,163,255,365, inhibitors 172
389,448,449,472,561,566 thrombi 81,245
as donor for man 9,239,339,420,430, Polyacrylate microcapsule membranes
439,448,457,517,532,541,549,551, 301
553,555,559,565 Polyethylene imine (PEl) 298
blood groups 367,368,439 Polylysine - alginate capsules (See
viral pathogens (See also "Viruses") "Alginate")
457 Porter, Kendrick 22
Non-Tcellcytotoxicity 151 Prednisone 562,563
Normoglycemia 282,284,290,298,301, Primate (See "Non-human primate")
303 Princeteau 9,10
582 Subject Index
Properdin 50 for insertion of host DNA in donor organs

Protein, staphylococcal 83,89 571
Proteins Rheumatoid factor 71
in canines 343 Ricordi. Camillo 289
incompatibility 395 "Right to life" 517
Proteolytic enzymes 433 Rights in medical ethics 515
Public opposition to xenotransplantation Rodents (See also "Mouse" and "Rat")
4, 7 139,313,323,331
Publications on xenotransplantation 23 anesthesia 142
drug toxicity 143
RS 61443 5,95,372,555 immunobiology 139,148
Rabbit (See "Animal models") immunodeficient strains 33,139,145
Rapamycin 5,95,372,555 immunohistochemical analysis 168
Rat (See also "Rodent") 103,139,145, immunosuppressive modalities 155
254,255,332 killer cells 150
Recognition non-Tcell cytotoxicity 151
direct 109 pancreatic islet 144
associative 109 role of spleen in rejection 153
Rejection skin grafting 140,142,143
accelerated 29,254,323 strain specificity 148
acute (cellular) 4,5,28,101,103,110, therapy for rejection 155
181,194,231,253,254,259,261,320, in xenografting 140,340
323,339,483,560 Ross, Donald 548
alternative mechanisms 30 Ruminants 568
chronic 5,53,127,181,262
crisis 326 Schwartzman-like reaction 53
CTL-independent 111 Selection of donor for man 532
discordant, difference from sensitized al- Selection of recipients for renal xenotrans-
lografts 384 plantation 531
effects of formed elements 410 Sera, immune 48,346
first-set 4,347 Serum, sheep 91
heart 231,233,243,551,552,554 Sheep (See also "Animal models" and
hyperacute (antibody-mediated, hu- "Human") 3,4,541,568
moral, vascular) 4,5,24,26,27,28,47, Sialic acid, effect on antibody binding 84
48,53,56,60,61,69,70,74,75,81,84, Simian (See "Nonhuman primates" and
111,150,181,194,197,207,209,225, "Viruses")
231,232,233,239,254,314,317,320, Skin (See also "Animal models") 55,139,
323,337,358,377,383,386,395,398, 140,143
400,405,482,501,555,560,564 Sorbents, antibody 89,90,95
kidney 181,194,196,197,207,381,536, Sources of infection in nonhuman primates
563 459
liver 253,254,256,257,258,259,261, Species
262,264,265,267,560,568,569 closely-related 232,281,313,339,365
mixed 5, 196, 238 disparate 232,275,281,313,377,382,389
pathogenesis 69,383,384 Specific pathogen free (SPF) animals 133
second-set 4,347 Spleen in xenograft rejection 153
Renal xenografting Splenectomy 88,93,95,239,267,268,282,
diagnosis of rejection in man 536 313,316,319,325,391
immunological studies in man 537 Splenomegaly 319
postoperative management in man 535 Staphylococcal protein A 83,89
results in man 535 Staphylococcal protein G 89
surgical techniques 13,14,377,532 Starzl, Thomas 20,22,562
Resource allocation 520 Stem cell engraftment 123
Respiratory pigments, different species Strain specificity in xenotransplantation
436 148
Retroviruses 467 Streptozotocin (STZ) 281,298
Subject Index 583
Stroma, pig erythrocyte 90,94 Ultraviolet (UV) irradiation 288
Subarachnoid space 277 Unger, Ernst 9,12
Substantia nigra neurons (fetal) 305 Ungulates 340,356
Support systems 518 University of Colorado 531,532
Surgical tcchnique University of Mississippi Medical Center
cardiac xenografting 324,369 541
renal xenografting 14,377,532
Swine (See "Pig") 490 Values in medical ethics 519
Swine leucocyte antigen (SLA) 501 "Vascular catastrophe" in xenograft rejec-
Synthetic blood group antigens 93 tion 381
Vascular lesions 75
T-cells Vascular resistance 410,567
activation 31 Vascular wall
CD4+ 29,30,31,103,104,105,106 inflammation, renal 1X5
CD8+ 31,103,104,105,279 necrosis, renal I X5, 190
depleted bone marrow 121, 122 Vascularization of rodent skin xenografts
depletion 166 143
donor-reactive 127 Vervet (African green) monkey (See
helper 30,102,106,110 "Nonhuman primates"
receptor 107 and "Animal models")
response 103 Virus transmission
subsets 31,334 horizontal 459
Thrombi vertical 459
fibrin 81 Viruses in nonhuman primates 370,443,
platelet XI 448,449,457
Thrombocyte aggregation IX5,384 Viscosity of blood 430
Thrombosis
intravascular 233 Western blot 72
renal 190 "White" graft failure, skin 55
Thymectomy 29 Wolf (See also "Animal models")
Thyroid 279 340
Tiger 340,345
Tolerance 32,33,57,121,123,130,133, Xenoantibodies (See" Antibodies")
285,570 Xenoantigens (See" Antigens")
Toxoplasma 6 Xenogeneic, definition 5, 181
Transfection 105,107 Xenograft 3
Transfusion, pretransplant 350,352 Xenoperfusion (See also "Ex vivo perfu-
Transgenic sion") 395,405,507
animals 174,556,571 animal models 9,90,91,208,348,395,
methods 105 396,397,400,407,409
models 107 apparatus 396,397,399
Tubular necrosis, renal 190, 191, 197 blood chemistry 399
Tulane University 10,531,532 historical background 396
Tumor necrosis factor (TNF) 298 methodology 395,406
Xenoresistance 131
Ultrastructure Xenospecificity, elimination by absorption
cardiac 243,247, 24X, 554 342
preparation of tissues 182,209,243
renal 195,207,209,213 Zoonoses, pig 489,490

Xeno Transplantation

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Xeno Transplantation

Uploaded by

Copyright:

Available Formats

D.K.C. Cooper E. Kemp K.

D.l.G. WHITE PH. D.,MRCPATH.

ISBN-13: 978-3-642-97325-3 e-ISBN-13: 978-3-642-97323-9

The cover of this book depicts a Lamassu, one of the "fabulous"

Fig.I. Homer described the Chimera as consisting of a lion's foreparts , a goat

more benign and desirable character than many of its mythologi-

Oklahoma, May 1991 DAVID K.c. COOPER

1. Hargreaves, J. Bestiary. Gothic Image Publications, Glastonbury, U. K.,

There was considerable clinical and experimental activity in the

Fig. I. Keith Reemtsma headed the group at Tulane University in New

search in xenotransplantation. Such promotion seemed to us to be

Our knowledge of the subject, however, is expanding rapidly

Oklahoma. May 1991 DAVID K.C. COOPER

1. Bannett, A. D .. Brynger. H .. McAlack. R. F.. Breimer, M. E .. Samuelson.

May1991 DAVID K.c. COOPER

Subject Index ........................................ 575

BINNS R M., M. A, Vet. M. B., Ph. D., M. R C. V. S.

MARINO LR, M.D.

MARQUET R. L., Ph. D.

SOON-SHIONG P., M. D., FRCS (C)

TAUBE D. H., M. A, M. B., FRCP

VAN DEN BOGAERDE 1. B., M. B., B. Ch., Ph. D.

WELSH K.I., Ph. D.

WHITE D. 1. G., M. A., Ph. D., MRCPath

WIGHT, D. G. D., M. D., MRCPath

WRIGHT R. A., Ph. D.

In regard to organs such as the liver, whose complexity is unlikely to be dupli-

antibody mediated, remains largely unknown, though experience in the realm

Xenotransplantation - A Brief History

The Mythology of Transplantation

Early Attempts at Renal Xenografting

Early in the twentieth century reports on cross-species grafting (then called

Y. Greffe rGnale. ~c..O\J"!.J -;, .. J ! J..........(

demonstrates that thrombosis or hemorrhage at the anastomosis is not in-

The Tulane University Chimpanzee-to-Man Renal Xenograft

In our renal allografting program at Tulane University in New Orleans, we had

BULLETIN DU LYON MEDICAL

(;R£"" DF. RF.IN!I At: PI.I DU COt;D" PAR 1I0UDURU

J'ai pratique I" greffd r~n .. le pllr luturea valcn).irct, dellE

Fig, 2,2, Report of Jaboulay's attempts at renal heterotransplantation

As this impasse was developing, we decided to explore the use of nonhu-

:\w; del' exp(:ril!lclltell-l,iologischen AI,teilllng des

Dr. Era., Cng.r· B.,lio.

The Chimpanzee as Donor

WITI( Til ..: (,OLL.AHOf( ... T I U~ U t>'

S A)It:~; L 1I1R ~ IIFELD. )1.0.

])J:;A~ LEWIS. A.D., )1.1>. Et:GENE H. POOL. A.B.• )[.D.

ARTHlR W. ELTI;\G. A.H .. )I.D.

D. APPLETO~ AND CO~lPA~Y

In each instance the donor received general endotracheal anesthesia with

A 43-year-old former dock worker with a history of hypertension since 1957

:rl L......... .............. . ~

Fig. 2.7. Post-transpla nt intra-

8 mllmin. There was no improvement with dietary management, and peri-

Fig. 2.8. Serial renograms post-transplant in Case 1

Fig. 2.9. Hemagglutination studies post-transplantation in Case 1

Fig. 2.10. Macroscopic appearance of one kidney at autopsy in Case I

Fig. 2.11. Photomicrograph of kidney at autopsy in Case I

A 23-year-old school teacher was admitted in November 1963 with chronic

Subsequent Clinical Studies

Fig. 2.14. Post-transplant intra-

Fig. 2.15. Hemagglutination studies post-transplant in Case 2

Dr. Tom Starzl began a series of baboon-to-man renal transplants which