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11 TH
Sleisenger and Fordtran’s EDITION
Gastrointestinal
and Liver Disease
PATHOPHYSIOLOGY | DIAGNOSIS | MANAGEMENT
EDITORS ASSOCIATE EDITORS

MARK FELDMAN, MD RAYMOND T. CHUNG, MD
Chairman of Internal Medicine Director of Hepatology, Vice Chief, Gastroenterology
Texas Health Presbyterian Hospital Dallas Division of Gastroenterology
Clinical Professor of Internal Medicine Massachusetts General Hospital and Harvard Medical School
University of Texas Southwestern Medical School Associate Member, Broad Institute
Dallas, Texas Boston, Massachusetts
LAWRENCE S. FRIEDMAN, MD DAVID T. RUBIN, MD

Professor of Medicine Joseph B. Kirsner Professor of Medicine
Harvard Medical School Chief, Section of Gastroenterology, Hepatology, and Nutrition
Professor of Medicine Department of Medicine
Tufts University School of Medicine University of Chicago
Boston, Massachusetts Chicago, Illinois
The Anton R. Fried, MD, Chair
Department of Medicine
Newton-Wellesley Hospital C. MEL WILCOX, MD, MSPH
Newton, Massachusetts Division of Gastroenterology and Hepatology
Assistant Chief of Medicine University of Alabama at Birmingham
Massachusetts General Hospital Birmingham, Alabama
Boston, Massachusetts
LAWRENCE J. BRANDT, MD
Professor of Medicine and Surgery
Albert Einstein College of Medicine
Emeritus Chief
Division of Gastroenterology
Montefiore Medical Center
Bronx, New York
Elsevier
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SLEISENGER AND FORDTRAN’S GASTROINTESTINAL AND LIVER DISEASE,

ELEVENTH EDITION ISBN: 978-0-323-60962-3
Volume 1: 978-0-323-76078-2
Volume 2: 978-0-323-76077-5
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Printed in Canada
Last digit is the print number: 9 8 7 6 5 4 3 2 1

We dedicate this 11th edition to you, our readers, as you
were always central in our thoughts as we wrote, edited,
and produced this textbook. We hope our book meets
your educational needs.
Contributors
Nezam H. Afdhal, MD, DSc Bruce R. Bacon, MD William Bernal, MD

Senior Physician in Hepatology Professor of Internal Medicine Professor
Department of Gastroenterology Division of Gastroenterology and Liver Intensive Therapy Unit
Beth Israel Deaconess Medical Center Hepatology King’s College Hospital
Boston, Massachusetts, United States Saint Louis University School of London, United Kingdom
Medicine
Rakesh Aggarwal, MD, DM Adil E. Bharucha, MBBS, MD
St. Louis, Missouri, United States
Director Professor of Medicine
Jawaharlal Institute of Postgraduate William F. Balistreri, MD Division of Gastroenterology and
Medical Education and Research Director, Pediatric Liver Care Center Hepatology
Puducherry, India Gastroenterology, Hepatology, and Mayo Clinic
Nutrition Rochester, Minnesota, United States
Taymeyah Al-Toubah, MPH
Cincinnati Children’s Hospital Medical
Gastroenterology and Oncology Taft P. Bhuket, MD
Center
H. Lee Moffitt Cancer Center Associate Clinical Professor of Medicine
Cincinnati, Ohio, United States
Tampa, Florida, United States Division of Gastroenterology
Todd H. Baron, MD University of California, San Francisco
Jaime Almandoz, MD
Professor of Medicine San Francisco, California
Assistant Professor
Division of Gastroenterology and Chief of Gastroenterology and
Department of Internal Medicine,
Hepatology Hepatology
Division of Endocrinology
University of North Carolina Director of Endoscopy
University of Texas Southwestern
Chapel Hill, North Carolina, United Alameda Health System
Dallas, Texas, United States
States Oakland, California, United States
Ashwin N. Ananthakrishnan, MD, MPH
Bradley A. Barth, MD, MPH Yangzom D. Bhutia, DVM, PhD
Associate Professor of Medicine
Professor Assistant Professor
Harvard Medical School
Department of Pediatrics Cell Biology and Biochemistry
University of Texas Southwestern Texas Tech University Health Sciences
Massachusetts General Hospital
Dallas, Texas, United States Center
Boston, Massachusetts, United States
Lubbock, Texas, United States
Lee M. Bass, MD
Karin L. Andersson, MD, MPH
Associate Professor of Pediatrics J. Andrew Bird, MD
Assistant Professor of Medicine
Gastroenterology, Hepatology, and Associate Professor
Nutrition Pediatrics, Division of Allergy and
Hepatologist
Ann and Robert H. Lurie Children’s Immunology
Hospital of Chicago University of Texas Southwestern
Northwestern University Feinberg Medical Center
School of Medicine Director
Farshid Araghizadeh, MD, MBA Chicago, Illinois, United States Food Allergy Center
Colon and Rectal Surgeon Children’s Medical Center
Alex S. Befeler, MD
Texas Digestive Disease Consultants Dallas, Texas, United States
Professor of Internal Medicine
(TDDC) and The GI Alliance (TGIA)
Medical Director of Liver Boris Blechacz, MD, PhD
Dallas–Fort Worth, Texas, United States
Transplantation Clinical Associate Professor of Internal
Louis J. Aronne, MD Department of Internal Medicine Medicine
Sanford I. Weill Professor of Metabolic Saint Louis University Gastroenterology and Hepatology
Research St. Louis, Missouri, United States Palmetto Health—University of South
Department of Medicine Carolina
Mark Benson, MD
Weill Cornell Medicine Columbia, South Carolina, United States
New York, New York, United States
Section of Gastroenterology and Diego V. Bohórquez, PhD
Fernando Azpiroz, MD, PhD Hepatology Assistant Professor
Chief University of Wisconsin School of Departments of Medicine and
Department of Gastroenterology Medicine and Public Health Neurobiology
University Hospital Vall d’Hebron Madison, Wisconsin, United States Duke University Medical Center
Professor of Medicine Durham, North Carolina, United States
Universitat Autònoma de Barcelona
Barcelona, Spain
vi
Contributors vii
Jan Bornschein, MD Eugene B. Chang, MD Paul A. Dawson, PhD

Translational Gastroenterology Unit Martin Boyer Professor Professor
John Radcliffe Hospital Department of Medicine Pediatrics— Gastroenterology,

Oxford University Hospitals University of Chicago Hepatology, and Nutrition
Oxford, United Kingdom Chicago, Illinois, United States Emory University
Atlanta, Georgia, United States
Christopher L. Bowlus, MD Joseph G. Cheatham, MD
Professor and Chief Associate Professor of Medicine Gregory de Prisco, MD
Division of Gastroenterology and Department of Medicine Diagnostic Radiologist
Hepatology Uniformed Services University Department of Radiology
University of California Davis Bethesda, Maryland Baylor University Medical Center
Sacramento, California, United States Program Director Director of Medical Education
Gastroenterology Fellowship American Radiology Associates
Lawrence J. Brandt, MD
Naval Medical Center San Diego Dallas, Texas, United States
Professor of Medicine and Surgery
San Diego, California, United States
Albert Einstein College of Medicine Jill K. Deutsch, MD
Emeritus Chief Shivakumar Chitturi, MD Clinical Fellow
Division of Gastroenterology Associate Professor Department of Internal Medicine
Montefiore Medical Center Australian National University Section of Digestive Diseases
Bronx, New York, United States Senior Staff Hepatologist Yale New Haven Hospital—Yale
The Canberra Hospital University School of Medicine
Robert Scott Bresalier, MD
Australian Capital Territory, Australia New Haven, Connecticut, United States
Professor of Medicine
Lydia and Birdie J Resoft Distinguished Daniel C. Chung, MD Kenneth R. DeVault, MD
Professor in GI Oncology Associate Professor of Medicine Professor of Medicine
Gastroenterology, Hepatology, and Harvard Medical School Mayo Clinic College of Medicine
Nutrition Division of Gastroenterology Jacksonville, Florida, United States
The University of Texas MD Anderson Massachusetts General Hospital
Adrian M. Di Bisceglie, MD
Cancer Center Medical Co-Director
Professor of Internal Medicine
Houston, Texas, United States Center for Cancer Risk Analysis
Department of Internal Medicine
Massachusetts General Hospital Cancer
Simon J.H. Brookes, PhD Saint Louis University
Center
Professor St. Louis, Missouri, United States
Human Physiology
John K. DiBaise, MD
College of Medicine, Flinders University Raymond T. Chung, MD
Adelaide, South Australia, Australia Director of Hepatology, Vice Chief,
Division of Gastroenterology and
Gastroenterology
Alan L. Buchman, MD, MSPH Hepatology
Professor of Clinical Surgery Mayo Clinic
Massachusetts General Hospital and
University of Illinois at Chicago Scottsdale, Arizona, United States
Medical Director
Associate Member, Broad Institute Philip G. Dinning, PhD
Intestinal Rehabilitation and Transplant
Boston, Massachusetts, United States Flinders Medical Centre
Center
Human Physiology
Chicago, Illinois, United States Marcello Costa, MD
Flinders University
Matthew Flinders Distinguished
Ezra Burstein, MD, PhD Adelaide, South Australia, Australia
Professor and Professor of
Professor
Neurophysiology J. Marcus Downs, MD
Departments of Internal Medicine and
Physiology Program Director
Molecular Biology
Flinders University Colon and Rectal Surgery
UT Southwestern Medical Center
Adelaide, South Australia, Australia Texas Health Resources
Clinical Professor of Surgery
Thomas G. Cotter, MD
Andres F. Carrion, MD Colon and Rectal Surgery
Gastroenterology Fellow
Assistant Professor of Clinical Medicine University of Texas Southwestern
Section of Gastroenterology,
Program Director, Transplant Medical School
Hepatology, and Nutrition
Hepatology Fellowship Dallas, Texas, United States
University of Chicago Medicine
Chicago, Illinois, United States Douglas A. Drossman, MD
Hepatology
Professor Emeritus of Medicine and
University of Miami Albert J. Czaja, MD
Psychiatry
Miami, Florida, United States Professor Emeritus of Medicine
Division of Digestive Disease and
Gastroenterology and Hepatology
Scott Celinski, MD Nutrition
Mayo Clinic College of Medicine and
Surgical Oncologist University of North Carolina
Science
Department of Surgery President
Rochester, Minnesota, United States
Baylor University Medical Center Center for Education and Practice of
Dallas, Texas, United States Brian G. Czito, MD Biopsychosocial Care
Professor Chapel Hill, North Carolina
Francis K.L. Chan, MBChB(Hons), MD,
Radiation Oncology President
DSc
Duke University Medical Center Drossman Gastroenterology PLLC
Durham, North Carolina, United States Durham, North Carolina, United States
Department of Medicine and
Therapeutics
Chinese University of Hong Kong
Hong Kong, China
viii Contributors
Kerry B. Dunbar, MD, PhD Michael B. Fallon, MD Alexander C. Ford, MBChB, MD

Section Chief, VA Gastroenterology Professor of Medicine Professor of Gastroenterology
Section Gastroenterology, Hepatology, and and Honorary Consultant
Department of Medicine– Nutrition Gastroenterologist
Gastroenterology and Hepatology University of Arizona Leeds Institute of Medical Research
VA North Texas Healthcare System– Chair St. James’s University of Leeds
Dallas VA Medical Center Department of Internal Medicine Leeds Gastroenterology Institute
Associate Professor of Medicine University of Arizona—Phoenix Leeds Teaching Hospitals Trust
Department of Medicine–Division of Phoenix, Arizona, United States Leeds, West Yorkshire, United Kingdom
Geoffrey C. Farrell, MD John S. Fordtran, MD
Professor, Hepatic Medicine Internal Medicine, Division of
Medical School
Australian National University Gastroenterology
Senior Staff Hepatologist Baylor University Medical Center
John E. Eaton, MD The Canberra Hospital Dallas, Texas, United States
Assistant Professor of Medicine Australian Capital Territory, Australia
Chris E. Forsmark, MD
Jordan J. Feld, MD, MPH Professor and Chief
Associate Professor of Medicine Division of Gastroenterology,
Hepatology
University of Toronto Hepatology, and Nutrition
Mayo Clinic
Research Director University of Florida
Toronto Centre for Liver Disease Gainesville, Florida, United States
Steven A. Edmundowicz, MD Senior Scientist
Lawrence S. Friedman, MD
Professor of Medicine Sandra Rotman Centre for Global
Interim Director, Division of Health
Gastroenterology and Hepatology Toronto General Hospital
University of Colorado Anschutz Toronto, Ontario, Canada
Tufts University School of Medicine
Medical Campus
Mark Feldman, MD Boston, Massachusetts
Aurora, Colorado, United States
Chairman of Internal Medicine The Anton R. Fried, MD, Chair
David E. Elliott, MD, PhD Texas Health Presbyterian Hospital Department of Medicine
University of Iowa Carver College of Dallas Newton-Wellesley Hospital
Medicine Clinical Professor of Internal Medicine Newton, Massachusetts
Department of Internal Medicine University of Texas Southwestern Assistant Chief of Medicine
Division of Gastroenterology and Medical School Massachusetts General Hospital
Hepatology Dallas, Texas, United States Boston, Massachusetts, United States
Iowa City VAHCS
Nielsen Q. Fernandez-Becker, MD Scott Fung, MD
Clinical Associate Professor of Medicine Associate Professor
Veterans Administration Health Care
Division of Gastroenterology and Department of Medicine
System
Hepatology University of Toronto
Iowa City, Iowa, United States
Stanford University Staff Hepatologist
B. Joseph Elmunzer, MD, MSc Redwood City, California, United States University Health Network
Peter B. Cotton Professor of Medicine Toronto General Hospital
Paul Feuerstadt, MD
and Endoscopic Innovation Toronto, Ontario, Canada
Attending Physician
Gastroenterology Vadivel Ganapathy, PhD
Hepatology
Gastroenterology Center of Connecticut Professor
Medical University of South Carolina,
Hamden, Connecticut Cell Biology and Biochemistry
Charleston
Assistant Clinical Professor of Medicine Texas Tech University Health Sciences
Charleston, South Carolina, United
Gastroenterology Center
States
Yale University School of Medicine Lubbock, Texas, United States
Charles O. Elson, MD New Haven, Connecticut, United States
Professor of Medicine and Microbiology John J. Garber, MD
Peter Fickert, Prof Instructor in Medicine
Basil I. Hirschowitz Chair in
Division of Gastroenterology and Harvard Medical School
Gastroenterology
Hepatology Assistant in Medicine
University of Alabama at Birmingham
Medical University of Graz Division of Gastroenterology
Birmingham, Alabama, United States
Graz, Austria Massachusetts General Hospital
Grace H. Elta, MD Boston, Massachusetts, United States
Robert E. Fleming, MD
Professor Emeritus
Professor of Pediatrics Praveen Ramakrishnan Geethakumari,
Formerly the H. Marvin Pollard
Saint Louis University School of MD, MS
Collegiate Professor
Medicine Assistant Professor
St. Louis, Missouri, United States Division of Medical Oncology
University of Michigan
Ann Arbor, Michigan, United States Department of Internal Medicine
Medical Center
Contributors ix
Marc G. Ghany, MD, MHSc David J. Hass, MD M. Nedim Ince, MD

Liver Diseases Branch Associate Clinical Professor of Medicine University of Iowa Carver College of
National Institute of Diabetes and Division of Digestive Diseases Medicine

Digestive and Kidney Diseases Yale University School of Medicine Iowa City, Iowa, United States
National Institutes of Health New Haven, Connecticut, United States Department of Internal Medicine
Bethesda, Maryland, United States Division of Gastroenterology and
David M. Hockenbery, MD
Hepatology
Pere Ginès, MD, PhD Member
Iowa City VAHCS
Chairman Clinical Research
Liver Unit Fred Hutchinson Cancer Research
Veterans Administration Health Care
Hospital Clinic Barcelona Center
System
Full Professor of Medicine Professor of Medicine
Iowa City, Iowa, United States
University of Barcelona Division of Gastroenterology
Principal Investigator University of Washington Rachel B. Issaka, MD, MAS
Institut d’Investigacions Biomediques Seattle, Washington, United States Assistant Member
August Pi i Sunyer (IDIBAPS) Clinical Research and Public Health
Christoph Högenauer, MD
Barcelona, Spain Science Divisions
Fred Hutchinson Cancer Research Center
Robert E. Glasgow, MD Department of Internal Medicine
Assistant Professor
Professor and Vice Chairman Medical University of Graz
Department of Medicine, Division of
Surgery Graz, Austria
Gastroenterology
University of Utah
Jacinta A. Holmes, MBBS, PhD University of Washington
Salt Lake City, Utah, United States
Division of Gastroenterology Seattle, Washington, United States
Gregory J. Gores, MD Massachusetts General Hospital
Johanna C. Iturrino, MD
Executive Dean for Research, Professor Boston, Massachusetts, United States
of Medicine Gastroenterology
Division of Gastroenterology and St. Vincent’s Hospital
Beth Israel Deaconess Medical Center
Hepatology University of Melbourne
Mayo Clinic Fitzroy, Victoria, Australia
Rochester, Minnesota, United States Theodore W. James, MD
Colin W. Howden, MD
Fellow
Peter H.R. Green, MD Hyman Professor of Medicine
Phyllis and Ivan Seidenberg Professor of Division of Gastroenterology
University of North Carolina
Medicine University of Tennessee Health Science
Chapel Hill, North Carolina, United
Columbia University Medical Center Center
States
New York, New York, United States Memphis, Tennessee, United States
Harry L.A. Janssen, MD, PhD
David A. Greenwald, MD Patrick A. Hughes, PhD
Director of Clinical Gastroenterology Centre for Nutrition and
and Endoscopy Gastrointestinal Diseases
University of Toronto
Division of Gastroenterology Adelaide Medical School
Toronto, Ontario, Canada
Mount Sinai Hospital University of Adelaide
New York, New York, United States South Australian Health and Medical Dennis M. Jensen, MD
Research Institute Professor of Medicine
C. Prakash Gyawali, MD, MRCP
Nutrition and Metabolism Professor of Medicine–Gastrointestinal
Adelaide, South Australia, Australia David Geffen School of Medicine at
UCLA
Department of Medicine Sohail Z. Husain, MD
Staff Physician
Washington University in St. Louis Professor of Pediatrics
Medicine-Gastrointestinal
St. Louis, Missouri, United States Division of Gastroenterology,
VA Greater Los Angeles Healthcare
Hazem Hammad, MD System
Stanford University School of Medicine
Assistant Professor of Medicine Key Investigator
Stanford, California, United States
Division of Gastroenterology and Director, Human Studies Core and
Hepatology Christopher D. Huston, MD Gastrointestinal Hemostasis Research
University of Colorado Anschutz Professor Unit
Medical Campus Medicine, Microbiology, and Molecular CURE Digestive Diseases Research
Aurora, Colorado, United States Genetics Center
University of Vermont College of Los Angeles, California, United States
Heinz F. Hammer, MD
Medicine
Associate Professor of Medicine Pamela J. Jensen, MD
Attending Physician
Department of Internal Medicine Department of Pathology
Medicine and Infectious Diseases
Medical University Texas Health Presbyterian Hospital
Fletcher Allen Health Care
Graz, Austria Dallas
Burlington, Vermont, United States
Stephen A. Harrison, MD
Visiting Professor of Hepatology
Radcliffe Department of Medicine
University of Oxford
Oxford, United Kingdom
x Contributors
D. Rohan Jeyarajah, MD Jonathan D. Kaunitz, MD Brian E. Lacy, MD, PhD

Chair of Surgery Professor of Medicine and Surgery Senior Associate Consultant
Assistant Chair of Clinical Sciences UCLA School of Medicine Division of Gastroenterology
Head of Surgery Attending Gastroenterologist Mayo Clinic
TCU and UNTHSC School of West Los Angeles Veterans Affairs Jacksonville, Florida, United States
Medicine Medical Center
Anne M. Larson, MD
Fort Worth, Texas Los Angeles, California, United States
Director, Gastrointestinal Services
Laurie Keefer, PhD Division of Gastroenterology/
Methodist Richardson Medical Center
Professor Hepatology
Director, HPB/UGI Fellowship
Medicine and Psychiatry University of Washington
Associate Program Director, General
Icahn School of Medicine at Mount Sinai Seattle, Washington, United States
Surgery Residency Program
Methodist Richardson Medical Center James Y.W. Lau, MD
Richardson, Texas, United States Ciarán P. Kelly, MD Professor of Surgery
Professor of Medicine Department of Surgery
Peter J. Kahrilas, MD
Gastroenterology The Chinese University of Hong Kong
Gilbert H. Marquardt Professor of
Harvard Medical School Director
Medicine
Fellowship Program Director Endoscopy Centre
Feinberg School of Medicine
Gastroenterology Prince of Wales Hospital
Northwestern University
Beth Israel Deaconess Medical Center Hong Kong, China
Northwestern Medicine Ryan Law, DO
Chicago, Illinois, United States Sahil Khanna, MBBS, MS Assistant Professor
Associate Professor of Medicine Division of Gastroenterology
Vishal Kaila, BS, MD
Gastroenterology and Hepatology University of Michigan
Resident
Mayo Clinic Ann Arbor, Michigan, United States
Internal Medicine
Texas Health Presbyterian Benjamin Lebwohl, MD, MS
Dallas, Texas, United States Arthur Y. Kim, MD Assistant Professor of Medicine and
Associate Professor of Medicine Epidemiology
Patrick S. Kamath, MD
Harvard Medical School Columbia University Medical Center
Division of Infectious Diseases New York, New York, United States
Hepatology Anthony J. Lembo, MD
Consultant Professor of Medicine
Gastroenterology and Hepatology Kenneth L. Koch, MD Department of Medicine
Mayo Clinic College of Medicine and Professor of Medicine Beth Israel Deaconess Medical Center
Science Department of Medicine Boston, Massachusetts, United States
Rochester, Minnesota, United States Section on Gastroenterology and
Cynthia Levy, MD
Hepatology
Gilaad G. Kaplan, MD, MPH Professor of Medicine
Wake Forest University School of
Professor of Medicine Division of Hepatology
Medicine
University of Calgary University of Miami
Winston-Salem, North Carolina, United
Calgary, Alberta, Canada Miami, Florida, United States
States
Purna Kashyap, MBBS Blair Lewis, MD
Benjamin Kulow, MD
Associate Professor of Medicine Medical Director
Colon and Rectal Surgeon
Physiology and Biomedical Engineering Carnegie Hill Endoscopy
Saint Luke’s Health System
Mayo Clinic Clinical Professor of Medicine
Kansas City, Missouri, United States
Rochester, Minnesota, United States Mount Sinai Medical Center
Rekha B. Kumar, MD, MS New York, New York, United States
Jennifer Katz, MD
Assistant Professor of Medicine James H. Lewis, MD
Endocrinology, Diabetes, and
Division of Gastroenterology Professor of Medicine
Metabolism
Montefiore Medical Center Director of Hepatology
Weill Cornell Medical College
Bronx, New York, United States Division of Gastroenterology
Attending Physician
Georgetown University Medical Center
David A. Katzka, MD Endocrinology, Diabetes, and
Washington, DC, United States
Professor of and Consultant in Medicine Metabolism
Gastroenterology New York Presbyterian Hospital Rodger A. Liddle, MD
Mayo Clinic New York, New York, United States Professor of Medicine
Rochester, Minnesota, United States Department of Medicine
Vidhya Kunnathur, MD
Duke University Medical Center
Debra K. Katzman, MD, FRCPC Assistant Professor
Durham, North Carolina, United States
Professor of Pediatrics Division of Digestive Diseases
Department of Pediatrics University of Cincinnati Steven D. Lidofsky, MD, PhD
The Hospital for Sick Children and Cincinnati, Ohio, United States Professor of Medicine
University of Toronto University of Vermont
Joann Kwah, MD
Toronto, Ontario, Canada Director of Hepatology
University of Vermont Medical Center
Albert Einstein College of Medicine
Burlington, Vermont, United States
Gastroenterology
Bronx, New York, United States
Contributors xi
Keith D. Lindor, MD Ricard Masia, MD, PhD Frederick H. Millham, MD, MBA
Senior Advisor and Professor Associate Director, Translational Chair, Surgery
Office of the University Provost Pathology South Shore Hospital

Arizona State University Medicine Surface Oncology Weymouth, Massachusetts
Gastroenterology and Hepatology Cambridge, Massachusetts, United States Associate Professor of Surgery (Part
Mayo Clinic Hospital Time)
Joel B. Mason, MD
Phoenix, Arizona, United States Harvard Medical School
Professor of Medicine and Nutrition
Mark E. Lowe, MD, PhD Divisions of Gastroenterology and
Harvey R. Colton Professor of Pediatric Clinical Nutrition Ginat W. Mirowski, DMD, MD
Science and Vice Chair Tufts University Adjunct Clinical Professor
Department of Pediatrics Director Department of Oral Pathology,
Washington University School of Vitamins and Carcinogenesis Laboratory Medicine, and Radiology
Medicine USDA Human Nutrition Research Indiana University School of Dentistry
St. Louis, Missouri, United States Center at Tufts University Professor of Clinical Dermatology
Boston, Massachusetts, United States (Clinical Track)
Cara L. Mack, MD
Department of Dermatology
Professor of Pediatrics Jeffrey B. Matthews, MD
Indiana University School of Medicine
University of Colorado School of Dallas B. Phemister Professor and
Indianapolis, Indiana, United States
Medicine Chairman
Children’s Hospital Colorado Department of Surgery Joseph Misdraji, MD
Aurora, Colorado, United States The University of Chicago Medicine Associate Professor of Pathology
Chicago, Illinois, United States Harvard Medical School
Ryan D. Madanick, MD
Associate Pathologist
Assistant Professor of Medicine Craig J. McClain, MD
Division of Gastroenterology and Professor of Medicine and Pharmacology
Hepatology and Toxicology
University of North Carolina School of Vice President for Health Affairs and Daniel S. Mishkin, MD, CM
Medicine Research Chief of Gastroenterology
Chapel Hill, North Carolina, United University of Louisville Atrius Health
States Director Boston, Massachusetts, United States
Gastroenterology
Willis C. Maddrey, MD Bijal Modi, MD
Robley Rex VA Medical Center
Special Assistant to the President Department of Internal Medicine
Louisville, Kentucky, United States
Professor of Internal Medicine Division of Hematology and Oncology
Arnold N. and Carol S. Ablon Stephen A. McClave, MD Texas Health Presbyterian Hospital
Professorship in Biomedical Science Professor and Director of Clinical Dallas
Adelyn and Edmund M. Hoffman Nutrition Dallas, Texas, United States
Distinguished Chair in Medical Department of Medicine
John Magaña Morton, MD, MPH, MHA
Science University of Louisville School of
Vice Chair for Quality
University of Texas Southwestern Medicine
Department of Surgery
Medical Center Louisville, Kentucky, United States
Chief
Shilpa Mehra, MD Bariatric and Minimally Invasive Surgery
Matthias Maiwald, MD, PhD Assistant Professor of Medicine Yale School of Medicine
Senior Consultant in Microbiology Department of Medicine Department of Surgery
Department of Pathology and Division of Gastroenterology New Haven, Connecticut, United States
Laboratory Medicine Albert Einstein College of Medicine
William Conan Mustain, MD
KK Women’s and Children’s Hospital, Bronx, New York, United States
Assistant Professor of Surgery
Singapore
Megha S. Mehta, MD Division of Colon and Rectal Surgery
Adjunct Associate Professor
Assistant Professor of Pediatrics University of Arkansas for Medical
Department of Microbiology and
University of Texas Southwestern Sciences
Immunology
Medical Center Little Rock, Arkansas, United States
Yong Loo Lin School of Medicine
National University of Singapore Filipe Gaio Nery, MD
Adjunct Associate Professor Shivang S. Mehta, MD Physician
Duke-NUS Graduate Medical School Pediatric Gastroenterology Fellow Departamento de Anestesiologia,
Singapore, Singapore Department of Pediatric Cuidados Intensivos e Emergência
Gastroenterology Centro Hospitalar do Porto–Hospital
Lawrence A. Mark, MD, PhD
University of Texas Southwestern Santo António, Porto
Associate Professor of Clinical
Medical Center Researcher, EPIUnit
Dermatology
Dallas, Texas, United States Instituto de Saúde Pública, Universidade
Department of Dermatology
do Porto, Porto
Indiana University School of Medicine Joanna M.P. Melia, MD
Researcher, Ciências Médicas
Indianapolis, Indiana, United States Assistant Professor of Medicine
Instituto de Ciências Biomédicas de Abel
Johns Hopkins University School of
Paul Martin, MD, FRCP, FRCPI Salazar
Medicine
Chief, Division of Gastroenterology and Porto, Portugal
Baltimore, Maryland, United States
Hepatology
University of Miami
Miami, Florida, United States
xii Contributors
Siew C. Ng, MBBS (Lond), PhD (Lond) Patrick R. Pfau, MD Christopher K. Rayner, MBBS, PhD
Professor of Medicine Professor, Chief of Clinical Professor
Department of Medicine and Gastroenterology Adelaide Medical School
Therapeutics Section of Gastroenterology and University of Adelaide
State Key Laboratory of Digestive Hepatology Consultant Gastroenterologist
Disease University of Wisconsin School of Department of Gastroenterology and
LKS Institute of Health Science Medicine and Public Health Hepatology
The Chinese University of Hong Kong Madison, Wisconsin, United States Royal Adelaide Hospital
Hong Kong, China Adelaide, South Australia, Australia
Angela K. Pham, MD
Mark L. Norris, BSc (Hon), MD Clinical Assistant Professor Ahsan Raza, MD
Associate Professor of Pediatrics Gastroenterology, Hepatology, and General and Colorectal Surgery
Pediatrics Nutrition Rapides Surgical Specialists
Children’s Hospital of Eastern Ontario University of Florida Alexandria, Louisiana, United States
University of Ottawa Gainesville, Florida, United States
Miguel D. Regueiro, MD
Ottawa, Ontario, Canada
Kimberly L. Pham, MD Chair and Professor of Medicine
John O’Grady, MD, FRCPI St. George’s University Grenada Department of Gastroenterology and
Professor West Indies, Grenada Hepatology
Institute of Liver Studies Cleveland Clinic, Digestive Disease and
Daniel S. Pratt, MD
King’s College Hospital Surgery Institute
Clinical Director, Liver Transplantation
London, United Kingdom Cleveland, Ohio, United States
Manisha Palta, MD Massachusetts General Hospital John F. Reinus, MD
Associate Professor Assistant Professor of Medicine Professor of Medicine
Radiation Oncology Harvard Medical School Department of Medicine
Duke University Boston, Massachusetts, United States Albert Einstein College of Medicine
Durham, North Carolina, United States Medical Director of Liver
David O. Prichard, MB, BCh, PhD
Transplantation
Stephen J. Pandol, MD Gastroenterologist
Montefiore-Einstein Center for
Professor Gastroenterology and Hepatology
Transplantation
Medicine Mayo Clinic
Cedars-Sinai Medical Center Rochester, Minnesota
Bronx, New York, United States
Los Angeles, California, United States
Michael Quante, PD, Dr
David A. Relman, MD
John E. Pandolfino, MD, MSCI Technische Universität München
Thomas C. and Joan M. Merigan
Hans Popper Professor of Medicine II Medizinische Klinik
Professor
Feinberg School of Medicine Klinikum rechts der Isar
Departments of Medicine and
Northwestern University München, Germany
Microbiology and Immunology
Division Chief
Eamonn M.M. Quigley, MD Stanford University
Professor of Medicine and Chief, Stanford, California
Northwestern Medicine
Gastroenterology and Hepatology Chief of Infectious Diseases
Chicago, Illinois, United States
David M. and Lynda K. Underwood Veterans Affairs Palo Alto Health Care
Darrell S. Pardi, MD, MS Center for Digestive Disorders System
Vice Chair Houston Methodist Hospital Palo Alto, California, United States
Division of Gastroenterology and Weill Cornell Medical College
Arvind Rengarajan, MD
Hepatology Houston, Texas, United States
Barnes-Jewish Hospital
Associate Dean
Balakrishnan S. Ramakrishna, MBBS, Department of Internal Medicine
Mayo School of Graduate Medical
MD, DM, PhD Washington University in St. Louis
Education
Head St. Louis, Missouri, United States
Mayo Clinic
Institute of Gastroenterology
Rochester, Minnesota, United States Joel E. Richter, MD
SRM Institutes for Medical Science
Professor and Director
Michelle Pearlman, MD Chennai, Tamil Nadu, India
Division of Digestive Diseases and
Mrinalini C. Rao, PhD Nutrition
Department of Internal Medicine,
Professor University of South Florida
Division of Digestive and Liver
Department of Physiology and Director
Diseases
Biophysics Joy McCann Culverhouse Center for
University of Illinois at Chicago Swallowing Disorders
Chicago, Illinois, United States University of South Florida
Vyjeyanthi S. Periyakoil, MD Tampa, Florida, United States
Satish S.C. Rao, MD, PhD
Director, Palliative Care Education and
Professor of Medicine Sumera H. Rizvi, MD
Training
Harold J. Harrison, MD, Distinguished Assistant Professor of Medicine
University Chair in Gastroenterology Division of Gastroenterology and
Stanford University School of Medicine
Medicine-Gastroenterology/Hepatology Hepatology
Stanford, California, United States
Augusta University Mayo Clinic
Augusta, Georgia, United States Rochester, Minnesota, United States
Contributors xiii
Syed Mujtaba Rizvi, MD Jayashree Sarathy, PhD Vijay H. Shah, MD

Assistant Professor Associate Professor Professor
Division of Medical Oncology Department of Biological Sciences Medicine, Physiology, and Cancer Cell
Department of Internal Medicine Program Director of Master of Science Biology
UT Southwestern Medical Center in Integrative Physiology Chair
Dallas, Texas, United States Benedictine University Division of Gastroenterology and
Lisle, Illinois Hepatology
Eve A. Roberts, MD, PhD
Visiting Research Professor Associate Chair of Research Medicine
Adjunct Professor
Department of Physiology and Mayo Clinic College of Medicine and
Pediatrics, Medicine, and Pharmacology
Biophysics Science
and Toxicology
University of Illinois at Chicago Rochester, Minnesota, United States
University of Toronto
Adjunct Scientist G. Thomas Shires, MD
Genetics and Genome Biology Program George S. Sarosi Jr., MD John P. Thompson Chair
Hospital for Sick Children Research Robert H. Hux MD Professor and Vice Surgical Services
Institute Chairman for Education Texas Health Presbyterian Hospital
Associate Department of Surgery Dallas
Division of Gastroenterology, University of Florida College of Dallas, Texas, United States
Hepatology, and Nutrition Medicine
Maria H. Sjogren, MD, MPH
The Hospital for Sick Children Staff Surgeon
Senior Hepatologist
Toronto, Ontario, Canada Surgical Service
Associate Fellow NF/SG VAMC
Walter Reed National Medical Center
History of Science and Technology Gainesville, Florida, United States
Bethesda, Maryland, United States
Program
Thomas J. Savides, MD
University of King’s College Phillip D. Smith, MD
Professor of Clinical Medicine
Halifax, Nova Scotia, Canada Professor of Medicine and Microbiology
University of Alabama at Birmingham
Martin D. Rosenthal, MD University of California San Diego
Birmingham, Alabama, United States
Assistant Professor La Jolla, California, United States
Surgery Elsa Solà, MD, PhD
Lawrence R. Schiller, MD
University of Florida Liver Unit
Attending Physician
Gainesville, Florida, United States Hospital Clinic
Gastroenterology Division
Associate Professor
Marc E. Rothenberg, MD, PhD Baylor University Medical Center
University of Barcelona
Professor of Pediatrics Dallas, Texas, United States
Researcher
Cincinnati Children’s Hospital Medical
Mitchell L. Schubert, MD Institut d’Investigacions Biomediques
Center
Professor of Medicine and Physiology August Pi i Sunyer (IDIBAPS)
Cincinnati, Ohio, United States
Virginia Commonwealth University Barcelona, Spain
Jayanta Roy-Chowdhury, MBBS Health System
Rhonda F. Souza, MD
Professor Chief, Division of Gastroenterology,
Co-Director, Center for Esophageal
Departments of Medicine and Genetics Hepatology, and Nutrition
Diseases
Director, Genetic Engineering and Gene McGuire Veterans Affairs Medical
Therapy Core Facility Center
Baylor University Medical Center
Albert Einstein College of Medicine Richmond, Virginia, United States
Cynthia L. Sears, MD Research
Namita Roy-Chowdhury, PhD Professor of Medicine and Oncology Baylor Scott and White Research
Professor Johns Hopkins University School of Institute
Departments of Medicine and Genetics Medicine Dallas, Texas, United States
Albert Einstein College of Medicine Baltimore, Maryland, United States
Cedric W. Spak, MD, MPH
Joseph H. Sellin, MD Clinical Assistant Professor
David T. Rubin, MD Professor Emeritus Infectious Diseases
Joseph B. Kirsner Professor of Medicine Division of Gastroenterology Baylor University Medical Center
Chief, Section of Gastroenterology, Baylor College of Medicine Staff Physician
Hepatology, and Nutrition Chief of Gastroenterology Infectious Diseases
Department of Medicine Ben Taub General Hospital Texas Centers for Infectious Disease
University of Chicago Houston, Texas, United States Associates
Chicago, Illinois, United States Dallas, Texas, United States
M. Gaith Semrin, MD, MBBS
Associate Professor Stuart Jon Spechler, MD
Pediatric Gastroenterology and Chief, Division of Gastroenterology
Nutrition Co-Director, Center for Esophageal
UT Southwestern Medical Center Research
Children Medical Center Dallas Department of Medicine
Dallas, Texas, United States Baylor University Medical Center at Dallas
Research
Baylor Scott and White Research Institute
xiv Contributors
James E. Squires, MD, MS Jan Tack, MD, PhD Dominique Charles Valla, MD
Assistant Professor Head, Division of Gastroenterology and Professor of Hepatology
Department of Pediatrics Hepatology Liver Unit
UPMC Children’s Hospital of Leuven University Hospitals Hôpital Beaujon, APHP,
Pittsburgh Professor of Medicine Clichy-la-Garenne
Pittsburgh, Pennsylvania, United States Translational Research Center for France
Gastrointestinal Disorders (TARGID) CRI, UMR1149
Neil H. Stollman, MD
Department of Clinical and Inserm and Université de Paris
Associate Clinical Professor
Experimental Medicine Paris, France
Department of Medicine, Division of
University of Leuven
Gastroenterology John J. Vargo II, MD, MPH
Leuven, Belgium
University of California San Francisco Associate Professor of Medicine
San Francisco, California Nicholas J. Talley, MD, PhD Gastroenterology and Hepatology
Chief Distinguished Laureate Professor Cleveland Clinic
Division of Gastroenterology Faculty of Health and Medicine Cleveland, Ohio, United States
Alta Bates Summit Medical Center University of Newcastle, Australia
Santhi Swaroop Vege, MD
Oakland, California, United States Newcastle, New South Wales, Australia
Professor of Medicine and Director
Sarah E. Streett, MD Jarred P. Tanksley, MD, PhD Pancreas Group
Clinical Associate Professor Resident Gastroenterology and Hepatology
Director IBD Education Radiation Oncology Mayo Clinic
Division of Gastroenterology and Duke University Rochester, Minnesota, United States
Hepatology Durham, North Carolina, United States
Axel von Herbay, MD
Stanford University
Narci C. Teoh, MD Professor of Pathology
Redwood City, California, United States
Professor of Medicine Faculty of Medicine
Jonathan R. Strosberg, MD Australian National University University of Heidelberg
Associate Professor Senior Staff Hepatologist Heidelberg Hans Pathologie
Gastrointestinal Oncology The Canberra Hospital Hamburg, Germany
Moffitt Cancer Center Australian Capital Territory, Australia
Margaret von Mehren, MD
Tampa, Florida, United States
Dawn M. Torres, MD Professor
Frederick J. Suchy, MD Program Director GI Fellowship Department of Hematology/Oncology
Children’s Hospital Colorado Department of Medicine Fox Chase Cancer Center
Professor of Pediatrics and Associate Walter Reed National Military Medical Philadelphia, Pennsylvania, United
Dean for Child Health Research Center States
Pediatrics Associate Professor of Medicine
David Q.-H. Wang, MD, PhD
University of Colorado School of Department of Medicine
Medicine Uniformed Services University of the
Departments of Medicine and Genetics
Aurora, Colorado, United States Health Sciences
Director, Molecular Biology and Next
Bethesda, Maryland, United States
Aravind Sugumar, MD Generation Technology Core
Instructor Kiran Turaga, MD, MPH Marion Bessin Liver Research Center
Gastroenterology and Hepatology Associate Professor Albert Einstein College of Medicine
Cleveland Clinic Foundation Department of Surgery Bronx, New York, United States
Cleveland, Ohio, United States The University of Chicago
Sachin Wani, MD
Shelby Sullivan, MD Associate Professor of Medicine
Associate Professor of Medicine Richard H. Turnage, MD Division of Gastroenterology and
Director, Gastroenterology Metabolic Executive Associate Dean for Clinical Hepatology
and Bariatric Program Affairs University of Colorado Anschutz
Division of Gastroenterology and Professor of Surgery Medical Campus
Hepatology University of Arkansas for Medical Aurora, Colorado, United States
University of Colorado Anschutz Sciences Medical Center
Frederick Weber, MD
Medical Campus University of Arkansas for Medical
Clinical Professor
Aurora, Colorado, United States Sciences
Little Rock, Arkansas, United States
Gyongyi Szabo, MD, PhD Hepatology
Mitchell T. Rabkin, MD Chair Michael F. Vaezi, MD, PhD, MS University of Alabama Birmingham
Chief Academic Officer Professor of Medicine and Birmingham, Alabama, United States
Beth Israel Deaconess Medical Center Otolaryngology
Barry K. Wershil, MD
and Beth Israel Lahey Health Division of Gastroenterology and
Professor
Faculty Dean for Academic Affairs Hepatology
Pediatrics
Harvard Medical School Vanderbilt University
Northwestern University Feinberg
Boston, Massachusetts, United States Director
School of Medicine
Center for Swallowing and Esophageal
Chief, Division of Gastroenterology,
Disorders
Vanderbilt University Medical Center
Pediatrics
Director
Ann & Robert H. Lurie Children’s
Clinical Research
Hospital of Chicago
Vanderbilt University Medical Center
Nashville, Tennessee, United States
Contributors xv
David C. Whitcomb, MD, PhD Christopher G. Willett, MD Anahit A. Zeynalyan, MD

Professor Professor and Chairman Resident
Medicine, Cell Biology and Molecular Radiation Oncology Internal Medicine

Physiology, and Human Genetics Duke University Baylor University Medical Center
University of Pittsburgh and UPMC Durham, North Carolina, United States Dallas, Texas, United States
Pittsburgh, Pennsylvania, United States
Joseph C. Yarze, MD
C. Mel Wilcox, MD, MSPH Assistant Professor of Medicine
Division of Gastroenterology and Harvard Medical School
Hepatology Associate Physician
University of Alabama at Birmingham Division of Gastroenterology
Birmingham, Alabama, United States Massachusetts General Hospital
Foreword
Even attempting to write a Foreword for the 11th edition been in the recent past and what we hope (and expect) to achieve
of Sleisenger and Fordtran’s Gastrointestinal and Liver Disease: in the future.
Pathophysiology/Diagnosis/Management, a textbook that has served A trusted book provides a helpful guide that is readily available
for many decades to prepare readers to respond to challenges at moments of uncertainty. A comparison of an individual chapter
presented by patients with gastrointestinal and liver disease, is from a past edition and what we have now further validates the
a daunting task and yet a great pleasure. Just having achieved an conclusion that progress is being made, and the future of our spe-
11th edition of a textbook is, in and of itself, a remarkable accom- cialty is encouraging. The three senior editors and three associate
plishment. Generations of gastroenterologists and hepatologists editors of the 11th edition are foremost authorities and widely
have relied on Sleisenger and Fordtran to provide comprehensive, recognized for their abilities to identify topics of interest and
up-to-date, reliable information. to persuade experts in these areas to share their knowledge. To
The 11th edition is a welcome addition to the previous editions, write an updated review of one’s field can be a Herculean task that
which have been widely acclaimed as important go-to sources requires not only knowledge but also courage. The editors have
of information regarding the broad array of disorders affecting surely succeeded. The careful selection of authors of individual
the gastrointestinal tract and the liver. Over the past half cen- chapters allows each to bring his or her own style regarding what
tury, these volumes have been mainstays in the libraries of those to emphasize; to lay out what we know, as well as what we need to
engaged in these fields. Since its inception 10 editions ago, this know, to diagnose and effectively treat specific problems; and to
now classic textbook has tracked the evolution of thinking in mul- provide suggestions and guidance as to how to manage patients
tiple areas and has served readers well. These days, there are ever- while integrating new observations into practice.
expanding ways for those of us interested in gastroenterology and With regard to the liver section, the current state of knowl-
hepatology to be stimulated, informed, educated, and refreshed. edge about hepatitis-inducing viruses and drug-induced liver dis-
Lectures, conversations with colleagues, and attendance at local, eases and the tsunami of interest in the many consequences of the
regional, and national meetings have their roles, and we all learn effects of excessive fat in the liver in the causation of chronic liver
from our patients. Perusal of relevant articles in medical journals diseases are breathtaking. These achievements have been well-
is increasingly difficult in an era in which the number of available chronicled journeys with opportunities (and hope) for even more
journals has increased remarkably. The practicing clinician, given effective therapeutic agents in the near future. Just one edition
present-day time constraints, will more than ever find this text- ago, we were on the threshold of having effective, widely appli-
book reliable, informative, and useful. In these two volumes are cable treatments for the several types of viral hepatitis; much of
overviews of what is known now and glimpses of what the future what we hoped for has been achieved. It is now likely that there
is likely to bring. A blend of skill, knowledge, practical experi- will be discovery of therapeutic approaches that will favorably
ence, and the ability to teach is required of the authors in order affect the broad array of fat-related liver injuries, including their
to achieve these goals. Overall, these efforts have been successful association with cardiovascular disorders. Widely available access
in presenting accurate and comprehensive updates in our fields of to advanced endoscopy has changed the approach to the evalua-
interest and serve us well as a look to our past, provide reflections tion and treatment of many disorders of the gastrointestinal tract,
regarding our present, and delineate problems yet to be solved. bile ducts, and pancreas. Furthermore, who could have foreseen
We are fortunate to live in exciting and rapidly changing just a few years ago how advances in biological therapies and
times in gastroenterology and hepatology. The sheer volume minimally invasive surgery would so redirect our treatments of
of new ideas presented in multiple journals is stimulating and a broad array of disorders or how important the gut microbiome
often overwhelming. Each of us must evaluate and assimilate would be in the pathogenesis of many disorders. Once we under-
new information while making efforts to appropriately incor- stand how to favorably alter the gut microbiome, major leaps for-
porate the new advances into our practices. To stay up to date ward can be expected.
and achieve our goals requires considerable effort and dedica- What is next? Gene editing and an understanding of intesti-
tion (Even COVID-19 is mentioned several times throughout nal microbiota, now in their infancy, will receive much deserved
the book.). There is comfort in having available a reliable and attention in the next few years. With each passing year, advances in
trusted guide to refresh and stimulate us. manipulation of the human genome and intestinal microbiota are
The 11th edition of Sleisenger and Fordtran provides a firm, becoming more precise and require constant, thoughtful oversight
authoritative platform regarding what is established knowledge to ensure that we do what we should do and not just what we can
and identifies where progress is being made to prepare us to do. In this edition, we have blueprints and predictions of the future
be better armed for the foreseeable future. We all need to be for many aspects of our specialty. It is important to discard old
informed of the likely validity and usefulness of new observa- ideas that have not proved effective while constantly re-examining
tions. It is vital that we recognize the degree of certainty of the the basis for what we think we know and appropriately altering
data that led to our conclusions. There have been (and will be) what we do.
definite game-changing advances and also many seemingly good We all marvel when we see what has been (and is) happening
ideas and approaches that turn out to be sidesteps. New concepts in medicine and the effects of these advances in gastroenterology
must be recognized, double-checked, processed, and then incor- and hepatology. Surely, the best is yet to come, and we all hope
porated into our thinking, subsequently affecting our actions. that what we are learning and applying now will stimulate us to
The breadth of subjects covered in depth in these two vol- create an even better future.
umes is impressive. I had the honor to write the Foreword to the
9th edition published in 2010. When comparing the expansion of Willis C. Maddrey, MD
knowledge from then to now, one can appreciate where we have Dallas, Texas
xvi
The Sleisenger and Fordtran Editors
Mark Feldman, MD Lawrence S. Friedman, MD Lawrence J. Brandt, MD
Editions 5-11 Editions 7-11 Editions 8-11
Raymond T. Chung, MD David T. Rubin, MD C. Mel Wilcox, MD
Edition 11 Edition 11 Edition 11
Marvin H. Sleisenger, MD John S. Fordtran, MD Bruce F. Scharschmidt, MD
Editions 1-7 Editions 1-5 Editions 5-6
xvii
Preface
Nearly a half century ago, in the summer of 1971, Drs. Marvin As one looks back 50 years, the advances made in our field
H. Sleisenger in San Francisco and John S. Fordtran in Dallas as a result of rigorous basic science and clinical research have
embarked on a new venture: planning, writing, and editing the been truly remarkable, and the future holds even greater prom-
inaugural edition of a new textbook for gastroenterologists. ise of discovery. Featured advances discussed in the 11th edition
The book received widespread praise for incorporating state- include improved diagnosis and treatment of chronic hepatitis B
of-the-art descriptions of the pathophysiology of the d isorders and C; evolution in the diagnosis and treatment of Helicobacter
discussed—a first for a medical textbook. Since the a uspicious pylori infection and the resulting benefits on the prevention and
debut of Gastrointestinal Disease: Pathophysiology/Diagnosis/ treatment of peptic ulcer disease and gastric neoplasia; improve-
Management, subsequent editions have been published every ments in the prevention of colorectal cancer through screening
4 to 5 years, and we are pleased that the 11th edition of this and surveillance; new approaches to the recognition and treat-
venerable textbook continues the tradition and standards set ment of Barrett esophagus and consequent prevention of esopha-
by the founding editors. To be sure, innumerable enhance- geal adenocarcinoma; the expanding use of biologic agents and
ments have been made since the 1st edition, such as the addi- novel small molecules to treat and prevent recurrences of IBD;
tion of chapters on liver diseases, the availability of the book recognition of an increasing number of immune and autoimmune
online and on hand-held devices, the introduction of monthly diseases affecting not only the stomach and hepatobiliary system
updates to bring attention to important new developments but also the pancreas and intestine; improvements in the ability
that occur between editions, the incorporation of videos of to risk stratify and treat patients with GI bleeding; and continuing
new diagnostic and therapeutic procedures, and the participa- progress in hepatic, pancreatic, and small bowel transplantation.
tion of authors from around the world to give the book a truly There have been remarkable advances in our understanding the
international flavor. gut microbiome, which is becoming the focus of interest in diverse
In the summer of 2017, the current editors met with the fields, such as IBS, IBD, obesity, hepatic encephalopathy, and oth-
publisher and reviewed the prior (10th) edition of the book ers, including non-GI disorders. We are particularly pleased to
in great detail. Most importantly, the core group of 3 senior have completely redesigned the section on IBD by reorganizing
editors invited 3 associate editors (Drs. Raymond T. Chung, and updating the discussions of pathophysiology, clinical presen-
David T. Rubin, and C. Mel Wilcox) to join them in order to tation, and management, all of which are evolving rapidly.
facilitate critical review of the chapters, to help select the most Sadly, the original co-founder of this textbook, Dr. Marvin H.
expert authors, and to provide greater content expertise. Each Sleisenger, passed away on October 19, 2017, at the age of 93.
associate editor worked closely with a senior editor. The result, Marvin will be greatly missed, and we trust that this 11th edition
we hope, is an easily readable, carefully edited, highly accurate, would have met with his approval and commendation.
and thorough review of the state of the art of gastrointestinal
and liver disease. The target audience is primarily practicing Mark Feldman, MD
gastroenterologists and hepatologists (adult and pediatric) and Lawrence S. Friedman, MD
trainees in gastroenterology. We hope the book will also be Lawrence J. Brandt, MD
useful to general internists, other specialists, and students at
all levels.
xviii
Acknowledgments
The editors and associate editors of the 11th edition of Sleisenger thank Dr. Willis C. Maddrey of the University of Texas South-
& Fordtran’s Gastrointestinal and Liver Disease are most grateful western for his eloquent Foreword, the second time he has been
to the more than 230 authors from countries in North America, called on to do this honor for Sleisenger & Fordtran. We remem-
Europe, Asia, and Australia who contributed their knowledge, ber with affection Dr. Marvin H. Sleisenger, who passed away as
expertise, and wisdom to the pages of the book. We are also the 11th edition of the book he co-created was being prepared,
appreciative of the talented staff at Elsevier who helped bring and pay tribute to Dr. John S. Fordtran for his continuing inspi-
this book to life, particularly Nancy Duffy, Dolores Meloni, and ration and contributions. We are deeply appreciative of the love
Deidre Simpson. A special call out goes to Cindy Thoms, who and support of our spouses: Barbara Feldman, Mary Jo Cappuc-
oversaw production of the book. We are most thankful to our cilli, Lois Brandt, Kim Wilcox, Diane Abraczinskas, and Rebecca
assistants, Sherie Strang, Alison Sholock, Amy Nash, and Amy Rubin. Finally, we thank our readers, to whom the book is dedi-
Majkowski, for outstanding secretarial support. We want to cated, for their confidence and trust in this textbook.
xix
Abbreviation List
AASLD American Association for the Study of Liver ESR Erythrocyte sedimentation rate
Diseases EUS Endoscopic ultrasonography
ACG American College of Gastroenterology FDA U.S. Food and Drug Administration
ACTH Corticotropin FNA Fine-needle aspiration
AE Angioectasia GAVE Gastric antral vascular ectasia
AFP Alpha fetoprotein GERD Gastroesophageal reflux disease
AGA American Gastroenterological Association GGTP Gamma glutamyl transpeptidase
AIDS Acquired immunodeficiency syndrome GI Gastrointestinal
ALF Acute liver failure GIST GI stromal tumor
ALT Alanine aminotransferase GU Gastric ulcer
AMA Antimitochondrial antibodies H & E Hematoxylin and eosin
ANA Antinuclear antibodies H2RA Histamine-2 receptor antagonist
ANCA Antineutrophil cytoplasmic antibodies HAV Hepatitis A virus
APACHE Acute physiology and chronic health HBV Hepatitis B virus
examination
HCC Hepatocellular carcinoma
APC Argon plasma coagulation
HCG Human chorionic gonadotropin
ASGE American Society for Gastrointestinal Endoscopy
HCV Hepatitis C virus
AST Aspartate aminotransferase
HDL High-density lipoprotein
ATP Adenosine triphosphate
HDV Hepatitis D virus
BICAP Bipolar electrocoagulation
HELLP Hemolysis, elevated liver enzymes, low platelets
BMI Body mass index
HEV Hepatitis E virus
BRBPR Bright red blood per rectum
Hgb Hemoglobin
CBC Complete blood count
HHT Hereditary hemorrhagic telangiectasia
CCK Cholecystokinin
HIV Human immunodeficiency virus
CEA Carcinoembryonic antigen
HLA Human leukocyte antigen
CDI Clostridioides difficile infection
HPV Human papillomavirus
CF Cystic fibrosis
HSV Herpes simplex virus
CFTR Cystic fibrosis transmembrane conductance
regulator Hp Helicobacter pylori
CMV Cytomegalovirus IBD Inflammatory bowel disease
CNS Central nervous system IBS Irritable bowel syndrome
CO2 Carbon dioxide ICU Intensive care unit
COX Cyclooxygenase IMA Inferior mesenteric artery
CT Computed tomography IMT Intestinal microbiota transplantation
CTA Computed tomography angiography INR International normalized ratio
DAA Direct-acting antiviral agent IV Intravenous
DIC Disseminated intravascular coagulation IVIG Intravenous immunoglobulin
DILI Drug-induced liver injury LDH Lactate dehydrogenase
DNA Deoxyribonucleic acid LDL Low-density lipoprotein
DU Duodenal ulcer LGI Lower gastrointestinal
DVT Deep vein thrombosis LGIB Lower gastrointestinal bleed
EBV Epstein-Barr virus LLQ Left lower quadrant
EGD Esophagogastroduodenoscopy LT Liver transplantation
EGF Epidermal growth factor LUQ Left upper quadrant
EMG Electromyography MELD Model for end-stage liver disease
ERCP Endoscopic retrograde cholangiopancreatography MEN Multiple endocrine neoplasia
xxv
xxvi Abbreviation List
MHC Major histocompatibility complex SBP Spontaneous bacterial peritonitis

MRA Magnetic resonance angiography SIBO Small intestinal bacterial overgrowth
MRCP Magnetic resonance cholangiopancreatography SLE Systemic lupus erythematosus
MRI Magnetic resonance imaging SOD Sphincter of Oddi dysfunction
NAFLD Nonalcoholic fatty liver disease TB Tuberculosis
NASH Nonalcoholic steatohepatitis TG Triglyceride(s)
NG Nasogastric TIPS Transjugular intraheptic portosystemic shunt
NPO Nil per os (nothing by mouth) TNF Tumor necrosis factor
NSAID(s) Nonsteroidal anti-inflammatory drug(s) TNM Tumor node metastasis
O2 Oxygen TPN Total parenteral nutrition
PBC Primary biliary cholangitis UC Ulcerative colitis
PCR Polymerase chain reaction UDCA Ursodeoxycholic acid
PET Positron emission tomography UGI Upper gastrointestinal
PPI Proton pump inhibitor UGIB Upper gastrointestinal bleed
PSC Primary sclerosing cholangitis UGIS Upper gastrointestinal series
PSE Portosystemic encephalopathy UNOS United Network for Organ Sharing
PUD Peptic ulcer disease US Ultrasonography
RA Rheumatoid arthritis USA United States of America
RLQ Right lower quadrant VLDL Very-low-density lipoprotein
RNA Ribonucleic acid WBC White blood cell
RUQ Right upper quadrant WHO World Health Organization
SBO Small bowel obstruction ZES Zollinger-Ellison syndrome
PART
I Biology of the Gastrointestinal Tract
1 Cellular Growth and Neoplasia

Ezra Burstein
CHAPTER OUTLINE MECHANISMS OF NORMAL TISSUE HOMEOSTASIS

MECHANISMS OF NORMAL TISSUE HOMEOSTASIS . . . . . . . 1 Cellular Proliferation
Cellular Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Tissue homeostasis is maintained by the delicate balance of cel-
Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 lular proliferation and differentiation, which provide new cellular
Senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 elements to replace dying cells as part of normal tissue function
Signaling Pathways That Regulate Cellular Growth . . . . . . . 3 or during tissue repair. At a fundamental level, neoplasia arises
INTESTINAL TUMOR DEVELOPMENT . . . . . . . . . . . . . . . . . . . 5 when cell proliferation escapes the homeostatic mechanisms
Multistep Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 that maintain this process in balance with senescence and pro-
Clonal Expansion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 grammed cell death. Cell proliferation occurs as cells divide, a
process that occurs through an orderly set of steps referred to as
Cancer Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 the cell cycle (Fig. 1.1). In preparation for cell division, there is a
Epithelial-Mesenchymal Transition . . . . . . . . . . . . . . . . . . . 5 period of biosynthetic activity called the G1 phase that is typically
NEOPLASIA-ASSOCIATED GENES . . . . . . . . . . . . . . . . . . . . . 6 associated with an increase of cell size. This phase is followed by
Oncogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 precise duplication of the genome, designated the S phase. After
Oncogenic Growth Factors and Growth Factor Receptors . . . 7 an intervening gap period designated as the G2 phase, mitosis
Nuclear Oncogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 occurs during the M phase.
Tumor Suppressor Genes . . . . . . . . . . . . . . . . . . . . . . . . . . 8 The commitment to proceed to DNA replication occurs at
the G1/S checkpoint or restriction (R) point. Cells may exit this
DNA Repair Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 cycle of active proliferation before reaching the R point and enter
Noncoding RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 a quiescent phase known as G0. Cells can subsequently reenter
Oncogenic Signaling Pathways . . . . . . . . . . . . . . . . . . . . . 10 the cell cycle from the G0 state (see Fig. 1.1). Another checkpoint
TUMOR MICROENVIRONMENT . . . . . . . . . . . . . . . . . . . . . . . 10 exists at the boundary between the G2 and M phases. The G2/M
checkpoint ensures that mitosis does not proceed prior to the
TUMOR METABOLISM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 repair of any damaged DNA after genome replication. Impaired
Inflammation and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . 10 function of these checkpoints is frequently observed in cancers.
Microbiome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Regulation of cell cycle progression is achieved principally by
BIOLOGICAL FEATURES OF TUMOR METASTASIS . . . . . . . . 11 a set of proteins known as cyclins and cyclin-dependent kinases
Angiogenesis and Lymphangiogenesis . . . . . . . . . . . . . . . 11 (CDKs). These proteins are expressed in specific parts of the cell
cycle and regulate the G1/S and G2/M checkpoints. During the
ENVIRONMENTAL INFLUENCES . . . . . . . . . . . . . . . . . . . . . . 11 G1 phase, cyclins D and E are most active.1 Overexpression of
Chemical Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . 11 cyclin D1 in fibroblasts results in more rapid entry of cells into
Dietary Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 the S phase, and, consistent with a role in cancer, cyclin D1 is fre-
MOLECULAR MEDICINE: CURRENT AND FUTURE quently overexpressed in a number of GI and non-GI malignan-
APPROACHES IN GASTROINTESTINAL ONCOLOGY . . . . . . . 12 cies.2 During the S phase, cyclin A is predominantly expressed,
Next Generation Sequencing . . . . . . . . . . . . . . . . . . . . . . . 12 and by the G2 phase cyclin B is the main regulator (see Fig. 1.1).
Each cyclin forms a complex with a CDK and function as cata-
Molecular Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 lysts for CDK activity in a cell cycle–dependent fashion (see Fig.
1.1). The cyclin-CDK complexes regulate cell cycle progression
through phosphorylation of key target proteins. For example,
Normal cellular proliferation and differentiation are essential to cyclin D1–dependent progression from G1 to S phase is the result
tissue homeostasis in all organs, including the digestive tract. The of cyclin D1/CDK4 phosphorylation of the tumor suppression
neoplastic process involves a fundamental disruption of these mech- pRb, the product of the retinoblastoma gene, as well as the Rb
anisms, which can give rise to cancer development and metastasis family members p130 and p107.3 These proteins sequester E2F
with the additional acquisition of other hallmarks of cancer. As a transcription factors that promote expression of factors required
group, malignancies of the GI tract are the leading cause of cancer- for S phase, and their phosphorylation by CDK4 leads to their
associated mortality, and it is therefore essential to understand the functional inhibition. Thus, loss of Rb expression also accom-
underlying biology that gives rise to tumor formation. This chapter plishes more rapid progression to S phase and is another genetic
reviews mechanisms of normal cell growth and the fundamental lesion seen in many tumors. An analogous circuit is found in the
cellular and molecular alterations that facilitate malignant transfor- G2/M transition, where cyclin A/CDK2 mediates the activation
mation. The basic concepts discussed in this chapter provide the of another transcriptional regulator, FoxM1, required for the
framework for discussion of specific GI neoplasms in later chapters. expression of factors involved in mitosis.4
1
2 PART I Biology of the Gastrointestinal Tract
Ink4A CDK4
Cyclin D1
P
pRb pRb P
P
E2F
G1/S
checkpoint
E2F
G0 G1 S
Cyclin D/E Cyclin A
Cyclin B
M G2
P
FoxM1
P
G2 /M
checkpoint
P
FoxM1 FoxM1
P
Cyclin A
Cip/Kip CDK2
Fig. 1.1 Regulation of the cell cycle by (cycs), cyclin-dependent kinases (cdks), and cdk inhibitors. In the
normal cell cycle, DNA synthesis (in which chromosomal DNA is duplicated) occurs in the S phase, whereas
mitosis (in which nuclei first divide to form a pair of new nuclei, followed by actual cellular division to form a
pair of daughter cells) takes place in the M phase. The S and M phases are separated by two gap phases: the
G1 phase after mitosis and before DNA synthesis, and the G2 phase following the S phase. During these gap
phases, the cell is synthesizing proteins and metabolites, increasing its mass, and preparing for the S phase
and M phase. Cell cycle progression is regulated primarily at two points, the G2/M and G1/S checkpoints,
through the coordinated activities of cyclins and CDKs, which in turn are negatively regulated by CDK inhibitors
(Ink4 and Cip/Kip families).
The cell cycle is also regulated by multiple CDK inhibi-

tors, which are classified into various classes and are referred by
Apoptosis
multiple names.5 CDK4 and CDK6 are inhibited by members Apoptosis is a form of programmed cell death that is geneti-
of the Ink4 family of inhibitors known as p16INK4a (encoded cally programmed and executed by specific proteases known
by the Cdkn2a gene), p15INK4b (Cdkn2b), p18INK4c (Cdkn2c), as caspases.9 Similar to other protease cascades, such as the
and p19INK4d (Cdkn2d)].6 Thus these factors also impinge on coagulation system, caspases become active upon cleavage of
Cyclin D1/CDK4 regulation of pRb, and consequent E2F activ- an inactive pro-form, typically through the action of another
ity and S phase entry. p16INK4A loss in cancer results in greater caspase or as a result of focal accumulation of inactive caspases.
activation of CDK4 and is frequently inactivated in GI cancers, a Apoptosis is an important mechanism that counterbalances cell
finding consistent with its function as a tumor suppressor gene.7,8 proliferation; thus, escape from normal apoptotic mechanisms
Members of the Cip/Kip family of CDK inhibitors are known plays a critical role in oncogenesis. Morphologically, apoptosis
as p21Cip1 (Cdkn1a), p27Kip1 (Cdkn1b), and p57Kip2 (Cdkn1c)] and is characterized by distinctive features that include chromatin
are more promiscuous and interfere with multiple cyclin/CDK compaction, condensation of the cytoplasm, nuclear fragmenta-
complexes, including CDK2. tion, and marked alterations at the plasma membrane, resulting
CHAPTER 1 Cellular Growth and Neoplasia 3
Death
Receptors 1
(TNFR1, Fas, etc.)
Bax
Cyto c Bak
Caspase-8 Cellular Stress
(Radiation,
chemotherapy, etc.)
Bcl-2
Caspase-9 Apaf-1
Bcl-xL Mitochondria
Mcl-1
Executioner Downstream
Caspases Targets leading to
(Casp-3, Casp-7) Cell Death
Fig. 1.2 Apoptosis (programmed cell death) counterbalances cellular proliferation to regulate overall tissue
growth. A complex interplay of proapoptotic and antiapoptotic molecules results in downstream activation of
caspases that mediate cell death. Some of these signals are initiated through cellular stress that can desta-
bilize mitochondrial membranes, and some are initiated through death receptors, including TNFR1 and Fas.
The mitochondrial step is regulated by the interplay between proapoptotic (Bax, Bak) and antiapoptotic (Bcl-2,
Bcl-xL) molecules. Upon mitochondrial permeabilization, cytochrome c release promotes the formation of the
apoptosome complex (APAF1, caspase 9, and cytochrome c). Activation of caspase-8 (downstream of death
receptor) or of caspase-9 (as a result of apoptosome formation), leads to activation of executioner caspases (3
and 7) which are responsible for targeting downstream targets that are responsible for cell death.
in compacted apoptotic bodies that are eventually phagocytosed

and eliminated.
Senescence
Apoptosis may be triggered by internal or external stimuli. Senescence is the process by which cells permanently lose their
Internal stimuli of apoptosis may include nutrient deprivation, ability to divide. Senescence may occur in response to the stress
hypoxia, DNA damage, or other stressors, including specific tox- induced by activation of oncogenes or DNA damage or after a fixed
ins, chemical signals, and pathogens. Apoptosis routinely occurs number of cellular divisions (replicative senescence). Associated
during normal development to facilitate tissue patterning. Simi- with the exit from the cell cycle, senescence is associated with a
larly, a number of stress situations, including tissue inflammation, secretory phenotype that includes a variety of proinflammatory
can trigger apoptosis. Apoptosis may also be stimulated by spe- factors. As a physiologic event, senescence limits dysregulated or
cific cell surface receptors belonging to the tumor necrosis factor excessive proliferation. However, when dysregulated, senescence
receptor superfamily, including tumor necrosis factor R1 and Fas, can also contribute to aging and depletion of stem cells.11 During
which are referred to as death receptors (Fig. 1.2). carcinogenesis, senescence is frequently bypassed or lost.
At the intracellular level, the last common event in all forms Replicative senescence is triggered shortening of telomeres,
of apoptosis is the activation of so-called executioner caspases, repetitive sequences at the end of chromosomes that protect
caspase 3 and 7, which mediate the cleavage of a large number genomic integrity. Telomeres shorten with each cell division,
of downstream targets that eventually precipitate cell death. and when they reach a critically short length, they initiate DNA
Proapoptotic signals frequently converge at the level of the damage signaling and cellular senescence. This phenomenon can
mitochondria, where they destabilize the mitochondrial mem- be routinely seen in vitro when primary cells undergo repeated
brane and collapse the electrical gradient required for aerobic rounds of replication, eventually acquiring critically short telo-
respiration (see Fig. 1.2). Besides the effects that result in cel- meres.12 To prevent senescence from being triggered by sus-
lular energetics, this process leads to the release into the cyto- tained replication, cancer cells activate the telomerase enzyme,
sol of proteins normally present in the intermembrane space which adds additional telomeres to the end of chromosomes.13
of the mitochondria, including cytochrome c, a component of
the respiratory chain. In the cytosol, cytochrome c helps in the
assembly of a multiprotein complex known as the apoptosome,
Signaling Pathways That Regulate Cellular Growth
which contains Apaf1 and facilitates the activation of caspase 9, Cellular proliferation is achieved through transition of cells from
which can directly activate caspases 3 and 7. On the other hand, G0 arrest into the active cell cycle (see Fig. 1.1). Although pro-
death receptors activate executioner caspases through receptor gression through the cell cycle is controlled by the regulatory
initiated intracellular signaling events that result in the upstream mechanisms just described, overall proliferation is also modulated
activation of caspase 8. by external stimuli. Growth factors that bind to specific trans-
The mitochondrial membrane permeabilization events that membrane receptors on the cell surface are especially important.
lead to apoptosome formation are controlled by proteins of the Also acting through transmembrane cell surface receptors, extra-
Bcl-2 family. On the one hand, Bax and Bak help form the pore, cellular matrix and cell-cell adhesion molecules (i.e., integrins,
whereas Bcl-2, Bcl-xL, and Mcl-1 inhibit pore formation. The cadherins, selectins, proteoglycans) can also have a significant
stoichiometric ratio between proapoptotic and antiapoptotic impact on cell proliferation. Alterations in cell-matrix or cell-
members of the Bcl-2 family can determine the balance between cell interactions are particularly important in contributing to the
cell survival and cell death.10 In cancer, alterations in the balance invasive phenotype of malignant cells.
of proapoptotic and antiapoptotic factors, including member of After ligand binding, the cytoplasmic tails of these transmem-
the Bcl-2 family, are common events. brane receptor proteins activate intracellular signaling cascades
that alter gene transcription and protein expression. Based on the Many receptors are members of the so-called 7-membrane–
nature of the intracellular signaling cascades that these recep- spanning receptor family. These receptors are coupled to guanine
tors initiate, they can be classified into three major categories: nucleotide binding proteins, also known as G proteins, and thus,
(1) tyrosine kinases, (2) serine and threonine kinases, and (3) G the receptors are referred to as G protein–coupled receptors. G
protein–coupled receptors (GPCRs). proteins undergo a conformational change that is dependent on
The receptors for many peptide growth factors contain intrin- the presence of guanosine phosphates.15 Activation of G proteins
sic tyrosine kinase activity within their intracellular tail. After can trigger a variety of intracellular signals, including stimula-
ligand binding, tyrosine kinase activity is stimulated, leading to tion of phospholipase C and the generation of phosphoinositides
phosphorylation of tyrosine residues in target proteins within (most importantly, inositol 1,4,5-triphosphate) and diacylglycerol
the cell. Most receptors also autophosphorylate tyrosine residues through hydrolysis of membrane phospholipids, as well as modu-
present in the receptors themselves to magnify signaling, and, in lation of the second messengers cyclic adenosine monophosphate
some cases, this also causes attenuation of their own activity to and guanosine monophosphate.16 Somatostatin receptors exem-
effect an intramolecular feedback regulatory mechanism. The plify a GPCR prevalent in the GI tract.
receptors for many peptide growth factors, including the receptor Binding of growth factors and cytokines to cell surface recep-
for EGF and related growth factors, belong to this receptor class. tors typically produces alterations in a variety of cellular functions
Other receptors on the cell surface possess kinase activity that influence growth. These functions include ion transport,
directed toward serine or threonine residues rather than tyrosine. nutrient uptake, and protein synthesis. However, the ligand-
These receptors also phosphorylate a variety of cellular proteins, receptor interaction must ultimately modify one or more of the
leading to a cascade of biological responses. Multiple sites of ser- homeostatic mechanisms discussed to affect cellular proliferation.
ine and threonine phosphorylation are present on many growth The Wnt pathway is one important example of a signaling
factor receptors, including the tyrosine kinase receptors, suggest- pathway that regulates a diverse number of homeostatic mecha-
ing the existence of significant interactions among various recep- nisms to control proliferation of intestinal epithelial cells (Fig.
tors present on a single cell.14 The transforming growth factor 1.3). Evolutionarily conserved among several species, Wnt sig-
(TGF)-α receptor complex is one important example of a serine- naling, as a rule, regulates proliferation in the stem cell niche
threonine kinase–containing transmembrane receptor. and is essential for epithelial homeostasis in the GI tract. From a
Wnt
Frizzled Receptor Plasma Membrane
β-catenin
Pi
GSK-3β
GSK-3β
Axin Axin
APC Dishevelled
APC
Pi
Cytosolic
β-catenin
Proteosome
Nucleus c-Myc
Cyclin D1
VEGF
Tcf4
Fig. 1.3 The Wnt signaling pathway is an important regulator of intestinal epithelial cell proliferation and tumori-
genesis. In the absence of a Wnt signal (left top), cytosolic β-catenin is regulated by the destruction complex,
consisting of APC, Axin, and glycogen synthase kinase-3β (GSK-3β). The destruction complex phosphorylates
α-catenin and targets it for degradation via the ubiquitin-proteosome pathway. In the presence of an active
Wnt signal (right top), α-catenin degradation is prevented and the protein is stabilized, leading to excess cyto-
plasmic α-catenin which is translocated to the nucleus. Nuclear α-catenin interacts with the Tcf-4 transcription
factor to regulate the expression of many key target genes. APC, Adenomatous polyposis coli; P, phosphate
group; Ub, ubiquitin; VEGF, vascular endothelial growth factor.
signaling perspective, its actions are largely the result of the accu- silencing. Other forms of epigenetic change involve the chemi-
mulation of α-catenin in the nucleus, where it binds with the tran- cal modification of the histone proteins that are required for the 1
scription factor Tcf-4 to activate a set of target genes.17 In normal assembly of the nucleosome and that control chromatin compac-
cells, α-catenin is largely associated with adherens junctions, and tion and DNA access. Although mutations in histones themselves
the cytoplasmic pool of this protein is rapidly degraded through are rare in cancer, mutations in the enzymes that modify histones
a phosphorylation and ubiquitination pathway. This is mediated are emerging as an important group of tumor-associated muta-
by the so-called destruction complex, which includes the tumor tions. It is important to note that involvement by these pathways
suppressor APC. When secreted Wnt ligands bind to cell surface is not mutually exclusive.
receptors of the Frizzled family, the constitutive degradation of
α-catenin is inhibited (disheveled) which results in the nuclear
accumulation of this factor, and the subsequent transcriptional
Clonal Expansion
activation of genes that promote cell proliferation. Inhibition of Clonal expansion is essential to tumor development.25 The acqui-
the Wnt signal in mice can be achieved by deletion of Tcf-4 or sition of a mutation that may provide a growth or survival advan-
overexpression of the Wnt inhibitor Dickkopf1, which results in tage to a cell is followed by clonal expansion of these mutated cells.
dramatic hypoproliferation of the intestinal epithelium.18,19 Wnt As this population grows, and particularly with the acquisition of
signaling is most active in the base of the crypt, and as differentia- genetic/epigenetic instability, a second round of clonal expansion
tion ensues, tissue homeostasis is maintained by growth-inhibit- occurs as a cell within this population sustains still another genetic
ing signals that counterbalance proliferative signals and promote alteration that further enhances its growth properties. This itera-
differentiation, including members of the TGF-α family such as tive process of selection, with accumulating genetic alterations,
BMP4.20 Specific members of this family have unique functions results in malignancy. Because of the nature of the clonal expan-
is tissue homeostasis, including promoting a differentiated and sion process, once frank malignancy has developed, it is often the
fibrogenic phenotype of mesenchymal cells, induction of specific case that multiple clones are present in the same tumor, with a
T cell subtypes, and myriad other activities. In broad terms, the different catalog of mutations harbored among various cancer
effects of TGF-α family members are mediated intracellularly cells. Referred to as tumor heterogeneity, this ongoing process may
through the Smad family of proteins, which are transcription fac- give certain cells selection advantages.26 Metastasis may be facili-
tors that are activated in response to ligand-receptor binding.21 tated by the evolution of a subset of tumor cells that acquire the
TGF-α induces transcription of the cell cycle inhibitors p15INK4B capability of traversing the circulatory system and thriving in a
and p21CIP1/WAF1 and is a potent growth-inhibiting factor that new environment.
mediates arrest of the cell cycle at the G1 phase. Furthermore, it
also enhances the inhibitory activity of p27KIP1 on the cyclin E/
CDK2 complex.22
Cancer Stem Cells
Recognition of tumor heterogeneity has led to the cancer stem cell
(CSC) hypothesis, which asserts that there exists a subset of tumor
INTESTINAL TUMOR DEVELOPMENT cells that have stem cell–like properties. CSCs are believed to be
the tumor-initiating cells from which clonal expansion occurs.
Multistep Formation Moreover, it is hypothesized that eradication of these cells is a key
Multiple sequential genetic alterations are required for the trans- therapeutic goal because failure to do so may result in relapse of
formation of normal intestinal epithelium to neoplasia. This mul- disease. Within this CSC hypothesis, there are 2 models.27 The
tistep nature of tumorigenesis is most directly illustrated by the first is a hierarchical model in which CSCs serve as progenitors
changes that accrue in the development of colonic neoplasia (see for all cells in in a given tumor, whereas other cells have limited
Chapter 127). The progression from normal epithelium through long-term reproductive potential. The basic evidence for this
adenomatous polyps to malignant neoplasia is paralleled by the model is the finding that only cells with specific surface markers
accumulation of genetic alterations that change key pathways can repopulate the tumor in xenotransplantation experiments. In
that control proliferation and tissue homeostasis. Studies on the the GI tract, analysis of putative CSCs demonstrate transcrip-
molecular pathogenesis of colon cancer have served as a paradigm tional programs and markers shared with normal intestinal stem
for the elucidation of genetic alterations in other GI cancers, cells, such as Lgr5 and EphB2, which identify and purify colon
including gastric and pancreatic cancer. CSCs.28 The second stochastic model posits that each cancer
Genomic instability is observed in almost all cancers in the GI cell has the same potential to be a CSC, but this determination
tract. This genetically unstable environment promotes the accu- is stochastically based on internal factors in addition to external
mulation of the multiple alterations that characterize GI cancers. environmental cues.
Instability of the genome may result from several mechanisms,
including changes in the genome DNA sequence or through mod-
ifications of the nucleotides to alter their functionality, a process
Epithelial-Mesenchymal Transition
called epigenetic change. In colon cancer, there are now 3 well- It has been noted that within tumors of epithelial origin, some
recognized forms of genetic/epigenetic instability that promote cells acquire features of mesenchymal cells. A similar process
carcinogenesis (Fig. 1.4), and they have been termed chromosomal occurs during normal embryogenesis, when polarized epithelial
instability, microsatellite instability (MSI), and CpG island methylator cells no longer recognize the boundaries imposed by adjacent
phenotype (CIMP).23,24 Chromosomal instability refers to altera- epithelial cells or their basement membrane and adopt features of
tions in chromosomal structure resulting in large chromosomal migratory mesenchymal cells. This phenomenon, designated epi-
deletions, duplications, and translocations, which in aggregate thelial-mesenchymal transition (EMT), endows cells with the ability
result in a state of aneuploidy. In contrast, MSI refers to frequent to move through tissue planes that normally serve as boundar-
alterations in tracts of repetitive DNA sequences (referred to ies for epithelial cells, such as the basement membrane, a dense
microsatellite DNA) and are often diploid or near-diploid on a matrix of collagen, glycoproteins, and proteoglycans. The trans-
chromosomal level (see later discussion on DNA repair). CIMP migration of tumor cells through the basement membrane likely
refers to the accumulation of an epigenetic modification, meth- involves production of key proteolytic activities. Alternatively,
ylation of guanine residues in so-called CpG-islands, areas rich the tumor cell may produce factors capable of activating pro-
in cytidine and guanine in gene promoter sites. This modifica- enzymes present in the extracellular matrix. For example, the
tion has a potent effect on gene transcription and results in gene tumor may produce urokinase, itself a protease, or plasminogen
7-10 years
Chromosomal Hyperproliferative Early Late

Normal Carcinoma
instability epithelium Adenoma Adenoma
APC COX2 KRAS TP53

overexpression
2-3 years
Microsatellite Hyperproliferative Early Late

Normal Carcinoma
instability epithelium Serrated Polyp Sessile Serrated Adenoma
Mutations in genes with polynucleotide tracks

Germline mutations in IGF2R, BAX, TGFB2R, etc. TP53
MMR genes followed
'second hit' (˜5%)
MLH1 promoter
methylation and
silencing (˜10%)
Unclear duration
CIMP Hyperproliferative Early Late

Normal Carcinoma
epithelium Serrated Polyp Sessile Serrated Adenoma
CpG island methylation and silencing of tumor

KRAS suppressor genes TP53
or BRAF
mutations
Fig. 1.4 Multistep models of colorectal cancer based on underlying genetic instability. As shown on the left,
there are 3 major pathways: chromosomal instability (top pathway), microsatellite instability (middle pathway),
and the CpG island methylation, or CIMP (lower pathway). The progression from normal colonic epithelium to
carcinoma is associated with the acquisition of several genetic and epigenetic alterations. In the chromosomal
instability pathway (top pathway), these alterations include the early loss of APC, followed by activation of on-
cogenes (e.g., KRAS) through a point mutation and inactivation of tumor suppressor genes (e.g., APC, TP53)
through a point mutation or deletion. An increasing aggregate number of mutations can be correlated with
progression from early benign adenoma to cancer, as reflected by analysis of polyps by size. In the microsatel-
lite instability model (middle pathway), mutations in DNA mismatch repair (MMR) genes create a mutator phe-
notype in which mutations accumulate in specific target genes (see section on DNA mismatch repair). Tumors
develop much more rapidly through this pathway than through the chromosomal instability pathway (2-3 years
compared to 7-10 years). Germline mutations in MMR genes account for 5% of all colorectal tumors. In the
CIMP pathway (lower pathway), the initiating event is hypothesized to be a BRAF or KRAS activating muta-
tion that somehow triggers extensive CpG island methylation, particularly of gene promoters, resulting in gene
silencing. Among the potential gene targets is MLH1, a component of the MMR pathway, and when silenced
as part of the CIMP pathway, the tumor evolves along a similar molecular as microsatellite unstable cancers
(MSI-H). Sporadic MLH1 methylation and silencing accounts for nearly 10% of sporadic colorectal cancers.
Alternatively, serrated adenomas arising in the CIMP pathway can undergo a pathway similar to that of chro-
mosomal instability to become microsatellite stable tumors.
activator. Having gained access to the interstitial stromal com- DNA repair genes, which prevent accumulation of new muta-
partment, tumor cells can then enter lymphatic and blood vessels tions. Activation of oncogenes or inactivation of tumor suppres-
and metastasize. sor genes contributes to malignant transformation. Although
In addition to these properties, it has been recognized that most of these genes encode for proteins, many cancer-promoting
cells that undergo EMT acquire not only invasive features but genes that harbor oncogenic and tumor suppressive functions
also CSC-like features.29 do not encode for proteins but rather for RNAs that modulate
One key feature of EMT is the loss of adherens junctions genomic function, so-called noncoding RNAs.
that normally maintain epithelial cell–cell interactions. The
molecular correlate of this phenomenon is the loss of expression
of E-cadherin, a critical component of the adherens junction.30
Oncogenes
Mutations in E-cadherin are common in many GI cancers, par- According to the Catalog of Somatic Mutations in Cancer
ticularly gastric cancer, where germline mutations in E-cadherin (COSMIC),31 there are close to 80 oncogenes with strong evidence
are also linked to hereditary diffuse gastric cancer. of involvement in cancer. Genes that encode a normal cellular pro-
tein, whose function may promote the neoplastic process (e.g., anti-
apoptotic function, cell proliferation stimulation, etc.), may function
NEOPLASIA-ASSOCIATED GENES as oncogenes when they are expressed at inappropriately high levels.
Genes that become altered during the neoplastic process belong A typical mechanism for this phenomenon is gene amplification,
to two distinct groups: (1) oncogenes, which actively confer a when tumors acquire multiple copies of a normal gene resulting in a
growth-promoting property, or (2) tumor suppressor genes, the dosage effect that leads to increased gene expression.
products of which normally restrain growth or proliferation. In other cases, a variety of mutations may lead to inappropri-
An important category within tumor suppressor genes includes ately high activity of a normal gene, leading to cancer-promoting
activities. Point mutations or large gene rearrangements result- Growth factor

ing in fusion proteins are examples of mutations that can lead receptors 1
to oncogene activation. For example, several genes that encode
tyrosine kinase–containing growth factor receptors become
oncogenes after a mutation results in unregulated tyrosine kinase
activity that is no longer dependent on the presence of the appro-
K-ras
priate ligand (e.g., EGF). Because of their tumor-promoting
activity, these mutations tend to be recurrent among specific can-
cer classes. The normal cellular genes from which the oncogenes
derive are designated proto-oncogenes. Most of these genes are
widely expressed in many different types of tumor cells. PI3K B-raf
Finally, another source of oncogenes are virally encoded pro-
teins that may affect cellular growth or survival.32 These factors,
while evolved to favor the viral cycle, may in some instances favor
neoplastic development and this is the reason why specific viruses AKT MEK
are associated with increased cancer risk. In addition, in the case
of retroviruses, the ability of the viral genome to insert itself in
the genome of the host can lead to disruptions in the expression
of genes in the vicinity of insertion sites, which at times, may have mTOR ERK
oncogenic activities.
The proteins encoded by oncogenes may affect any of the
hallmarks of cancer, such as stimulate growth factor pathways,
promote tumor invasion, prevent cell death, or have other tumor- Cell growth
promoting actions. With regards to promoting growth factor Proliferation
pathways, oncogenes may encode for (1) growth factors or their Survival
receptors, or for (2) intracellular signal transduction molecules
downstream of the receptor itself, including transcription factors
that mediate the actions of the growth factor at the level of the
nucleus. Fig. 1.5 Signal transduction downstream of growth factor receptors,
where K-ras plays a major role. Oncogenic K-ras can activate multiple
signaling pathways. Molecules that are frequently mutated in colorectal
Oncogenic Growth Factors and Growth Factor cancer are noted by a red arrow and include K-ras (40%), B-raf (10%),
Receptors and PI3K (15%). AKT, Cellular homolog of v-Akt oncogene; ERK,
The transforming effects of enhanced expression of a variety of extracellular signal regulated kinase; MEK, MAPK/ERK kinase; mTOR,
growth factors have been demonstrated both in vitro and in vivo. mammalian target of rapamycin; PI3K; phosphoinositide-3 kinase.
Several growth factor–related proteins encoded by oncogenes
have now been recognized, including the family of Wnt and Sis
proteins, which encodes the α chain of platelet-derived growth To date, almost all ras mutations in GI malignancies occur in
factor. Cancer cells may engage in autocrine signaling to promote the K-ras oncogene. The highest mutation frequency is found in
their growth, or coax the adjacent stroma to hypersecrete such tumors of the exocrine pancreas (>90%).33 Ras genes activated
growth-stimulating factors. More frequently, a variety of recep- through point mutation have been identified in approximately
tors are upregulated in expression or dysregulated leading to con- 50% of colonic cancers as well as a subset of serrated tumors (see
stitutive action. Among them, are receptor tyrosine kinases of the Fig. 1.4).34
EGF receptor family (ERBB1-4), which are frequently upregu- Most oncogenic mutations in ras cause biochemical changes
lated in a variety of GI cancers. that maintain it in the active, guanosine triphosphate–bound state
by reducing guanosine triphosphatase activity or by destabiliz-
ing the inactive guanosine diphosphate–bound form. However,
Signal Transduction–Related Oncogenes several ras mutants retain significant guanosine triphosphatase
Intermediate steps that effectively translate ligand-receptor activity; therefore, other mechanisms that convert ras to a trans-
binding to an intracellular signal are essential in mediating func- forming protein may be involved.35
tional responses of the cell. Mutations in genes that encode key A functional consequence of ras activation is the phosphoryla-
proteins that participate in signal transduction can also lead to tion and activation of key downstream serine/threonine kinases.
cellular transformation (Fig. 1.5). In this regard, the largest fam- One important target of ras is B-raf. In colon cancers without
ily of oncogenes encodes proteins with protein kinase activity. an identifiable K-ras mutation, 20% possess an activating B-raf
Many members of this large oncogene group are expressed by mutation,36 consistent with the concept that activation of an
neoplasms of the GI tract, and these include the Src nonrecep- oncogenic pathway can be achieved through an alteration in any
tor tyrosine kinase that associates with the inner surface of the of several sequential components of a particular pathway (see
plasma membrane. Fig. 1.5).
G proteins regulate signaling of the large family of GPCRs
through the exchange of guanosine triphosphate with guano-
sine diphosphate. In this regard, the ras family of genes, which
Nuclear Oncogenes
encodes a family of proteins related to the G proteins, are among Many cellular oncogenes encode proteins that localize to the
the most commonly detected oncogenes in GI tract cancers. The nucleus. In essence, these nuclear oncogene products are the final
ras family contains 3 genes: H-ras, K-ras, and N-ras. These factors mediators of signal transduction pathways that are also affected
are essential to transduce signals from various growth receptor by cytoplasmic and plasma membrane–bound oncoproteins,
signaling cascades and point mutations that result in activating because they act as transcription factors that regulate expression
amino acid substitutions at critical hot spot positions convert the of certain genes that enhance cellular proliferation and suppress
normal gene into an oncogene. normal differentiation.
The role of nuclear oncogenes is illustrated by the myc family. only one additional hit is required, leading to the younger age of
The c-Myc protein product is involved in critical cellular func- onset and the potential for tumor multiplicity that accompanies
tions like proliferation, differentiation, apoptosis, transformation, these syndromes.
and transcriptional activation of key genes.37 Frequently, c-Myc Although this 2-hit model has been generally observed, there
is overexpressed or amplified in many GI cancers. c-Myc has been are exceptions. Some tumor suppressors may function to increase
found to be a transcriptional target of the α-catenin/TCF-4 com- cancer risk when only one allele is mutated. Moreover, some
plex in colorectal cancers (see Fig. 1.3), which may explain the cancer genetic syndromes display somatic recessive mode of
overexpression of c-Myc observed in this cancer type.38 inheritance because genetic risk is conferred only when biallelic
inactivating mutations are present. Another important feature of
tumor suppressor genes is that they do not function identically
Tumor Suppressor Genes in every tissue type. Consequently, inactivation of a particular
Mutations of tumor suppressor genes are associated with all GI tumor suppressor gene is tumorigenic only in certain tissues. For
cancers, and a number of these genes and their products have example, the tumor suppressor genes RB1 and VHL play crucial
been identified and characterized (Table 1.1). Unlike gain-of- roles in retinoblastomas and renal cell cancer, respectively, but
function mutations, which are characteristic of oncogenes, muta- are rarely mutated in GI malignancies. Tumor suppressor genes
tions in tumor suppressor genes are loss-of-function mutations shown to have a critical role in the pathogenesis of GI malignan-
and are therefore biallelic. cies, APC, TP53, and SMAD4, are described later. Furthermore,
Initial recognition of the existence of tumor suppressor genes we will discuss DNA repair pathways that, when lost, can give rise
was derived from genetic analyses of cancer-prone families. In to neoplasia and therefore function as tumor suppressor factors.
the GI tract, hereditary colon cancer, gastric cancer, and pancre-
atic cancer syndromes are the best described and are discussed
elsewhere in this text (see Chapters 54, 60, and 127). In these
Adenomatous Polyposis Coli Gene
syndromes, there is a marked increase in risk for a particular Genetic linkage analysis revealed markers on chromosome 5q21
tumor in the absence of other predisposing environmental fac- that were tightly linked to polyp development in affected mem-
tors. Tumors arise typically at a younger age than they do in the bers of kindreds with familial adenomatous polyposis (FAP) and
general population, and multiple primary tumors may develop Gardner’s syndrome.40 Further work led to identification of the
within the target tissue. gene responsible for FAP, the APC gene.41-43 The full spectrum
From a genetic standpoint, cancer genetic syndromes most of adenomatous polyposis syndromes attributable to APC is dis-
often have an autosomal dominant mode of mendelian inheri- cussed in detail in Chapter 126. Somatic mutations in APC have
tance. Based on observations in hereditary retinoblastoma, also been found in most sporadic colon polyps and cancers.44,45
Knudson proposed the “2-hit” hypothesis,39 which explains Mutations in APC are characteristically identified in the earliest
the relationship between sporadic and familial forms of cancer. adenomas, indicating that APC plays a critical role as the gate-
Whereas sporadic tumors are initiated by somatic biallelic inac- keeper in the multistep progression from normal epithelial cell to
tivating mutations of a tumor suppressor gene, tumors in familial colon cancer (see Fig. 1.4).
cancer syndromes are accelerated by the inheritance of a monoal- The APC gene comprises 15 exons and encodes a predicted
lelic mutation of a tumor suppressor gene present in all cells in protein of 2843 amino acids, or approximately 310 kDa. Most
affected family members. When this germline mutation is fol- germline and somatic APC gene mutations result in a premature
lowed by a somatic mutation in the remaining normal allele of stop codon and therefore a truncated APC protein product and
the tumor suppressor gene, this gives rise to the development of loss of function. As discussed earlier, APC is a negative regulator
a neoplastic clone that eventually gives rise to a tumor (Fig. 1.6). of the Wnt signaling pathway and its inactivation results in a state
Because of the germline mutation, the likelihood of full inactiva- that resembles constitutive activation of Wnt. Intracellularly,
tion of the tumor suppressor is diminished substantially because
Acquired
Germline somatic
TABLE 1.1 Mutations Associated with Hereditary Gastrointestinal mutation mutation
Cancer Syndromes
TSG X
Disorder Gene(s) Mutated Inherited

cancer Tumor
FAP, AFAP, Gardner syndrome APC syndrome
Lynch syndrome (HNPCC) MLH1, MSH2, PMS2, MSH6, Acquired Second
EPCAM (through disruption of the somatic somatic
neighboring MSH2 gene) mutation mutation
MAP MUTYH X X
Peutz-Jeghers syndrome STK11 Sporadic

Tumor
cancer
Cowden’s disease PTEN
Juvenile polyposis SMAD4, BMPR1A
Hereditary diffuse gastric cancer CDH1 Time
Hereditary pancreatic cancer ATM, BRCA1, BRCA2, PALB2, , Tumor suppressor gene.
PALLD, CDKN2A, PRSS1,
Fig. 1.6 Knudson’s 2-hit hypothesis. In an inherited cancer syndrome,
SPINK1, PRSS2, CTRC, CFTR
one chromosome has an inactive tumor suppressor gene (TSG) locus
MEN1 Menin because of a germline mutation. The counterpart TSG on the remaining

AFAP, Attenuated FAP; APC, adenomatous polyposis coli; FAP, familial paired chromosome is subsequently inactivated by a somatic mutation,
adenomatous polyposis; HNPCC, hereditary nonpolyposis colorectal leading to tumor formation. In contrast, in a sporadic cancer, the two
cancer; MAP, MUTYH-associated polyposis; MEN1, multiple alleles of the TSG need to become inactivated through two indepen-
endocrine neoplasia, type 1; MUTYH, mutY homolog. dent somatic mutations, an event that is less likely to occur within a
single cell.

this is manifested by stabilization of α-catenin, which mediates DNA fragment, fill in the gap with the correct nucleotide, and
the transcriptional effects of Wnt activation and the subsequent finally reseal the remaining nick. The family of DNA mismatch 1
oncogenic phenotype (see Fig. 1.3). Interestingly, another mecha- repair genes includes two basic molecular components, a mis-
nism to achieve this signaling outcome are mutations in α-catenin match recognition complex composed of MSH2 and MSH6, and
itself that render the protein impervious to APC-dependent deg- an excision inducing complex composed of MLH1 and PMS2.
radation. Mutations in any of these genes result in defective mismatch
repair, and when inherited due to a germline mutation, they
give rise to Lynch syndrome, also known as hereditary nonpolypo-
TP53 Gene sis colorectal cancer.55,56 Complete loss of a mismatch repair factor
This is the most commonly mutated gene in human cancer,46 and leads to very high rates of DNA mutations, and mismatch repair
point mutations in TP53 are found with high frequency in all can- defective tumors accumulate a high burden of cancer somatic
cers of the GI tract.47 In fact, point mutations in TP53 have been mutations, typically over 2000 somatic mutations, resulting in a
identified in as many as 50% to 70% of sporadic colon cancers large number of tumor-specific neoantigens.57 Affected cells are
(see Fig. 1.4). Interestingly, these mutations arise relatively late called replication error positive, in contrast to the replication error–
in the oncogenic process as the gene is mutated in only a small negative phenotype.58,59 Because microsatellite DNA sequences
subset of colonic adenomas.48 are primarily affected by this type of genetic instability, the tumor
Named for a 53-kDa-sized gene product, p53 is a nuclear cells display insertions or deletions in these stretches of DNA
phosphoprotein that plays a key role in cell cycle regulation and when compared to nontumor tissue, a phenomenon referred to
apoptosis.47 In the nucleus, p53 functions as a transcription fac- as microsatellite instability. Mechanistically, the absence of DNA
tor which can be induced by conditions of cellular stress, such as repair does not directly cause cancer but creates a milieu that per-
ionizing radiation, growth factor withdrawal, or cytotoxic ther- mits accumulation of mutations in a variety of genes that contain
apy. Induction of p53 arrests cells at the G1 phase to facilitate repetitive DNA sequences, such as the TGF-α type II receptor,
DNA repair, senescence, or trigger apoptosis. These responses IGF type II receptor, BAX, and E2F-4, among others.
are mediated in part by its transcriptional targets such as the Loss of mismatch repair genes represents an important
p21CIP1/WAF1 inhibitor of the cell cycle or the proapoptotic gene, mechanism for the accumulation of mutations within a tumor
PUMA.49 Interestingly, it is often the case that TP53 mutations (see Fig. 1.4). While 5% of colon cancer are due to Lynch syn-
occur as the combination of a genomic deletion encompassing drome, i.e., germline mutations in the mismatch repair system,
one allele, together with a missense mutation in the second allele twice as many tumors (10%) display similar molecular charac-
that targets specific hotspots within the protein. Recent evidence teristics without a germline mutation in any of the mismatch
indicates that the genomic deletions function not only by remov- repair genes. These tumors are most often driven by somatic
ing TP53 but through the loss of adjacent genes with tumor sup- loss of function in this system, most often as a result of silencing
pressive activities.50 Furthermore, the second type of mutations, of MLH1 gene expression as a result of an epigenetic change
resulting in specific missense mutations are thought to contribute in the promoter region of this gene called DNA methylation.
gain-of-function tumorigenic activities.51 In addition to the TP53 MLH1 promoter hypermethylation is most often observed in
point mutations in sporadic cancers, germline TP53 mutations lesions that are serrated adenomas by histology and that also
have been observed in the Li-Fraumeni syndrome, an autosomal carry B-Raf mutations (see Fig. 1.4). Finally, it has been recog-
dominant familial disorder in which breast carcinoma, soft tissue nized that another mechanism that can lead to a state of high
sarcoma, osteosarcoma, leukemia, brain tumor, colon cancer, and mutation burden is the loss of exonuclease proofreading activity
adrenocortical carcinoma can develop in affected persons.52 of the replicative DNA polymerase Pol-ε or Πολ–δ, through a
variety of missense mutations.60
Another important DNA repair pathway involved in carci-
SMAD4 Gene nogenesis is mediated by the MUTYH gene. It encodes a DNA
SMAD4 is a tumor suppressor gene located on chromosome 18q glycosylase that participates in the repair of oxidized guanine
and is deleted or mutated in most pancreatic adenocarcinomas nucleotides, such as 8-oxoguanine residues, that may inappropri-
and a subset of colon cancers. Smad4, the protein encoded by this ately pair with adenines, ultimately leading to somatic G:C→T:A
gene is an essential intracellular mediator of factors belonging to mutations if uncorrected. Biallelic mutations in MUTYH results
the TGF-α superfamily. Smad4 functions as a transcription fac- in an adenomatous polyposis syndrome that resembles FAP,
tor and is an obligate partner of other members of the Smad pro- except that its mode of inheritance is autosomal recessive (see
tein family.53 Mutant Smad4 lacks these properties and among Chapter 126).61,62 Interestingly, G:C→T:A mutations in the APC
other effects, leads to loss of TGF-α inhibition of proliferation. gene were almost universally found in the polyps of patients with
Germline mutations in SMAD4 result in the juvenile polyposis germline MUTYH mutations, indicating that there are impor-
syndrome (see Chapter 126). tant similarities in the molecular pathogenesis of polyps in the
MUTYH and FAP syndromes.
DNA Repair Genes
DNA replication itself and various types of DNA damaging agents
Noncoding RNAs
can introduce errors into the genome. These errors include spon- Our genomes harbor a variety of genes whose products are RNAs
taneous mismatching of nucleotides during normal DNA replica- that do not encode for a protein. The RNA products, termed non-
tion, oxidative damage of nucleotides, and complete double-strand coding RNAs, consist of a broad category of active RNA molecules
breaks. Therefore, a variety of cellular mechanisms have evolved that can mediate a variety of effects. The categories of noncoding
to prevent or correct DNA errors. One type of error that devel- RNAs are rapidly expanding and include so-called microRNAs
ops during replication may occur in repetitive mononucleotide or and long noncoding RNAs, which are frequently dysregulated in
dinucleotide stretches of DNA, so-called microsatellite regions.54 cancers. The microRNAs play a critical role in silencing of other
These repetitive regions are prone to DNA mismatches, which if RNA transcripts via RNA degradation or translational inhibition
not resolved, can result in short insertions or deletions. The cel- and typically regulate dozens of target RNAs at a time. Their
lular machinery devoted to correct these errors is referred to the biogenesis involves conventional gene transcription, followed by
mismatch repair system. The enzymes bind mismatched DNA, processing of the resulting RNA by a variety of nuclease cleavage
cut the DNA strand with the mismatched nucleotide, unwind the events, resulting ultimately in the generation of small interfering
RNAs (siRNAs) by the protein Dicer. These siRNAs bind to its mesenchymal cells, its vasculature, a variety of immune cells
complementary mRNA sequences, and this binding determines recruited to the tumor and particularly in tumors of the intes-
the specificity for RNA targets. Long noncoding RNAs may per- tinal tract, and tumor-associated microbiota which contribute
form diverse functions like gene silencing, splicing, and extension significantly to the tumor microenvironment. In addition, these
of telomeres. elements acting in concert lead to a metabolic environment, such
as the oxygen and nutrient supply of the tumor, that often plays
a significant role in the evolution of the tumor at the primary site
Oncogenic Signaling Pathways and its potential for distant metastasis.
Individual oncogenes or tumor suppressor genes do not necessar-
ily induce cellular transformation directly but typically function
in concert with one another as components of larger oncogenic
TUMOR METABOLISM
signaling pathways already discussed. Some of the pathways that Tumor cells exhibit abnormal metabolic profiles to facilitate their
are particularly relevant for GI tumorigenesis include the Wnt growth and anabolic needs. Observations in 1924 from Nobel
and Ras signaling pathways. These are pathways that regulate Laureate Otto Heinrich Warburg revealed that tumor cells dis-
normal tissue homeostasis but become oncogenic when the sig- played dramatic increases in aerobic glycolysis and diminished
nals are transduced in an aberrant or amplified manner. The key mitochondrial respiration. This metabolic state, known as the
features of Wnt signaling are illustrated in Fig. 1.3. α-Catenin is Warburg effect, has been validated and is a hallmark feature of
translocated from the inner plasma membrane to the cytoplasm. most malignancies.66 It is becoming increasingly clear that inte-
There, it forms a macromolecular complex with the APC pro- gration of the genetic lesions that characterize cancer formation
tein Axin and glycogen synthase kinase-3α. Phosphorylation of is responsible for the changes in cellular metabolism that accom-
α-catenin by glycogen synthase kinase-3α triggers its degradation. pany cellular transformation. Many of the genes implicated in GI
In the presence of an active Wnt signal, α-catenin is stabilized cancers (p53, K-Ras, PI3K, mTOR, HIF, Myc) can in fact regulate
and enters the nucleus, where it interacts with the transcription metabolic pathways. Moreover, germline mutations in metabolic
factor Tcf-4 to up-regulate a number of key target genes, includ- regulators (e.g., subunits of succinate dehydrogenase) that are not
ing c-Myc, cyclin D1, and vascular endothelial growth factor (VEGF). classical oncogenes or tumor suppressor genes have been associ-
As discussed earlier, Wnt signaling is essential for regulating ated with a high risk of tumorigenesis (pheochromocytoma and
proliferation of normal intestinal epithelium, and dysregulated paraganglioma).67,68 The selection advantage of increased glycol-
Wnt signaling is an almost universal feature of all colorectal can- ysis in cancer cells may include greater tolerance to hypoxic envi-
cers. The latter can result from a mutation in the APC, Axin, or ronments and shunting of metabolic byproducts (e.g., lactate) to
α-catenin genes, although alterations in the APC tumor suppres- other biosynthetic pathways. These altered metabolic pathways
sor gene are the most common. An alteration in just one of these are promising new targets for therapy.
components is sufficient to activate the entire pathway. Thus, it is
essential to consider individual genetic alterations in the context
of the overall signaling pathway in which they function.
Inflammation and Cancer
Because pathways are typically not linear, additional levels of Immune cells recruited to the tumor microenvironment can result
complexity arise. There is frequent overlap among pathways, and in a variety of effects. On the one hand, tumor immune surveil-
the distinction between pathways can be somewhat arbitrary. For lance is well recognized and immunosuppressed states increase
example, mutations in the K-ras oncogene result in activation of the risk of cancer development. On the other hand, a number of
multiple distinct signaling pathways, including Raf/ERK/MAPK, cellular elements of hematopoietic origin can promote primary
PI3K/Akt, and nuclear factor-κB, all of which play an important tumor growth, prevent effective immune surveillance, or pro-
role in tumorigenesis (see Fig. 1.5). Crosstalk between these mote the acquisition of features of neoplastic cells that facilitate
effector pathways serves to modulate the cellular responses fur- metastasis. Myeloid cells with immature characteristics, so-called
ther. For example, Akt, a target of PI3K, can phosphorylate Raf myeloid-derived suppressor cells, are an important example of
and thereby regulate signaling through the MAPK pathway.63 this phenomenon.69
Finally, each of these signaling pathways regulates multiple bio- In addition, a number of chronic inflammatory conditions
logical processes related to tumorigenesis,64 including cell cycle increase the site-specific risk of cancer; examples of this include
progression, apoptosis, senescence, angiogenesis, and invasion. ulcerative colitis (see Chapter 115), chronic gastritis (see Chap-
Another pathway that plays a particularly important role ter 52), chronic pancreatitis (see Chapter 59), Barrett’s esopha-
in GI tumors is the cyclooxygenase-2 (COX-2) pathway. The gus (see Chapter 47), and chronic viral hepatitis (see Chapters
enzyme COX-2 is a key regulator of prostaglandin synthesis that 79 and 80). The influences of inflammation on the development
is induced in inflammation and neoplasia. Although no mutations of neoplasia are multifaceted and complex. Cytokines produced
of COX-2 have been described, overexpression of COX-2 in by inflammatory cells can lead to activation of antiapoptotic and
colonic adenomas and cancers is associated with tumor progres- pro-proliferative signals in tumor cells mediated by transcrip-
sion and angiogenesis (see Fig. 1.4), primarily through induction tion factors such as nuclear factor-κB and STAT3.70,71 Immune
of prostaglandin E2 synthesis. Inhibition of COX-2 with a vari- cells may also promote remodeling of the vascular network and
ety of agents (aspirin, nonsteroidal anti-inflammatory drugs, or promote angiogenesis (discussed later). Inflammation may also
COX-2 selective inhibitors such as celecoxib) is associated with a induce DNA damage from cytokine-stimulated production of
reduced risk of colorectal adenomas and cancer.65 reactive oxygen species.
TUMOR MICROENVIRONMENT Microbiome

Cancer is ultimately a complex tissue consisting not only of neo- The human body possesses over 100 trillion microbes and the
plastic cells harboring a number of genetic lesions, as outlined largest concentration of these organisms are present in the GI
previously, but the composite of a number of cellular components tract. The interaction between these organisms and the host is
that endow the tumor with all of its properties. Indeed, the conan area of great interest, particularly for a broad range of autoim-
tribution of non-neoplastic cells to the behavior and evolution mune, metabolic, and neoplastic disorders.72 Interestingly, colonic
of a tumor is increasingly recognized. Cellular elements with tumors are associated with specific subsets of bacteria, and the
recognized contributions to the behavior of the tumor include tumor associated microbial species have the capacity of inducing
colonic tumors in specified animal models.73 Fusobacterium nuclea-

tum, an organism typically found in the oral cavity, is an exam-
Chemical Carcinogenesis 1
ple of this behavior as it can be found in association with colon Many compounds that have carcinogenic potential often require
tumors and, when introduced into colon cancer models driven by metabolic modification by host enzymes, a process called meta-
germline APC mutations, can drive colon tumorigenesis.74 bolic activation. The initial compound, the procarcinogen, is con-
verted by host enzymes to an electrophilic derivative, which then
chemically modifies DNA. Mutations result from errors that occur
BIOLOGICAL FEATURES OF TUMOR METASTASIS during DNA replication as a result of distorted base pairs. Factors
The establishment of distant metastases requires multiple pro- that influence the potency of any chemical carcinogen include the
cesses, many of which involve alterations in interactions between equilibrium between activation of the procarcinogen and deacti-
tumor cells and normal host cells. To metastasize, a cell or group vation or degradation of the carcinogen.80 Deactivation typically
of cells must detach from the primary tumor, gain access to the occurs through a conjugation reaction, usually in the liver.
lymphatic or vascular space, adhere to the endothelial surface at These principles are exemplified by experimental colonic
a distant site, penetrate the vessel wall to invade the second tissue carcinomas that arise in rodents fed cycasin, a glucosylated com-
site, and finally proliferate as a second tumor focus. Angiogenesis pound present in the cycad nut. The glucose residue of cycasin
is necessary for proliferation of the primary tumor and tumor is cleaved in the rat liver by α-glucosidase to form methylazoxy-
metastases. Tumor cells must also overcome host immune cell methanol, which is subsequently deformylated by enzymes in the
killing. As a result, few circulating tumor cells (<0.01%) suc- liver and colon to give rise to methyldiazonium, a carcinogen.
cessfully initiate metastatic foci. A “survival of the fittest” view These same metabolites are formed through hepatic enzymatic
of metastasis has been proposed, in which selective competition modification of the compound dimethylhydrazine and result in
favors metastasis of a subpopulation of cells from the primary colon cancer in the rat.
site.75 In favor of this view is the fact that the mutational land- In humans, regular tobacco use is strongly associated with a
scape of the primary and distant tumor sites are often distinct, higher risk of multiple GI cancers, including pancreatic and colon
indicating that only specific tumor clones acquire the ability to cancer. Among active smokers with long-term tobacco use, the
metastasize. risk for pancreatic cancer can be elevated twofold. Multiple car-
cinogenic agents including arsenic, benzene, and ethylene oxide
have been identified in cigarettes, but the chemicals linked spe-
Angiogenesis and Lymphangiogenesis cifically to the development of pancreatic or colon cancer have
Angiogenesis is essential to sustain continued growth of the pri- not yet been defined.
mary tumor. If new vessels are not developed as the primary tumor
expands, cells most distant from available vessels are deprived of
an adequate source of nutrition and oxygen, and central necrosis
Dietary Factors
occurs. Neovascularization is also an important permissive factor Chemical mutagenesis may be especially important in the devel-
in facilitating metastatic dissemination of tumors.76 A number of opment of cancers within the GI tract and related organs. The
protein growth factors produced by malignant tumor cells and mucosal surfaces from which most primary cancers in the GI tract
stromal cells have been found to be potent stimuli of angiogenesis, develop are exposed to a complex mixture of dietary constituents
including VEGF-A, basic fibroblast growth factor, and TGF-α. that are potential carcinogens or procarcinogens. The ability of
VEGF-A is perhaps the most critical factor that is up-regulated in dietary factors to act as mutagens in humans was demonstrated
most tumor types, including colorectal cancer. Multiple genetic directly in 1995. The frequency of contamination of food with
pathways implicated in GI carcinogenesis modulate VEGF-A aflatoxins, a fungal metabolite, parallels the incidence of hepa-
expression, including Wnt and mutant ras.77 tocellular carcinoma in various areas of the world.81 Studies
Angiogenesis occurs in an ordered series of events. Endo- demonstrating that aflatoxins cause mutations in the TP53 gene
thelial cells in the parent vessel are stimulated to degrade the in hepatocellular carcinoma have provided a compelling link
endothelial basement membrane, migrate into the perivascular between genes and the environment.81
stroma, and initiate a capillary sprout. The sprout develops into Nitrates present in many foods appear to be additional dietary
a tubular structure that in turn develops into a capillary network. constituents that may act as procarcinogens in the GI tract. Diet-
In vitro models that recapitulate the early events of angiogenesis derived nitrates can be converted by bacterial action in a hypo-
indicate that this process involves a balance between proteases chlorhydric stomach to nitrites and subsequently to mutagenic
and protease inhibitors in a manner similar to that during tumor nitrosamines.82 These events may underlie the documented cor-
invasion. Indeed, functional parallels between tumor invasion and relation between dietary intake of foods high in nitrates and the
angiogenesis are evident in their mutual requirement for cellular incidence of gastric cancer in different populations.
motility, basement membrane proteolysis, and cell growth. Other dietary factors may modulate the biological potency of
In addition to angiogenesis, lymphangiogenesis plays an dietary procarcinogens. Variations in the relative and absolute
important role in tumor metastasis. Some important clues into amounts of dietary fats may lead to alterations in the composi-
the molecular basis of tumor lymphangiogenesis have been tion of the colonic microflora and their metabolic characteristics,
obtained. VEGF-C or VEGF-D bind to the VEGF receptor-3 resulting in modulation of the production of enzymes that con-
on lymphatic endothelial cells to stimulate formation of new lym- vert dietary constituents into potentially mutagenic compounds.
phatic vessels.78 This results in the development of new lymphatic Changes in dietary fiber content can alter the transit time of
channels within the tumor mass and, consequently, enhanced dis- luminal contents in the bowel, thereby changing the duration
semination of tumor cells to regional lymph nodes.79 of exposure of the mucosa to potential mutagens. Bile salt con-
tent may be an additional luminal factor that can modulate the
biological effect of procarcinogens. Deconjugated bile salts may
ENVIRONMENTAL INFLUENCES promote carcinogenesis through mucosal injury and enhanced
Fundamentally, cancer is a genetic disorder and genetic mutation epithelial proliferation.
is the common denominator of agents or mechanisms that con- These mechanisms could explain well-documented correla-
tribute to the development of neoplasia. Environmental factors tions between the intake of various dietary constituents and the
play an important role in tumorigenesis insofar as they affect the incidence of colorectal cancer in certain populations (see Chap-
progression of the underlying genetic lesions. ter 127). Populations that have a high fiber intake and resulting
fast colonic transit times generally exhibit a lower incidence of misexpression of intronic sequences. These types of changes are
colorectal cancer than populations with low fiber intake and relatively easy to interpret and adjudicate. Missense changes are
delayed transit. The incidence of colorectal cancer in Japanese those that result in a change in the amino acid encoded by the
immigrants to the United States who consume a Western diet is codon. Given the normal genetic variation present in the human
much higher than that of native Japanese who consume a tradi- population, understanding whether these changes are deleteri-
tional Japanese diet.83 ous can be quite difficult to accomplish. When the effect of such
variants is not known, these are referred as “variants of unknown
significance.” Important limitations of exome sequencing at the
MOLECULAR MEDICINE: CURRENT AND FUTURE present time include variants of unknown significance adjudi-
APPROACHES IN GASTROINTESTINAL ONCOLOGY cation, detection of copy number variants and large rearrange-
ments, and the potential for intronic or promoter mutations not
Next Generation Sequencing detectable by exome capture strategies.
DNA sequencing relies on polymerase-mediated strand synthesis
and the detection of the incorporated nucleotides throughout the
successive steps of the chemical reaction, by a variety of physico-
Molecular Diagnostics
chemical methods. The ability to monitor billions of reactions Genetic testing is a powerful tool to identify high-risk families
simultaneously, so-called massively parallel sequencing, along and define the cancer risk for individual family members. Today,
with the ability to computationally assemble short sequence sequencing panels that assess most of the genes associated with
reads into a continuous long read, have revolutionized sequenc- familial cancer syndromes are commercially available. Application
ing technologies. These new approaches, often referred to as of genetic testing must take into consideration the sensitivity and
next generation sequencing (NGS), are finding their way into specificity of the assay as well as issues of patient confidentiality
the clinical care of patients with cancer in a variety of settings.84 and potential impact on medical insurability. Because these tests
First, sequencing of germline DNA is increasingly used to define rely on target enrichment, it is important to be aware of their
if a patient may have a cancer genetic syndrome. Secondly, these potential limitations. For these reasons, genetic counseling is an
technologies can be applied to determine the mutational land- essential component of the genetic testing process.
scape of a tumor to guide treatment decisions. In addition to genetic germline testing, molecular phenotyping
The extent of DNA sequencing may involve the entire of tumors for the purpose of guiding therapeutic decisions is impor-
genome. Whole genome sequencing uses DNA from a defined tant. To detect tumors due to defects in mismatch repair, testing for
source without any step of enrichment or selection. Another MSI can be performed on archived colon tumor samples.85 In addi-
method is to subject the sample to preliminary step of enrich- tion, loss of immunohistochemical staining for any of the 4 pro-
ment, where areas of interest are extracted from the sample, teins required for mismatch repair (MLH1, PMS2, MSH2, MSH6)
using hybridization methods and primer libraries, with the goal may provide similar information. Studies have demonstrated that
of decreasing the complexity of the sample and increasing the the MSI status of a colon tumor is predictive of the response to
number of reads possible during the sequencing reaction. With 5-fluorouracil–based chemotherapy.86,87 More recently, it has been
greater number of reads available, so-called reading depth, the shown that mismatch repair–deficient tumors, due to their high
accuracy of sequencing increases and the cost is also reduced. The burden of somatic mutations and tumor neoantigens, are highly
most common enrichment method is to focus on the areas of the responsive to immune checkpoint inhibition therapy.88
genome known to harbor genes, collectively referred to as the Therapies that target specific signaling pathways are likely to
exome, which corresponds to about 1% of the entire genome. For increase as our molecular understanding of GI cancers increases.
certain applications, subsets of genes from the entire exome may Antibodies that target EGF receptors and block the EGF recep-
be the only ones enriched for sequencing, and this is the basis for tor signaling pathway have proved therapeutic benefit in colorec-
NGS-based diagnostic tests that focus on gene panels relevant tal cancer. However, their benefit has been shown only in cancers
to cancer. Because NGS involves short reads that are computa- lacking activating mutations in K-ras. Testing for K-ras muta-
tionally assembled into predicted long reads, this technology is tions in colorectal cancers is now standard of care before admin-
insensitive to gene inversions, large insertions, or generally copy istration of such targeted therapy. In addition, small molecule
number variants that affect one allele. tyrosine kinase inhibitors of the c-KIT oncogene now consti-
tute routine treatment of GI stromal tumors (see Chapter 33).89
Molecular techniques may also find a role in the staging of dis-
Cancer and Tumor Genomics ease. For example, capture of small numbers of circulating tumor
As genetic information is obtained from sequencing analy- cells prior to the discovery of metastasis may yield prognostic and
sis, understanding the potential impact of the genetic changes therapeutic benefits.90 Finally, as more tests for genetic markers
observed becomes an important challenge. Single nucleotide vari- become available, monitoring for disease recurrence after surgery
ants refer to changes in a single base pair of the genetic code may become another important application.
compared to a reference sequence. Nonsense mutations refer to the
introduction of a premature stop codon. Single nucleotide vari- Full references for this chapter can be found on www.expertconsult.com .
ants at splice-acceptor or donor sites may result in exon loss or

2
2 Mucosal Immunology and Inflammation

Charles O. Elson, Phillip D. Smith
CHAPTER OUTLINE
toxins from products that may benefit the body such as molecules
derived from food or commensal bacteria. To achieve homeo-
IMMUNOGLOBULINS OF THE MUCOSAL SURFACE ��13
stasis, unusual cell types, immunoglobulins (Igs), and secreted
PHYSIOLOGY OF MUCOSAL IMMUNE CELLS 15
��
mediators function in a coordinated fashion. In contrast to the
FUNCTIONAL ANATOMY OF THE MUCOSAL systemic immune system, whose focus is to act quickly within
IMMUNE SYSTEM ��15 seconds of encountering a foreign antigen, the mucosal immune
system is poised to respond but is predominantly tolerant,3,4
Peyer Patches and M Cells ��15
rejecting harmful antigens but allowing beneficial/harmless ones
Intestinal Epithelial Cells��16 to persist without evoking harmful immune responses such as
Recognition of Pathogen-Associated Molecular Patterns allergic reactions or inflammation.
by Pattern Recognition Receptors ��18 Billions of activated plasma cells, memory T cells, memory B
ANTIGEN PRESENTATION IN THE GUT 18
��
cells, macrophages, and dendritic cells reside in the LP, but sig-
nificant active inflammation is not present. This phenomenon has
EFFECTOR COMPARTMENTS WITHIN THE GUT IMMUNE been called controlled or physiologic inflammation (Fig. 2.1). Impor-
SYSTEM 19
��
tantly, entry of immune cells into the LP and cell activation is

Intraepithelial Lymphocytes 19
��
antigen driven. Thus germ-free mice have few immune cells in

Lamina Propria Lymphocytes and Mononuclear Cells 19
��
their LP, but within hours to days after colonization with nor-
T Cell Differentiation��19 mal intestinal commensals, a massive influx and activation of cells
Innate Lymphoid Cells 20
��
occurs.5-7 A similar process begins in human neonates, whose
Dendritic Cells 20
��
intestine is colonized at birth, initiating the development of the
Macrophages ��21 mucosal immune system and stimulating innate and adaptive sys-
Oral Tolerance 21 temic immunity. The microbiota expands and changes in infancy,
resulting in trillions of gut bacteria that are in communication
��
Chemokine Role in Homeostasis and Inflammation��22

with host cells in every GI organ and have profound effects on the
intestinal vascular, nervous, and immune systems. Humans have
co-evolved with their microbiota and have developed multiple
mechanisms of response, including intestinal epithelial cells and
Mucosal immunity refers to immune responses that occur at muco- innate, adaptive, and regulatory immune components.8
sal sites. The demands on the mucosal immune system are quite An abnormal composition of gut microbiota has been impli-
distinct from their systemic counterparts. At mucosal sites, the cated in multiple metabolic and immune diseases, including IBD.
“outside world” is typically separated from the inner world by a Many, if not most, of the gene variants conferring susceptibil-
single layer of epithelium. The mucosal immune system exists at ity to IBD are linked to cells and functions involved in each of
a number of sites, including the intestinal tract, respiratory tract the components that regulate host-microbiota interactions.9 The
(especially the upper respiratory tract), urogenital tract, mam- microbiota itself is the subject of the next chapter. Despite the
mary glands, eyes, and ears. Each of these sites encounters a dis- persistence of the antigenic drive of the intestinal microbiota,
tinctive array of environmental stimuli and has evolved its own set intestinal lymphocyte effector cells fail to develop into aggressive
of cell populations. Nevertheless, these different compartments inflammation-producing cells. Bacteria or their products play a
can interact and share some cell populations and together form role in this persistent state of suspended activation10 contributing
a common mucosal immune system. This chapter focuses on the to the control of inflammation in the intestinal mucosa.
intestinal mucosal immune system.1
The intestinal mucosa forms the largest compartment of the
mucosal immune system and is unique in several aspects.2 Relative
IMMUNOGLOBULINS OF THE MUCOSAL SURFACE
to other mucosal sites, the intestine contains billions to trillions Immunoglobulin A, an immunoregulatory antibody.
of microorganisms, mainly bacteria. These organisms and their Secretory IgA (SIgA) is the hallmark of mucosal immune
products, along with ingested food, represent an enormous anti- responses (Fig. 2.2). IgG is the most abundant isotype in the sys-
genic load that must be tolerated to maintain mucosal homeosta- temic immune system, but IgA is the most abundant antibody in
sis. This unusual environment and the demands associated with mucosal secretions.3,11,12 Given the numbers of IgA+ plasma cells
it have resulted in the development of a distinct immune system and the 3-5 grams of IgA produced daily in humans, IgA is the
composed of inductive lymphoid follicles named gut-associated most abundant antibody in the body.
lymphoid tissue (GALT) and effector cells distributed in the epi- SIgA is a dimeric form of IgA produced by plasma cells in
thelium (intra-epithelial lymphocytes, IELs) and in the lamina the LP and transported into the gut lumen through the intes-
propria (LP) as mononuclear cells (LPMCs). Collectively, these tinal epithelium by a specialized pathway (Fig. 2.3). Two IgA
cells comprise the largest number of cells in the immune system. molecules (homodimers) are bound together by J chain (pro-
The specific characteristics and peculiarities of the mucosal duced by plasma cells). Subsequently, the homodimer binds
immune cells reflect the unique milieu in which the cells function. to a highly specialized glycoprotein, the polymeric Ig receptor
To maintain mucosal homeostasis in the intestinal mucosa, one (pIgR), previously called secretory component, a 55-kd glyco-
of the most important tasks of the immune system is to differenti- protein produced by epithelial cells.11 The pIgR is expressed on
ate potentially harmful antigens such as pathogenic bacteria and the basolateral membrane of the intestinal epithelial cell (IEC)
13
IELs
↓Cellular responses
Bacteria
DC
Tight
junctions Defensins Treg
Goblet HBD-2, 3, 4
cell SlgA
J Lymphocytes
Macrophage
LPMC
Plasma cell MAdCAM-1
α4β7
integrin
Blood vessels
Fig. 2.1 Mechanisms for damping mucosal immune responses. The intestine uses a number of distinct mech-
anisms to dampen mucosal immune responses. The major source of antigen in the intestine is the commensal
bacterial flora, but both innate and adaptive responses control local responses. Physical barriers like mucins
secreted by goblet cells and tight junctions between epithelial cells prevent invasion by luminal flora (circle
inset). Defensins like HBD-2, -3, and -4 are thought to maintain sterility of the crypt, whereas secretory im-
munoglobulin A produced by local plasma cells prevents attachment and invasion by luminal bacteria, thereby
reducing antigenic load. Even with antigenic challenge, intestinal lymphocytes, macrophages, and dendritic
cells are programmed to not respond as a consequence of decreased expression of pattern recognition recep-
tors (e.g., Toll-like receptors) and a decrease in the ability of lymphocytes to be activated through their antigen
receptor. Egress of circulating lymphocytes from blood vessels such as high endothelial venules is directed by
the integrin α4β7, which recognizes the addressin MAdCAM-1, is also shown. DC, dendritic cell; HBD, human
β-defensin; IELs, intraepithelial lymphocytes; LPMC, lamina propria mononuclear cells; MAdCAM, mucosal
addressin cell adhesion molecule; SIgA, secretory immunoglobulin A, a dimer with a connecting J chain; Treg,
T regulatory cells (formerly known as suppressor T cells).
classical complement components but rather binds luminal bac-

pIgR terial antigens, preventing their uptake by epithelium and pro-
moting their agglutination and subsequent removal.12,15-18 This
J chain process, referred to as “immune exclusion,” includes agglutina-
tion, entrapment, and clearance of antigen due to specific inter-
Light action with the secreted antibody,14 as opposed to nonspecific
chain mechanisms of exclusion exerted by the epithelium (e.g., mucus
production, proteolytic digestion, defensin secretion). SIgA also
may exert specific protective immunity against certain pathogens
Heavy chain via more direct mechanisms such as the suppression of bacterial
virulence19, as well as non-antigen specific binding to bacterial
glycan residues on free or bound pIgR, or the SIgA complex.20,21
Fig. 2.2 Secretory immunoglobulin (Ig)A complex. Two IgA molecules M cells in Peyer patches (discussed later) selectively bind SIgA
are linked by a J chain and stabilized by polymeric Ig receptor (pIgR) to and SIgA immune complexes,22,23 and uptake of antigen-IgA
form dimeric secretory IgA. complexes via M cells is a potential mechanism to dampen local
inflammatory responses.24
and binds only to dimeric IgA or IgM (also polymerized with IgM is another antibody capable of binding the pIgR. Like
J chain). Once bound to the pIgR on the IEC, SIgA is actively IgA, IgM uses J chain produced by plasma cells to form poly-
transported within vesicles to the apical membrane of the IEC. mers—in the case of IgM, a pentamer. pIgR binds to the Fc por-
The vesicle fuses with the apical membrane, and the pIgR/ tion of the antibody formed during polymerization. The ability
IgA complex is released into the intestinal lumen. Within the of IgM to bind pIgR may be important in patients with IgA defi-
lumen, pIgR serves to protect the SIgA dimer from degradation ciency, where secretory IgM (SIgM) compensates for the absence
by luminal proteases and gastric acid.13 of IgA in the lumen.
SIgA binds to mucus, enhancing its ability to bind and trap SIgA is the major antibody isotype produced in the mucosa,
microbial products.14 In addition to its unique form, SIgA is also but IgG is present in the mucosa as well.25,26 The neonatal Fc
unusual in that it is anti-inflammatory in nature. It does not bind receptor (FcRN) expressed by IECs can serve as a bidirectional
CHAPTER 2 Mucosal Immunology and Inflammation 15
A mucus coat, produced by goblet cells, lines the intestinal

tract and is composed of a mixture of glycoproteins (mucins) 2
heavily glycosylated with O-linked oligosaccharides and N-glycan
chains linked to a protein backbone.32 At least 21 different mucin
Secretory IgA genes, encoding secreted and membrane bound mucins, each with
pIgR a distinct carbohydrate and amino acid composition,33,34 have
been identified in the human genome. The major colonic mucins
are MUC1, MUC2, MUC3A, MUC3B, MUC4, MUC13, and
MUC17. MUC2 is a secreted mucin and serves as the primary
Intestinal
component of intestinal mucus, whereas the other mucins are
Epithelial Cell
membrane bound. The membrane-bound mucins participate in
processes such as cell signaling, adhesion, growth, and immune
modulation. Mucus protects the intestinal epithelium by several
mechanisms, including its stickiness and competitive binding of
its glycoprotein receptors to decrease microorganism penetra-
tion.35 In the colon there are two mucus layers, a loose outer layer
containing bacteria and a dense inner layer devoid of bacteria.36
The mucus stream also moves luminal contents away from epi-
thelial cells. Further, intestinal infection and inflammation are
Polymeric Ig receptor associated with disruption or dysfunction of the mucus barrier,
which may be accompanied by altered innate and adaptive host
immune responses to the microbiota.37
Below the mucus layer, the epithelium acts as a physical bar-
IgA plasma cell
rier that normally prevents antigen penetration through epithe-
Dimeric IgA
lial cells (the transcellular route) and through intercellular spaces
(the paracellular route), the latter regulated by tight junction (TJ)
complexes (e.g., zona occludens) and the subjunctional space.38
IgA Tight junctions have the greater role in preventing macromo-
lecular diffusion across the epithelium, because these junctions
exclude almost all molecules present in the lumen.39 The barrier
J chain formed by the TJ is a dynamic structure that may be modified
by various cytokines and growth factors. The cytokines IFN-γ,
TNF-α, IL-1β, IL-4, IL-6, and IL-13 increase intestinal TJ per-
meability, whereas IL-10, IL-17, and TGF-β decrease intestinal
Fig. 2.3 Assembly and secretion of dimeric immunoglobulin A (IgA). TJ permeability,40 a feature that may limit intestinal inflamma-
IgA and J chain produced by IgA-committed plasma cells (bottom) tion such as that in IBD.41
dimerize to form polymeric IgA, which covalently binds to membrane-
bound polymeric Ig receptor produced by intestinal epithelial cells FUNCTIONAL ANATOMY OF THE MUCOSAL IMMUNE
(top). This complex is internalized, transported to the apical surface of
epithelial cell, and secreted into the lumen. pIgR, polymeric immuno-
SYSTEM
globulin receptor. Several key features of the mucosal immune system facilitate the
maintenance of homeostasis and clearance of pathogens. The
mucosal immune system is comprised of inductive and effec-
tor compartments. Peyer patches and other lymphoid follicles
transporter of IgG27,28 and is important in control of certain comprise the inductive compartment and are known as GALT.
neonatal infections and IgG metabolism. In patients with IBD, Lymphoid cells in the epithelium (IELs) and the LP (LPLs) are
marked increases in IgG within the LP and lumen have been innate and antigen-experienced memory cells and comprise the
detected.29 effector compartment. Cell populations and the immune response
IgE production may play an important role in the intestinal in the epithelium, subepithelial region, LP, Peyer patches, and
response to helminths and in food allergy (see Chapter 10). CD23 mesenteric lymph nodes (MLNs) may differ substantially. Cells
(low-affinity IgE Fc receptor) has been reported to be expressed residing in these compartments differ not only topographically
by gut epithelial cells, and one model has suggested that it may but also phenotypically and functionally.
facilitate antigen uptake and resultant mast cell degranulation
in food allergy. In this condition, IgE transcytosis and mast cell
degranulation may be associated with fluid and electrolyte loss
Peyer Patches and M Cells
into the lumen, an event intimately associated with an allergic The follicle-associated epithelium (FAE) is a specialized epithe-
reaction in the lung and gut.30,31 lium overlying the Peyer patch and isolated lymphoid follicles.
The M (microfold) cells in the FAE, in contrast to the adjacent
absorptive epithelium, have few microvilli, a limited mucin over-
PHYSIOLOGY OF MUCOSAL IMMUNE CELLS layer, a thin elongated cytoplasm, and a shape that forms a pocket
The cells, structures, and mediators that separate the intesti- surrounding subepithelial lymphocytes, macrophages, T cells, B
nal lumen from the LP function as a physical barrier, which cells, and dendritic cells (DCs) (Fig. 2.4). M cells are highly spe-
constantly interacts with its ever-changing luminal environ- cialized for phagocytosis and transcytosis and are capable of tak-
ment. The intestinal barrier changes not only on a day-to-day ing up large particulate antigens from the lumen and transporting
basis but also through the years. Many barrier mechanisms are them intact into the subepithelial space.42-45 These cells contain
not fully developed at birth, and evidence in animal studies few lysosomes, and thus little or no processing of antigen occurs.46
supports increased antigen transport in neonates compared to The apical surface of M cells expresses several unique lectin-like
adults. molecules that help promote binding to specific pathogens such
as poliovirus.47 Antigens that bind to the M cell and are trans- The M cell is a conduit to Peyer patches and lymphoid folli-
ported to the underlying Peyer patches generally elicit a positive cles. Antigens transcytosed across the M cell and into the sub-epi-
(SIgA) response. Thus M cells appear to be critical for the ini- thelial pocket are taken up by macrophages and DCs and carried
tial positive aspects of mucosal immunity.48,49 However, certain into the Peyer patch. Once antigens enter the Peyer patch, TGF-
pathogens or their toxins may exploit M cells and use M cell tran- β-secreting T cells promote B cell isotype switching to IgA.
scytosis to penetrate the intestinal mucosa. Induction of M cell differentiation is dependent on direct contact
between the epithelium and Peyer patch lymphocytes,45,50 medi-
ated, at least in part, by the expression of NOTCH receptors and
ligands.51 In the absence of Peyer patch B cells, M cells are not
present, as M cells have not been identified in B cell–deficient
E animals, which lack Peyer patches. The Peyer patches have T
cell-dependent areas and B cell-dependent/germinal centers typi-
cal of lymph nodes, but only efferent lymphatics.
E E After activation in the Peyer patch, lymphocytes are induced
to express specific integrins (α4β7) that provide a homing signal
for mucosal sites where the endothelial ligand is MadCAM-1.52,53
L Lymphocytes exit the Peyer patch, traffic to the MLN and then
the thoracic duct, into the main intestinal lymphatic drainage
system, which empties into the circulation (Fig. 2.5). There,
mucosally activated cells with their mucosal “addressins” circu-
late in the bloodstream to exit in high endothelial venules in vari-
ous mucosal sites.54 Cells bearing α4β7 molecules exit in the LP,
where they undergo terminal differentiation. Chemokines and
L
their receptors (discussed later) as well as adhesion molecules and
ligands help direct this trafficking pattern.
L
Intestinal Epithelial Cells

Intestinal epithelium is composed of a single layer of columnar
cells. These IECs (enterocytes) are derived from the stem cell
zone at the base of crypts, from which they migrate up the vil-
lus and differentiate into absorptive epithelium or secretory cells,
L including goblet cells, enteroendocrine cells, Paneth cells, and
tuft cells.45 In addition to their function as a physical barrier,
IECs contribute to both innate and adaptive immunity in the gut
mucosa and play a key role in maintaining intestinal homeosta-
Fig. 2.4 M cell. Transmission electron micrograph from noncolumnar sis.45
region of a Peyer patch epithelium shows a cross-sectional view of a The absorptive epithelial cells maintain a physical barrier
microfold (M) cell, as well as associated microvillus-covered intestinal via their tight junctions, which are comprised of claudins, zonal
epithelial cells and at least 3 lymphoid cells (L). Note the attenuated occludens, other occludens, and junctional adhesion molecules.
cytoplasm of the M cell (between arrows) that bridges the surface Tight junctions seal the space between epithelial cells and dynam-
between microvillus-covered epithelial cells, forming tight junctions with ically regulate solute transport. Some DCs can express tight junc-
them and producing a barrier between lymphoid cells and the intestinal tion proteins and can extend processes between epithelial cells
lumen (×9600). B, B cell; E, intestinal epithelial cell. (From Owen RL, to sample antigens in the lumen.55 As shown in Figure 2.6, IECs
Jones AL. Epithelial cell specialization within human Peyer’s patches: communicate with immune cells, particularly IELs, and produce
an ultrastructural study of intestinal lymphoid follicles. Gastroenterology various chemokines that regulate cell trafficking into the mucosa,
1974; 66:189–203.) as discussed below.
Intestine
Tonsil/Eye/Ear
Villi
Lumen
Follicle-associated
epithelium Mammary
gland
Peyer’s patch
Genitourinary
tract Fig. 2.5 Mucosal lymphocyte migration. Following
Circulation antigenic stimulation, T and B lymphocytes migrate
Respiratory from the intestine (Peyer patch) to the draining
Mesenteric tract mesenteric lymph nodes, where they further dif-
lymph node ferentiate and then reach the systemic circulation
Thoracic via the thoracic duct. Cells bearing appropriate
duct
mucosal addressins then selectively home to muco-
sal surfaces that constitute the common mucosa-
associated lymphoid tissue, including the intestinal
immune system.
Luminal 2
bacteria
Food or bacterial
antigen
Inflammation
↑paracellular
transport
Tight junction
Stress response/
homeostasis? γδ TCR MICA/MICB αβ TCR Homeostasis?
CD2
CD4+ or CD8+ IEL
Autoregulatory CD4+ IEL
or suppressor β2m MHC
population? αβ TCR CD1d class I αβ TCR Cytolytic or
CD8 CD8 suppressor
gp180 MHC
CD8+ CD1d activity?
class II CD86 CD8+ IEL
CD28− IEL β2m gp180 CD58
αβ TCR αβ TCR
CD28 CD28+
αβ TCR αβ TCR
CD4+ CD8 CD2 CD4+ LPL
CD25+ LPL
CD8+ CD4+ LPL
CD28− LPL
Tolerance?
Autoregulatory Inflammation?
or suppressor
population?
Fig. 2.6 A normal intestinal epithelial cell (IEC). The IEC is shown to express classic MHC molecules (classes
I and II) that have the potential to present conventional antigen to local T cell populations and a broad array of
nonclassic class I molecules (e.g., CD1d, MICA/MICB, and β2m [shown in the figure] and MR-1, ULBP, and
HLA-E), which have the potential to present unconventional antigens to unique T cell populations. In addi-
tion, alternate pathways of activation appear to be functional in the intestine (e.g., activation via a CD58-CD2
interaction), and classic co-stimulatory molecules are not expressed on IECs, although CD86 may be induced
in patients with UC. Other members of the B7 family are expressed, such as PO-L1 (CD274) and ICOS-L
(CD275), and may play a role in local T cell activation. β2 microglobin (β2m) associates with MHC class I,
CD1d, HLA-E, HLA-G, and FcRn. β2m, β2 microglobulin; gp180, membrane glycoprotein 180 (a CD8 ligand);
IEL, intraepithelial lymphocyte; LPL, lamina propria lymphocyte; MHC, major histocompatibility complex; MICA/
MICB, MHC class I-related chains A and B; TCR, T cell receptor.
Paneth cells GFI-1, is important in Paneth cell development by suppressing

Paneth cells are a secretory population derived from epithelial a pro-enteroendocrine cell transcription factor, neurogenin-3
stem cells and reside in the base of the crypts in the small intes- (NEUROG3). Decreased numbers of Paneth cells and of defen-
tine.45 These cells can be readily identified on stains due to the sins 5 and 6 are associated with ileal Crohn disease. Because of
large eosinophilic granules that occupy most of their cytoplasm their highly secretory nature with strong synthesis of various
(see Chapter 98). These granules contain high concentrations of molecules, Paneth cells are dependent on the unfolded protein
antimicrobial peptides, including α-defensins, lysozyme, C-type response to maintain intracellular homeostasis. Interference with
lectins, and phospholipase-A2. They secrete these antimicrobial the unfolded protein response in mice has led to ileitis.56,57
peptides into the lumen of the crypt, thus keeping it relatively
sterile. Defensins are released upon stimulation by various bacte-
rial ligands, including endotoxin, which induce Paneth cells via
Goblet Cells
Toll-like receptors (TLRs) and nuclear oligomerization domain Goblet cells are a secretory type of epithelial cell that is special-
2 (NOD2). Paneth cells co-localize with stem cells at the base of ized in the production of mucins and other molecules. Two types
crypts and have been shown to provide important factors preserv- of mucin layers are present in the gut. One is an unattached or
ing stem cell function in addition to the antimicrobial peptides. loose layer, which is characteristic of the small intestine. The sec-
Paneth cell development is regulated by transcription factors ond mucus layer, which is attached and impenetrable to bacteria,
in the WNT pathway and the NOD signaling cascade such as is present in the distal colon. In addition to their physical prop-
the transcription factor MATH-1. A transcriptional repressor, erties, mucins interact with antimicrobial peptides to maintain a
high concentration of the latter close to the epithelium. A variety as well as their contribution to innate and adaptive T and B cell
of cytokines increase Muc-2 expression, including IL-1β, IL-4, responses in both intestinal inflammation and homeostasis, has
IL-6, IL-13, and TNF-α. Several metalloproteinases, including been demonstrated in several murine models.70,71
ADAM-10 and ADAM-17, as well as meprin-23, are involved in In contrast, some bacteria induce anti-inflammatory cytokine
the release of mucin proteins into the intestinal lumen. Goblet production (e.g., IL-10) and increase expression of peroxisome
cells also selectively produce trefoil factor-3 (TFF-3), which can proliferator–activated receptor (PPAR)-γ by IECs.72,73 Further-
influence mucus viscosity and is important in epithelial repair more, other bacterial products (e.g., from Bacteriodes thetaiotaomi-
after injury. Mice deficient in TFF-3 are highly susceptible to cron) help promote the barrier and IEC differentiation.74
dextran sulfate sodium (DSS) colitis due to an inability to heal
the lesions. Innate immune cells also interact with goblet cells
increasing their number by stimulating enterocytes to differen-
ANTIGEN PRESENTATION IN THE GUT
tiate into goblet cells. ILC2-derived IL-13 and ILC3-derived Effective immune responses to antigenic proteins require the help
IL-22 have been implicated in increasing goblet cell hyperplasia of T lymphocytes. This in turn depends on the antigen being pre-
and mucin production. sented by APCs that internalize, digest, and couple a small frag-
Goblet cells also serve to transmit antigen across the epithelial ment of the antigen to a surface major histocompatibility complex
layer through goblet-associated antigen passages (GAPs). Solu- [MHC] heterodimer that eventually interacts with either CD4+
ble antigens and bacteria have been shown to cross goblet cells T cell receptor (MHC class II) or CD8+ T cell receptor (MHC
and be presented to subjacent CD103+ CX3CR1− tolerogenic class I). Multiple cells in the intestinal mucosa can act as APCs,
DCs. CD103+ DCs then migrate to the mesenteric lymph node including DCs, macrophages, and B cells. The ability of these
where they induce antigen-specific T regulatory (Treg) cells. cells to present antigen to CD4 T cells depends on the expres-
Thus GAPs appear to contribute to the maintenance of intestinal sion of class II MHC on their surface. Class II MHC molecules
homeostasis.58 are also present on the epithelium of the normal small intestine
and to a lesser extent colonocytes in both humans and rodents.
In vitro studies have demonstrated that isolated enterocytes from
Tuft Cells rat and human small intestine can present antigens to previously
Tuft cells are a type of epithelial cell that is infrequent in the primed T cells,75,76 raising the possibility that the intestinal IECs
normal intestine but are increased in number during parasite may present peptides to T cells that are localized below the epi-
infection. Tuft cells are identified by a distinct morphology, thelium. Thus IECs are capable of both antigen processing and
characterized by microvilli projecting from the apical membrane, presentation in the appropriate context to cells within the LP.
and a distinguishing phenotype, characterized by ATOH1 and Interestingly, bidirectional lymphocyte-epithelial crosstalk exists
Neurog 3 expression. Tuft cells express genes associated with in the LP, and LP lymphocytes (LPLs) promote mucosal barrier
taste receptors (see Chapter 4) and are able to discern helminths function via Notch-1 signaling and induction of IEC differentia-
in the intestinal lumen via these chemosensors. Tuft cells pro- tion, polarization, and barrier function.77 Importantly, increased
duce the cytokine IL-25, which activates type 2 innate lymphoid expression of MHC class II molecules by IECs has been reported
cells (ILC2) to produce IL-13 that in turn stimulates mucin pro- in IBD,78,79 which likely increases the potential of IECs to acti-
duction and helps clear the parasite. ILC2-produced IL-13 pro- vate lymphocytes.80,81
vides a positive feedback loop by increasing the number of tuft Interestingly, drugs used to treat IBD (e.g., 5-aminosalicylate
cells.59,60 [5-ASA] preparations) may reduce IEC MHC class II expres-
sion.82 In addition to MHC class II expression, IECs in normal
Recognition of Pathogen-Associated Molecular subjects and IBD patients express a variety of co-stimulatory
molecules required for T cell activation (see Fig. 2.6). These
Patterns by Pattern Recognition Receptors molecules include intercellular adhesion molecule (ICAM)-1,
Classical antigen-presenting cells (APCs) in the systemic immune which binds to leukocyte function associated antigen (LFA)-l on
system possess the innate capacity to recognize highly conserved the T cell and ICOS ligand and PD-L1. CD86 (B7-2), which
pathogen-associated molecular patterns (PAMPs) on bacteria and binds to CD28 and CTLA-4,83 is expressed by IECs in ulcerative
viruses. Receptors for PAMPs are expressed on both the APC colitis. Interestingly, unique expression of these co-stimulatory
surface (e.g., TLRs) and intracellularly (e.g., nuclear oligomer- molecules by IECs may be involved in the distinct regulation of
ization domain [NOD] receptors). Although IECs are exposed to mucosal responses. Small intestinal IECs do not express CD80
large numbers of luminal bacteria, they retain the ability to recog- (B7-1),84 and thus activation of naive T cells by IECs is improb-
nize some components of these bacteria. IEC pro-inflammatory able, aiding in the downregulation of T cell responses. However,
responses are downregulated in the normal setting. For example, increased expression during intestinal inflammation may serve to
IECs do not respond to bacteria lipopolysaccharide (LPS) due to augment T cell stimulation.85
the absence of TLR4, the LPS receptor. However, the expression MHC class I and non-classical class I molecules are also
of other pattern recognition receptors is maintained, including expressed by IECs. Thus antigen presentation to certain T
expression of TLR5, which recognizes bacterial flagellin.61 TLR5 cell populations is possible and has been reported by several
is expressed basolaterally and is positioned to identify organisms groups.86-88 Specifically, CD1d expressed on human IECs is able
such as Salmonella species that have invaded the epithelial layer.62 to present antigen (in a complex with CEACAM5) to CD8+ T
After invasion and engagement of TLR5, the intestinal epithe- cells.89-91 CD1d-restricted natural killer T (NKT) cells, effec-
lium is induced to secrete a broad array of cytokines and chemo- tor memory cells that share characteristics of innate and adaptive
kines that attract inflammatory cells to the local environment to lymphocytes, are among the earliest responders in immune reac-
control the spread of infection. tions and affect activation of other immune cell lineages such as
Intracellular NOD1 and NOD2 have been shown to contribute NK cells, T cells, and B cells. NKT cells participate in immune
to intestinal inflammation. About 25% of Crohn disease patients responses in infectious, malignant, and immune-mediated dis-
have mutations in the NOD2/CARD15 gene, interfering with eases.92 Other non-classical class I molecules are expressed by
their ability to mount an appropriate immune response to bacterial IECs. The role of MICA, a stress-induced MHC-related mol-
stimuli (see Chapter 115).63-68 In addition, TLRs that are normally ecule expressed on normal IECs and recognized by the NKG2D-
weakly expressed by IECs are expressed at higher levels on IECs activating receptor on CD8+ T cells, T cells, and NK cells, may
from patients with IBD.69 Expression of different TLRs by IECs, be of specific importance, since Crohn disease patients have
increased numbers of CD4+NKG2D+ T cells with a Th1 cyto- Lamina Propria Lymphocytes and Mononuclear
kine profile in the intestinal mucosa.93 2
In humans, IECs specifically activate CD8+ Treg cells, which Cells
are involved in local tolerance and interaction with CD8+ IECs. The LP is the major effector site in gut mucosa, containing an
The role of IECs in the regulation of mucosal immunity is best abundance of antigen-experienced memory T cells. LPLs are
demonstrated in studies with IBD tissues. IECs derived from IBD more prone to undergo apoptosis than their peripheral counter-
patients, in contrast to those derived from normal subjects, stimu- parts, a potential regulatory mechanism that limits the inflam-
late CD4+ T cells in vitro rather than regulatory CD8+ cells.80,81,94 matory effects of activated lymphocytes. In inflammatory bowel
Furthermore, oral antigen administration does not result in toler- diseases such as Crohn disease, LPLs resist apoptosis.
ance in IBD patients but causes active immunity.95 Clearly the mucosal LP operates under a distinct set of rules
compared to the systemic immune system, reflected in its func-
EFFECTOR COMPARTMENTS WITHIN THE GUT tional anatomy (no organized structure) and its responses and
regulation. Highly specialized cells mediate these effects, some
IMMUNE SYSTEM detected only in the LP.
Two lymphocyte populations, IELs and LPLs, reside in the Lamina propria mononuclear cells (LPMCs) are a heteroge-
intestinal mucosa. The compartmentalization of these two dis- neous group of cells107 (see Fig. 2.1). A prevalent cell type is the
tinct cell populations correlates with their ability to respond to IgA+ plasma cell, but more than 50% of LPMCs are T cells and
distinct microenvironmental cues. B cells (together comprising the LPL population), in addition to
macrophages and DCs. In contrast to IELs, LPLs express the
mucosal addressin, but similar to IELs, LPLs express an acti-
Intraepithelial Lymphocytes vated memory phenotype and do not proliferate in response to
IELs form one of the main branches of the intestinal immune engagement of the TCR. Alternate pathways of LPL activation
system, balancing protective immunity with support of epithe- are mainly through CD2 and CD28.103,108
lial barrier integrity. In the small intestine, IELs are more than In the healthy mucosa, LPMCs are downregulated for the
98% T cells and mostly CD8+,96-98 including CD8+α T cells, ability to respond to antigen stimulation via the TCR and have
as well as CD4+CD8+ double-positive, and CD4−CD8− double- an increased tendency to undergo apoptosis if activated inap-
negative cells. Greater numbers of these cells also express the propriately, dampening responses to normal luminal contents.
γδ T cell receptor (TCR), in contrast to the αβ TCR expressed The mechanism underlying the increased apoptosis may relate
by CD8+ T cells in systemic immune system.99 Roughly half to engagement of the death receptor Fas and its ligand on acti-
of murine small bowel IELs express the γδ TCR,100 whereas vated LPLs and the imbalance between the intracellular anti- and
both the murine and human large intestine contain primarily pro-apoptotic factors, Bcl2 and Bax. Defects in this pro-apoptotic
αβ CD4+ or αβ CD8+ T cells similar to those present in the balance have been reported in Crohn disease.109,110
systemic immune system. Together, the above-described mechanisms contribute to
Based on their phenotype, IELs are classified into two sub- controlled/physiologic inflammation, which characterizes healthy
sets: induced IELs (iIELs), including TCRαβ T cells selected in intestinal mucosa. When regulatory mechanisms are disrupted,
the thymus by conventional MHC class I and II, and natural uncontrolled inflammation occurs, as in the mucosa of patients with
IELs, including TCRαβ CD8+αα, TCRγδ double-positive, and IBD.
TCRγδ double-negative cells.101 Both subpopulations are cyto-
lytic, killing via granzyme or by engagement of Fas, and secrete
Th1 cytokines. However, iIELs can transfer protection against
T Cell Differentiation
a variety of pathogenic organisms, whereas natural IELs are In GALT (see Fig. 2.5), B and T lymphocytes interact with anti-
unable to transfer immunologic protection and do not possess gen sampled via M cells in the overlying FAE. Activation and
immunologic memory. This difference may be due to natural maturation of T lymphocytes from naive Th0 cells to distinct Th
IEL activation by IECs in situ by non-classical MHC mol- subpopulations is strongly influenced by the microenvironment,
ecules rather than by the polymorphic MHC-expressed mol- particularly the microbiota, and by responses to pathogens. Viral
ecules on professional APCs that activate iIELs.100 Both IEL infections induce CD4 Th1 cells, whereas parasitic colonization
subsets express molecules present on natural killer (NK) cells. induces the CD4+ Th2 subset. CD4+ Th17 effector cells respond
IELs express a variety of activation markers and are CD45RO+ to extracellular bacteria and fungi. The microbiota shapes the
(memory cells). IELs also express the integrin, which is induced mucosal T cell response. For example, in mice, the commensal
by TGF-β and E-cadherin on IECs.102 Isolated, IELs are dif- known as segmented filamentous bacteria (Candidatus arthromitis)
ficult to activate through their TCR and barely proliferate, even selectively induces Th17 CD4 cells.8
in response to potent stimuli,98 and may be activated by alterna- DCs, professional APCs within the GALT and their secreted
tive pathways (e.g., via CD2). mediators skew T lymphocytes to one of several effector cells.
iIELs secrete an array of cytokines different from the ones Th1 cells secreting IL-2, IFN-γ, and TNF-α develop when
secreted by their peripheral blood counterparts.98,103-105 A broad DCs secrete the IL-12/p35-40 heterodimer,111 which induces
spectrum of cytokines are produced by IELs, including IFN-γ, activation and phosphorylation of the transcription factor
TNF-α, IL-2, IL-4, IL-6, IL-10, TGF-β, keratinocyte growth STAT-4 (signal transducer and activator of transcription factor
factor (KGF), and IL-17, with important effects on intestinal bar- 4). STAT-4 in turn induces IFN-γ expression and production.
rier function and local immune responses.106 IFN-γ induces activation of STAT-1 and consequently of T box
Functionally, IELs may kill epithelial cells that have under- expressed in T cells (T-bet), which is the master transcription
gone stress due to infection, transformation, or invasion by factor that induces Th1 cytokine and IL-12 receptor β2 produc-
other cells.100 Alternatively, IELs have been proposed to sup- tion, while simultaneously suppressing Th2 cytokine production.
press local immune cells, although the evidence that they actu- Thus a cycle promoting Th1 and suppressing Th2 responses is
ally function in luminal antigen recognition is weak. IELs do created. Activation of T-bet is possibly an essential step for Th1-
not traffic in and out of the epithelium. Rather, epithelial cells mediated mucosal diseases, such as those seen in some patients
move over the IELs as the epithelial cells move from the crypt with Crohn disease. Another important Th1-promoting cytokine
to the villus surface. Thus IELs likely serve as sentinels for epi- is IL-18. IL-18 mediates its effects on T cells through augmenta-
thelial integrity. tion of IL-12Rβ2 chain expression, AP-1(c-fos/c-jun)-dependent
transactivation of the IFN-γ promoter, and activation of nuclear the SCFA.129-131 SCFA such as butyrate, rather than glucose, are
factor κB (NF-κB.111 the major nutritional fuel of enterocytes. Thus depletion of bac-
In contrast, when IL-4 is secreted by DCs or other mucosal teria producing SCFA, which has been identified in IBD, could
cells, Th2 cytokine production (IL-4, IL-5, IL-6, IL-9, IL-10, have multiple detrimental effects, both on the epithelium and on
IL-13) occurs by activation of STAT-6 followed by activation Treg function. In mice, deficiency of the effector cytokines of
of the master transcription factor GATA-3. GATA-3 is capable Tregs, such as TGF-β and IL-10, results in colitis. Deficiency of
of promoting the expression of several Th2 cytokines, including IL-10 and/or its receptor, by inactivating mutations in humans,
IL-4, IL-5, and IL-13. In addition to IL-4, IL-13 also plays an results in early onset IBD.132 Tregs and their effector cytokines
important role in Th2 development and IgE synthesis in an IL-4– are thus crucial for maintaining homeostasis in the intestine by
independent fashion. These cytokines appear to contribute to the potentially controlling pathogenic innate and adaptive responses.
development of food allergies (see Chapter 10). IL-5 induces B The biology of T cell lineages in the LP is complex, related in
cells expressing surface IgA to differentiate into IgA-producing part to the plasticity of these cell populations. Under specific cir-
plasma cells. IL-6 causes a marked increase in IgA secretion, with cumstances, Th17 cells may become Th1 cells. Moreover, regu-
little effect on either IgM or IgG synthesis. latory Foxp3+ cells expressing Th17 cytokines and having potent
A third important LP CD4 subset are Th17 cells. The Th1- suppressor activity in vitro were recently identified in humans.125
polarizing cytokine IL-12, composed of the p40 and p35 sub- These findings suggest that a degree of plasticity in vivo exists in
units, has similarities with the Th17-polarizing cytokine IL-23, all known T cell subsets, reflected in their capacity to produce
composed of p40 and a unique p19 subunit. The possibility that specific cytokines depending on the microenvironment. Th17
some of the inflammatory activity previously attributed to an cells play a homeostatic role in gut mucosa,133 which may con-
IL-12–driven Th1 pathway might actually be an IL-23–driven tribute to the failure of anti-IL-17A monoclonal antibody ther-
Th17 pathway was supported by studies showing that intestinal apy in active Crohn disease.134,135 Addressing the complexity of
inflammation was still possible when IL-12 was inhibited, and the LP milieu with its vast amounts of mediators and effectors,
that inhibition of IL-23, rather than IL12, ameliorated inflam- including the microbiota, will likely contribute to better designed
mation.112-116 In Crohn disease, expression of both IL-12 and therapeutic strategies to modify intestinal inflammation.
IL-23 is increased, and inhibition of the common p40 subunit
of IL-12 and IL-23 was beneficial in clinical studies in Crohn
disease patients117,118 Th17 cells express retinoid-related orphan
Innate Lymphoid Cells
receptor-γt (RORγt), which is the master transcription factor for The recently identified innate lymphoid cells (ILCs) produce T
these cells. In addition to RORγt, human Th17 cells express IL- helper (Th) cell–associated cytokines but do not express a T cell
23R, CCR6, and CD161, whereas they lack CXCR3, a chemokine receptor or cell-surface markers that are associated with other
receptor characteristic of Th1 cells.119-122 The main effector cyto- immune cell lineages. Thus ILCs are lineage marker-negative,
kines secreted by Th17 cells are IL-17A, IL-17F, IL-21, IL-22, and their immune response is not antigen-specific. ILCs are
IL-26, TNF-α, and the chemokine CCL20. Human Th17 cells effectors of innate immunity and regulators of tissue modeling.
differentiate under the influence of IL-1β, IL-6, IL-21, IL-23, ILCs have several subpopulations with distinct cytokine expres-
and TGF-β.121 In humans, not all Th17 cells produce IL-22, and sion patterns that resemble the helper T cell subsets Th1, Th2,
a Th22 subset of CD4 helper T cells that produces IL-22 but and Th17.136
not IL-17 has been identified.122 IL-17 promotes recruitment Group 1 ILCs include ILC1 cells and NK cells. ILC1 cells
and activation of neutrophils, whereas IL-22 promotes mucosal express the transcription factor T-bet and respond to IL-12 by
healing through epithelial proliferation and increased mucus pro- producing IFN-γ. They differ from NK cells in that they do not
duction.123 express the NK cell markers CD16 and CD94 and lack perforin
Regulatory T cells (Tregs) are abundant in the intestine and, and granzyme B. ILC1 cells are increased in the inflamed intes-
similar to CD4 effector cells, are also comprised of subsets, which tine of Crohn disease patients, suggesting a role for ILC1 cells in
are distributed unevenly along the length of the bowel, reflect- the pathogenesis of intestinal inflammation.
ing the different microenvironments.124 The major Treg subset Group 2 ILCs include ILC2 cells, which are also termed natu-
expresses the transcription factor, Foxp3. Foxp3+ Tregs are gen- ral helper cells, nuocytes, and innate helper 2. Their transcription fac-
erated in the thymus (tTreg) and also are called “natural Tregs.” tors are retinoic acid receptor-related orphan receptor-α (RORα)
Foxp3+ Tregs also can be generated in the periphery from naïve and GATA3, and they have key roles in anthelminthic responses
CD4 T cells and are termed “peripheral Tregs” (pTreg) or and allergic lung inflammation.
induced Tregs. A subset of pTregs is present in colonic mucosa Group 3 ILCs include ILC3 and lymphoid tissue inducer
in humans and mice and express the transcription factor RORγt, (LTi) cells. Some cells of this group express the NK cell-activat-
typical of Th17 cells.125Interestingly, the Foxp3+ RORγt+ Treg ing receptor NKp46, which depends on the transcription factor
cells are induced in suckling mice by the microbiota, which is RORγt, and lack the cytotoxic effectors perforin and granzyme.
taken up via goblet-associated passages.58 Foxp3− T regulatory 1 Group 3 ILCs express IL-22 but not IFN-γ or TNF. A subset of
cells (Tr1) also are present in gut mucosa in fairly high abundance ILC3 express MHC class II and serve to regulate adaptive CD4+
and selectively produce high amounts of IL-10. Foxp3+ Tregs T cell responses to microbiota antigens.137 The contribution of
produce TGF-β only or TGF-β plus IL-10 as their inhibitory ILCs to mucosal homeostasis and intestinal inflammation is a
effector cytokines. subject of intensive research.
Certain microbiota or their products can induce the differ-
ent types of Tregs in mice. For example, the polysaccharide A
component of B. fragilis selectively induces Foxp3+ IL-10+ CD4
Dendritic Cells
T cells.126,127 An assortment of microbiota Clostridia have been DCs play a central role in tolerance and immunity in the intes-
shown to induce Foxp3+ Tregs in mouse colonic mucosa,128 and tinal mucosa. DCs continuously migrate within lymphoid tissues
this effect is at least partially due to the production of short-chain and present self-antigens, likely from dying apoptotic cells to
fatty acids (SCFA; acetate, propionate, butyrate) that these organ- maintain self-tolerance, as well as non-self-antigens.138 In the LP
isms produce during fermentation. SCFA have genome-encoded of the mouse distal small intestine, DCs express the chemokine
receptors on innate and adaptive immune cells in mice, which receptor CX3CR1 and form transepithelial dendrites that allow
tend to dampen immune responses.129 Humans have the same direct sampling of luminal antigen.139 IECs expressing CCL25
SCFA receptors and presumably have similar Treg responses to (the ligand for CCR9 and CCR10) may attract DCs to the small
intestinal mucosa, whereas CCL28 (the ligand for CCR3 and The second contribution to the inflammation anergy char-
CCR10) attracts DCs to colonic mucosa.140-142 acteristic of intestinal macrophages is dysregulated NF-κB sig- 2
DCs process internalized antigens more slowly than macro- naling. LP stromal cell factors, particularly TGF-β, potently
phages,80,143 possibly contributing to local tolerance.81,144 Tol- downregulate monocyte TRIF, MyD88, and TRAF6 proteins,
erance induction by DCs is associated with (1) their degree of leading to the inability of monocytes newly recruited into the
maturation at the time of antigen presentation to T cells (imma- lamina propria to phosphorylate NF-κB p65. Intestinal macro-
ture DCs activate Tregs), (2) downregulation of co-stimulatory phages also express increased levels of mRNA for suppressor of
molecules CD80 and CD86, (3) production of the suppressive cytokine signaling (SOCS1), which promotes the degradation
cytokines IL-10, TGF-β, and IFN-α, and (4) interaction with of MAL (MyD88 adaptor-like protein), and increased levels of
the co-stimulatory molecule CD200.145,146 Murine CD103+ DCs sterile Armadillo motif-containing protein (SARM), which inhib-
are able to perform all stages of antigen processing, including its TRIF signaling. MyD88 is a critical element in the NF-κB
uptake, transportation, and presentation of bacterial antigens.147 activation pathway for all TLRs, except TLR3, and TRIF medi-
In the mouse LP, CD103+ DCs share the burden of immuno- ates TLR3-induced RANTES and IFN-γ production, as well
surveillance with CX3CR1+ macrophages, and impaired function as TLR4-mediated MyD88-independent signaling. In addition,
of these subpopulations may contribute to the development of intestinal macrophages are unable to activate NF-κB through
IBD.148 mitogen-activated protein kinase (MAPK) pathways involving
phosphorylated(p) p38, p-ERK, or p-JNK, pathways dependent
on TRAF6. These dysregulations lead to the marked inability
Macrophages of intestinal macrophages to activate NF-κB and thus release of
Lamina propria macrophages are part of the innate immune sys- NF-κB pathway-dependent pro-inflammatory cytokines.
tem that orchestrates initial responses to microorganisms and The third mechanistic component of inflammation anergy is
their products. Among all body tissues, macrophages are most active TGF-β signaling. Intestinal macrophage TGF-β RI and
numerous in the gastrointestinal mucosa, residing in high num- RII are activated by local stromal TGF-β to induce the Smad
bers in the LP. In this critical location, macrophages (1) protect signal cascade. Smad4, a key component of the cascade, associates
against pathogens and noxious substances that breach the epi- with the phosphorylated heterodimeric Smad2/3 complex and
thelium, (2) contribute to tolerance to commensal bacteria and then translocates into the nucleus, initiating gene transcription
food antigens, and (3) maintain tissue homeostasis by scavenging for IκBα, which sequesters NF-κB in the cytoplasm. In contrast
apoptotic and dead cells. Intestinal macrophages mediate these to blood monocytes, intestinal macrophages do not express the
innate functions through powerful phagocytic and bactericidal pathway inhibitor Smad7, causing constitutive expression of IκBα
capabilities. and blockade of NF-κB signal transduction, thereby inhibiting
Innate cell responses to microbes are initiated within minutes NF-κB-mediated responses.
and are directed toward PAMPs, the conserved carbohydrate, Together, these overlapping mechanisms, induced mainly by
lipid, and nucleic acid molecules present on microbes. Macro- stromal TGF-β, cause profound inflammation anergy in human
phages recognize PAMPs through predetermined repertoires of resident intestinal macrophages. Recent studies also indicate that
pattern recognition receptors (PRRs) that include the prototypic stimulus-exposed intestinal macrophages do not polarize into
germline-encoded transmembrane TLRs and cytosolic sensors, classical and alternatively activated (M1, M2) macrophages char-
including nucleotide-binding oligomerization domain (NOD)- acteristic of mouse tissue macrophages. In the setting of infec-
like receptors. The predetermined nature of PRRs facilitates tion or a disrupted epithelium, immunostimulatory microbes and
rapid innate responses to microbial antigens but limits the diver- microbial products that breach the epithelium are rapidly phago-
sity of ligands to which macrophages can respond. cytosed by intestinal macrophages that provide potent, but non-
After infancy, intestinal macrophages are derived from and inflammatory, host defense. Similarly, intestinal macrophages
replenished by circulating pro-inflammatory monocytes, which clear apoptotic cells and debris in a non-inflammatory manner.
recruit to the LP. In the gut LP, however, macrophages display a Thus intestinal macrophages play a fundamental role in promot-
unique innate receptor phenotype with very limited pro-inflamma- ing the absence, or near absence, of inflammation that character-
tory capabilities, termed inflammation anergy, despite the presence izes healthy human intestinal mucosa.
of potent phagocytic and bacteriocidal activity. Three important
features contribute to the inflammation anergy. First, intestinal
macrophages in healthy mucosa do not express the receptors for
Oral Tolerance
LPS (CD14), IgA (CD89), IgG (CD16, 32, and 64), CR3 (CD11b/ One of the manifestations of the highly regulated mucosal immune
CD18), CR4 (CD11c/CD18), growth factor receptors for IL-2 system is oral tolerance.149,150 Oral tolerance is the antigen-specific
(CD25) and IL-3 (CD123), the integrin leukocyte function-associ- non-response to antigens administrated orally.150This also occurs
ated antigen-1 (LFA-1) (CD11a), and TREM-1. Intestinal macro- at other mucosal surfaces, where it is termed mucosal tolerance.
phages also express very low levels of chemokine receptors CCR5 The immune system regulates the response to the vast array of
and CXCR4, the co-receptors for R5 and X4 HIV-1. The mecha- antigens introduced via the oral route, specifically antigens that
nism by which the expression of these receptors is suppressed is not avoid complete digestion. Notably, up to 2% of dietary proteins
known, but since monocytes, the source of intestinal macrophages, enter the draining enteric vasculature fully intact. Non-response
express the receptors, local factors likely contribute to this unique to these antigens is achieved by oral tolerance. The intestinal
phenotype, possibly through the induction of epigenetic regula- mucosal immune system’s ability to discriminate between harm-
tion, as newly recruited monocytes take up residence in the lam- ful and harmless, or even beneficial, antigens and to generate
ina propria. Still, intestinal macrophages express some receptors a differential immune response toward each type of antigen is
involved in the recognition of, and interaction with, potentially present in humans and investigated extensively in animal mod-
harmful microbes, notably TLR1 and TLR3–9, as well as TGF-β els.95,151,152 Disruption of oral tolerance/mucosal tolerance, may
RI and RII, which mediate recruitment and active Smad signal- result in food allergies, celiac disease, and IBDs and has been
ing. The unique receptor phenotype of intestinal macrophages implicated in systemic immune-mediated diseases.
has profound functional implications. For example, the absence of An important difference between oral tolerance to food anti-
CD14 is consistent with the inability of intestinal macrophages to gens and mucosal tolerance to the microbiota is that the former
recognize LPS, a feature well suited to macrophages residing in attenuates intestinal and systemic immune responses, whereas
a microenvironment potentially rich in immunostimulatory LPS. the latter attenuates only mucosal immune responses.150 Factors
affecting the induction of oral tolerance include the host’s age, LP of patients with IBD, the number of such cells is significantly
genetic factors, nature of the antigen, and the tolerogen’s form reduced, supporting a role for these epithelial-induced T regula-
and dose. Part of the explanation for oral tolerance relates to tory cells in the control of intestinal inflammation.175
digestion itself, where large macromolecules are degraded so that Lastly, oral tolerance may also be influenced by the cell serv-
potentially immunogenic substances are rendered non-immuno- ing as the antigen-presenting cell, as well as by the site of antigen
genic or tolerogenic. uptake. In mice, orally administered reovirus type III is taken up
Oral tolerance is difficult to achieve in the neonate, likely by M cells expressing reovirus type III–specific receptors (see Fig.
related to the relatively permeable intestinal barrier in the new- 2.2).11 This induces an active IgA response. In contrast, reovirus I
born, as well as the immaturity of the mucosal immune system. infects IECs and induces tolerance. Thus whether a specific anti-
Within 3 months of age, oral tolerance can be induced, and gen enters the mucosa through M cells or IECs may dictate the
many previous antibody responses to food antigens are sup- type of immune response generated (IgA vs. tolerance). Interest-
pressed. The limited diet in the newborn may further serve to ingly, poliovirus, one of the few oral vaccines effective in man,
protect the infant from generating a vigorous response to food binds to M cells, which may account for its ability to stimulate
antigens. Interestingly, the intestinal microbiota has been shown active immunity in the gut.176
to affect the development of oral tolerance. Continuous expo-
sure to microbial molecules such as LPS during pregnancy and
early infancy was associated with a lower prevalence of atopy and
Chemokine Role in Homeostasis and Inflammation
asthma in children.153,154 The effects of the microbiota on oral Many of the chemokines secreted in the GALT are produced
tolerance are probably mediated through modulation of cytokine by IECs, evidence for epithelial cell participation in regulating
responses,155 the positive effect on intestinal barrier function and intestinal immune responses. Of the chemokines secreted, those
restitution of tight junctions,156 suppression of intestinal inflam- produced by IECs have the capacity to attract inflammatory cells
mation via downregulation of TLR expression, and secretion of such as lymphocytes, macrophages, and DCs, thus contribut-
metabolites that may inhibit inflammatory cytokine production ing to normal mucosal homeostasis (Table 2.1). The production
by mononuclear cells. of most of these chemokines is increased during infection and
The nature and form of the antigen also impact the induction inflammation.
of tolerance. Protein antigens are the most tolerogenic compared The chemokine CCL5 (regulated on activation, normal T cell
with carbohydrates and lipids.156 Regarding the form of the anti- expressed and secreted [RANTES]) is secreted predominantly
gen, ovalbumin (OVA) given in soluble form is quite tolerogenic, by macrophages but can also be produced by human IECs.177
whereas aggregated OVA has reduced capacity to induce toler- RANTES may have a role in innate as well as adaptive muco-
ance. The site of antigen sampling also may affect tolerance, since sal immunity,178 and increased RANTES expression has been
exposure (prior sensitization) to an antigen through an extraintes- demonstrated in the mucosa of patients with ulcerative coli-
tinal route reduces the development of mucosal tolerance. tis.179-182 The CXC chemokines, including monokine induced by
The dose of antigen administered is also considered critical interferon-γ (MIG, CXCL9), IFN-γ-inducible protein 10 (IP-10,
to the form of oral tolerance generated. In mouse models, high CXCL10), a chemokine that appears to promote Th1 responses,
doses of antigen are associated with clonal deletion or anergy and IFN-γ-inducible T cell α-chemoattractant (ITAC, CXCL11)
of T cells. In this setting, transfer of T cells from tolerized to are constitutively expressed by lymphocytes, endothelial cells,
non-tolerized animals does not lead to transfer of tolerance. Low and human colonic IECs.183,184 Their expression and polarized
doses of antigen, on the other hand, have been found to acti- basolateral secretion increase after IFN-γ stimulation. CXC che-
vate regulatory/suppressor T cells,157-159 but the effect of anti- mokines attract Th1 cells expressing high levels of CXCR3,185
gen dose on oral tolerance remains to be defined. Treg cells of contribute to NK T cell chemotaxis and increased cytolytic
both CD4 and CD8 lineages have a role in oral tolerance. CD4+ responses,186 and activate subsets of DCs.187
Treg cells appear to be activated in the Peyer patch and secrete In contrast to the inflammation-related CXCR3 receptor,
TGF-β, which is a potent suppressor of T and B cell responses, a tissue-specific chemokine receptor, CCR9, is constitutively
while promoting the production of IgA by inducing a genetic expressed on small intestinal IELs and LPLs.188-190 Its ligand,
switch from IgM to IgA in B cells.160,161 Production of TGF-β the chemokine thymus-expressed chemokine (TECK, CCL25) is
and IL-10 by Treg cells elicited by low-dose antigen administra- differentially expressed in the jejunal and ileal epithelium, where
tion helps explain an associated phenomenon of oral tolerance decreasing levels of expression from the crypt up to the villous
termed bystander suppression. Oral tolerance is antigen-specific, have been reported.191 CCL25 expression by IECs has been
but the effector arm is antigen non-specific. When an irrelevant shown to be increased in the inflamed small intestine of patients
(bystander) antigen is co-administered systemically with the with Crohn disease, with increased CCR9 expression by periph-
tolerogen, suppression of T and B cell responses to the irrelevant eral blood lymphocytes and decreased expression by LPLs.189
antigen also will occur (hence bystander suppression), because Fractalkine (CX3CL1) is a unique chemokine expressed by
secreted TGF-β and IL-10 can suppress the response to the co- IECs that combines the properties of chemokines and adhe-
administered antigen. T regulatory 1 cells, which produce only sion molecules. CX3CL1 attracts NK cells, monocytes, CD8+
IL-10, a potent immunosuppressive cytokine, may also partici- T lymphocytes, and to a lesser extent CD4+ T lymphocytes,
pate in bystander suppression and oral tolerance.162-164 In mice, which express the specific receptor CX3CR1.192 Its expression is
the deletion of CD4+ Treg cell activity results in IBD, whereas increased in Crohn disease, specifically in the basolateral aspect
its expansion ameliorates murine colitis.165-167 In IBD patients, of IECs.193,194
the number of Treg cells is generally greater than in controls, Macrophage-derived chemokine (MDC, CCL22) is constitu-
and a peripheral-to-intestinal shift has been suggested.168-170 tively expressed and secreted by colonic IECs and attracts CCR4+
Whether the failure of these cells to protect against IBD is due Th2 cytokine-producing lymphocytes. Polarized basolateral
to an intrinsic defect or microenvironmental effects is still being secretion of MDC/CCL22 from stimulated colonic IEC lines has
investigated.171 been reported.195 The specific recruitment of lymphocytes that
Antigen-specific CD8+ T cells may play a role in oral tol- preferentially secrete anti-inflammatory cytokines supports an
erance,172,173 as well as in the regulation of mucosal immune important role for the intestinal epithelium in orchestrating nor-
responses. Specifically, in vitro activation of human CD8+ periph- mal mucosal homeostasis, and adds to the accumulating evidence
eral blood T cells by normal IECs results in the expansion of that these cells possess the ability to regulate mucosal immune
CD8+CD28− T cells with regulatory activity.174 Moreover, in the responses.
TABLE 2.1 Chemokines, Their Receptors, Cells That Produce Them, and Target Cell(s)
2
Chemokine Receptor Produced by Target Cell References
CCL5 (RANTES) IEC T cells 177
Mϕ Eosinophils
Leukocytes
CXCL9 (MIG) CXCR3 Colon IECs Th1 CXCR3+ 183,184
Endothelial cells NK
Lymphocytes DC
CXCL10 (IP10) CXCR3 Colon IECs Th1 CXCR3+ 183,184
Endothelial cells
Lymphocytes
CXCL11 (ITAC) CXCR3 Colon IECs Th1 XCXR3+ 183,184
Endothelial cells
Lymphocytes
CCL25 (TECK) CCR9 IEC CD8+ E7 188-191
CX3CL1 (Fractalkine) CX3CR1 IEC CD8>CD4 monocytes 192-194,200
NK cells
CCL28 (MEC) CCR3 Colon IEC CD4 Tm eosinophils 200
CCR10
CCL22 (MDC) CCR4 Colon IEC CD4 Th1 195
CCL20 (MIP3α) CCR6 IEC DCs 196-199
CD4 Tm
CXCL12 CXCR4 IEC CD4 Th1 CD45RO+ 201-205
CXCR7 Plasma cells
CXCL8 (IL-8) CXCR1>CXCR2 IEC Neutrophils 206
Mϕ
Neutrophils

DC, Dendritic cell; IEC, intestinal epithelial cell; Mϕ, macrophage; NK, natural killer.

The chemokine macrophage inflammatory protein-3α (MIP3, are pathogens because they have evolved mechanisms to breach
CCL20) is unique in its ability to specifically attract immature the mucosal barrier. In healthy mucosa, resident macrophages
DCs as well as memory CD4+ T lymphocytes.196-198 CCL20 potently phagocytose and kill such microbes in a non-inflam-
is also expressed and produced by human small intestinal ECs matory manner, but in disease conditions, the mechanism(s)
(mainly in the follicle-associated epithelium) and by colonic responsible for inflammation anergy are disrupted allowing the
IECs and may be the mediator of lymphocyte adhesion to the macrophages to retain the pro-inflammatory profile of their
α4β7 ligand MAdCAM-1.196 MIP3α expression and secretion is monocyte progenitors. However, once IECs are invaded, they
increased in colonic IECs derived from IBD patients.199 Muco- produce large amounts of chemokines such as IL-8, which attract
sal memory T cells, as well as IECs, express CCR6, the cognate neutrophils and monocytes from the blood into the gut at the
receptor for MIP3α. site of infection. Such phagocytes are inflammatory and produce
Mucosal defenses, including microbiota itself, provide protec- more chemokines, as well as other cytokines, rapidly acquiring a
tion from intestinal pathogens. The microbiota competes with, critical mass and killing the invading bacteria, thus resolving the
and provides resistance to, colonization by transient bacteria and infection.
pathogens in food and water. Some enteric pathogens induce
host inflammation that in turn kills anaerobes in the gut, thus Full references for this chapter can be found on www.expertconsult.com.
opening a niche for the aerotolerant pathogen. Certain bacteria

3 The Enteric Microbiota
Eugene B. Chang, Purna Kashyap
CHAPTER OUTLINE of the microorganisms. Each of these contributes to the stabil-

ity of the ecosystem and drives specific interactions with the host.
We are making advances in understanding the role of each of these
CHARACTERISTICS OF THE HUMAN INTESTINAL components, although our primary focus has been on bacteria. We
MICROBIOME�� 24 have made big strides in our ability to characterize the microbiome
Spatial Variation in the Intestinal Microbiome�� 24 and its impact on the host following the advent of next-generation
Temporal Changes and Resilience of the Intestinal sequencing (NGS) and advanced experimental tools (Box 3.2).
Microbiome�� 25 Composition of the intestinal microbiota varies significantly among
FACTORS AFFECTING INTESTINAL MICROBIOME individuals, and so it is not surprising that it has been difficult to
identify a universal “healthy” intestinal microbiota in terms of spe-
VARIABILITY AND RESILIENCE �� 25
cific microbial members. This variation primarily reflects differences
Age�� 25 in the relative abundance of the 4 dominant phyla: Bacteroidetes,
Sex�� 25 Firmicutes, Proteobacteria, and Actinobacteria.4, 5 In contrast to
Genetics�� 27 composition, a subset of microbial functional properties does appear
Geography and Diet�� 27 to be conserved among individuals, including central metabolic
Exercise �� 28 pathways and nutrient metabolism, of, for example, carbohydrates
Medications �� 28 and proteins. There are still significant inter-individual differences
Other Lifestyle Factors�� 29 in the microbial functions, however, such as in drug metabolism,
Microbe-Microbe Signaling�� 29 pathogenicity islands, and nutrient transporters. There are several
factors that shape the intestinal microbiome (Fig. 3.1), but there is
THE EFFECT OF HOST–INTESTINAL MICROBIOME no 1 crucial factor. Diet appears to have the most prominent effects,
INTERACTIONS ON HOST PHYSIOLOGY�� 29 but the membership and functions of the intestine microbiome
Interactions Between the Intestinal Microbiome and result from complex interplay among the different factors.
Immune System �� 29
Interactions Between the Intestinal Microbiome and Spatial Variation in the Intestinal Microbiome
Gastrointestinal Tract�� 29
The Microbiome-Gut-Brain Axis�� 30 The microbiota composition varies along the gastrointestinal
(GI) tract, from mouth to anus through both its longitudinal and
THE ROLE OF THE INTESTINAL MICROBIOME IN HUMAN radial axes.6 There are several factors that determine the localiza-
DISEASE�� 30 tion of bacteria within different niches in the intestine, including
Metabolic Function�� 30 oxidation-reduction potential, chemical and nutrient gradients,
INFLAMMATORY DISEASES�� 30 host immune activity, and the mucus layer (Fig. 3.2).7 The high-
est bacterial density, perhaps due to increased nutrient availabil-
CANCER�� 31 ity and slower transit, is in the colon (see Fig. 3.1). In contrast, the
FUNCTIONAL GASTROINTESTINAL harsh chemical environment and relatively rapid transit through
DISORDERS �� 31 the small intestine contribute to lower abundance and diversity of
microbiota. In addition, contact between bacteria and the surface
THE ROLE OF THE INTESTINAL MICROBIOME IN epithelium differs between the colon and small intestine. Colonic
MODULATION OF DRUG RESPONSE�� 31 mucus has 2 layers: the inner layer is devoid of bacteria, whereas
THERAPEUTIC MODULATION OF THE INTESTINAL the looser outer layer is populated by bacteria.8 In contrast, the
MICROBIOME�� 32 small intestine has a single, incomplete mucus layer. Here, anti-
microbial factors (e.g., REGIIIγ) appear to be more important
NONBACTERIAL MEMBERS OF THE INTESTINAL than the mucus in segregating microbes from the epithelium.9,10
MICROBIOME�� 32 The radial gradient of oxygen from the mucosa to the lumen
FUTURE DIRECTIONS�� 33 results in differences in taxonomic membership, genetics, and
function between mucosa-associated microbes and lumen-asso-
ciated microbes.11 There is an increased proportion of oxygen-
tolerant organisms within the Proteobacteria and Actinobacteria
phyla in the mucosa, highlighting the effect of oxygen availability.
In addition, groups of bacteria that primarily use amino acids12
CHARACTERISTICS OF THE HUMAN INTESTINAL are associated with the mucus layer, which is a nutrient source
MICROBIOME driving differences across the intestine.
The intestinal microbiome is a diverse ecosystem comprising The majority of studies characterizing the intestinal micro-
microorganisms (bacteria, archaea, fungi, and viruses including bac- biota analyze stool samples, treating fecal microbes as surrogates
teriophages), their genomes (i.e., genes), and the surrounding envi- for those present in the colon. Although this approach is sim-
ronmental conditions. The population of microorganisms alone in pler and more accessible to a wider scientific community, it only
a particular niche is referred to as microbiota (Box 3.1) and is often allows insight into the microbial side of host-microbe interac-
used interchangeably with microbiome, which includes the genomes tions. A more regionally targeted sampling approach is needed to
24
CHAPTER 3 The Enteric Microbiota 25
composition in samples obtained longitudinally from an individ-

BOX 3.1 Glossary of Terms Used to Describe Relationships 3
ual is more similar to each other than to those obtained from a
Among Individual Organisms Within the different individual. Thus although relative abundances of indi-
Microbiota and Between the Microbiota and Host vidual microbes can change, the overall community function and
membership community remain intact. Similarly, an unfavorable
Allochthonous: Organisms found in a place other than their origin. microbial community can also be stable and contribute to chronic
Autochthonous: Organisms that are indigenous to their present disease or states of poor health. Resilience is a key property of
location. microbial community states, and is defined as the amount of stress
Commensal: Strictly speaking, the term commensal (derived from or perturbation a microbial community can tolerate before it shifts
cum mensa, “to share a table”) describes a relationship between to a different steady state (see Fig. 3.1). A high degree of resilience
2 organisms in which 1 organism benefits and the other is unaf- is desirable to maintain healthy states, but not in an unhealthy
fected. In most instances, however, the term commensal is used condition. The competition among microbes and positive and
to describe the in situ microbes colonizing a particular niche negative feedback to maintain levels of individual microbes fur-
without doing harm, but may include organisms that provide a ther contributes to stability.13 Certain perturbations such as with
benefit to each other or to the host. short courses of antibiotics can result in a transiently disrupted
Microbiome: The microorganisms, their genomes (i.e., genes), and microbial community structure (see Fig. 3.1), which often returns
the surrounding environmental conditions.181 to the original state.14 A persistent perturbation such as long-term
Microbiota: The population of microorganisms (bacteria, archaea, change in diet/antibiotic administration (see Fig. 3.1), or pertur-
lower and higher eukaryotes, and viruses) organisms in a par- bation during a vulnerable phase such as early childhood or the
ticular niche.180 peripartum period,15, 16 can result in disordered assembly with a
Pathobiont: Usually refers to an organism that is a potential shift to a disease-promoting state that is resistant to change.
pathogen, but only causes disease under a given set of circum-
stances such as when the microbiome is perturbed. An example
is Clostridioides difficile, which can be carried in the intestine
FACTORS AFFECTING INTESTINAL MICROBIOME
of healthy individuals, but usually only causes a problem after VARIABILITY AND RESILIENCE
antibiotic treatment.
Pathogen: Any pathologic (disease-causing) organism.
Age
Pharmabiotic: Any biological entity mined from human microbiota The most dramatic and influential changes in intestinal micro-
and with a proven biological effect. These entities could include biota composition occur during the first years of life. Although
live or dead microbes, cell wall components, purified proteins or there is some evidence suggesting that microbes are acquired
lipids, individual metabolites (e.g., neurotransmitters), or active in utero, most microbial acquisitions start at the time of birth
enzymes. when an infant’s intestine is seeded with microbes from mother’s
Prebiotic: A nondigestible compound that, through its metaboliza- vagina.17 By contrast, the intestines of infants delivered by cesar-
tion by microorganisms in the intestine, modulates functional ean section are colonized by their mothers’ skin bacteria.17 Mode
capacity of the microbial community, thus conferring a beneficial of feeding is also linked to differences in microbiota composi-
physiological effect on the host.182 tion, with formula feeding linked to a decreased abundance of
Probiotic: Live microorganisms that when administered in adequate Bifidobacteria relative to that found in breastfed infants.18 The
amounts confer a health benefit on the host. infant continues to acquire microbes both from the environ-
Symbiont: Any organism participating in a symbiotic (mutually ment and from different body sites of the mother; interestingly,
beneficial) relationship. microbes acquired from the mother are more persistent and bet-
Synbiotic: A nondigestible compound that contains both prebiotics ter adapted to infant intestine.19
and probiotics and combines nutrients appropriate to stimulate Early development of the intestinal microbiome is critical in
the specific beneficial microbe in the synbiotic. educating the mucosal20, 21 and systemic immune response.22-24
Disturbance of host-microbe interactions when the immune sys-
tem is maturing, i.e., with antibiotic exposure in infancy, is linked
to a higher risk of conditions such as asthma, type I diabetes, and
understand the microbial effects in regulating host metabolism, obesity later in life.17
digestion and absorption, local immune systems, and in causing The intestinal microbiota composition and function contin-
or contributing to diseases such as IBD, IBS, food allergy, celiac ues to change throughout life. By 3 years of age, an individual’s
disease, and colon cancer.11 microbiome more or less resembles that of an adult,25, 26 although
the pre-adolescent intestinal microbiome is enriched in func-
tions such as vitamin synthesis that support development.27 In
Temporal Changes and Resilience of the Intestinal general, microbial membership and functional diversity increase
with age,4 although older adults living in long-term care facilities
Microbiome often harbor intestinal microbiomes that are distinct from and
A healthy stable state is characterized by a diverse intestinal less diverse than those of persons living in the community.28
microbiota that develops from compositional and functional
changes in the early years of life. There is such significant inter-
personal variation, however, that a “healthy” state is difficult to
Sex
define. Perhaps the best approximation for this definition would Women display higher levels of microbiota diversity and func-
be one that promotes health by providing critical functions essen- tional richness than men,4 and a decreased abundance of Bacteroides
tial to the host.13 The concept of enterotypes based on metage- and Prevotella species.29 Although the implications of these differ-
nomics sequencing that stratifies healthy communities into 3 ences remain unclear, animal studies have provided some clues.
groups (Bacteroides, Prevotella, or Ruminococcus) does not hold up Colonization with commensal bacteria prevents development
when expanded to larger “healthy” populations where it becomes of diabetes in male but not female mice predisposed to develop-
clear that the range of inter-personal variability is a continuous ing type 1 diabetes.30, 31 This protective effect is dependent on
spectrum of stable configurations.13 The intestinal microbial androgen receptor activity and can be transferred to female mice
ecosystem is generally stable over time, i.e., intestinal microbiota by transplanting intestinal microbes from adult male to immature
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he must sit always on a throne, and wear a crown, according to the
traditions of Mr. Gex.
Now that the procession was over, all might have gone well if
Tiburce had held out as he began; but alas! in an evil moment, he
yielded to temptation and fell.
They were on their way back to Tante Modeste, quite satisfied with
all they had seen, when they came upon a crowd gathered around
the door of a fashionable club. From the balcony above a party of
young men, who were more generous than wise, were throwing
small change, dimes and nickels, into the crowd, that the rabble
might scramble for them; and there right in the midst of the seething
mass was Tite Souris, her domino hanging in rags, her wings gone,
and her whole appearance very dilapidated and disorderly; but the
demon of greed was gleaming in her eyes, and her teeth were
showing in a fierce, white line, while she plunged and struggled and
battled for the root of all evil.
Tiburce’s first intention was to make a detour of the crowd; but just
as he was about to do so the gleam of a dime on the edge of the
sidewalk caught his eye, and, overcome by the spirit of avarice, he
forgot everything, and dropped Lady Jane’s hand to make a dive for
it.
Lady Jane never knew how it happened, but in an instant she was
whirled away from the Paichoux, swept on with the crowd that a
policeman was driving before him, and carried she knew not where.
At first she ran hither and thither, seizing upon every domino that
bore the least resemblance to her companions, and calling Tiburce,
Sophie, Nanette, in heartrending tones, until quite exhausted she
sank down in a doorway, and watched the crowd surge past her.
CHAPTER XX
LADY JANE DINES WITH MR. GEX
F OR some time Lady Jane sat in the doorway, not knowing just
what to do. She was very tired, and at first she was inclined to
rest, thinking that Tiburce would come back and find her there; then
when no one noticed her, and it seemed very long that she had
waited, she felt inclined to cry; but she was a sensible, courageous
little soul, and knew that tears would do no good; besides it was very
uncomfortable, crying behind a mask. Her eyes burned, and her
head ached, and she was hungry and thirsty, and yet Tiburce didn’t
come; perhaps they had forgotten her altogether, and had got into
the milk-cart, and gone home.
This thought was too much to bear calmly, so she started to her
feet, determined to try to find them if they were not coming to search
for her.
She did not know which way to turn, for the crowd confused her
terribly. Sometimes a rude imp in a domino would push her, or twitch
her sleeve, and then, as frightened as a hunted hare, she would dart
into the first doorway, and wait until her tormentor had passed. She
was such a delicate little creature to be buffeted by a turbulent
crowd, and had it not been for the disguise of the domino she would
soon have found a protector amongst those she fled from.
After wandering around for some time, she found herself very near
the spot she started from; and, thankful for the friendly shelter of the
doorway, she slipped into it and sat down to think and rest. She
wanted to take off her mask and cool her hot face, but she did not
dare to; for some reason she felt that her disguise was a protection;
but how could any one find her when there were dozens of little
figures flitting about in pink dominos?
While she sat there thinking and wondering what she should do,
she noticed a carriage drive up to the next door, and two gentlemen
got out, followed by a young man. When the youth turned his face
toward her, she started up excitedly, and holding out her hands she
cried out pitifully, “It’s me; it’s Lady Jane.”
The young fellow glanced around him with a startled look; he
heard the little cry, but did not catch the words, and it moved him
strangely; he thought it sounded like some small creature in pain, but
he only saw a little figure in a soiled pink domino standing in the next
doorway, some little street gamin, he supposed, and without further
notice he passed her and followed his companions up the steps.
It was the boy who gave Lady Jane the blue heron, and he had
passed her without seeing her; she had called to him, and he had
not heard her. This was too much, she could not bear it, and
withdrawing again into her retreat she sat down and burst into a
passion of tears.
For a long while she cried silently, then she fell asleep and forgot
for a time all her troubles. When she woke a rude man was pulling
her to her feet, and telling her to wake up and go home; he had a
stick and bright buttons on his coat. “A young one tired out and gone
to sleep,” he muttered, as he went on his way.
SHE CRIED OUT PITIFULLY, “IT’S LADY JANE”
Then Lady Jane began to think that that place was no longer a
safe refuge; the man with the stick might come back and beat her if
she remained there, so she started out and crept along close to the
high buildings. She wondered if it was near night, and what she
should do when it got dark. Oh, if Tante Modeste, Tiburce, or
Madelon would only come for her, or Tante Pauline,—even she
would be a welcome sight, and she would not run away from Raste,
although she detested him; he pulled her hair and teased her, and
called her “My Lady,” but still if he should come just then she would
not run away from him, she would ask him to take her home.
At that moment some one behind her gave her domino a violent
pull, and she looked around wildly; an imp in yellow and black was
following her. A strand of her bright hair had escaped from her hood
and fallen over her back; he had it in his hand, and was using it as a
rein. “Get up, my little nag,” he was saying, in a rude, impertinent
voice; “come, trot, trot.” At first she tried to jerk her hair away; she
was so tired and frightened that she could scarcely stand, but she
turned on her tormentor and bade him leave her alone.
“I’m going to pull off your mask,” he said, “and see if you ain’t Mary
O’Brien.” He made a clutch at her, but Lady Jane evaded it; all the
spirit in her was aroused by this assault, and the usually gentle child
was transformed into a little fury. “Don’t touch me,” she cried; “don’t
touch me,”—and she struck the yellow and black imp full in the face
with all her strength.
Now this blow was the signal for a battle, in which Lady Jane was
sadly worsted, for in a few moments the boy, who was older and of
course stronger, had torn her domino from her in ribbons, had
snatched off her mask, and pulled the hood from her head, which
unloosened all her beautiful hair, allowing it to fall in a golden shower
far below her waist, and there she stood with flashing eyes and
burning cheeks, quivering and panting in the midst of a strange, rude
crowd, like a little wild hunted animal suddenly brought to bay.
At that moment she saw some one leap on to the banquette, and
with one well-aimed and dexterous kick send her enemy sprawling
into the gutter, while all the bystanders shouted with laughter.
It was Gex, little Gex, who had come to her rescue, and never did
fair lady cling with greater joy and gratitude to the knight who had
delivered her from the claws of a dragon, than did Lady Jane to the
little horny hand of the ancient professeur of the dance.
For a moment she could not speak; she was so exhausted with
her battle and so overcome with delight that she had no voice to
express her feelings.
Gex understood the situation, and with great politeness and
delicacy led her into a pharmacy near, smoothed her disordered
dress and hair, and gave her a glass of soda.
This so revived the little lady that she found voice to say: “Oh, Mr.
Gex, how did you know where I was?”
“I didn’t, I didn’t,” replied Gex tremulously. “It vas vhat you call one
accident. I vas just going down the Rue Royale, vas just turning the
corner, I vas on my vay home. I’d finished my Mardi-gras, all I vant of
the noise and foolishness, and I vas going back to Rue des Bons
Enfants, vhen I hears one leetle girl cry out, and I look and saw the
yellow devil pull down my leetle lady’s hair. Oh, bon, bon, didn’t I
give him one blow!—didn’t I send him in the gutter flying!”—and Gex
rubbed his hands and chuckled with delight. “And how lucky vas I to
have one accident to find my leetle lady, vhen she vas in trouble!”
Then Lady Jane and Mr. Gex turned down Rue Royale, and while
she skipped along holding his hand, her troubles all forgotten, she
told him how it happened that she had been separated from Tiburce,
and of all her subsequent misadventures.
Presently, Gex stopped before a neat little restaurant, whose
window presented a very tempting appearance, and, looking at Lady
Jane with a broad, inviting smile, said, “I should like to know if my
leetle lady vas hungry. It is past four of the clock, and I should like to
give my leetle lady von Mardi-gras dinner.”
“Oh, thank you, Mr. Gex,” cried Lady Jane, delightedly, for the
smell of the savory food appealed to her empty stomach. “I’m so
hungry that I can’t wait until I get home.”
“Vell, you sha’n’t; this is one nice place, vairy chic and fashionable,
fit for one leetle lady, and you shall see that Gex can order one fine
dinner, as vell as teach the dance.”
When the quaint little old man, in his antiquated black suit, a relic
of other and better days, entered the room, with the beautiful child,
rosy and bareheaded, her yellow hair flying out like spun silk, and
her dainty though disordered dress plainly showing her superior
position, every eye was turned upon him, and Gex felt the stirrings of
old pride and ambition, as he placed a chair with great ceremony,
and lifted Lady Jane into it. Then he drew out his spectacles with
much dignity, and, taking the card the waiter handed him, waited,
pencil poised, for the orders of the young lady.
“If you please,” he said, with a formal bow, and an inviting smile,
“to tell me vhat you prefair.”
Lady Jane frowned and bit her lips at the responsibility of deciding
so important a matter; at length she said, with sparkling eyes and a
charming smile:
“If you please, Mr. Gex, I’ll take some—some ice cream.”
“But first, my leetle lady,—but first, one leetle plat of soup, and the
fish with sauce verte, and one leetle bird,—just one leetle bird vith
the petit pois—and one fine, good, leetle salad. How vould that suit
my leetle lady?”
“And ice cream?” questioned Lady Jane, leaning forward with her
little hands clasped primly in her lap.
“And after, yes, one crême à la glace, one cake, and one leetle
bunch of raisin, grape you say,” repeated Gex, as he wrote
laboriously with his old, stiff fingers. “Now ve vill have one fine leetle
dinner, my leetle lady,” he said, with a beaming smile, when he had
completed the order.
Lady Jane nodded an affirmative, and while they waited for their
dinner her bright eyes traveled over everything; at length they rested
on Mr. Gex with unbounded admiration, and she could not refrain
from leaning forward and whispering:
“Oh, Mr. Gex, how nice, how lovely you look! Please, Mr. Gex,
please don’t wear an apron any more.”
“Vell, if my leetle lady don’t vant me to, vell, I von’t,” replied Gex,
beaming with sudden ambition and pride, “and, perhaps, I vill try to
be one fine leetle gentleman again, like vhen I vas professeur of the
dance.”
CHAPTER XXI
AFTER THE CARNIVAL
I T was nearly dark, and the day had been very long to Pepsie,
sitting alone at her window, for Madelon must remain all day and
until late at night on the Rue Bourbon. A holiday, and especially
Mardi-gras, was a day of harvest for her, and she never neglected a
chance to reap nickels and dimes; therefore Pepsie began to look
anxiously for the return of the merry party in the milk-cart. She knew
they were not to remain to see the night procession; at least, that
had not been the intention of Tante Modeste when she left, and she
could not imagine what had detained them. And Tite Souris,—
ungrateful creature! had been told to return as soon as the
procession was over, in order to get Pepsie’s dinner. Owing to the
excitement of the morning, Pepsie had eaten nothing, and now she
was very hungry, as well as lonesome; and even Tony, tired of
waiting, was hopping about restlessly, straining at his cord, and
pecking the floor viciously.
Madame Jozain had returned some time before, and was even
then eating her dinner comfortably, Pepsie had called across to know
if she had seen anything of the Paichoux and Lady Jane; but
madame had answered stiffly that she had been in her friend’s
gallery all the time, which was an intimation that she had been in no
position to notice a milk-cart, or its occupants. Then she observed
indifferently that Madame Paichoux had probably decided to remain
on Canal Street in order to get good positions for the night
procession.
Pepsie comforted herself somewhat with this view of the case, and
then began to worry about the child’s fast. She was sure Tante
Modeste had nothing in the cart for the children to eat, and on Mardi-
gras there was such a rush that one could hardly get into a
restaurant, and she doubted whether Tante Modeste would try with
such a crowd of young ones to feed. At length when she had thought
of every possible reason for their remaining so late, and every
possible plan by which they could be fed, she began to think of her
own hunger, and of Tite Souris’s neglect, and had worked herself up
to a very unenviable state of mind, when she saw her ungrateful
handmaid plunging across the street, looking like a much-abused
scarecrow, the remnants of her tatters flying in the wind, and her
long black legs, owing to the unexpected abbreviation of her skirts,
longer and thinner than ever, while her comical black face wore an
expression impossible to describe.
“Oh, Miss Peps’,” she gasped, bursting into Pepsie’s presence like
a whirlwind, “Ma’m Paichoux done sont me on ahead ter tell yer how
Miss Lady’s done got lost.”
“Lost, lost?” cried Pepsie, clasping her hands wildly and bursting
into tears. “How, where?”
“Up yon’er, on Cunnul Street. We’s can’t find ’er nowhar.”
“Then you must have let go of her,” cried Pepsie, while her eyes
flashed fire. “I told you not to let go of her.”
“Oh laws, Miss Peps’, we’s couldn’t holp it in dat dar scrimmage;
peoples done bus’ us right apart, an’ Miss Lady’s so littl’ her han’ jes
slip outen mine. I’se tried ter hole on, but’t ain’t no use.”
“And where was Tiburce? Did he let go of her too?”
“He war dar, but Lor! he couldn’t holp it, Mars’ Tiburce couldn’t, no
more en me.”
“You’ve broken my heart, Tite, and if you don’t go and find her I’ll
hate you always. Mind what I say, I’ll hate you forever,” and Pepsie
thrust out her long head and set her teeth in a cruel way.
“Oh laws, honey! Oh laws, Miss Peps’, dey’s all a-lookin’, dey’s
gwine bring ’er back soon; doan’t git scart, dat chile’s all right.”
“Go and look for her; go and find her! Mind what I tell you; bring
her back safe or—” Here Pepsie threw herself back in her chair and
fairly writhed. “Oh, oh! and I must stay here and not do anything, and
that darling is lost, lost!—out in the streets alone, and nearly dark.
Go, go and look for her; don’t stand there glaring at me. Go, I say,”
and Pepsie raised her nutcracker threateningly.
“Yes, Miss Peps’, yes, I’ll bring ’er back shore,” cried Tite, dodging
an imaginary blow, as she darted out, her rags and tatters flying after
her.
When she had gone Pepsie could do nothing but strain her eyes in
the gathering darkness, and wring her hands and weep. She saw the
light and the fire in Madame Jozain’s room, but the door was closed
because the evening was chilly, and the street seemed deserted.
There was no one to speak to; she was alone in the dark little room
with only Tony, who rustled his feathers in a ghostly sort of way, and
toned dismally.
Presently, she heard the sound of wheels, and peering out saw
Tante Modeste’s milk-cart; her heart gave a great bound. How foolish
she was to take on in such a wild way; they had found her, she was
there in the cart, safe and sound; but instead of Lady Jane’s blithe
little voice she heard her Uncle Paichoux, and in an instant Tante
Modeste entered with a very anxious face.
“She hasn’t come home, has she?” were Tante Modeste’s first
words.
“Oh, oh!” sobbed Pepsie, “then you haven’t brought her?”
“Don’t cry, child, don’t cry, we’ll find her now. When I saw I couldn’t
do anything, I took the young ones home, and got your uncle. I said,
‘If I have Paichoux, I’ll be able to find her.’ We’re going right to the
police. I dare say they’ve found her, or know where she is.”
“You know I told you—” moaned Pepsie, “you know I was afraid
she’d get lost.”
“Yes, yes; but I thought I could trust Tiburce. The boy will never get
over it; he told me the truth, thank Heaven; he said he just let go her
hand for one moment, and there was such a crowd. If that fly-away
of a Tite had kept on the other side it wouldn’t have happened, but
she ran off as soon as they got on the street.”
“I thought so. I’ll pay her off,” said Pepsie vindictively.
“Come, come, Modeste,” called Paichoux from the door, “let’s be
starting.”
“Oh, uncle!” cried Pepsie, imploringly, “do find Lady Jane.”
“Certainly, child, certainly, I’ll find her. I’ll have her back here in an
hour or so. Don’t cry. It’s nothing for a young one to get lost Mardi-
gras; I dare say there are a dozen at the police stations now, waiting
for their people to come and get them.”
Just at that moment there was a sound of voices without, and
Pepsie exclaimed: “That’s Lady Jane. I heard her speak.” Sure
enough, the sweet, high-pitched little voice chattering merrily could
be distinctly heard; and at the same instant Tite Souris burst into the
room, exclaiming:
“Her’s here, Miss Peps’, bress der Lor’! I’s done found her”; and
following close was Lady Jane, still holding fast to little Gex.
“Oh, Pepsie! Oh, I was lost!” she cried, springing into her friend’s
arms. “I was lost, and Mr. Gex found me; and I struck a boy in the
face, and he tore off my domino and mask, and I didn’t know what to
do, when Mr. Gex came and kicked him into the gutter. Didn’t you,
Mr. Gex?”
“Just to think of it!” cried Tante Modeste, embracing her, and
almost crying over her, while Paichoux was listening to the modest
account of the rescue, from the ancient dancing-master.
“And I had dinner with Mr. Gex,” cried Lady Jane joyfully; “such a
lovely dinner—ice cream, and grapes—and cake!”
“And one leetle bird, vith a vairy fine salad, my leetle lady,—vasn’t
it—one vairy nice leetle bird?” interrupted Gex, who was unwilling to
have his fine dinner belittled.
“Oh, yes; bird, and fish, and soup,” enumerated Lady Jane, “and
peas, Pepsie, little peas.”
“Oh, mon Dieu! oh, leetle lady!” cried Gex, holding up his hands in
horror, “you have it vairy wrong. It vas soup, and fish, and bird. M.
Paichoux, you see the leetle lady does not vell remember; and you
must not think I can’t order one vairy fine dinner.”
“I understand,” said Paichoux, laughing. “I’ve no doubt, Gex, but
what you could order a dinner fit for an alderman.”
“Thank you, thank you, vairy much,” returned Gex, as he bowed
himself out and went home to dream of his triumphs.
CHAPTER XXII
PAICHOUX MAKES A PURCHASE
“J UST to think,” said Pepsie to her mother, the next morning,

“Madame Jozain wasn’t the least anxious last night about Lady.
I don’t believe she cares for the child, or she’d never be willing to let
her stay away from her the most of the time, as she does. She’s
always fussing about her great, overgrown son, if he’s out of her
sight.”
“And no wonder,” returned Madelon. “Poor woman, she has
trouble enough with him. She keeps it to herself and pretends to be
proud of him; but, my dear, he’s a living disgrace to her. I often hear
him spoken of on the Rue Bourbon; he dresses fine and never
works. Where does he get his money, ma petite? If people are poor
and don’t work they must steal. They may call it by some other
name, but I call it stealing. Madame Jozain can’t make money
enough in that little shop to support herself and keep that boy in
idleness. We mustn’t be too hard on her. She has trouble enough, I
can see it in her face; she looks worn out with worry. And we’ll do all
we can for that little darling. It’s a pleasure; she’s so sweet and
grateful. I only wish I could do more. I’d work my fingers to the bone
for you two, my darling.”
“Bonne maman,” said Pepsie, clinging to her neck, and kissing her
fondly, “have you thought of what I asked you—have you, mama?”
“Yes, my dear, I have, I’ve thought of it a great deal; but I don’t see
my way clear quite yet.”
“Why, you’ve got the money in the bank, mama?”
“I can’t touch that money, my dear; it’s for you. If anything should
happen to me, and you were left alone.”
“Hush, hush, mama; I shouldn’t need any money then, for I should
die too.”
“No, my dear, not if it was the good God’s will that you should live.
I don’t want to spend that; I want to feel that you’ve something. A
piano costs a great deal of money; besides, what would your uncle
and aunt think if I should do such a thing?”
“They’d think you did it because I wanted you to,” returned Pepsie
slyly.
“That would be a reason certainly,” said Madelon, laughing, “and
I’ll try to do it after a while. Have a little patience, dear, and I think I
can manage it without touching the money in the bank.”
“Oh, I hope you can, mama, because Mam’selle Diane says Lady
learns very fast, and that she ought to practise. I hate to have her
kept back for the need of a piano, and Madame Jozain will never get
one for her. You know you could sell it afterward, mama,”—and
Pepsie went on to show, with much excellent reasoning, that Lady
Jane could never make a great prima donna unless she had
advantages. “It’s now, while her fingers are supple, that they must be
trained; she ought to practise two hours a day. Oh, I’d rather go
without the money than to have Lady kept back. Try, bonne maman,
try to get a piano very soon, won’t you?”
And Madelon promised to try, for she was devoted to the child; but
Pepsie had begun to think that Lady Jane was her own—her very
own, and, in her generous affection, was willing to sacrifice
everything for her good.
And Madelon and Pepsie were not the only ones who planned and
hoped for the little one with almost a mother’s love and interest.
From the first day that Lady Jane smiled up into the sad, worn face
of Diane d’Hautreve, a new life had opened to that lonely woman, a
new hope, a new happiness brightened her dreary days; for the
child’s presence seemed to bring sunshine and youth to her. Had it
not been for her mother, she would have kept the gentle little
creature with her constantly, as the sweetest hours she knew, or had
known for many a weary year, were those she devoted to her lovely
little pupil. It was a dream of delight to sit at the tinkling piano with
Lady Jane nestled close to her side, the sweet, liquid notes mingling
with hers, as they sang an old-fashioned ballad, or a tender lullaby.
And the child never disappointed her; she was always docile and
thoughtful, and so quiet and polite that even Diane’s mother,
captious and querulous though she was, found no cause for
complaint, while the toleration with which she had at first received
Lady Jane was fast changing into affection. The more they became
interested in her, the more they wondered how she could be kin to
such a woman as Madame Jozain; for Mam’selle Diane had been
obliged to show how exclusive she could be in order to keep
madame where she belonged.
At first Madame Jozain had annoyed them greatly by trying to
intrude upon their seclusion; and it had taken several polite, but
unmistakable rebuffs to reach her that they were d’Hautreves, and
that the child would be received gladly where the aunt must not
expect to enter.
Madame swallowed her mortification and said nothing, but she
bided her time to take her revenge. “I’ll show them before long that I
know how poor they are; and that funny little story I got out of Tite
Souris, about Mam’selle Diane cleaning her banquette with a veil
over her face—every one in the neighborhood shall know it. Poor,
proud, old thing, she thought she could insult me and I wouldn’t
resent it!”
And while Madame was planning her little revenge, and rehearsing
her grievances to herself, Madame d’Hautreve and Mam’selle Diane
were wondering if something couldn’t be done to get the child out of
the clutches of such an aunt.
“It seems dreadful,” Mam’selle Diane would say, sadly, “to leave
her with that woman. I can’t think she has any right to her; there’s a
mystery about it, and it ought to be investigated. Oh, mama dear, if
we had some money I’d hire a lawyer to find out. If she really is the
child’s next-of-kin, I suppose she has a legal right to her, and that no
one could oblige her to relinquish that right; but one might buy the
child; I think she is just the woman to be moved by money. Oh,
mama, if our claim had only gone through! If we’d only got what we
ought to have had, I would try—if you had no objections—to get the
child.”
“Dear, dear, Diane, how absurd you are! What would you do with
her?”
“Why, you could adopt her, mama, and I could have the care of
her.”
“But, my child, that is all romancing. We have no money, and we
never shall have any. It is useless to think of that claim, it will never
be considered; and even if we had money, it would be a great risk to
take a child we know nothing of. I think with you that there’s some
mystery, and I should like to have it looked into, yet I don’t think it’s
worth while worrying about; we have troubles enough of our own.”
“Oh, mama, we need not be selfish because we are poor,” said
Diane, gently.
“We can’t help it, child; selfishness is one of the results of poverty.
It is self, self, constantly; but you are an exception, Diane. I will give
you the credit of thinking more of others’ interest than of your own.
You show it in everything. Now, about that bird. Madame Jourdain
should have paid you for it, and not thrown it on your hands.”
“Oh, mama, she couldn’t sell it,” said Mam’selle Diane, rejectedly.
“It wouldn’t be right to expect her to lose the price of it. She says it
didn’t ‘take’ as well as the ducks.”
“Well, she might have thrown in the wool,” insisted Madame
d’Hautreve, querulously, “she might have given the wool against your
time.”
“But she didn’t ask me to experiment with a new model, mama
dear. It wasn’t her fault if I didn’t succeed.”
“You did succeed, Diane. It was perfect; it was most life-like, only
people haven’t the taste to recognize your talent.”
“Madame Jourdain said that her customers didn’t like the bird’s
bill, and they thought the neck too long,” returned Mam’selle Diane,
humbly.
“There, there; that shows how little the best educated people know
of ornithology. It is a species of crane; the neck is not out of
proportion.”
“They thought so, mama, and one can’t contend with people’s
tastes and opinions. I shall not try anything new again. I shall stick to
my ducks and canaries.”
“You know I advised you to do so in the first place. You were too
ambitious, Diane, you were too ambitious!”
“Yes; you are right, mama, I was too ambitious!” sighed Mam’selle
Diane.
One morning in August, about a year from the time that Madame
Jozain moved into Good Children Street, Tante Modeste was in her
dairy, deep in the mysteries of cream-cheese and butter, when
Paichoux entered, and laying a small parcel twisted up in a piece of
newspaper before her waited for her to open it.
“In a moment,” she said, smiling brightly; “let me fill these molds
first, then I’ll wash my hands, and I’m done for to-day.”
Paichoux made no reply, but walked about the dairy, peering into
the pans of rich milk, and whistling softly.
Suddenly, Tante Modeste uttered an exclamation of surprise. She
had opened the paper, and was holding up a beautiful watch by its
exquisitely wrought chain.
“Why, papa, where in the world did you get this?” she asked, as
she turned it over and over, and examined first one side and then the
other. “Blue enamel, a band of diamonds on the rim, a leaf in
diamonds on one side, a monogram on the other. What are the
letters?—the stones sparkle so, I can hardly make them out. J, yes,
it’s a J, and a C. Why, those are the very initials on that child’s
clothes! Paichoux, where did you get this watch, and whose is it?”
“Why, it’s mine,” replied Paichoux, with exasperating coolness. He
was standing before Tante Modeste, with his thumbs in his waistcoat
pockets, whistling in his easy way. “It’s mine, and I bought it.”
“Bought it! Where did you buy a watch like this, and wrapped up in
newspaper, too? Do tell me where you got it, Paichoux,” cried Tante
Modeste, very much puzzled, and very impatient.
“I bought it in the Recorder’s Court.”
“In the Recorder’s Court?” echoed Tante Modeste, more and more
puzzled. “From whom did you buy it?”
“From Raste Jozain.”
Tante Modeste looked at her husband with wide eyes and parted
lips, and said nothing for several seconds; then she exclaimed, “I
told you so!”
“Told me what?” asked Paichoux, with a provoking smile.
“Why, why, that all those things marked J. C. were stolen from that
child’s mother; and this watch is a part of the same property, and she
never was a Jozain—”
“Not so fast, Modeste; not so fast.”
“Then, what was Raste Jozain in the Recorder’s Court for?”
“He was arrested on suspicion, but they couldn’t prove anything.”
“For this?” asked Tante Modeste, looking at the watch.
“No, it was another charge, but his having such a valuable watch
went against him. It seems like a providence, my getting it. I just
happened to be passing the Recorder’s Court, and, glancing in, I
saw that precious rascal in the dock. I knew him, but he didn’t know
me. So I stepped in to see what the scrape was. It seems that he
was arrested on the suspicion of being one of a gang who have
robbed a number of jewelry stores. They couldn’t prove anything
against him on that charge; but the watch and chain puzzled the
Recorder like the mischief. He asked Raste where he got it, and he
was ready with his answer, ‘It belonged to my cousin who died some
time ago; she left it to my mother, and my mother gave it to me.’”
“‘What was her name?’ asked the Recorder.
“‘Claire Jozain,’ the scamp answered promptly.
“‘But this is J. C.,’ said the Recorder, examining the letters closely.
‘I should certainly say that the J. came first. What do you think,
gentlemen?’ and he handed the watch to his clerk and some others;
and they all thought from the arrangement of the letters that it was J.
C., and while this discussion was going on, the fellow stood there
smiling as impudent and cool as if he was the first gentleman in the
city. He’s a handsome fellow, and well dressed, and the image of his
father. Any one who had ever seen André Jozain would know that
Raste was his son, and he’s in a fair way to end his days in Andre’s
company.”
“And they couldn’t find out where he got the watch?” interrupted
Tante Modeste impatiently.
“No, they couldn’t prove that it was stolen. However, the Recorder
gave him thirty days in the parish prison as a suspicious character.”
“They ought not to have let him off so easily,” said Tante Modeste
decidedly.
“But you know they couldn’t prove anything,” continued Paichoux,
“and the fellow looked blue at the prospect of thirty days. I guess he
felt that he was getting it pretty heavy. However, he put on lots of
brass and began talking and laughing with some flashy-looking
fellows who gathered around him. They saw the watch was valuable,
and that there was a chance for a bargain, and one of them made
him an offer of fifty dollars for it. ‘Do you think I’m from the West?’ he
asked, with a grin, and shoved it back into his pocket! ‘I’m pretty hard
up, I need the cash badly; but I can’t give you this ticker, as much as
I love you.’ Then another fellow offered him sixty, and he shook his
head. ‘No, no, that’s nowhere near the figure.’
“‘Let me look at the watch,’ I said, sauntering up. ‘If it’s a good
watch I’ll make you an offer.’ I spoke as indifferently as possible,
because I didn’t want him to think I was anxious, and I wasn’t quite
sure whether he knew me or not. As he handed me the watch he
eyed me impudently, but I saw that he was nervous and shaky. ‘It’s a
good watch,’ I said after I examined it closely; ‘a very good watch,
and I’ll give you seventy-five.’

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Sleisenger and Fordtran's

Gastrointestinal and Liver Disease 11th

EDITORS ASSOCIATE EDITORS

LAWRENCE S. FRIEDMAN, MD DAVID T. RUBIN, MD

SLEISENGER AND FORDTRAN’S GASTROINTESTINAL AND LIVER DISEASE,

Copyright © 2021 by Elsevier, Inc. All rights reserved.

Library of Congress Control Number: 2020934045

Senior Content Strategist: Nancy Duffy

Last digit is the print number: 9 8 7 6 5 4 3 2 1

Nezam H. Afdhal, MD, DSc Bruce R. Bacon, MD William Bernal, MD

Jan Bornschein, MD Eugene B. Chang, MD Paul A. Dawson, PhD

John Radcliffe Hospital Department of Medicine Pediatrics— Gastroenterology,

Kerry B. Dunbar, MD, PhD Michael B. Fallon, MD Alexander C. Ford, MBChB, MD

Marc G. Ghany, MD, MHSc David J. Hass, MD M. Nedim Ince, MD

National Institute of Diabetes and Division of Digestive Diseases Medicine

D. Rohan Jeyarajah, MD Jonathan D. Kaunitz, MD Brian E. Lacy, MD, PhD

Office of the University Provost Pathology South Shore Hospital

Syed Mujtaba Rizvi, MD Jayashree Sarathy, PhD Vijay H. Shah, MD

David C. Whitcomb, MD, PhD Christopher G. Willett, MD Anahit A. Zeynalyan, MD

Medicine, Cell Biology and Molecular Radiation Oncology Internal Medicine

Mark Feldman, MD Lawrence S. Friedman, MD Lawrence J. Brandt, MD

Editions 5-11 Editions 7-11 Editions 8-11

Raymond T. Chung, MD David T. Rubin, MD C. Mel Wilcox, MD

Edition 11 Edition 11 Edition 11

Marvin H. Sleisenger, MD John S. Fordtran, MD Bruce F. Scharschmidt, MD

Editions 1-7 Editions 1-5 Editions 5-6

MHC Major histocompatibility complex SBP Spontaneous bacterial peritonitis

1 Cellular Growth and Neoplasia

CHAPTER OUTLINE MECHANISMS OF NORMAL TISSUE HOMEOSTASIS

Cyclin D/E Cyclin A

The cell cycle is also regulated by multiple CDK inhibi-

in compacted apoptotic bodies that are eventually phagocytosed

Frizzled Receptor Plasma Membrane

Chromosomal Hyperproliferative Early Late

APC COX2 KRAS TP53

Microsatellite Hyperproliferative Early Late

Mutations in genes with polynucleotide tracks

CIMP Hyperproliferative Early Late

CpG island methylation and silencing of tumor

activities. Point mutations or large gene rearrangements result- Growth factor

Disorder Gene(s) Mutated Inherited

Peutz-Jeghers syndrome STK11 Sporadic

TUMOR MICROENVIRONMENT Microbiome

colonic tumors in specified animal models.73 Fusobacterium nuclea-

ants at splice-acceptor or donor sites may result in exon loss or

2 Mucosal Immunology and Inflammation

tantly, entry of immune cells into the LP and cell activation is

antigen driven. Thus germ-free mice have few immune cells in

Chemokine Role in Homeostasis and Inflammation���������22

Plasma cell MAdCAM-1

classical complement components but rather binds luminal bac-

A mucus coat, produced by goblet cells, lines the intestinal

Intestinal Epithelial Cells

Paneth cells GFI-1, is important in Paneth cell development by suppressing

opening a niche for the aerotolerant pathogen. Certain bacteria

CHAPTER OUTLINE of the microorganisms. Each of these contributes to the stabil-

composition in samples obtained longitudinally from an individ-

“J UST to think,” said Pepsie to her mother, the next morning,

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