Internet Access: Wireless Internet is available for delegates carrying a laptop and can be accessed in the meeting room

. Laptops must be wi-fi enabled. Username: transplant Password: meeting

C2: Inside Front Cover

Ad Genentech/Roche

The Transplantation Society • www.tts.org

TABLE OF CONTENTS
Committees Message from the President of TTS Message from the Conference Chairs Conference Information Floor Plans Local Information Evening Event Conference Learning Objectives Instructions to Oral and Poster Presenters Invited Speakers & Chairs Wednesday • Feb. 24 • Pre-Conference Workshop Thursday/Friday • Program Quick-View Thursday • Feb. 25 • Detailed Program Friday • Feb. 26 • Detailed Program 2 3 4 5 6 7 8 9 10 11 13 14 16 36

Posters Presenter Index Sponsor Acknowledgements

51 71 72

The Transplantation Society International Headquarters 1255 University Street, Suite 325 Montreal, QC, Canada  H3B 3B4 Phone: 514-874-1717 Fax:  514-874-1716  Email: info@tts.org Web: www.tts.org

Printed in Canada, February 2010

Transplantomics and Biomarkers in Organ Transplantation

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COMMITTEES
LOCAL ORGANIZING COMMITTEE Minnie Sarwal, Conference Chair Stanford, CA, USA Mark M. Davis, Conference Co-Chair Stanford, CA, USA Atul Butte, Conference Co-Chair Stanford, CA, USA SCIENTIFIC PROGRAM COMMITTEE Minnie Sarwal, Conference Chair Stanford, CA, USA Tom Blydt-Hansen Winnipeg, MB, Canada Atul Butte, Conference Co-Chair Stanford, CA, USA Anthony Jevnikar London, ON, Canada Allan D. Kirk Atlanta, GA, USA Roslyn B. Mannon Birmingham, AL, USA Elaine Reed Los Angeles, CA, USA David Rush Winnipeg, MB, Canada Daniel Salomon San Diego, CA, USA Manikkam Suthanthiran New York, NY, USA

THE TRANSPLANTATION SOCIETY REPRESENTATIVES Jeremy Chapman, TTS President Westmead, Australia Kathryn Wood, TTS Past President 2006-2008 Oxford, United Kingdom

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

MESSAGE FROM THE PRESIDENT OF TTS
Transplantation has evolved over the last 60 years through the combined efforts of clinicians and scientists. The problems that our early predecessors faced included only very limited understanding of the immunological barriers, however this was combated with clinical experimentation and by slowly accrued experience. Science provided some answers as we came into the 1960’s during which the cross match test and immunosuppressive drugs yielded some initial triumphs over the biology of transplantation. Slowly we have used our scientific knowledge to bring forward both an understanding of the clinical problems that face us and some solutions. The tools of our trade have been crude in comparison to the sophistication of the immune system and the responses to injury - we have histology, some biochemical measures, some techniques for detection of antibody specificity, imaging techniques mostly based on the scientific developments of the 1940’s and slow application of information technology. If we were to be honest in our appraisal of the current state of affairs - clinical transplantation remains mired in the mud of our application of old technology. This conference is about bringing together those with expertise in the new technologies of genomics, proteomics and metabalomics and those who carry expertise in clinical application of technologies in transplantation. Forgive us the title ‘Transplantomics‘ as it is an attempt to bring these concepts into a single word. The Transplantation Society stands for development in the science and clinical practice of transplantation. It is our expectation that smaller concentrated workshops and meetings such as this will help to stimulate the translation of these new technologies into clinical practice over the years ahead. We thank our generous sponsors Roche/Genentech, Bristol-Myers Squibb, Novartis, Pfizer, Astellas and Genzyme who have accepted the challenge of assisting TTS with funding this first Transplantomics conference and hope that you will enjoy this opportunity to meet with your colleagues here in San Francisco. The 2nd International Conference on Transplantomics will be held next year and we would welcome proposals for the venue.

Jeremy Chapman President, The Transplantation Society

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MESSAGE FROM THE CONFERENCE CHAIRS

Dear colleagues,

Welcome to the 1st International Conference on Transplantomics and Biomarkers in Organ Transplantation and welcome to the beautiful city of San Francisco, California. Minnie Sarwal Conference Chair As host of the meeting, The Transplantation Society (TTS) has called for this first-ever “Transplantomics” meeting in order to establish a meeting platform that brings together disciplines in transplantation research in genomics, proteomics, informatics and clinical transplantation with the goal to create more dialogue and collaboration across disciplines and researchers. The agenda has been drafted with the objectives to maximize outstanding presentations as a basis for discussion, time for questions and answers, discussions and networking. The poster reception will be the perfect opportunity to explore specific subjects with your colleagues. Mark M. Davis Conference Co-Chair

Enjoy this conference!

Atul Butte Conference Co-Chair

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

CONFERENCE INFORMATION
Certificate of Attendance An attendance certificate can be requested via e-mail to info@tts-transplantomics.org. This conference is not CME-accredited. Conference Evaluation A conference evaluation form will be distributed during the morning coffee break on Friday. Please complete and return to the registration desk. Emergency / First Aid For any emergency or first aid services inside the hotel, please dial “0” from any house phone. Exhibit Display Our Platinum sponsor Genentech / Roche will have a table-top display in the Cyril Magnin Foyer on February 25 and 26 from 10:00-18:00. Information The Conference Service Desk is located in the registration area and is in service for the entire duration of the Conference. Should you require any assistance outside of service desk hours, please call 1-514-991-3851. Internet Access Wireless Internet is available for delegates carrying a laptop and can be accessed in the meeting room. Laptops must be wi-fi enabled. Username: transplant Password: meeting Lost and Found Any lost and found items will be held at the Conference Service Desk for the duration of the event. For any unclaimed items after the conference, please contact The Transplantation Society. Luggage We recommend that you leave your luggage with the Bellman at your hotel. The Transplantation Society does not take any responsibility for suitcases that are left in the meeting area, including the registration desk. Poster Displays Posters are on display in the Cyril Magnin Foyer on February 25 and February 26. Delegates can visit posters at their convenience. Poster presenters are encouraged to be at their posters during break times. An official poster reception will take place on February 25 from 17:00-18:00. Public Notice The Parc 55 Hotel is a smoke-free environment. Smoking is permitted only outside the building. Registration Hours Wednesday, February 24 (outside Divisadero) Thursday, February 25 Friday, February 26 (outside Cyril Magnin) 13:00-18:00 07:00-18:00 07:00-17:00

Transplantomics and Biomarkers in Organ Transplantation

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ASHBURY BOARDROOM AND SUTRO CREATES A PRIVATE

FLOOR PLANS

Ashbury

Divisadero

Haight

Sutro

Guest Reception

cityhouse

Level Two - Divisadero

Level Four – Cyril Magnin I & II

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

LOCAL INFORMATION
About San Francisco The City and County of San Francisco is the fourth most populous city in California and the 12th most populous city in the United States, with a 2008 estimated population of 808,976. It is the second most densely populated major city in the U.S. and is the financial, cultural, and transportation center of the larger San Francisco Bay Area, a region of more than seven million people. The city is located at the northern end of the San Francisco Peninsula, with the Pacific Ocean to the west and San Francisco Bay to the north and east. San Francisco inspires images recognized the world over: the Golden Gate Bridge, Fisherman’s Wharf, Chinatown, cable cars, that beautiful city by the Bay. San Francisco heightens an appreciation for ‘the good life’. Known for its fine restaurants and shops, San Francisco has something for every appetite and budget. Home to the highest concentration of arts organizations in the country, audiences are captivated by everything from street minstrels and sidewalk art shows to the highly acclaimed San Francisco Opera and Symphony and a multitude of fascinating museums. Stroll through Golden Gate Park past the Arboretum and Botanical Gardens, the Children’s Playground (complete with carousel) and the National AIDS Memorial Grove. Take the ferry to Alcatraz or Sausalito, head north to Muir Woods and see the magnificent redwoods, or explore wine country in Napa and Sonoma just an hour’s drive from the city. Two days or two decades here reveal only that there is more to learn, more to see, more to do. Getting Around San Francisco Public Transportation: Muni rail lines serve only San Francisco. Bus, historic streetcar, and Metro trips for adults cost $2.00, including a free transfer. Fares can be paid with any US coins. Exact change is required. Cable car trips cost $5.00 per single ride. There are single-ride ticket booklets for multiple rides (booklets of 10 single tickets) and passports which offer unlimited travel on the transit system. Ride streetcars, buses and cable cars as many times a day as you wish with your Passport. Available for 1 day, 3 consecutive days or 7 consecutive days. 1day Cable Car passes, available from Cable Car conductors, are good only on Cable Cars.

Taxis: Taxis are easy to spot in downtown San Francisco, particularly during rush hour. Taxis can be easily hailed - just look for the lighted sign on the top that indicates if the taxi is available for hire. Major hotels around Union Square and Fisherman’s Wharf usually have taxis in line for hire. PIER 39 in Fisherman’s Wharf is also a good place to hail a cab. Extra travel charges may be added for luggage or late-night traveling, so ask the driver before leaving. Taxi Fares: First 1/5th mile or flag $3.10; each additional 1/5th mile or fraction thereafter,45¢; each minute of waiting, or traffic time delay, 45¢; airport surcharge $2.00.

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EVENING EVENT

Forbes Island Fantasy Fine Dining aboard the World’s Only Floating Island
Thursday, February 25 19:00 to 22:00 (bus departure from hotel at 18:45, return at 22:30) Dress Code: Casual • Tickets available for $85 per person (limited capacity)
Forbes Island provides a fantasy dining experience, beneath the sea and on the white sand patios with incredible views of San Francisco Bay, Downtown, Alcatraz, Sea Lions, Coit Tower & the Golden Gate Bridge. You surely will want to be at this dinner! We recommend reading about the 7 Wonders of Forbes Island / www.forbesisland.com.
1) The English Tudor paneling on the interior walls of the island is decorated with tight fitted molding which is secured with no nails or glue. The molding, if pieced together, would span more than 10,000 feet or nearly 2 miles. 2) The antique ship's wheel in the wheelhouse was from the Brigatine, Regina Maris, built in 1906 and she rounded Cape Horn 17 times. 3) The 40 foot Lighthouse is the only privately built Lighthouse in the United States and is equipped with an authentic Fresnel Lens on loan from the US Lighthouse Society. The Fresnel Lens was built in France in 1820 and was first lit using whale oil. 4) The island is powered by an actual Sea Mule motor, which is the world's largest outboard motor and was used extensively in World War II by the US Navy to propel ocean going barges. It can rotate 360 degrees and is a 250 horse power motor. 5) Forbes Island displaces 700 tons of water, the structure was made with 280 tons of concrete, 120 tons of rocks surround the perimeter, 90 tons of sand top the shores, and 40 tons of topsoil are the earth for the plants and palms. 6) During the spring the Washatona Palm Trees are the nesting grounds for hundreds of Starling blackbirds and the rocks around the perimeter of the island are the nesting grounds of several seagulls. 7) Forbes Island launched on December 23, 1980. On launch day the island had 3 staterooms all with private baths, 56 portholes throughout, and the waterfall cascaded into a hot tub. Shortly thereafter it was featured on "Lifestyles of the Rich and Famous" and "That's Incredible".

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

CONFERENCE LEARNING OBJECTIVES
The overall objective of the meeting is to present the latest progress on using high throughput and informatics approaches to improve translational biomedical research in organ transplantation.

After attending this conference, delegates will be able: n To review and gain an understanding of key lessons learnt by translational bioinformatics researchers in organ transplantation and to better set up cross-talk between groups; n To identify the current challenges of translational research omics (transplantomics) in organ transplantation and to define the future directions; n To articulate challenges and opportunities in transplantomics; n To learn a framework for developing, deploying and assessing the success and applications of translational bioinformatics initiatives in organ transplantation; n To understand the techniques for implementing specific clinical decision support interventions; n To appreciate how translational bioinformatics may be deployed to enhance clinical and translational research; n To identify areas of interaction among computational biology, genomics research, electronic health records, health information exchanges, HLA disparity testing and public health.

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INSTRUCTIONS TO ORAL PRESENTERS
THERE IS NO SPEAKER READY ROOM. Speakers must arrive with their final PowerPoint presentation onsite. All presentation files will be uploaded directly onto the meeting room computer (PC) and will be erased at the end of the day. Only authorized presentations will be recorded and posted online. If you speak in the morning block, please bring your presentation to the AV technician in the meeting room at least 30 minutes before the session starts or during the coffee break. If you speak in the afternoon block, please bring your presentation to the AV technician during the lunch break and at least 30 minutes before session starts. As the schedule is very compact, we want to make sure that the presentations for each block are pre-loaded and tested. While we recommend using the session computer, we also allow the use of a speaker’s own computer. SPEAKERS MUST ADVISE THE TECHNICIAN BEFORE THE SESSION IF USING THEIR OWN COMPUTER.

INSTRUCTIONS TO POSTER PRESENTERS
Participants will view posters during lunches and breaks, therefore authors are encouraged to be present at their posters during those times. Authors are responsible for the setting up and the removal of their posters according to the following schedule: Mounting time: Removal: Room: Thursday, February 25, 2010 Friday, February 26, 2010 Foyer Cyril Magnin 07:00 to 08:00 by 17:00

Posters not removed by the specified time on the last day of their presentation will be removed and discarded by Meeting staff. TTS cannot accept liability for lost or damaged posters. TTS will not mail posters to authors after the meeting.

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

INVITED SPEAKERS & CHAIRS
Dany Anglicheau Associate Professor, Kidney Transplant Unit, Necker Hospital, Paris, France Atul Butte Assistant Professor, Medicine, Stanford Medical Informatics, Stanford University, Stanford, CA, USA Jeremy Chapman Director, Acute Interventional Medicine and Renal Services, Westmead Hospital, SWAHS, Westmead, Australia Christopher Contag Co-Director, Molecular Imaging Program at Stanford, Department of Pediatrics and of Microbiology and Immunology, Stanford University, Stanford, CA, USA Mark M. Davis Professor, Microbiology & Immunology, Stanford University, Stanford, CA, USA Ronald W. Davis Professor of Biochemistry and Professor of Genetics, Member of the Stanford Comprehensive Cancer Center, Stanford University, Stanford, CA, USA Stuart Flechner Director, Clinical Research, Section of Renal Transplantation, Cleveland Clinic; Professor of Surgery, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, USA Federico Goodsaid Associate Director for Operations in Genomics; Office of Clinical Pharmacology; Office of Translational Science; Center for Drug Evaluation and Research; U.S. Food and Drug Administration; Silver Spring, MD, USA Philip F. Halloran Director, Alberta Transplant Applied Genomics Centre; Distinguished University Professor, University of Alberta; Editor-in-Chief, American Journal of Transplantation, Edmonton, AB, Canada Peter S. Heeger Director, Clinical Research Program, Transplantation Institute, Mount Sinai School of Medicine, New York, NY, USA Ajay Israni Assistant Professor of Medicine, Adjunct Professor of Epidemiology & Community Health, Hennepin County Medical Center, University of Minnesota, Minneapolis, MN, USA Kathleen Kelly Associate Professor and Technical Director, Department of Pathology & Laboratory Medicine, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA Allan D. Kirk Professor of Surgery and Pediatrics, Emory University and Scientific Director of the Emory Transplant Center, Atlanta, GA, USA Graham Lord Professor of Medicine, Nephrology, Transplantation and Internal Medicine; Director of Translational Research Development and Deputy Director of NIHR Comprehensive Biomedical Research Centre, Guy’s and St. Thomas’ Hospital and King’s College London, London, UK Roslyn B. Mannon Director of Research, Alabama Transplant Center; Professor, Department of Medicine, Division of Nephrology; Department of Surgery, Division of Transplantation, University of Alabama at Birmingham, Birmingham, AL, USA Francesco M. Marincola Chief Infectious Disease and Immunogenetics Section (IDIS), Department of Transfusion Medicine, Clinical Center; Associate Director, Trans-NIH Center for Human Immunology, National Institutes of Health; Director, CC/CHI FOCIS Center of Excellence, Bethesda, MD, USA Steven G.E. Marsh Deputy Director of Research, Anthony Nolan Research Institute and Professor of Immunogenetics, UCL Cancer Institute, Royal Free Campus, London, UK

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INVITED SPEAKERS & CHAIRS
Valeria Mas Associate Professor, Departments of Surgery and Pathology; Director, Molecular Transplant Research Laboratory, Division of Transplant, Virginia Commonwealth University, Richmond, VA, USA Bruce McManus Professor, Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada Herwig-Ulf Meier-Kriesche University of Florida, Gainesville, FL, USA Thalachallour Mohanakumar Jacqueline G. & Wm. E. Maritz Professor, Surgery, Pathology and Immunology; Director, Histocompatibility & Immunogenetics; Director, Islet Isolation Core, Washington University, St. Louis, MO, USA Thomas Mueller, Division of Nephrology and Immunology, University of Alberta, Edmonton, AB, Canada Maarten Naesens Department of Nephrology and Renal Transplantation, University Hospitals Leuven, Leuven, Belgium; Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA Philip O’Connell Director, Transplantation and CTRR, Westmead Millennium Institute for Medical Research, Westmead, Australia Richard N. Pierson III Professor of Surgery, University of Maryland School of Medicine, Baltimore, MD, USA Caius G. Radu Assistant Professor, Department of Molecular and Medical Pharmacology, UCLA Crump Institute, Los Angeles, CA, USA Elaine Reed Professor and Director of Immunogenetics, Transplant/Immunogenetics Testing, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Michael Reich Broad Institute of MIT and Harvard, Boston, MA, USA David Rush Professor and Head, Section of Nephrology; Director, Transplant Manitoba Adult Kidney Program, Health Sciences Centre, Winnipeg, MB, Canada Alberto Sanchez-Fueyo Hospital Clinic Barcelona, University of Barcelona, Barcelona, Spain Minnie Sarwal Professor of Pediatrics and Immunology, Stanford University, Stanford, CA, USA Richard Smith Biological Sciences Division, Pacific Northwest National Laboratory, Richland, VA, USA Manikkam Suthanthiran Stanton Griffis Distinguished Professor of Medicine, Chief, Nephrology and Transplantation Medicine, New York Presbyterian Hospital-Cornell, New York, NY, USA Robert Tibshirani Associate Chairman and Professor of Health Research and Policy, and Statistics, Stanford University, Stanford, CA, USA David Wishart Professor, Departments of Biological Sciences and Computing Science, University of Alberta; Senior Research Officer and Co-director, Nanobiology Program, NRC’s National Institute for Nanotechnology (NINT), Edmonton, AB, Canada Kathryn Wood Associate of the Oxford Stem Cell Institute, Professor of Immunology, University of Oxford, Oxford, UK

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

PRE-CONFERENCE WORKSHOP – WEDNESDAY • FEB 24 DIVISADERO ROOM CURRENT AND NEW TECHNOLOGIES IN GENOMICS AND PROTEOMICS
Sponsored by Invitrogen
Chair: Holden Maecker, Director, Human Immune Monitoring Center, Stanford University, Stanford, CA, USA

1. CURRENT TECHNOLOGIES
15:00 FLOW CYTOMETRY Smita Ghanekar, BD Biosciences

15:30

LUMINEx Holden Maecker, Stanford University

16:00

GENE ARRAYS Sharoni Jacobs, Agilent

2. NEW TECHNOLOGIES
17:00 NANOIMMUNOASSAYS Alice Fan, Stanford University

17:20

MICROFLUIDIC SINGLE CELL ASSAYS Caroline Dando, Fluidigm Corporation

17:40

CyToF Sean Bendall, Stanford University

Transplantomics and Biomarkers in Organ Transplantation

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THURSDAY • FEB 25 CYRIL MAGNIN I & II

PROGRAM QUICK-VIEW

NOVEL APPLICATIONS FOR GENOMIC TECHNOLOGIES IN ORGAN TRANSPLANT INJURY Session is sponsored through an unrestricted educational grant by Pfizer. Co-chairs: Jeremy Chapman and Kathryn Wood 08:00 08:10 08:40 09:00 09:20 09:40 10:00 Opening Message • Jeremy Chapman, Australia Advances in Genome Analysis Technologies • Ronald W. Davis, USA Reclassifying Graft Injury by Genomics • Philip F. Halloran, Canada Deconvoluting the Peripheral Blood Transcriptome in Graft Rejection • Minnie Sarwal, USA Noninvasive Diagnosis of Renal Allograft Status • Manikkam Suthanthiran, USA Exploring Genomic Medicine Using Translational Bioinformatics • Atul Butte, USA NETWORKING COFFEE BREAK, POSTERS & EXHIBITS (FOYER) NOVEL APPLICATIONS FOR GENOMIC TECHNOLOGIES IN ORGAN TRANSPLANT INJURY (cont.) Co-chairs: Philip F. Halloran and Manikkam Suthanthiran 10:30 11:00 11:20 11:40 12:00 Immune-Monitoring in Health and Infection • Mark M. Davis, USA The Evolution of Non-Immune Histological Injury • Maarten Naesens, USA The Genomics of Fibrosis Progression • Valeria Mas, USA RNA Silencing in Gene Regulation • Christopher Contag, USA LUNCH, POSTERS & EXHIBITS (FOYER) PREDICTING GRAFT RISK BY TRANSPLANTOMICS Session is sponsored through an unrestricted educational grant by Bristol-Myers Squibb Co-chairs: Philip O'Connell and Thomas Mueller 13:30 14:00 14:20 14:40 15:00 15:20 15:40 16:00 17:00 19:00 The Impact of Genome-wide Variation in Renal Transplant Donors and Recipients on Graft Survival • Graham Lord, UK Challenges in Association Studies of Single Nucleotide Polymorphism Associated and Renal Transplant Outcomes • Ajay Israni, USA MicroRNA and RNA Profiles with Graft Rejection • Dany Anglicheau, France Predictors of Transplant Vasculopathy and Glomerulopathy • Roslyn B. Mannon, USA A Signature for Operational Liver Transplant Tolerance • Alberto Sanchez-Fueyo, Spain Can Immune Accommodation Be Predicted Using "-omics" Tools? • Bruce McManus, Canada NETWORKING COFFEE BREAK, POSTERS & EXHIBITS (FOYER) MINI-ORAL PRESENTATIONS (See detailed program on page 34) Co-chairs: Allan D. Kirk and Roslyn B. Mannon POSTER RECEPTION DINNER AT FORBES ISLAND (BUS DEPARTS FROM HOTEL AT 18:45)

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

PROGRAM QUICK-VIEW
PERSONALIZING MEDICINE THROUGH OMICS Co-chairs: Herwig-Ulf Meier-Kriesche and Richard N. Pierson III 08:30 09:00 09:30 10:00

FRIDAY • FEB 26 CYRIL MAGNIN I&II

Conducting Transplant Trials for Biomarker Discovery • Allan D. Kirk, USA Tools for Integrative Genomics • Michael Reich, USA Path from Exploratory Biomarkers for Qualified Biomarkers in the Clinic • Federico Goodsaid, USA NETWORKING COFFEE BREAK, POSTERS & EXHIBITS (FOYER) THE ANTIBIOME AND TRANSPLANTATION Co-chairs: Atul Butte and Stuart Flechner

10:30 11:00 11:20 11:40 12:00

Common Signatures of Rejection • Francesco M. Marincola, USA Cross Talk between AlloImmunity and AutoImmunity in Organ Transplantation • Thalachallour Mohanakumar, USA Phosphoproteomics and the Endothelium • Elaine Reed, USA Antibody Repertoires to Non-HLA Antigens in Kidney Transplant Recipients • Peter Heeger, USA LUNCH, POSTERS & EXHIBITS (FOYER) PROTEINS AND METABOLITES IN TRANSPLANTATION Chair: Elaine Reed

13:30 14:00 14:20 14:40 15:00

Population Proteomics and Protein Biomarker Discovery • Richard Smith, USA Metabolomics in Monitoring Organ Transplant • David Wishart, USA Metabolomics in Renal Transplantation • David Rush, Canada HLA Diversity in Transplantation • Steven G.E. Marsh, UK NETWORKING COFFEE BREAK, POSTERS & EXHIBITS (FOYER) NOVEL APPLICATIONS OF OMICS Session is sponsored through an unrestricted educational grant by Novartis. Chair: Minnie Sarwal

15:30 16:00 16:30 17:00

A Method to Systematically Identify Cell-Type-Specific Differential Gene Expression from Complex Tissues • Robert Tibshirani, USA Molecular Imaging in Transplantation • Caius Radu, USA Use of Nanoparticles for Augmenting Mucosal Immunity • Kathleen Kelly, USA CONFERENCE WRAP-UP Chair: Jeremy Chapman

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THURSDAY • FEB 25

DETAILED PROGRAM

08:00-12:00 NOVEL APPLICATIONS FOR GENOMIC TECHNOLOGIES IN ORGAN TRANSPLANT INJURY
Session is sponsored through an unrestricted educational grant by Pfizer. Co-chairs: Jeremy Chapman, Westmead, Australia Kathryn Wood, Oxford, UK 08:00 OPENING MESSAGE Jeremy Chapman, President, The Transplantation Society ADVANCES IN GENOME ANALYSIS TECHNOLOGIES Ronald W. Davis, Professor of Biochemistry and Professor of Genetics, Member of the Stanford Comprehensive Cancer Center, Stanford University, Stanford, CA, USA New biological and engineering technology are advancing very fast. They will have a major impact on diagnostic medicine. Learning Objectives: 1. Biochemical/Genetic assays can be multiplexed where the cost of millions of assays is the same as one current assay. 2. Direct DNA sequencing of predetermined Targeted Regions (exons and control regions) using new DNA sequencing Instruments currently under development will soon become cheaper and much faster. 3. New engineering/biology interfaces will allow many new point of use real time assays to be developed at low cost.

08:10

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

DETAILED PROGRAM
08:40

THURSDAY • FEB 25

RECLASSIFYING GRAFT INJURY BY GENOMICS Philip F. Halloran, Director, Alberta Transplant Applied Genomics Centre; Distinguished University Professor, University of Alberta; Editor-in-Chief, American Journal of Transplantation; Edmonton, AB, Canada Learning Objectives: 1. To understand how the molecular features of a biopsy add to the other features such as anti HLA, clinical state, biopsy histopathology, in predicting diagnosis and outcomes. 2. To examine the general implications of these findings for understanding disease mechanisms. 3. To discuss the platforms by which this new knowledge can be made available to clinicians. A new global view of the molecular changes in kidney transplant biopsies is emerging from data driven approaches. This approach uses iterative loops to define the relationship of molecular features to the other phenotypes: clinical (function, proteinuria), HLA antibody, biopsy histology, and outcomes in approximately 650 kidneys with microarray results. Analysis incorporates annotation in mouse models, class comparison and classifier approaches, principal component analysis. The troubled kidneys display striking stereotyping of changes reflecting key events: T cell and macrophage infiltration, IFNG effects, active injury response and dedifferentiation, and time dependent cumulative burden of injury changes. The Other diseases can then be defined by relative shifts in the molecular features of this stereotyped disturbance. Specificity for T cell mediated rejection is captured by selective changes in T cell and macrophage genes and alternative macrophage activation, whereas specific for antibody-mediated rejection is derived from endothelial and NK cell transcripts. The injury response is the best correlate of both functional disturbance and probability of graft loss. Time dependent changes in injured kidneys include B cell, plasma cell, and mast cell transcripts, but these have little impact in multivariate analysis when time is factored in, since late biopsies have an intrinsic high risk due to the diseases operating. Transcripts of potential diagnostic utility for ABMR include those associated with DSA in biopsies for cause. The results indicate that: 1. TCMR is an intense inflammatory process with an excellent prognosis when ABMR is absent. 2. ABMR is a disease of endothelium and NK cells that is often C4d negative and not diagnosed. 3. The strong reflection of progression is the ongoing injury response, driven by disease processes that are unresponsive to therapy. To progress, one must have a potentially progressive disease, but the true predictor of progression is the injury response.

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THURSDAY • FEB 25
09:00

DETAILED PROGRAM

DECONVOLUTING THE PERIPHERAL BLOOD TRANSCRIPTOME IN GRAFT REJECTION Minnie Sarwal, Professor of Pediatrics and Immunology, Stanford University, Stanford, CA, USA Learning Objectives: 1. To discuss why the transplant field needs non-invasive means to predict for alloimmune injury to customize immunosuppression. 2. Microarrays as a means to discover new biomarkers for acute rejection. 3. The process of biomarker validation and cross-validation to develop a sensitive and specific predictor for graft rejection. Early detection of acute renal allograft rejection (AR) remains a major clinical concern and unmet need in organ transplantation. Current blood and biofluid non-invasive screening and monitoring methods cannot detect early and subclinical graft injury. The clinical current practice only allows for the screening of established tissue rejection by the detection of inflammatory infiltrates on kidney biopsy samples, despite the fact that this methodology is limited by sampling variability, procedural morbidity and cost. The optimal monitoring approach in organ transplantation would be to noninvasively evaluate the risk for graft rejection before the onset of renal dysfunction such that immunosuppression could be proactively titrated to limit graft injury, and immunosuppression delivery could be customized to the patients’ immunosuppression threshold and thus limit patient morbidity from infectious and malignant complications. Transcriptional profiling studies on biopsy specimens have confirmed that significant, coordinated expression changes occur in many genes with established AR. However, when these studies are applied to peripheral blood, the expression of the rejection response is substantially weaker than the corresponding response in the organ. The gene signature for AR is also influenced by biological and experimental variance, resulting in low signal to noise ratio. This talk discusses the issues in biomarker discovery in blood relating to AR and the results of a recent study that has identified a highly specific and sensitive gene-based biomarker panel in blood that can predict the onset of AR, well before any injury can be detected by organ dysfunction or graft biopsy. This approach provides a skeleton for further studies for biomarker discovery of disease phenotypes in organ transplantation.

09:20

NONINVASIVE DIAGNOSIS OF RENAL ALLOGRAFT STATUS Manikkam Suthanthiran, Stanton Griffis Distinguished Professor of Medicine; Chief, Nephrology and Transplantation Medicine; New York Presbyterian Hospital-Cornell, New York, NY, USA Development of predictive, diagnostic and prognostic biomarkers of allograft status and outcome is important and challenging, and may be rewarded with individualized therapy of the organ graft recipient.

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

DETAILED PROGRAM

THURSDAY • FEB 25

Learning Objectives: 1. To improve understanding of allograft rejection. 2. To improve understanding of molecular markers of allograft rejection. 3. To improve understanding of biomarkers of allograft status. In order to noninvasively characterize renal allograft status and outcome we performed urinary cell mRNA profiling studies and report that: (a) Urinary cell levels of mRNA for perforin and granzyme B are significantly higher in renal allograft recipients with a biopsy confirmed episode of acute rejection than in the patients without an episode of acute rejection (Li et al. NEJM 2001); (b) CD103 mRNA levels are higher in urinary cells from renal allograft recipients with a biopsy confirmed episode of acute rejection than in the patients without acute rejection (Ding et al. Transplantation 2003); (c) Both IP-10 and CxCR3 mRNA levels are highly associated with acute rejection (Tatapudi et al. Kidney International 2004); (d) Whereas mRNA for FOxP3, CD25, CD3ε-chain and perforin are all higher during an episode of acute rejection than in the group with CAN or normal biopsy group, levels of mRNA for FOxP3 alone: (1) inversely correlate with serum creatinine levels measured at the time of biopsy in the acute rejection group; (2) predict the reversal of acute rejection, and (3) identify subjects at risk for graft failure within six months after the incident episode of acute rejection (Muthukumar et al. NEJM 2005). We subscribe to the definition proposed by the NIH Biomarker Definition Working Group: “A characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic responses, or pharmacological responses to a therapeutic intervention”. In this light, mRNA profiles, measured at the time of allograft biopsy and predicting acute rejection with a high specificity and sensitivity serve as a diagnostic biomarker; mRNA levels, measured during an episode of acute rejection and predicting responsiveness to anti-rejection therapy serve as a prognostic biomarker. The ongoing NIH sponsored Cooperative Clinical Trials in Transplantation should validate or refute the hypothesis that urinary cell mRNA profiles function as predictive biomarkers (mRNA levels in sequential samples will predict the subsequent development of acute rejection or maintenance of stable graft function) and noninvasive diagnostic biomarkers (mRNA levels measured at the time of diagnostic allograft biopsies will predict allograft histopathology) of acute rejection of renal allografts. These trials are also designed to test the hypothesis that sequential urinary cell mRNA profiles predict renal allograft status (stable graft function vs. acute rejection) and function. Should future studies demonstrate that the measured biomarker is causatively involved in the biologic process studied (e.g., acute rejection), the biomarker can be designated as a mechanistic biomarker, and help accomplish the objective of developing mechanism based therapy.

Transplantomics and Biomarkers in Organ Transplantation

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THURSDAY • FEB 25
09:40

DETAILED PROGRAM

ExPLORING GENOMIC MEDICINE USING TRANSLATIONAL BIOINFORMATICS Atul Butte, Assistant Professor, Medicine, Stanford Medical Informatics, Stanford University, Stanford, CA, USA Learning Objectives: 1. How publicly-available molecular measurements can be used to probe disease mechanisms. 2. How RNA is the new proteomics. 3. How bioinformatics has moved from a service-provider role to a science role. With the end of the United States NIH budget doubling and completion of the Human Genome Project, there is a need to translate genome-era discoveries into clinical utility.  The difficulties in making bench-to-bedside translations have been described: comprehensive molecular studies on patients are expensive, and hospitals are not phenotypers.  The nascent field of translational bioinformatics may help.   I will show how we build and apply tools that convert the billions of points of molecular, clinical, and epidemiological data measured by biomedical investigators and clinicians over the past decade into insights into diagnostic and therapeutic potential.  I will highlight how using publicly-available molecular data enables the discovery of new gene variants and biomarkers for diseases like transplantation rejection and diabetes, suggests novel roles for drugs in the treatment of disease, and for the first time allows us to probe the inner commonality across disease. 

10:00 NETWORKING COFFEE BREAK, POSTERS & EXHIBITS (FOYER)

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

DETAILED PROGRAM

THURSDAY • FEB 25

10:30-12:00 NOVEL APPLICATIONS FOR GENOMIC TECHNOLOGIES IN ORGAN TRANSPLANT INJURY (continued)
Co-chairs: Philip F. Halloran, Edmonton, AB, Canada Manikkam Suthanthiran, New York, NY, USA

10:30

IMMUNE-MONITORING IN HEALTH AND INFECTION Mark Davis, Professor, Microbiology & Immunology, Stanford University, Stanford, CA, USA Learning Objectives: 1. Why has the translation of basic immunology into the clinic been so slow? 2. What are some possible solutions to this problem? 3. What might this mean with respect to transplantation? There has been an explosive growth in our understanding of basic immunological mechanisms, cell types and functions over the past fifty years, but the application of this knowledge to human health has ranged from "minute" to "undetectable". There are many reasons for this, including the diversity of human beings, likely differences between mice and humans, and problems with the experimental design of animal models of disease. The key question though, is how to move forward, and at Stanford we have launched a series of initiatives designed to fill in critical gaps in our understanding of human immunology and establish metrics of immunological health. With such metrics we can begin to systematically diagnose correctly functioning immune systems from aberrant ones and develop corrective therapies for the later. We might also be able to predict who will reject an organ with standard therapy versus who will not. We also see this as a way to develop a systems biology approach to analyzing the immune system, which could reveal a wealth of new insights into how this complex network of cells and molecules organizes itself and responds to both pathological and non-pathological microorganisms.

Transplantomics and Biomarkers in Organ Transplantation

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THURSDAY • FEB 25
11:00

DETAILED PROGRAM

THE EVOLUTION OF NON-IMMUNE HISTOLOGICAL INJURY Maarten Naesens, Department of Nephrology and Renal Transplantation, University Hospitals Leuven, Leuven, Belgium; Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA Learning Objectives: 1. Progression of histological injury of a kidney allograft is associated with important gene expression alterations over time, and future histological phenotypic changes are preceded by changes on the molecular level. 2. The variability of the clinical background and the complexity of the histological picture need to be taken into account when patient samples are used for transcriptomic analysis. 3. Even in the absence of acute rejection, it becomes clear that the innate and adaptive immune systems play an important role in the evolution of histological injury over time after renal transplantation. In renal transplantation, slowly progressive chronic tubulo-interstitial damage jeopardizes long-term renal allograft survival. Both immune and non-immune mechanisms contribute to this progressive renal allograft scarring, as is demonstrated in protocol biopsy studies. Until now the specific tools to prevent this progression of chronic histological damage have not yet been identified. Micro-array technology can be applied on biopsy samples in order to get a better insight in these molecular changes and pathways, and could yield promising targets for timely intervention. In this presentation, protocol biopsy results are used to demonstrate the pathways involved in the evolution of histological injury over time. Previous and recent micro-array data are reviewed that shed light on the risk factors and mechanisms of chronic histological damage. As is demonstrated in these studies, it has to be emphasized that great care should be given to careful patient selection, unbiased and detailed histological scoring and strict sample classification. The complexity of the histological picture and the important clinical variability has to be taken into account when transcriptomic data are analyzed in clinical samples. The complexity of the clinical parameters, the complex physiology and immunology, the complex histological outcome, the multitude of data per sample and the complex data analysis can only lead to biologically and clinically meaningful results with a multidisciplinary, holistic approach. Finally, it is becoming clear that immune and non-immune factors are impossible to separate from each other after transplantation, and many non-immune phenomena also lead to immune activation. Immune activation is already evident in biopsies of deceased donor kidneys prior to implantation compared to living donor kidneys. When rejection-free renal allograft biopsies obtained posttransplantation are compared to pre-implantation samples, there is also a major shift in global gene

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

DETAILED PROGRAM

THURSDAY • FEB 25

expression, with highly significant overrepresentation of immune genes involved in mostly adaptive immune responses. In post-transplantation samples of patients who did not experience delayed graft function, clinical or subclinical rejection episodes, there is a highly significant association of established, ongoing and most importantly, future chronic histological damage with regulation of adaptive immunity but also innate immune response genes. These findings underscore the complexity of the immunological processes in human kidney transplantation, and corroborate the idea that inflammation that is quantitatively below the diagnostic threshold of acute T-cell mediated rejection is involved in early subclinical stages of progressive renal allograft damage. Timely intervention aimed at influencing these early immune responses could well be the clue to slow or abort the progression of chronic renal graft scarring and improve long-term graft survival.

Transplantomics and Biomarkers in Organ Transplantation

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THURSDAY • FEB 25
11:20

DETAILED PROGRAM

THE GENOMICS OF FIBROSIS PROGRESSION Valeria Mas, Associate Professor, Departments of Surgery and Pathology; Director, Molecular Transplant Research Laboratory, Division of Transplant, Virginia Commonwealth University, Richmond, VA, USA Learning Objectives: 1. Understand the molecular events involved in fibrogenesis in allograft response to chronic injury. 2. Evaluate the utility of early systematic molecular signatures study of protocol biopsy tissues to identify those patients at high risk of allograft fibrosis progression. 3. Analyze the critical importance of prospective studies with appropriate sample size for power of calculation in biomarker discover of fibrogenesis post-transplantation. Fibrosis is the replacement of normal tissue by scar tissue as consequence of a reactive or reparative process called fibrogenesis. Stimuli to injury may include cell necrosis, apoptosis, inflammatory cell infiltration, and extra cellular matrix (ECM) alterations. The response to injury triggers the production of cytokines and other extracellular signals, including reactive oxygen species. These stimuli induce a fibrogenic response, resulting in an accumulation of ECM proteins within the organ as consequence of an imbalance between the deposition and degradation of ECM components. Although self-reliant scar tissue has no effect on long-term outcomes, fibrogenesis will result in organ failure if injury persists or if response to injury is excessive. Protocol biopsies have played an important role demonstrating that fibrosis occurs before graft dysfunction is present. Transcriptional changes may be detectable prior to histological apparent fibrosis, and discrimination of inflammatory infiltrates according to the group of expressed genes, promises to both improve diagnoses and optimize treatment strategies. Systematic analysis of gene-expression patterns will provide a window into the biology and pathogenesis of allograft fibrosis development. Innovative data of early detection of molecular patterns associated with fibrosis graft development in early protocol biopsies will be presented. Gene expression analysis results will be presented for two different conditions (Loss of graft function with interstitial fibrosis (IF) and tubular atrophy (TA) in kidney transplant recipients (KTR) and HCV recurrence post-liver transplantation (LT)). For loss of graft function with IF/TA, gene expression (microarrays) in protocol biopsies was performed. Whether the pre-existing histological changes in the time zero biopsies were involved in post-transplant IF/TA progression was considered in the analysis. For HCV recurrence post-LT, formalin fixed embedded tissues (FFPE) protocol biopsies were evaluated using Whole Genome DASL Assay (Illumina). Prediction modeling systems were established combining molecular and clinical data for predicting risk of allograft fibrosis development.

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

DETAILED PROGRAM
11:40

THURSDAY • FEB 25

RNA SILENCING IN GENE REGULATION Christopher Contag, Co-Director, Molecular Imaging Program at Stanford, Associate Professor, Department of Pediatrics and of Microbiology and Immunology, Stanford University, Stanford, CA, USA Learning Objectives: 1. Participants should learn the strengths and limitations of the various imaging modalities used to study transplantation biology. 2. Participants should learn about optical imaging and the opportunities for advancing transplantation biology. 3. The molecular basis of how several mediators of successful tissue and cell engraftment enable long term survival will be identified and the participants should understand the basic mechanisms. Tissue and cell transplantation hold tremendous potential for the development of new therapeutic strategies, however the nature of these tissues and cells that make them so valuable, also makes them very difficult to study. The development of imaging tools that enable visualization of the fates and function or transplants will accelerate and refine studies in transplantation biology. In stem cell therapies the aim is to transfer relatively small numbers of undifferentiated cells that then sense the tissue environment and respond with a directed proliferation into a large number of cells dedicated to a specific function. Therefore to study these processes effectively, tools need to be used that have a broad dynamic range to initially detect small numbers of cells in vivo and then monitor the tremendous cellular expansion and differentiation associated with tissue restoration. This requires the use of molecular markers that are linked to cellular metabolism, are replicated during the cellular proliferation such that the signals are not diluted or lost, and that are stable during the differentiation process with tolerance to significant changes in cell physiology. These signals need to be detected externally and monitored temporally. The emerging technologies in the field of molecular imaging have been used for these purposes and are providing new insights into stem cell and transplantation biology.

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THURSDAY • FEB 25
12:00 LUNCH, POSTERS & EXHIBITS (FOYER) 13:30-16:00

DETAILED PROGRAM

PREDICTING GRAFT RISK BY TRANSPLANTOMICS
Co-chairs: Philip O’Connell, Westmead, Australia Thomas Mueller, Edmonton, AB, Canada Session is sponsored through an unrestricted education grant from Bristol-Myers Squibb.

13:30

THE IMPACT OF GENOME-WIDE VARIATION IN RENAL TRANSPLANT DONORS AND RECIPIENTS ON GRAFT SURVIVAL Graham Lord, Professor of Medicine, Nephrology, Transplantation and Internal Medicine; Director of Translational Research Development and Deputy Director of NIHR Comprehensive Biomedical Research Centre; Guy’s and St. Thomas’ Hospital and King’s College London, London, UK Learning Objectives: 1. Understand the theory of genome-wide association scans. 2. Understand ways of analyzing the interaction of two genomes in a single patient. 3. Learn what is known about the genetics of allograft dysfunction. Genetic interactions between donor and recipient genomes in the context of renal transplantation determine early and late renal allograft dysfunction and subsequent transplant failure. Renal transplant failure is largely genetically determined and the majority of the donor and recipient genes that cause allografts to fail are unknown. Genome wide association scans in this patient population will help us to define the genetic variation in donor and recipient genomes that determine long term renal allograft outcome and the variation in donor and recipient genomes that determine short term renal allograft dysfunction. Furthermore, we are able to discover variation in the recipient genome that correlates with end-stage renal failure. Resolving these scientific questions will rapidly enable early translational studies of organ allocation and graft monitoring with a realistic prospect of improving patient outcomes within a relatively short timeframe.

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

DETAILED PROGRAM
14:00

THURSDAY • FEB 25

CHALLENGES IN ASSOCIATION STUDIES OF SINGLE NUCLEOTIDE POLYMORPHISM ASSOCIATED AND RENAL TRANSPLANT OUTCOMES Ajay Israni, Assistant Professor of Medicine, Adjunct Professor of Epidemiology & Community Health, Hennepin County Medical Center, University of Minnesota, Minneapolis, MN, USA Learning Objectives: 1. To understand issues of study design related to single nucleotide polymorphisms (SNPs) in transplantation. 2. To understand challenges of analyzing outcomes data in genetic epidemiology studies. 3. Consider the future direction for studies of genomic variation in transplantation. The Genomics of Transplantation is a NIH funded genetic ancillary to an ongoing prospective cohort of renal transplant recipients. This ongoing, international, multi-center cohort is Deterioration of Kidney Allograft Function (DeKAF) study (PI, Arthur Matas). We will discuss the issues relevant to the design of our Genomics of Transplantation study. Our study includes 2 projects, genetic epidemiology project (Project Leader, Ajay Israni) and pharmacogenomics (Project Leader, Pamala Jacobson). All study subjects are prospectively enrolled, kidney transplant recipients and their living donors, at the 6 study sites of DeKAF. Target enrollment is 4,000 recipients in the test and validation cohorts. Sites are: University of Minnesota and Hennepin County Medical Center, Minneapolis, MN; University of Alberta, Edmonton, Canada; University of Iowa, Iowa City, IA; University of Alabama, Birmingham, AL; and the Mayo Clinic, Rochester, MN. Genotyping will employ a custom Affymetrix chip with 3,500 functional single nucleotide polymorphisms (SNPs) in the Genotyping Core (Core Leader, William Oetting). The genetic epidemiology project will study the relationship SNPs of recipient and donor genes with chronic allograft dysfunction (CGD) and estimated glomerular filtration rate (eGFR). The project will also compare the frequency of SNPs that are associated with CGD and eGFR among African Americans (AA) and non-AA, given the increased risk of allograft loss among AA. The pharmacogenomics project will test the hypothesis that SNPs of drug metabolizing enzymes, transporters, drug targets and biological pathways are associated with immunosuppressant exposure, adverse effects and clinical outcomes. The results from the test cohort of both projects will be discussed.

Transplantomics and Biomarkers in Organ Transplantation

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THURSDAY • FEB 25
14:20

DETAILED PROGRAM

MICRORNA AND RNA PROFILES WITH GRAFT REJECTION Dany Anglicheau, Associate Professor, Kidney Transplant Unit, Necker Hospital, Paris, France Learning Objectives: 1. Learn a framework for developing microRNA as new biomarkers of kidney allograft outcome. 2. Discuss the implication of miRNAs as new biomarkers of allograft rejection. 3. Gain an understanding of the application of urinary cell mRNA levels as markers of IFTA and graft outcome. The overall objective of this talk is to present new biomarkers of acute T-cell mediated rejection of the kidney allograft and of interstitial fibrosis/tubular atrophy, the hallmark of chronic allograft nephropathy, by the analysis of microRNA (miRNA) or messenger RNA (mRNA) profiles in biopsy samples or urinary cells. We have identified a molecular signature of acute rejection based on a high throughput miRNA quantification of kidney allografts. Our investigation identified a subset of 17 miRNAs that are differentially expressed in acute rejection biopsies compared to normal allograft biopsies at a Pvalue < 0.01, and the presence or absence of acute rejection could be accurately predicted using miRNA expression patterns. The identification of differentially expressed miRNAs during an episode of acute rejection could lead to the development of new non-invasive biomarkers. Through the analysis of mRNA expression levels in urinary cells, we have recently developed new hypothesis-driven mRNA biomarkers that can noninvasively diagnose interstitial fibrosis/tubular atrophy. In addition, a combination of mRNAs involved in epithelial-to-mesenchymal transition/fibrogenesis and alloimmune response appeared predictive of the subsequent function of kidney allograft with normal histology.

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

DETAILED PROGRAM
14:40

THURSDAY • FEB 25

PREDICTORS OF TRANSPLANT VASCULOPATHY AND GLOMERULOPATHY Roslyn B. Mannon, Director of Research, Alabama Transplant Center; Professor, Department of Medicine, Division of Nephrology; Department of Surgery, Division of Transplantation, University of Alabama at Birmingham, Birmingham, AL, USA Learning Objectives: 1. To understand the methodology utilizing low density real time PCR arrays. 2. To recognize the potential factors mediating transplant glomerulopathy. 3. To identify novel strategies to diagnose and monitor for late kidney allograft injury. Long-term kidney allograft survival continues to improve modestly, despite dramatic improvements in acute rejection rates and short term patient and graft survivals (1). Identification of biomarkers of allograft failure and the development of tools for their interpretation is of critical interest, both in providing disease detection in a more sensitive and specific fashion, and in allowing sufficient lead time for intervention. Additionally, such markers may allow for risk assessment and medical-regimen tailoring that is personalized to provide optimum outcomes. Transplant glomerulopathy (TG) is a disease of the kidney allograft initiated by endothelial injury. Morphologically, there is widening of the subendothelial space with accumulation of debris, mesangial interpositioning and matrix deposition in the glomerular capillary wall, and capillary wall double-contouring in the absence of immune complex deposition (2). The etiology of TG is under considerable scrutiny. Prior studies implicated an antibody mediated response (3-5), but this has not been consistently demonstrated (6-7). Accompanying this lesion may be evidence of chronic injury, including interstitial fibrosis and tubular atrophy (IF/TA), the hallmarks of chronic allograft nephropathy (8). Clinical presentation often occurs a year or more after transplantation, although in the context of protocol kidney biopsies, light microscopic changes may be seen earlier, with associated proteinuria, hypertension, and a progressive decline in function culminating in graft loss (9). Importantly, there is no specific effective therapeutic strategy beyond augmentation of immunosuppression. Thus, identifying pathogenic mediators not only for therapeutic purposes but also for early identification may lead to improved outcomes. We have assessed the potential of a novel diagnostic method utilizing custom low density gene expression arrays and machine learning algorithms in an effort to determine the transcriptional features associated with TG and to begin to identify biomarkers that may be indicative of TG. Initial data analysis using conventional statistical methods confirms the pro-inflammatory state of this lesion. Incorporation of these data utilizing machine-learning software, however, has derived statistically significant yet substantially novel associations between individual transcripts. Moreover,

Transplantomics and Biomarkers in Organ Transplantation

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THURSDAY • FEB 25

DETAILED PROGRAM

the resulting model provides insight into the probable pathogenesis of TG and a set of potential biomarkers to test and characterize recipients at risk for disease. These results highlight the hypothesis-generating potential of this method by elucidating potential pathways for investigation and the decision-supportive utility of defined, quantitative classification models of disease versus health states. References:
1. Meier-Kriesche HU, Schold JD, Srinivas TR, Kaplan B: Lack of improvement in renal allograft survival despite a marked decrease in acute rejection rates over the most recent era, Am J Transplant 2004, 4:378-383 2. Racusen L: Chronic transplant glomerulopathy: need for further assessment, Clin J Am Soc Nephrol 2007, 2:1108-1109 3. Mauiyyedi S, Pelle PD, Saidman S, Collins AB, Pascual M, Tolkoff-Rubin NE, Williams WW, Cosimi AA, Schneeberger EE, Colvin RB: Chronic humoral rejection: identification of antibody-mediated chronic renal allograft rejection by C4d deposits in peritubular capillaries, J Am Soc Nephrol 2001, 12:574-582 4. Regele H, Bohmig GA, Habicht A, Gollowitzer D, Schillinger M, Rockenschaub S, Watschinger B, Kerjaschki D, Exner M: Capillary deposition of complement split product C4d in renal allografts is associated with basement membrane injury in peritubular and glomerular capillaries: a contribution of humoral immunity to chronic allograft rejection, J Am Soc Nephrol 2002, 13:2371-2380 5. Sis B, Campbell PM, Mueller T, Hunter C, Cockfield SM, Cruz J, Meng C, Wishart D, Solez K, Halloran PF: Transplant glomerulopathy, late antibody-mediated rejection and the ABCD tetrad in kidney allograft biopsies for cause, Am J Transplant 2007, 7:1743-1752 6. Akalin E, Dinavahi R, Dikman S, de Boccardo G, Friedlander R, Schroppel B, Sehgal V, Bromberg JS, Heeger P, Murphy B: Transplant glomerulopathy may occur in the absence of donor-specific antibody and C4d staining, Clin J Am Soc Nephrol 2007, 2:1261-1267 7. Al Aly Z, Yalamanchili P, Cortese C, Salinas-Madrigal L, Bastani B: C4d peritubular capillary staining in chronic allograft nephropathy and transplant glomerulopathy: an uncommon finding, Transpl Int 2005, 18:800-805 8. Gloor JM, Sethi S, Stegall MD, Park WD, Moore SB, DeGoey S, Griffin MD, Larson TS, Cosio FG: Transplant glomerulopathy: subclinical incidence and association with alloantibody, Am J Transplant 2007, 7:2124-2132 9. Cosio FG, Gloor JM, Sethi S, Stegall MD: Transplant glomerulopathy, Am J Transplant 2008, 8:492-496

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

DETAILED PROGRAM
15:00

THURSDAY • FEB 25

A SIGNATURE FOR OPERATIONAL LIVER TRANSPLANT TOLERANCE Alberto Sanchez-Fueyo, Hospital Clinic Barcelona, University of Barcelona, Barcelona, Spain Learning Objectives: 1. Operationally tolerant liver recipients exhibit characteristic expression patterns in peripheral blood. 2. Innate type immune cells appear to be involved in the maintenance of the tolerant state in human liver recipients. 3. Transcriptional profiles from tolerant liver and kidney recipients show minimal overlap. A fraction of liver transplant recipients can discontinue all immunosuppressive therapies without undergoing rejection (operational tolerance). However, accurate identification of these recipients remains a challenge. We have employed both Affymetrix microarrays and real-time PCR technologies to explore in peripheral blood samples the differential gene expression between tolerant recipients and those requiring indefinite immunosuppressive therapy, and ultimately to design a clinically applicable molecular test of tolerance in liver transplantation. To do so we have studied peripheral blood transcriptional patterns from 80 liver transplant recipients in whom a previous attempt at immunosuppression withdrawal had been attempted, and from 96 recipients enrolled in a prospective trial of immunosuppression withdrawal. This has resulted in the discovery and validation of several gene signatures comprising a modest number of genes capable of identifying tolerant and non-tolerant recipients with high accuracy. Multiple peripheral blood lymphocyte subsets and functional pathways contribute to the tolerance-associated transcriptional patterns, but NK-related genes appear to exert a predominant influence. These patterns substantially differ from the expression profile identified in tolerant kidney recipients. We conclude that transcriptional profiling of peripheral blood can be employed to identify liver transplant recipients who can discontinue immunosuppressive therapy and that innate immune cells are likely to play a major role in the maintenance of operationally tolerance in liver transplantation.

Transplantomics and Biomarkers in Organ Transplantation

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THURSDAY • FEB 25
15:20

DETAILED PROGRAM

CAN IMMUNE ACCOMMODATION BE PREDICTED USING “-OMICS” TOOLS? Bruce McManus, Professor, Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada Learning Objectives: 1. To convey the overall discovery rationale and strategies in the Biomarkers in Transplantation initiative within the PROOF Centre of Excellence. 2. To share current data and early insights regarding the ability to predict rejection or its absence in kidney and heart allograft recipients. 3. To foster discussion and collaboration in regards to prediction and the use of “-Omic” tools. Introduction: Organ transplantation remains the main treatment for end-stage heart and kidney failure. The Biomarkers in Transplantation (BiT) team and the PROOF Centre of Excellence have undertaken an unbiased discovery strategy to identify and ultimately validate biomarker panels diagnostic and predictive of immune rejection in prospectively enrolled heart and kidney failure patients receiving transplanted allografts. Utilizing high-performance genomics, proteomics and metabolomics platforms, the BiT team has internally validated peripheral blood-derived diagnostic biomarker panels of acute and chronic rejection. These panels are currently undergoing external validation and qualification in a Canada-wide trial. Predictive Biomarkers: The challenges of precision, accuracy, timeliness and invasiveness of current patient monitoring approaches post-transplant provide significant impetus to a search for “-Omics” markers for prediction of those at risk of immune rejection. Indeed, a significant goal in transplant immunology remains the prediction and prevention of organ rejection as opposed to the diagnosis and treatment. The realization that humans are immunologically distinct, dating back to discoveries of ABO blood groups and MHC antigens, has led to extensive research directed towards predicting and preventing organ rejection. The application of “-Omics” technologies to the task of predicting immune accommodation, tolerance or rejection is a natural extension of these efforts. Kidney Transplantation: Biomarkers of early graft accommodation would offer an important option for post-transplant monitoring and permit timely and effective therapeutic intervention. The BiT team investigated longitudinal PAXgene-based gene expression patterns in peripheral blood of quiescent kidney transplant recipients to better understand early phases of accommodation. We have generated a comprehensive signature of immediate gene expression changes in whole blood after renal transplantation reflecting a variety of immune and inflammatory processes, as well as regenerative processes occurring after the transplantation procedure. Monitoring longitudinal changes in biological processes as reflected in peripheral gene expression may provide insights into the mechanisms of immunological accommodation, and thus facilitate personalization of immunosuppressive regimens.

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

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THURSDAY • FEB 25

Heart Transplantation: End-stage heart failure is characterized by a peripheral blood molecular signature reflective of the homogeneity of the final common pathways of failure. Reversion of the levels of these markers post-transplant to baseline may be reflective of “normalized” graft performance and could impact patient outcomes. The BiT team analyzed gene expression profiles of a subset of patients, demonstrating that approximately one third of end-stage genomic markers revert to normal levels post-transplant. These molecular signatures may give insight into both the pathogenesis of heart failure and the processes of tolerance and accommodation occurring following transplantation. Conclusion: Limitations of current diagnostic and predictive approaches has led many in transplantation science to assess “-Omics” platforms for more sensitive and specific, clinically helpful biomarkers. While the notion that graft integrity post-transplant may be predicted pre-surgery has existed for many years, current approaches may bring the field closer to this yet elusive goal.

15:40 NETWORKING COFFEE BREAK, POSTERS & EXHIBITS (FOYER)

Transplantomics and Biomarkers in Organ Transplantation

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THURSDAY • FEB 25
16:00 MINI-ORAL PRESENTATIONS
Co-chairs: Allan D. Kirk, Atlanta, GA, USA Roslyn B. Mannon, Birmingham, AL, USA

DETAILED PROGRAM

This session has one combined Q&A period at the end of the session. Delegates are encouraged to visit the presenters at their posters during the Poster Reception (immediately after this session). Abstracts are located on pages 51 to 68. 16:00 P-01 – EARLY EVENTS IN THE ALLOGRAFT IDENTIFY MARKERS THAT PREDISPOSE AND INITIATE THE DAMAGE INVOLVED IN THE PROGRESSION TO INTERSTITIAL FIBROSIS (IF) AND TUBULAR ATROPHY (TA) Daniel Maluf, Kellie Archer, Mariano Scian, Anne King, Davis Massey, Benjamin Whitehill, Marc Posner, Valeria Mas. Hume-Lee Transplant Center, Virginia Commonwealth University, Richmond, VA, USA. P-09 – INNATE AND ADAPTIVE IMMUNE GENE ExPRESSION DYNAMICS AND PROGRESSION OF CHRONIC HISTOLOGICAL DAMAGE OF RENAL ALLOGRAFTS Maarten Naesens, Li Li, Tara Sigdel, Purvesh Khatri, Neeraja Kambham, Oscar Salvatierra, Minnie Sarwal. Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA. P-08 – ExPRESSION SIGNATURES OF ExTRACELLULAR MATRIx RELATED TRANSCRIPT SETS LINKED RENAL ALLOGRAFT INTERSTITIAL FIBROSIS/TUBULAR ATROPHY WITH ACUTE TCELL MEDIATED REJECTION Silke Rödder1, Andreas Scherer2, Hans-Peter Marti1. 1University of Bern, Inselspital Bern, Department of Nephrology and Hypertension, Switzerland; 2Spheromics, Kontiolahti, Finland. P-18 – ASSESSMENT OF KIDNEY ORGAN QUALITY AND OUTCOME USING THE TRANSCRIPTOME OF THE IMPLANT BIOPSY Thomas Mueller1, Motaz Obeidat1, Gordon Broderick1, Wenjie Wang2, Phillip Halloran1, Valerie Luyckx1. 1Division of Nephrology and Immunology, Department of Medicine, University of Alberta, Edmonton, AB, Canada; 2Division of Nephrology, Department of Medicine, University of Calgary, Calgary, AB, Canada. P-05 – A PERIPHERAL BLOOD 12 GENE-SET FOR DIAGNOSIS OF PEDIATRIC LIVER ALLOGRAFT TOLERANCE Li Li, Anita Talisetti, Sue Hsieh, Ken Cox, Carlos Esquivel, Waldo Concepcion, Minnie Sarwal. Pediatic Department, Stanford University, Stanford, CA, USA.

16:05

16:10

16:15

16:20

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

DETAILED PROGRAM
16:25

THURSDAY • FEB 25

P-14 – THE EUROPEAN TOLERANCE INVESTIGATION PLATFORM: A UNIFIED RESOURCE FOR TOLERANCE DATA MINING Patrick Miqueu1, Coline Thomas2, Hilke Schmidts3, Olivier Rivain2, Michel Goldman4, Lucienne Chatenoud1, Kathryn Wood5, Hans-Dieter Volk3. 1INSERM U580, Hôpital Necker-Enfants Malades, Université Paris Descartes, Paris, France; 2EA 4275, Faculty of Pharmaceutical Sciences, University of Nantes, Nantes, France; 3Institute for Medical Immunology and BerlinBrandenburg Center for Regenerative Therapies, Charité University Medicine, Berlin, Germany; 4Institute for Medical Immunology, Université Libre de Bruxelles, Brussels, Belgium; 5 Transplantation Research Immunology Group, Nuffield Department of Surgery, University of Oxford, Oxford, UK. P-16 – META-ANALYSIS OF SOLID ORGAN TRANSPLANT DATA SETS IDENTIFIES DIFFERENTIALLY ExPRESSED MIRNAS COMMON IN HEART, KIDNEY AND LUNG ALLOGRAFTS Purvesh Khatri, Richard Hayden Jones, Atul Butte, Minnie Sarwal. Department of Pediatrics, School of Medicine, Stanford University, Stanford, CA, USA. P-19 – ExPERIMENTAL DESIGN AND QUALITY ASSURANCE OF HIGH-THROUGHPUT CLINICAL TRANSPLANTOMICS STUDIES: A CRUCIAL STEP TOWARD ROBUST BIOMARKER DISCOVERY Zhong Gao1, Adam Asare1, Vicki Seyfert-Margolis1, Deborah Phippard1, Vincent Carey2. 1 Immune Tolerance Network / UCSF, Tolerance Assays and Data Analysis, Bethesda; 2Harvard Medical School, Channing Laboratory, Boston, MA, USA P-21 – PROTEIN MICROARRAYS IDENTIFY NOVEL NHLA ANTIBODIES SPECIFIC TO CHRONIC RENAL ALLOGRAFT INJURY Tara Sigdel, Li Li, Hong Dai, Poonam Sansanwal, Szu-Chuan Hsieh, Minnie Sarwal. 1 Department of Pediatrics, Stanford University, Stanford, CA, USA. P-24 – AUTOANTIGEN BIOMARKER DISCOVERY THROUGH IMMUNOLOGICAL PROFILING WITH FUNCTIONAL PROTEIN MICROARRAYS Dawn Mattoon1, Mary Brodey1, Gengxin Chen1, Barry Schweitzer1, Thien Dinh1, Dhavel Patel2. 1 Life Technologies, Cupertino, CA; 2Novartis; USA.

16:30

16:35

16:40

16:45

17:00-18:00 19:00

POSTER RECEPTION (FOYER CYRIL MAGNIN) DINNER AT FORBES ISLAND
(bus departure at 18:45 from hotel, return at 22:30; Please present your ticket upon boarding the bus.)

Transplantomics and Biomarkers in Organ Transplantation

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FRIDAY • FEB 26

DETAILED PROGRAM

08:30-10:00 PERSONALIZING MEDICINE THROUGH OMICS
Co-chairs: Herwig-Ulf Meier-Kriesche, Gainesville, FL, USA Richard N. Pierson III, Baltimore, MD, USA

08:30

CONDUCTING TRANSPLANT TRIALS FOR BIOMARKER DISCOVERY Allan D. Kirk, Professor of Surgery and Pediatrics, Emory University and Scientific Director of the Emory Transplant Center, Atlanta, GA, USA Learning Objectives: 1. Describe the nature of clinical sample collection for -omics analysis of clinical trials. 2. Describe the informatics logistics for -omics sample collection. 3. Describe strategies for intigrating -omics into observational and interventional clinical tr

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

DETAILED PROGRAM
09:00 TOOLS FOR INTEGRATIVE GENOMICS Michael Reich, Broad Institute of MIT and Harvard, Boston, MA, USA Learning Objectives:

FRIDAY • FEB 26

1. Learn how integrative genomics is contributing to our understanding of biology and the mechanisms of disease. 2. Learn about tools developed at the Broad Institute for the analysis of integrative genomics data. Integrative genomics provides unprecedented power to increase our understanding of basic biological processes and determine the mechanisms of disease. This approach – the combining of evidence from multiple data modalities such as gene expression, copy number, epigenetic, and mutation data to find the genomic causes of a disease state – has resulted in the identification of novel mutations, the discovery of causal relationships between genomic aberrations and clinical pathologies, and other important insights in the short time it has been in practice. To take advantage of this wealth of data, new tools are needed that can span data modalities and support the very large datasets characteristic of integrative efforts. The Broad Institute has produced a number of software tools to facilitate integrative genomics investigations, including GenePattern, a suite of over 120 tools for the analysis of gene expression, copy number, proteomics, flow cytometry, and other data, along with extensive capabilities for combining these tools to create complex, reproducible methodologies; and the Integrative Genomics Viewer (IGV), a flexible, scalable, high-performance tool for the concurrent visualization of multiple large scale datasets. These freely available tools are used by tens of thousands of researchers worldwide to improve our understanding of cancer, immunology, microbial genomics, stem cell biology, and other fields.

Transplantomics and Biomarkers in Organ Transplantation

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FRIDAY • FEB 26
09:30

DETAILED PROGRAM

THE PATH FROM ExPLORATORY BIOMARKERS TO QUALIFIED BIOMARKERS IN THE CLINIC Federico Goodsaid, Associate Director for Operations in Genomics, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA Learning Objectives: 1. Understand the need for biomarker qualification in the acceptance of new biomarkers. 2. Recognize how and why regulatory agencies have developed Biomarker Qualification Processes. 3. Describe the general features of the path for Biomarker Qualification at the FDA. Exploratory biomarkers are used throughout drug development and are tested exhaustively in multiple clinical studies. Their acceptance by clinicians depends on the data available to support their use as well as on guidelines integrating these biomarkers within the standard of care. Their therapeutic application is also identified in drug labels. How can we determine whether exploratory biomarkers are ready for clinical applications? How can we determine whether they are acceptable for regulatory decision-making? Several paths have been followed in the past to qualify biomarkers. The FDA has developed a Biomarker Qualification Process to provide a regulatory path for the qualification of biomarkers which may impact the use of more than one drug. This qualification path captures the consensus on data available to support a specific context of use for a biomarker. A consensus on a context of use allows the uniform application of a biomarker across different submissions, reviewers and clinical areas in regulatory review, as well as a predictable integration into regulatory submissions. This Biomarker Qualification Process is supported by draft guidelines at the US and ICH levels. Its application at the EMEA is supported by legislation in the European Union and is being tested by the PMDA in Japan. Case studies in biomarker qualification highlight the usefulness of this Process and its impact on drug development and regulatory review.

10:00 NETWORKING COFFEE BREAK, POSTERS & EXHIBITS (FOYER)

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

DETAILED PROGRAM
10:30-12:00 THE ANTIBIOME AND TRANSPLANTATION
Co-chairs: Atul Butte, Stanford, CA, USA Stuart Flechner, Cleveland, OH, USA

FRIDAY • FEB 26

10:30

COMMON SIGNATURES OF REJECTION Francesco M. Marincola, Chief Infectious Disease and Immunogenetics Section (IDIS), Department of Transfusion Medicine, Clinical Center; Associate Director, Trans-NIH Center for Human Immunology, National Institutes of Health & Director, CC/CHI FOCIS Center of Excellence, Bethesda, MD, USA Learning Objectives: 1. Learn about biomarkers that are relevant to cancer rejection during immune therapy. 2. Lean about a common mechanism that seems to be responsible for the rejection of tissues through immune activation. 3. Discuss how this information could be used to improve treatment. COMMON SIGNATURES OF REJECTION Ena Wang and Francesco M. Marincola Infectious Disease and Immunogenetics Section (IDSI), Department of Transfusion Medicine, Clinical Center and Trans-NIH Center for Human Immunology (CHI), National Institutes of Health, Bethesda, MD, USA. Fundamental strides in the understanding of the molecular basis of tumor rejection were made in the last decade thanks to observational studies performed at relevant time points in human cancerous tissues. The following concepts emerged: immune surveillance against tumors is a likely occurrence. When cancer cells evolve to escape the ongoing immune defense, the neoplastic process reaches a clinically observable phase. By necessity, at this clinical stage, escape mechanisms override anticancer mechanisms for tumors to be observable. When cancers become established, two molecular phenotypes can usually be observed: one is characterized by a tumor microenvironment infiltrated by immune cells bearing transcriptional signatures consistent with a status of partial activation. Although incapable of dramatically affecting tumor growth, immune infiltration bears a favorable prognostic and/or predictive connotation on the natural history of the disease or its responsiveness to therapy. In this presentation, we will discuss the significance of transcriptional signatures observed in pre-treatment biopsies as predictive of responsiveness to biological therapy. Moreover, we will discuss the transcriptional signatures observable during and after therapy documenting the switch from chronic to acute inflammation that leads to tumor rejection. Finally, we will discuss how mechanisms leading to tumor rejection, largely overlap those associated with other aspects of immune-mediated tissue-specific destruction such as allograft rejection, graft versus host disease, acute clearance of pathogen and autoimmunity. These include overlapping yet distinct themes that

Transplantomics and Biomarkers in Organ Transplantation

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FRIDAY • FEB 26
are consistently present when TSD occurs: • the STAT-1/IRF-1/T-bet/IFN-γ, IL-15 path • the Granzyme A/B, TIA-1 pathway • the CxCR3 ligand chemokine pathway • the CCR5 ligand chemokine pathway

DETAILED PROGRAM

Understanding the basic mechanisms that can switch a chronic inflammatory process incapable of eradicating its cause into an acute reaction with the power of destroying completely the triggering cause, may shed insights that may guide the development of novel therapeutic strategies.

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

DETAILED PROGRAM
11:00

FRIDAY • FEB 26

CROSS TALK BETWEEN ALLOIMMUNITY AND AUTOIMMUNITY IN ORGAN TRANSPLANTATION Thalachallour Mohanakumar, Jacqueline G. & Wm. E. Maritz Professor, Surgery, Pathology and Immunology; Director, Histocompatibility & Immunogenetics; Director, Islet Isolation Core, Washington University, St. Louis, MO, USA Learning Objectives: 1. What are the self antigens which are known at the present time to which immune responses have been identified in patients with chronic rejection following human lung, heart and kidney transplantation? 2. What are the approaches to detect immune responses to alloantigens and self antigens following organ transplantation? 3. What are the mechanisms by which alloimmunity may induce autoimmunity? Chronic rejection is a major cause for morbidity and mortality resulting in poor longterm survival of the allografts. Our studies began with chronic rejection following human lung transplantation (LTx) ie, bronchiolitis obliterans syndrome (BOS) which develops in approximately 50% at 5 and over 90% at 10 years. We demonstrated that development of anti-HLA Abs (DSA) and Abs to non-HLA antigens (self-antigens, K-alpha 1 tubulin (K-α1T) and Collagen V (ColV)) precede the development of BOS. To define mechanisms by which alloimmune responses may induce the development of autoimmune responses which leads to chronic rejection, we serially analyzed LTx sera from 103 LTx for the development of Ab to HLA and self-antigens (K-α1T, ColV). 42.7% developed DSA and 30.1% developed Abs to K-α1T and ColV. Detection of DSA preceded development of Abs to self-antigens and the auto-Abs persisted even when DSA was undetectable. Further, lymphocytes from BOS+ LTx proliferated upon stimulation with K-α1T and ColV. BOS+ LTx demonstrated higher frequency of T cells secreting IL-17 and IFNγ with decreased IL-10. To confirm and define mechanisms, we developed an animal model of obliterative airway disease (OAD) by administration of anti-MHC Ab to native lung. Results from this model of OAD confirmed our findings in LTx which allowed us to conclude that alloimmune responses to mismatched donor HLA can induce autoimmune responses to self-antigens which are characterized by activation of Th17 cells leading to increased IL-17 and autoimmune responses. Therefore, autoimmune responses induced by alloimmunity plays a pivotal role in BOS pathogenesis and strategies to prevent the development of autoimmunity may be important in preventing chronic rejection. Cross talk between allo- and auto-immunity and their potential role in chronic rejection of human heart (self-antigens, myosin and vimentin) and renal transplants (self-antigens, Col IV, Vimenin) will also be presented.

Transplantomics and Biomarkers in Organ Transplantation

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FRIDAY • FEB 26
11:20

DETAILED PROGRAM

PHOSPHOPROTEOMICS AND THE ENDOTHELIUM Elaine Reed, Professor and Director of Immunogenetics, Transplant/Immunogenetics Testing, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Learning Objectives: 1. Attendees of the symposium will learn different approaches to investigate biomarkers that are being used to detect ongoing rejection as well as attempts to identify markers that predict the development of graft injury and eventual loss. 2. Foster interactions within clinical disciplines to develop new approaches to detect and treat rejection. 3. Showcase new approaches to monitor the efficacy of immunosuppression protocols to improve patient care. The morphologic classification of antibody-mediated rejection (AMR) has limited sensitivity and reproducibility. Therefore, a new direction in the diagnosis of transplant rejection is the switch from morphological classification to an approach based on molecular biomarkers. In light of the development of new immunosuppressive agents inhibiting signaling pathways, this goal is even more important for both diagnosis and selection of appropriate treatment strategies. Numerous studies have demonstrated that the production of antibodies to donor HLA antigens after transplantation is a major risk factor for the development of AMR. The pathological effect of donor specific antibody binding to the transplanted organ is likely to involve signaling pathways elicited by ligation of class I and class II molecules on the surface of endothelial cells (EC). Ligation of HLA molecules by antibodies induces phosphorylation of FAK and Src and prompts the assembly of mTORC1 and mTORC2, resulting in concomitant activation of cell survival and proliferation signaling pathways. mTORC1 stimulates cell proliferation by activating Erk, p70 S6 ribosomal protein and S6 kinase (S6K). mTORC2 stimulates cell survival by activating Akt and upregulating the anti-apoptotic proteins Bcl-2 and Bcl-xL. With the availability of phosphorylation-specific antibodies that can detect activated signaling molecules, we investigated the significance of phosphorylated S6K, S6RP, and Erk in AMR by immunohistochemical staining of paraffin embedded cardiac biopsy tissue. We found a strong association between the diagnosis of AMR in cardiac transplants and the presence of phosphorylated forms of S6 ribosomal protein and S6 kinase in the capillary endothelium of the graft (p<0.0001).Protein phosphorylation also correlated with the presence of circulating donor specific HLA antibodies (p<0.001). These data indicate that phosphorylation of S6 kinase and S6 ribosomal protein are useful biomarkers for the diagnosis of AMR and support a role for antibody-mediated HLA signaling in the process.

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

DETAILED PROGRAM
11:40

FRIDAY • FEB 26

ANTIBODY REPERTOIRES TO NON-HLA ANTIGENS IN KIDNEY TRANSPLANT RECIPIENTS Peter Heeger, Director, Clinical Research Program in Transplanation Institute, Mount Sinai School of Medicine, New York, NY, USA Learning Objectives: 1. To list the strengths and weaknesses of antibody repertoire analysis using protein microchips. 2. To explain the hypothesis that antibodies reactive to non HLA antigens can contribute to the development of chronic allograft injury. 3. To distinguish pathogenic mechanisms from biomarker analysis in the context of transplant studies. Emerging evidence indicates that autoreactive antibodies contribute to chronic allograft injury but the prevalence and specificity of antibodies reactive to non-HLA antigens are poorly defined. We tested the hypothesis that following solid organ transplantation, exposure of neo/self antigens in a proinflammatory environment breaks self-tolerance, and the induced autoantibodies contribute to chronic allograft injury. We used a protein microarray (>8000 proteins) to compare serum antibody repertoires, first in cross-sectional cohorts of transplant recipients with and without chronic allograft injury. We found stronger reactivity >300 antigensin kidney transplant recipients compared to those on dialysis, and stronger reactivity to >100 antigens in recipients with transplant glomerulopathy (TGP) compared to those with stable kidney function. Selected antibodies, including those reactive to EGFR and CDK9 were confirmed by dot blot assays to be more prevalent in patients with TGP. In a second design, we analyzed antibody repertoires before and after transplant in kidney transplant recipients with stable function and with TGP. While antibody repertoires of normal volunteers did not change over 12 months, transplantation induced a statistically significant change in antibody repertoires (p=0.03 vs controls). 44% of the reactivities detected posttransplant in the stable recipients were new compared with those found prior to transplantation, and the numbers of new reactivities increased between 6-12 months. In the TGP cohort, we found shared reactivities in the posttransplant samples to more than 100 new targets compared to the pretransplant samples. Potentially important antigens include ion transporters and histone F2A2, a described target of lupus nephritis. Together, our findings support the hypothesis that kidney transplantation induces formation of serum antibodies reactive to self/non-HLA antigens, which may contribute to chronic injury. In addition to providing insight into mechanisms of human allograft injury, screening antibody repertoires with protein arrays has the potential to identify novel biomarkers associated with allograft failure.

12:00 LUNCH, POSTERS & EXHIBITS (FOYER)

Transplantomics and Biomarkers in Organ Transplantation

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FRIDAY • FEB 26
Chair: Elaine Reed, Los Angeles, CA, USA

DETAILED PROGRAM

13:30-15:00 PROTEINS AND METABOLITES IN TRANSPLANTATION

13:30

POPULATION PROTEOMICS AND PROTEIN BIOMARKER DISCOVERY Richard Smith, Biological Sciences Division, Pacific Northwest National Laboratory, Richland, VA, USA Emerging proteomics technologies are promising biomarker discovery tools because of their ability to measure thousands of proteins from complex biological samples, e.g., human blood and urine. Platforms for proteomics measurements are now largely based on mass spectrometry, and continue to advance rapidly. Yet, these technologies have met with limited success to date in establishing new clinical biomarkers. A major challenge is to provide sufficient sensitivity for sufficiently broad coverage of the proteome in combination with the throughput needed to account for the range of human biological variation. Population studies present a tremendous challenge for the throughput of current discovery proteomics platforms that are typically capable of analyzing, at most, a few samples per day. Compounding this challenge is the range of protein abundances in blood, and the sensitivity needed to detect clinically relevant analytes expected to be present in low ng/mL to pg/mL concentrations. In this presentation, the challenges relevant to candidate protein/proteome biomarker discovery and verification will be discussed, and illustrated in the context of a range of studies using currently available platforms. New developments and platform advances will be described for both broad discovery proteomics studies, and also for targeted proteomics studies where a subset of proteins are selected in advances for analysis with greater sensitivity and throughput than achievable in broad discovery measurements. Present capabilities and new developments will be illustrated in the context of several biomarker related applications.

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

DETAILED PROGRAM
14:00

FRIDAY • FEB 26

METABOLOMICS IN MONITORING ORGAN TRANSPLANTS David Wishart, Professor, Departments of Biological Sciences and Computing Science, University of Alberta; Senior Research Officer and Co-director, Nanobiology Program, NRC’s National Institute for Nanotechnology (NINT), Edmonton, AB, Canada Learning Objectives: 1. To understand what metabolomics is and the kinds of technologies that are used in metabolomics studies. 2. To understand how metabolomics complements other “omics” methodologies. 3. To learn about new, small molecule biomarkers that can be used to monitor organ function, early stage rejection and immunosuppressive drug toxicity. Metabolomics is an emerging field of “omics” science that allows one to rapidly identify and quantify hundreds of metabolites in organs, tissues and biofluids. It offers a complementary picture to what can be revealed via genomics, transcriptomics, proteomics or histology. Because metabolic changes typically happen within seconds or minutes after an “event” whereas transcript, protein abundance or tissue changes may take place over days or weeks, metabolomic measurements may offer a particularly useful and inexpensive diagnostic tool to monitor donor organ viability or detect organ rejection. While the concept of metabolomics is relatively new to organ transplantation, the idea of measuring metabolites as a quick, non-invasive probe of organ function is not. Indeed, serum creatinine has long been used to assess pre- and post-operative organ function. However, with the development of improved metabolomic techniques, the ability to identify much more specific biomarkers for organ function, organ viability and immunosuppressive toxicity has greatly improved. In this presentation I will give a brief introduction to metabolomics and discuss some of its strengths and limitations. I will also provide a short overview of the results from a number of metabolomic studies on organ transplants with a special focus on using metabolomics to monitor kidney and heart transplants. Many of these studies have identified a number of lesser-known, organ-specific metabolites that appear to be good diagnostic indicators of organ function, early stage rejection and drug toxicity.

Transplantomics and Biomarkers in Organ Transplantation

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FRIDAY • FEB 26
14:20

DETAILED PROGRAM

METABOLOMICS AND RENAL TRANSPLANTATION David Rush, Professor and Head, Section of Nephrology; Director, Transplant Manitoba Adult Kidney Program, Health Sciences Centre, Winnipeg, MB, Canada Learning Objectives: 1. To review the technique of urine 1H-NMR spectroscopy. 2. To discuss its use in serial cases post-renal transplantation. 3. To review preliminary findings of the DeKAF study. Of all the “omics” or systems biology approaches to biomarker discovery, metabolomics has theoretically the greatest potential, as it examines biological processes that actually take place, as opposed to the examination of genes that may or may not be transcribed or of proteins that may be non-functional. The metabolomics of various biotissues and biofluids have been studied in a variety of conditions. Our group has been studying the urine metabolome of renal transplant patients using 1 H-NMR spectroscopy for the last 10 years. Our initial studies focused on the differentiation between the urine metabolome of patients with grafts with normal histology from that of patients with subclinical acute rejection. More recently, and as part of the NIAID-sponsored DeKAF study, we have examined the urine metabolome of patients with late-onset new graft dysfunction where a clinically indicated biopsy showed interstitial fibrosis and tubular atrophy (IF/TA), IF/TA plus inflammation, or transplant glomerulopathy. For each of these conditions, the urine spectra of many (n=50-100) samples with contemporary biopsies has allowed for the development of “classifiers” that were accurate with ~90% sensitivity and specificity for the histological diagnosis. Validation studies are underway, and preliminary data are encouraging. With subclinical rejection, resolution of inflammation on repeat protocol biopsy shows ‘normalization’ of the urine 1H-NMR spectrum, and persistence of the spectrum of rejection if inflammation is not eliminated. Our data suggest that the 1 H-NMR spectroscopy determination of the urine metabolome in renal transplant patients has great potential as a non-invasive test of kidney histology, and may become a useful method to assess the safety of immunosuppressive minimization strategies.

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

DETAILED PROGRAM
14:40

FRIDAY • FEB 26

HLA DIVERSITY IN TRANSPLANTATION Steven G.E. Marsh, Deputy Director of Research, Anthony Nolan Research Institute, Royal Free Hospital, London, UK Learning Objectives: 1. Understanding the current level of HLA diversity. 2. Understanding the methodology for HLA typing. 3. Understanding the new HLA nomenclature. Successful outcomes of transplantation are still highly dependant on the accurate matching of HLA molecules between donor and recipient. Unfortunately, HLA genes are the most polymorphic genes within the human genome. To date over 4300 alleles have been recognised and surprisingly the rate at which new alleles are discovered is continuing to accelerate. Over 1000 alleles were described in 2009 alone. This rapid expansion is primarily due to the use of sequence-based HLA typing methods for tissue typing. The immunogenetic and transplantation communities face major challenges in both in defining HLA diversity and in the informatic approaches to dealing with the complex and often ambiguous HLA data. These and related issues will be discussed.

15:00 NETWORKING COFFEE BREAK, POSTERS & EXHIBITS (FOYER)

Transplantomics and Biomarkers in Organ Transplantation

47

FRIDAY • FEB 26
15:30-17:00 NOVEL APPLICATIONS OF OMICS

DETAILED PROGRAM

Session sponsored through an unrestricted educational grant by Novartis. Chair: Minnie Sarwal, Stanford, CA, USA

15:30

A METHOD TO SYSTEMATICALLY IDENTIFY CELL-TYPE-SPECIFIC DIFFERENTIAL GENE ExPRESSION FROM COMPLEx TISSUES Robert Tibshirani, Associate Chairman and Professor of Health Research and Policy, and Statistics, Stanford University, Stanford, CA, USA Accurate analysis of gene expression patterns from many tissues is hampered by the variation in relative cell subset frequency from one sample to another and the different expression patterns of each cell type. Blood is a mixed tissue containing many different cell subsets, which vary in relative frequency between individuals, both in humans and in animal models. Despite this, and at a loss of sensitivity for differential expression, analysis of gene expression from peripheral blood is frequently used in both basic and clinical research as it is easily accessible and is thought to be reflective of immune state. Here, we will describe a statistical methodology aimed at harnessing quantitative information on mixed tissue composition to control for tissue heterogeneity and yield increased sensitivity and specificity from gene expression studies. We apply the method to a study of rejection in kidney transplants. This work is in collaboration with Shai Shen-Orr, Dale Bodian, Atul Butte, Mark Davis, Trevor Hastie, Purvesh Khatri, Balasubramanian Narasimhan, Nicholas Perry, Minnie Sarwal, Lihua Ying.

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

DETAILED PROGRAM
16:00

FRIDAY • FEB 26

MOLECULAR IMAGING IN TRANSPLANTATION Caius Radu, Assistant Professor, Department of Molecular and Medical Pharmacology, UCLA Crump Institute, Los Angeles, CA, USA Learning Objectives: 1. Overview of molecular imaging modalities. 2. Direct and indirect PET imaging techniques to monitor immune responses. 3. Potential applications of PET in transplantation. In recent years, Positron Emission Tomography (PET) has emerged as a powerful non-invasive molecular imaging tool. PET provides quantitative measurements of the 3D distribution of positron emitting radio-nuclides. PET scanners routinely measure radioactivity concentrations in the 10-12 M range, making PET the most sensitive imaging technology applicable to both preclinical and clinical studies. Briefly, positrons released from nuclear decay of isotope-labeled probes collide with electrons in surrounding tissues resulting in 511keV annihilation photons emitted approximately 180° apart. These annihilation photons are detected by specialized PET detectors and various mathematical algorithms are used to reconstruct tomographic images of probe biodistribution throughout the body. MicroPET scanners approach a spatial resolution of ~1 mm3 while clinical scanners have an intrinsic resolution of ~4-5 mm3. While PET was initially developed as a clinical diagnostic tool, the invention of the microPET has made it possible to more easily test new PET imaging strategies in small animals, before translation to the clinic. This talk will describe the advances and challenges in using PET to visualize immune responses in vivo. I will highlight the utilization of novel probes and transgenic reporters to monitor and quantify adaptive immune responses and proceed to address efforts that will improve reporter imaging for basic science and clinical applications. While mainly focused on PET imaging, this talk will also highlight other imaging modalities, including optical imaging and computed tomography, with a special emphasis on the concept of multimodality imaging.

Transplantomics and Biomarkers in Organ Transplantation

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FRIDAY • FEB 26
16:30

DETAILED PROGRAM

USE OF NANOPARTICLES FOR AUGMENTING MUCOSAL IMMUNITY Kathleen Kelly, Associate Professor and Technical Director, Department of Pathology & Laboratory Medicine, David Geffen School of Medicine at UCLA, UCLA, Los Angeles, CA, USA Learning Objectives: 1. Discuss the application of Nanotechnology in clinical practice and research. 2. Recognize the contribution of dendritic cells for inducing immunity. 3. Understand the role of an inflammasome in immunity. Mucosal immune responses provide superior protection against disease but the ability of the immune system to protect mucosal surfaces against invasion by pathogens is a poorly understood process. Currently there are no FDA-approved adjuvants capable of stimulating robust mucosal immunity. Protective immunity requires activation of the innate immune system and adjuvants provide this critical stimulation. However, the mechanism whereby adjuvants activate innate immunity was not known until recently. Adjuvants initiate adaptive immune responses by activating a class of innate pathogen receptors called pattern recognition receptors (PRRs), specifically Toll-like receptors and Inflammasomes. Nanoparticles have the unique ability to deliver immunogenic peptides and activate PRRs. This has accelerated vaccine design by engineering nanoparticle to selectively activate protective immunity. To test the utility of vaults as mucosal vaccine delivery platforms, we chose an infection that relies on cell-mediated mucosal immune responses for elimination and is a significant burden on health care; Chlamydia trachomatis infection. C. trachomatis is a prominent cause of STI, with approximately 92 million cases occurring annually and is an instigator of female reproductive dysfunction. T helper immune cells (Th1) must be present within vaginal tissues in order to eradicate infection. We encapsulated an immunogenic protein, the major outer membrane protein (MOMP) of Chlamydia muridarum, within hollow, vault nanocapsules (MOMP-vaults) that were engineered to bind IgG for enhanced immunity. Our data indicate that vaults engineered to deliver antigens may in fact act as “smart adjuvants” directing Th1 mediated immunity in mucosal tissues without inducing excessive inflammation. Thus, natural nanoparticles called vaults can be engineered to act as “smart” adjuvants and serve as platforms to induce mucosal immunity against mucosal infections.

17:00 CONFERENCE WRAP UP
Jeremy Chapman, TTS President, Westmead, Australia Minnie Sarwal, Conference Chair, Stanford, CA, USA Atul Butte, Conference Co-Chair, Stanford, CA, USA

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February 24-26, 2010 • 1st International Conference

The Transplantation Society • www.tts.org

POSTERS
P-01 – EARLY EVENTS IN THE ALLOGRAFT IDENTIFY MARKERS THAT PREDISPOSE AND INITIATE THE DAMAGE INVOLVED IN THE PROGRESSION TO INTERSTITIAL FIBROSIS (IF) AND TUBULAR ATROPHY (TA) Daniel Maluf, Kellie Archer, Mariano Scian, Anne King, Davis Massey, Benjamin Whitehill, Marc Posner, Valeria Mas. HumeLee Transplant Center, Virginia Commonwealth University, Richmond, VA, USA. Early gene expression (GE) changes might signal allograft injury post-transplantation (Tx) and identify kidney recipients (KRs) at risk of IF/TA progression. Prospective GE profiling of allograft biopsies (Bx)(N=120) from 30 KRs at pre-implantation (PI), post-reperfusion (PR), 3 and 9 mo post-Tx was evaluated. Time-dependent associations among GE profiling, histological allograft damage and graft stressors were evaluated. GE on PI biopsies with and without glomerusclerosis (GSC) was evaluated. KRs were classified as with (N=12) or without (N=18) IF/TA (Banff score system) at 9 mo post-KTx. The RMA method was used to obtain probe set (Pset) expression summaries. For comparing PI to 3 mo Bx GE profiles, Pset level paired t-tests were performed. For 3 mo GE analysis between the biopsies with and without IF/TA at 9 mo post-KTx a two sample t-test was used. P-values were used in estimating the false discovery rate (FDR) using the q-value method. Allograft Bx from 20 KRs were used as a validation group. Six significant Psets (P<0.001) were identified when analyzing the samples with and without GSC at PI time. 668 probe sets were significant when comparing T0 vs. 3mo post-KTx (FDR<5%). The top molecular and cellular functions associated with these genes were cell death and protein synthesis. Apoptosis signaling was up regulated in the PI biopsies. Growth factors signaling were also up regulated. Cytotoxic T lymphocyte mediated apoptosis of target cells, CD28 signaling in T helper cells and T helper cells differentiation signaling were up regulated in 3mo Bx. GE at 3mo post-KTx (IF/TA vs. no-IF/TA at 9mo post-KTx,) showed 143 significant Psets (p<0.001). HGF and FGF signaling and THBS1 expression were up-regulated in IF/TA Bxs. IL10 signaling were up-regulated in patients without IF/TA at 9 mo post-KTx. GE profiling changes in early protocol graft biopsies was associated with progression to IF/TA.

P-02 – DISTINGUISHING ABMR FROM TCMR USING ROC CURVE DIAGNOSTICS J Sellarés1, J Reeve1, B Sis1, B Kaplan2, A Matas3, P Halloran1. 1Department of Medicine, University of Alberta, Edmonton, Canada; 2Department of Medicine, Nephrology Section, Arizona Health Science Centre, Tucson, AZ, USA; 3Department of Surgery, University of Minnesota, Minneapolis, Minnesota, MN, USA. Antibody mediated rejection (ABMR) and T cell mediated rejection (TCMR) share an intense inflammatory disturbance but the molecular phenotype of ABMR remains poorly understood. To address this problem, we studied 403 biopsies for cause from kidney transplant patients, analyzed with microarray chips.The area under the curve (AUC) in a receiver operating characteristic (ROC) curve can be used to assess the diagnostic value of a biomarker over the full range of that marker’s values. In addition, modifications including partial areas under the curve and user-defined sensitivity/specificity cutoffs make ROC curve statistics an attractive alternative to the more commonly used t-test. To assess the reliability of ROC curve based diagnostics, we used Monte Carlo cross-validation, where the full dataset was split into 1000 training:test set splits. In each training set, the probeset with the highest AUC was chosen, and evaluated in the corresponding test set. The test set AUC was then recorded. Also recorded were the proportion of times a probeset had the best AUC in the training sets, and the average ranking of each probeset in the training sets. Each of these methods has been advocated as a good method for finding diagnostically relevant genes. We assessed the genes for two separate tasks: 1) Distinguishing C4d+ ABMR from TCMR, and 2) C4d(+ or –) from TCMR. C4d-(negative) ABMR is a new category whose importance is now gaining recognition in the transplant community. For task 1, the top genes included CD5 and GZMK (TCMR expression > ABMR expression) and SOx7, and CDH13 (ABMR >TCMR). For task 2, the top genes included COL3A1 and CCL18 (TCMR >

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ABMR)and SLC5A11 and SOx7 (ABMR > TCMR). AUCs in the test sets for these genesranged from 72-82%. Sox7 emerges as one of the best genes for distinguishing TCMR from ABMR using either definition of ABMR.

P-03 – ASSIGNING A MOLECULAR PROBABILITY OF T CELL MEDIATED REJECTION IN KIDNEY TRANSPLANT BIOPSIES Jeff Reeve1, Joana Sellarés1, Banu Sis1, Michael Mengel1, Bert Kasiske3, Bruce Kaplan4, Arthur Matas2, Phil Halloran1. 1 Alberta Transplant Applied Genomics Centre, Department of Medicine, University of Alberta, Edmonton, AB, Canada; 2 Department of Surgery, University of Minnesota, Minneapolis, Minnesota, MN, USA; 3 Department of Medicine, University of Minnesota and Hennepin County Medical Center, Minneapolis, Minnesota, MN, USA; 4Department of Medicine, Nephrology Section, Arizona Health Science Centre, University of Arizona, Tucson, AZ, USA. T cell mediated rejection (TCMR) in kidney transplants is currently defined by an international consensus, the Banff system, based on histopathological lesion thresholds. Attempts have recently been made to define “true” or “canonical” TCMR using gene sets from model rejection systems using mice. While both histology- and gene set-based methods will generate diagnoses highly concordant with the true disease state, each makes decisions using arbitrary definitions, and the absolute truth can never be known with certainty. Since each allows only two categories (TCMR/no TCMR), samples close to the interface will somtimes be misclassified. We developed a machine-learning classifier whose goal was to assign a continuous molecular “probability of TCMR” to each biopsy sample. Individual gene expression values from 403 kidney biopsy microarrays were used by Predictive Analysis of Microarrays (PAM) to build the classifier. We addressed the problem of the imperfect gold standard by using an iterative approach as follows: to be included in the next iteration’s gold standard definition of TCMR, a sample must be TCMR by the previous gold standard and have a PAM probability of TCMR of >0.5. This process resulted in the removal of samples that were called TCMR by the gold standard, but that were unlikely to be true TCMR according to the classifier, resulting in a more robust gold standard. In both the histopathology and gene set classifiers, the process converged within 3 iterations to produce a stable set of index cases. Furthermore, the probabilities of TCMR assigned by the histology and gene set classifiers were highly correlated with each other (r = 0.97), despite different samples being used as index cases. The resulting continuous probability of TCMR helps the clinician assess the certainty with which calls can be made in diagnostically difficult cases.

P-04 – A HIGHLY SPECIFIC NOVEL 3 GENE-SET CAN NON-INVASIVELY PREDICT OPERATIONAL RENAL ALLOGRAFT TOLERANCE Li Li, Sue Heish, John Scandling, Tara Sigdel, Minnie Sarwal. Stanford University, Stanford, CA, USA. Objectives: To improve on the sensitivity and specificity of a peripheral blood transcriptional biomarker gene-panel for diagnosis and prediction of operational tolerance in a heterogeneous cohort of 63 adult kidney transplant recipients. Methods: 31 peripheral blood samples were analyzed by oligonucleotide Agilent arrays from 3 different phenotypes from different transplant centers: 16 operational tolerant (TOL) kidney transplant patients without medications for more than 1 year, 10 with chronic graft injury and on triple maintenance immunosuppression (CAN), and 5 healthy donors (HD). Samples were used as a training set to define a minimum gene-set for diagnosis of operational tolerance which were cross validated on an 20 independant samples, including 9 TOL, previously run on cDNA microarrays (PNAS, 2007). Q-PCR was run on 21 genes to build a model for prediction of tolerance by logistic regression and tested on an independent sample set containing 7 TOL patients. Bioinformatics analysis including GeneSpring, AILUN, SAM, PAM, logistic regression and IPA were used.

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Results: Twenty-one unique genes were identified (FDR<5%) by prediction analysis of microarrays (PAM) and statistical analysis of microarray (SAM) as a minimum gene for TOL. Genes overlapping across cDNA and Agilent platforms, among the 21 set, could back predict 9 TOL samples run on cDNA arrays with 100% sensitivity and specificity. These genes are enriched in immune cell trafficking and 13/21 genes are regulated by TNF, IL6 and IL4. Multinomial logistic regression modeling by Q-PCR for all samples identified 3 genes as a minimal gene set for TOL in the array data-set with 93% sensitivity and PPV, and 90% specificity and NPV. The 3-gene model predicted TOL with 100% sensitivity and NPV, 80% specificity, and 88% PPV, in a blinded independent data-set containing 7 TOL patients. 2 out of the final 3 genes are highly expressed in B cells. Conclusion: A highly regulated minimal gene-set in peripheral blood has been validated across multiple patients groups and transplant centers. This gene-set can be used as a non-invasive monitoring tool for screening patients with stable operational tolerance after kidney transplantation. Serial measurement of expression for this gene-set in peripheral blood opens the door for deliberate immunosuppression minimization after transplantation, and should be further tested for its utility in also monitoring patients on tolerance induction protocols in kidney transplantation.

P-05 – A PERIPHERAL BLOOD 12 GENE-SET FOR DIAGNOSIS OF PEDIATRIC LIVER ALLOGRAFT TOLERANCE Li Li, Anita Talisetti, Sue Hsieh, Ken Cox, Carlos Esquivel, Waldo Concepcion, Minnie Sarwal. Pediatic Department, Stanford University, Stanford, CA, USA. Objectives: To investigate shared and unique pathways that drive pediatric and adult liver transplant tolerance. To identify the monitoring biomarkers of liver TOL specific to pediatric and adult liver transplant tolerance. Methods: 46 unique whole blood samples from 4 demographically matched patient phenotypes were run on Agilent Whole Human Genome 44K microarrays: 7 operational tolerant pediatric liver transplant patients were on no medications from 9.3-17.1 years (P-TOL). Additional liver transplant recipient groups were: 13 with biopsy proven acute rejection (AR), 7 on low-dose prograf monotherapy/ minimal immunosuppression (MIS), and 13 stable patients on dual immunosuppression. Additionally we also processed samples from 6 healthy donors (HD). Standardized bioinformatic analyses were applied and significant TOL genes were mapped by AILUN to published data on Affymetrix arrays from an operational tolerance study in adult liver transplant recipients (A-TOL; Llordella et al). Results: Twelve unique genes were identified (FDR<5%) by prediction analysis of microarrays (PAM) as a minimum gene set to cross-validate and predict P-TOL with 100% sensitivity and 85% specificity. These genes are enriched in liver regeneration and 11/12 genes are regulated by NFkB1 and SMAD3. The tolerant specific genes are highly expressed in T cells, CD34+ endothelial and NK cells. 65% MIS and STA patients were predicted as TOL based on prediction probability scores >50%. These genes also correctly predicted 76% of the 17 A-TOL samples and 95% of the 21 non-TOL samples in the adult study. The most significant 100 genes from the A-TOL published study could not back predict any of the P-TOL samples in the tolerance class. There is no association between gene expression and age either at the sample time or age at the transplant for these 12 gene set. Conclusion: Specific peripheral transcriptional programs can be identified in operational tolerance in pediatric recipients of liver allografts, distinct from those previously identified in adult operationally tolerant liver recipients, and may provide a means to non-invasively monitor patients in a serial manner for immunosuppression minimization. These genes are highly expressed in specific peripheral blood lymphocyte subsets, and their coordinated regulation by specific cytokines may support the maintenance of operational tolerance in children, following liver transplantation.

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P-06 – GENDER SPECIFIC DIFFERENCES IN ALLOGRAFT BIOLOGICAL AGE IMPACT ON POST TRANSPLANT ORGAN FUNCTION Liane McGlynn, Marc Gingell Littlejohn, David Kingsmore, Marc Clancy, Paul Shiels. Department of Surgery, Faculty of Medicine, University of Glasgow, Glasgow, UK. Donor age is a key predictor of post transplant organ function but lacks the predictive value required for targeted intervention. CDKN2A transcription levels, however, act as a biomarker for allograft function at 6months and 1year post transplant. Consequently, we have employed renal CDKN2A transcription levels pre-transplant as a prognostic biomarker of post transplant organ function. Pre-implantation deceased donor renal allograft biopsies (n=61) were assayed for CDKN2A expression and any association with recipient urinary protein/creatinine ratio (UPCR- a sensitive marker of tubulo-glomerular damage and proven predictor of later graft failure) and white cell count (WCC) post transplant. Patients whose donor organ expressed high CDKN2A levels had significantly elevated UPCR at 6months (p=0.025), 1year (p=0.035) and 2years post transplant (p=0.028). Conversely, these same patients had reduced WCC at 6months (p=0.028), 1year (p=0.009) and 2years post transplant (p=0.020). When patients were categorised by donor sex, the association between UPCR, WCC and CDKN2A was lost in patients receiving male organs. However, in patients receiving female organs, high CDKN2A expression remained significantly associated with increased UPCR (p=0.012, p=0.033, p=0.009) and decreased WCC (p=0.095, p=0.004, p=0.008) at 6months, 1year, 2years post transplant. This study confirms that allograft biological age, as assayed by CDKN2A transcript levels, is a novel prognostic determinant for renal function post transplant and that this is influenced by donor gender. In female organs elevated CDKN2A transcription is associated with increased biological age and correspondingly poorer organ function post transplant. This was not observed for male organs and suggests there are gender specific drivers of biological ageing. CDKN2A expression may provide valuable pre-transplant prognostic information on organ quality, allowing improved patient counselling and providing the possibility for targeted intervention strategies.

P-07 – ILLUMINA-MICROARRAY ANALYSIS OF MYCOPHENOLIC ACID (MPA -INDUCED) CELL DEATH IN INSULINPRODUCING CELL LINE AND PRIMARY ISOLATED ISLET CELLS FROM RAT: NEW MECHANISMS INTO THE APOPTOSIS PATHWAYS INVOLVED Yun-Jong Park1, Joon Ye Kim1, Yuri Cho1, Hyung Joon Ahn1,2, Dong Jin Joo1,3, Yu Seun Kim1,3. 1The Research Institute for Transplantation, Yonsei University College of Medicine; 2Department of Surgery, Kyunghee University School of Medicine; 3 Department of Surgery, Yonsei University College of Medicine; Seoul, South Korea. MPA, an IMPDH inhibitor, is one of the effective immunosuppressive drugs. However, MPA may induce cellular toxicity and impair cellular function in β-cells. The mechanisms underlying cell death following MPA treatment have not been fully explored yet. RhoGTPases has been reported for playing a critical role such as differentiation and gene expression, and they worked as a molecular switch on many cellular events. Among RhoGTPase subfamily, activated Rac and Cdc42 were reported to induce JNK (c-jun N-terminal kinase) activation through MEK pathway following MPA treatment. This could lead apoptosis by JNK activation through PKC (protein kinase C) activation-dependent pathway. In that pathway, activation of p38 MAPK, Akt and ERK1/2 by TNF-α signaling are closely involved. In this study, using a microarray approach, we examined gene expression patterns in MPA-treated INS-1E and primary isolated rat islets. Through western-blot analysis, we found that RhoGDI-α expression was altered time dependently after MPA treatment, and then these mechanisms affected RhoGTPases, especially Rac1 activation in downstream pathway. We examined the relationship between RhoGDI-α expression and JNK activation during MPA-induced apoptosis. MPA decreased the cellular expression of RhoGDI-α and enhanced the activation of JNK. Also, decrease of RhoGDI-α was inhibited when we treated cells with GTP and MPA

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simultaneously. Furthermore, we found that, using MTT assay, altered expression of RhoGDI-α (by using siRNA and gene over-expression methods) directly affected the cell death rate, and then regulated the apoptosis pathway through Rac1 activation and downstream of MAPKs pathway. In conclusion, MPA induced cell death in insulin secreting cell line and primary isolated pancreatic β-cells via down-regulation of RhoGDI-α linked with increased Rac1 activation, and they caused JNK activation. This RhoGDI-α/Rac1/JNK pathway could be the main therapeutic target for the prevention of MPA-induced islet apoptosis.

P-08 – ExPRESSION SIGNATURES OF ExTRACELLULAR MATRIx RELATED TRANSCRIPT SETS LINKED RENAL ALLOGRAFT INTERSTITIAL FIBROSIS/TUBULAR ATROPHY WITH ACUTE T-CELL MEDIATED REJECTION Silke Rödder1, Andreas Scherer2, Hans-Peter Marti1. 1University of Bern, Inselspital Bern, Department of Nephrology and Hypertension, Switzerland; 2Spheromics, Kontiolahti, Finland. Renal allograft T cell-mediated acute rejection (AR) and interstitial fibrosis / tubular atrophy (IF/TA) commonly share excessive extracellular matrix remodelling, which is regulated by the Zn-protease family of metzincins, including matrix metalloproteases, tissue inhibitors of metalloproteases and their related genes. Metzincins (METS, n=81) and metzincins and related genes (MARGS, n=160) were deregulated in renal allograft biopsies with AR (n=10) and IF/TA (n=22), and microarray profiling identified METS and MARGS based AR and IF/TA gene signatures 1, 2. Here, we illustrate the differences and conformities between AR and IF/TA METS and MARGS signatures applying differential gene expression analyses, together with pathway and biological process analyses. Expression changes of METS and MARGS were also linked to patient histology. A five-way VENN diagram of all deregulated (log2 fold change vs. Banff Normal 3(N), p<0.05) METS and MARGS in AR, AR+IF/TA, IF/TA (I, II, III) identified 2 METS and 7 MARGS to be commonly deregulated in all patient groups. Interestingly, no gene was solely deregulated in AR. In contrast, 4 MARGS, 1 METS showed unique IF/TAI deregulation and could be associated to renal injury and hypertrophy by Ingenuity Pathway Analyses (IPA). IF/TAII and -III patients showed the greatest number of commonly deregulated genes. 5 MARGS, 1 METS were solely deregulated in IF/TAII, and 34 MARGS, 16 METS in IF/TAIII. METS and MARGS, deregulated in AR were associated to the canonical pathway “hepatic fibrosis”, and higher expression of METS in our severe and moderate IF/TA patients was significantly associated to the occurrence of AR prior to IF/TA diagnosis. THBS2, previously identified as IF/TA marker 2 belonged to the top scoring candidates associated to connective tissue disorders, apoptosis and cancer identified by IPA in IFTA. QRT-PCR of microdissected functional kidney compartments showed significant upregulation of THBS2 in proximal tubuli of AR and in glomeruli, proximal tubuli and tubular interstitium of IF/TA patients. Serum ELISA revealed increasing expression of THBS2 comparing AR and IF/TA, both significantly different from N patients. In conclusion, METS and MARGS expression in IF/TA was linked to AR, and AR THBS2 upregulation may be indicative of IF/TA development.

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P-09 – INNATE AND ADAPTIVE IMMUNE GENE ExPRESSION DYNAMICS AND PROGRESSION OF CHRONIC HISTOLOGICAL DAMAGE OF RENAL ALLOGRAFTS Maarten Naesens, Li Li, Tara Sigdel, Purvesh Khatri, Neeraja Kambham, Oscar Salvatierra, Minnie Sarwal. Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA. Background: In renal transplantation, slowly progressive chronic tubulo-interstitial damage jeopardizes long-term renal allograft survival. Both immune and non-immune mechanisms are thought to contribute to this progressive renal allograft scarring, but the most promising targets for timely intervention have not yet been identified. Methods: In the current study we seek to determine the driving force behind progressive histological damage of renal allografts, without donor pathology, delayed graft function and clinical or subclinical acute graft rejection. We used microarrays to examine whole genome expression profiles using tissue from 161 unique human renal allograft protocol biopsies obtained within the first two years from two independent patient cohorts: a cross-sectional cohort [N= 89] and a longitudinal cohort [N=72]. Results: In this highly cross-validated study, we demonstrate a major shift in global gene expression when rejection-free renal allograft biopsies obtained post-transplantation were compared with pre-implantation samples, with highly significant overrepresentation of immune genes involved in mostly adaptive immune responses, i.e. T- and B-cell associated transcript sets. In post-transplantation samples, we demonstrate the highly significant association of established, ongoing and most importantly also future chronic histological damage with regulation of adaptive immunity (T-cell and B-cell transcript sets) but also innate immune response genes (dendritic cell, NK-cell, mast cell and granulocyte transcripts), even in the absence of classic types of acute T-cell mediated or antibody-mediated rejection as defined in the current Banff classification. Conclusion: Progressive chronic histological damage after kidney transplantation is associated with significant regulation of both innate and adaptive immune responses, months before the histological lesions appear. This study therefore underscores the complexity of the immunological processes in human kidney transplantation, and corroborates the hypothesis that quantitative inflammation below the diagnostic threshold of classic T-cell or antibody-mediated rejection is involved in early subclinical stages of progressive renal allograft damage.

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P-10 – ExPRESSION OF THE PRO-APOPTOTIC MOLECULES BNIP3, BNIP3L AND BAx INDICATES THAT HUMAN ISLETS ARE ExPOSED TO NUTRIENT AND OxYGEN DEPRIVATION DURING ISOLATION Peter Campbell1, Jonathon Chee1, Lina Mariana1, Tom Loudovaris1, Shane Grey2, Helen Thomas1, Thomas Kay1. 1 St. Vincent’s Institute, Fitzroy, Victoria; 2Garvan Institute, Darlinghurst, NSW; Australia. Allogeneic human islet transplantation replaces the insulin producing beta cells and corrects blood glucose levels in type 1 diabetic recipients. Islets isolated from cadaveric donors are exposed to a number of insults during the isolation procedure including mechanical stress, hypoxia, temperature variations and enzyme impurities. These insults may lead to functional impairment and activation of cell death pathways resulting in loss of viable beta cell mass in the first days after transplantation. We investigated the expression of the Bcl-2 family of pro- and anti-apoptotic molecules in human islets post isolation. These molecules are activated in a death stimulus-specific manner, therefore knowledge of their expression may help to understand the mechanism of the cellular responses involved in loss of islet homeostasis after transplantation. Microarray analysis and real time PCR were performed using RNA isolated from human islets post isolation. There was high expression of the pro-apoptotic molecule Bax, indicating islets may be undergoing apoptosis. The hypoxia-inducible BH3only proteins BNIP3 and BNIP3L were highly expressed, even in islets meeting the quality control criteria for transplantation, suggesting oxygen deprivation occurs during isolation. The autophagy-inducing molecule Beclin 1 was also highly expressed indicating degradation of islet cell components under conditions of nutrient starvation. There was no correlation of cell death molecule expression with quality control assays, donor characteristics or isolation variables. Therefore expression of pro-apoptotic members of the Bcl-2 family indicates that islets are exposed to a lack of nutrients and oxygen during isolation and this may contribute to their death after transplantation. Inhibition of these molecules during islet isolation may prevent the initial graft loss after transplantation.

P-11 – THE RESEARCH OF FOxP3 GENE-TRANSFERRED CD4+CD25- T-CELLS ON IMMUNE TOLERANCE IN RAT CARDIAC ALLOGRAFTS Zhenya Shen, Ning Zhong, Michun He, xiaomei Ten, Wenxue Ye, Yanqiu Hu. Department of Cardiovascular Surgery and Clinical Immunology Laboratory, First Affiliated Hospital of Soochow University, Jiangsu, China. Objective: To investigate the immune suppression effect of Foxp3 gene-transferred CD4+CD25- T-cells on acute rejection of rat cardiac allografts and the involved mechanisms. Methods: Rat model of abdomen heterotopic heart transplantation were established. Recipients (Wistar rat) were divided into four groups. Group 1: 1 ml PBS was injected before transplantation, Group 2: 1×106 fresh CD4+CD25+ regulatory T cells were injected into recipients before transplantation. Group 3: CSA was used for intragastric administration one week before transplantation. Group 4: 1×106 CD4+CD25- T cells transfected with Foxp3 gene were injected into recipients before transplantation. Abdominal touch was carried out 3 times one day, 8 Wistar rats from every group were operated to observe the abdominal graft condition. The level of IL-6 and TGF-β1 of peripheral blood in the recipients were detected. Results: The Foxp3 gene-transferred CD4+CD25- T cell and isolated CD4+CD25+ T cell have the similar ability in suppressing the lymphocyte proliferation in vitro. Mean survival time of the grafted heart of group 1 and group 2 was shorter obviously than that of group 2 and group 4. There was no manifest difference between group 2 and group 4. The level of IL-6 of group 1 was higher than that of other groups, but there is no difference in group 2, group 3 and group 4. The level of TGF-β1 of group 2 and group 4 was much higher than group 1 and group 3, but there were no difference between group 2 and group 4. Conclusion: Foxp3 gene-transferred CD4+CD25- T cells can suppress the proliferation of responsive cell, and was successfully used to induce immune tolerance in rat cardiac allografts. There was no apparent difference comparing with CD4+CD25+ T cell.
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P-12 – MONITORING OF PHARMACOLOGICAL TREATMENT AND CLINICAL EVOLUTION OF RENAL TRANSPLANT, BY METABONOMICS, NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY PROFILING AND MULTIVARIATE STATISTIC Antonio Vivi2, Maria Tassini2, Marco Calderisi2, Mario Carmellini1, Johan Trygg3, Rasmus Madsen3, Hans Stenlund3, Torbjörn Lundstedt4. 1Department of Surgery and Bioengineering, University of Siena, Siena, Italy; 2Nuclear Magnetic Resonance Centre of Siena University, Siena, Italy; 3Department of Chemistry, Computational Life Science Cluster (CLiC), Umeå University, Umeå, Sweden; 4AcureOmics AB, Umeå, Sweden. 1H Nuclear Magnetic Resonance spectroscopy (NMR) of body fluids has been successfully applied to investigate numerous diseases and toxic processes. Metabonomic refers to analytical techniques, like NMR spectroscopy and to the intensive use of multivariate statistic (Chemometrics). Projection based methods such as Principal Component Analysis (PCA) and Orthogonal Projection to Latent Structures (OPLS) are applied. An important challenge posed by NMR spectroscopy of bio-fluids is how to efficiently recover metabolic information that allows diagnosis or classification of the post-trasplant events or pharmacological toxicity. Proper elaboration of this data can bring to identification of a “recovery trajectory” model, i.e. evolution of metabolic profile during recovery time, that could be the starting point for the interpretation of postoperative courses of patients. The main strength of the proposed approach is that each patient is used as his own reference, thus the focus is on variation around an individual starting point. We already demonstrated the feasibility of identifying a profile reflecting biochemical changes that occur in the first two weeks after a kidney transplant (Chemometrics and Intelligent Laboratory Systems (vol.98, 45 – 50, 2009)), the recovery during the first two weeks could be seen as a discrete two-stage progress. Since each patient is used as his own reference, it is possible to build models that are more sensitive then standard multivariate pattern recognition models, that treat data from a large group of patients altogether. In particular, the use of recently developed OPLS-DA analysis of NMR spectroscopy data, allow us to estimate a joint intra-person effect that is representative for the majority of the individuals. A larger dataset with more than 30 patients that have been followed also after hospital discharge (follow-up). There is the evidence that the metabolic profile trajectory go on all along the patient life and could effectively be used to monitor its status.

P-13 – TLR4 SIGNALING IS INVOLVED IN IGA-STIMULATED MESANGIAL CELL ACTIVATION Da Hye Lee1, Beom Jin Lim2, Yu Seun Kim1,3, Hyeon Joo Jeong2. 1The Research Institute for Transplantation; 2Department of Pathology; 3Department of Surgery; Yonsei University College of Medicine, Seoul, Korea. Background: Deposition of polymeric IgA in renal mesangium is the hallmark of IgA nephropathy. However, the molecular mechanisms of IgA-mediated mesangial responses and the initiation of inflammatory injury remain still poorly understood. Renal TLR4 expression is involved in patients with IgA nephropathy. However, it is not known whether TLR4 pathway is involved in this mesangial activation.

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Method: Murine mesangial cell line was stimulated with LPS (1ug/ml) or IgA (20ug/ml). TLR4 expression was measured by real time RT-PCR and western blotting. Intracellular responses to LPS or IgA were assessed by Western blotting for ERK1/2, JNK, p38 MAP kinase, and Iκ-Bα. Secretion of MCP-1 were assessed by ELISA. The effects were tested using small interfering RNA (siRNA) of TLR4 (25nM). Result: LPS and IgA up-regulated the level of TLR4 mRNA and protein levels in cultured mesangial cell line at 24hr. LPS induced rapid phosphorylation of MAP kinases (ERK1/2, JNK, p38) and degradation of Iκ-Bα. IgA also induced rapid phosphorylation of MAP kinases, but Iκ-Bα degradation was not observed. LPS and IgA induced secretion of MCP-1 at 6hr. LPS- or IgA-induced mesangial cellular TLR4, MAPKs activation and MCP-1 secretion were inhibited by transfection of TLR4 siRNA. Conclusion: Activation of MAPKs and secretion of MCP-1 by IgA are mediated, in part, by TLR4 in mesangial cells. TLR4 seems to be involved in mesangial cell injury by induction of pro-inflammatory cytokines in IgA nephropathy.

P-14 – THE EUROPEAN TOLERANCE INVESTIGATION PLATFORM: A UNIFIED RESOURCE FOR TOLERANCE DATA MINING Patrick Miqueu1, Coline Thomas2, Hilke Schmidts3, Olivier Rivain2, Michel Goldman4, Lucienne Chatenoud1, Kathryn Wood5, Hans-Dieter Volk3. 1INSERM U580, Hôpital Necker-Enfants Malades, Université Paris Descartes, Paris, France; 2EA 4275, Faculty of Pharmaceutical Sciences, University of Nantes, Nantes, France; 3Institute for Medical Immunology and BerlinBrandenburg Center for Regenerative Therapies, Charité University Medicine, Berlin, Germany; 4Institute for Medical Immunology, Université Libre de Bruxelles, Brussels, Belgium; 5Transplantation Research Immunology Group, Nuffield Department of Surgery, University of Oxford, Oxford, UK. The European Tolerance Investigation Platform is a unique resource organizing the data pertaining to innovative clinical trials in the field of transplantation tolerance, launched in the framework of the European Consortium: RISET (Reprogramming the Immune System for the Establishment of Tolerance). Allowing transversal statistical analyses, this platform aims at the qualification and discovery of fit-for-purpose biomarkers. Twelve pilot clinical investigations, including weaning, minimization and tolerance induction trials in kidney, liver and bone marrow transplantation, have been integrated into a relational database. With more than 530 exhaustively monitored patients, around 4000 samples have been collected and tested by dedicated biological assays including ‘omics’ technologies (Agilent microarrays, T cell repertoires analysis), functional investigations (screening of HLA alloantibodies, IFN-gamma ELISPOT, CTLp frequencies), virology assays and exploratory biomarkers (HMOx-1 polymorphism, expression of tolerance related genes). Cross-comparisons of clinical and immunological data allow both confirmation of findings and new discoveries. Integrating multiple gene expression datasets, where genes behave similarly across several independent experiments, improves for example the statistical confidence of a novel biomarker. The collaborative culture established between clinicians and scientists within the RISET consortium allows sharing and reanalysis of rare and expensive datasets. Promoting the development of a data federating infrastructure and knowledge management tools, such as clinical decision support algorithms, will make possible an individually-tailored approach to post-transplant management.

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P-15 – NOVEL CELL-TYPE SPECIFIC DECONVOLUTION OF WHOLE-BLOOD GENE ExPRESSION PROFILES IN RENAL ACUTE REJECTION Purvesh Khatri, Shai Shen-Orr, Robert Tibshirani, Atul Butte, Minnie Sarwal. Department of Pediatrics, School of Medicine, Stanford University, Stanford, CA, USA. Background: Although, a set of blood-based biomarkers have been identified for acute rejection, the expression profile of different blood cell-types in rejection is unknown. Methods: We developed a novel statistical deconvolution method for identifying cell-type specific gene expression profiles using whole blood microarray expression data and relative frequencies of individual cell types in each sample. Whole blood gene expression data from 24 renal transplant patients were analyzed on Affymetrix HGU133 plus 2 whole genome expression arrays. Of the 24 patients, 15 samples were from biopsy-proven acute rejection patients and 9 were from stable patients. Simultaneous Complete Blood Counts (CBCs), containing relative frequencies of monocytes, lymphocytes, eosinophils, basophils, and neutrophils, were available for each sample. Results: Whole blood gene expression analysis using SAM did not identify any significantly differentially expressed genes between AR and STA patients at FDR of 10%. We estimated expression profiles for each cell type in each sample using statistical deconvolution. Significance analysis of the estimated expression profiles identified 213 genes significantly upregultaed in AR in one of the cell types at an FDR of 5%. Furthermore, while hierarchical clustering using whole blood expression data does not classify the samples correctly, the deconvoluted gene expression profile in specific cell subsets could distinctly cluster the samples in two groups (Fig. 1). In addition, we downloaded 132 microarrays of a specific blood cell type from NCBI GEO. As shown in Fig. 1, the estimated cell-specific expression profile in STA group is highly correlated with measured expression profiles in that cell type, whereas the estimated cell-specific expression profile in AR group does not correlate with the measured cell-specific expression profile, which clearly show that during rejection gene expression profiles of a specific cell-type are clearly disrupted. Conclusion: Our novel statistical deconvolution method is able to identify the specific subset of blood cells that correlates with acute rejection in renal allografts. (figure1)

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P-16 – META-ANALYSIS OF SOLID ORGAN TRANSPLANT DATA SETS IDENTIFIES DIFFERENTIALLY ExPRESSED MIRNAS COMMON IN HEART, KIDNEY AND LUNG ALLOGRAFTS Purvesh Khatri, Richard Hayden Jones, Atul Butte, Minnie Sarwal. Department of Pediatrics, School of Medicine, Stanford University, Stanford, CA, USA. Background: Recently, differentially expressed miRNAs in kidney and liver allograft rejection have been identified. The aim of our study was to investigate if there is a set of miRNAs that exhibit regulatory effects in acute rejection (AR), irrespective of the transplanted organ. Method: We downloaded 5 allograft biopsy datasets from GEO (3 heart, 1 kidney and 1 lung datasets). Using miRBase, the genes in each sample in each dataset were divided into target and non-target genes. The genes in both groups were ranked by their expression values and difference in the average ranks for both groups was computed such that high difference in average ranks (called RE-score of a miRNA) indicate lower expression of target genes. We then calculated a tscore for each miRNA measuring the difference in RE-scores in AR versus stable (STA) samples, where higher t-score indicate higher regulatory effect of the corresponding miRNA in AR. The significance of t-scores was computed using sample label permutations for both two-tail and one-tail tests. Results: All 5 datasets showed overall trend of higher t-scores (Fig. 1A), suggesting that many miRNAs have strong regulatory effects in AR. Using a two-tail test, we identified 40 miRNAs in the lung dataset, 278 miRNAs in the kidney and 40 miRNAs in all 3 heart datasets with statistically significant t-scores. Furthermore, there were 40 miRNAs that were statistically significant (FDR [lt] 0.2) in all 5 datasets. Interestingly, hierarchical clustering of the kidney dataset using the REscores of these 40 miRNAs classifies the samples into two distinct clusters with one sample misclassified (Fig. 1B). Similarly, using one-tail test, we identified 128 miRNAs with statistically significant positive t-scores in all 5 datasets. Interestingly, these 128 miRNAs include 9 miRNAs that were recently found to be differentially expressed in acute rejection of renal allografts, suggesting that they may also be differentially expressed during acute rejection in other solid organs. Conclusion: Our analysis of solid organ transplant datasets suggests there may be a common set of miRNAs that play significant role in rejection of allografts. (figure1)

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P-17 – URINE METABOLITE PROFILES ASSOCIATED WITH ACUTE REJECTION IN PEDIATRIC RENAL TRANSPLANTS Tom Blydt-Hansen1, Rajmund Somorjai2, Kirk Marat3. 1Department of Pediatrics and Child Health (Nephrology), University of Manitoba; 2Institute for Biodiagnostics, National Research Council of Canada; 3Department of Chemistry, University of Manitoba; Winnipeg, MB, Canada. Renal biopsy is used for surveillance and diagnosis of rejection. Non-invasive testing could reduce morbidity and possibly identify rejection risk at an earlier stage. Nuclear magnetic resonance (NMR) spectroscopy may be used to identify metabolite profiles associated with acute rejection in children after renal transplantation. 195 urine samples from 39 pediatric patients were obtained at the time of renal biopsy for surveillance and clinical indication to identify rejection. Biopsies were graded according to Banff criteria and classified as no-, borderline- or acuterejection (NR=108, BR=65, AR=22). NMR spectra (500 MHz) were obtained after correction to pH=7.0. Association of samples with AR was tested using Hierarchical cluster analysis. Classifiers for AR were developed using Genetic-algorithmbased optimal region selection methodology on whole spectra. Hierarchical analysis yielded 5 principal clusters. Samples with AR were associated with clusters 2 and 4 (p=0.019). Of these, AR in surveillance biopsies (subclinical) was associated with cluster 2, whereas clinically suspected AR was associated with cluster 4 (p=0.012). Comparing AR vs. NR (excluding BR), the classification methodology achieved a balanced optimized sensitivity/specificity of 90.7% and 90.9%. Optimizing the classifier for 100% sensitivity resulted in modest reduction in specificity to 82.4%. Applying the classifier to the 65 BR samples, 36% and 43% were classified as AR, using the balanced and sensitivity- optimized classifiers, respectively. In a subset (NR=68, BR=40, AR=14), BR & NR were combined vs. AR, yielding sensitivity/specificity of 92.9% and 93.5%. It was not possible, however, to optimize sensitivity when the BR group was included with NR. This approach is noninvasive and inexpensive and may be useful in prospective surveillance for rejection. Unsupervised analysis shows distinct clustering of samples with subclinical and overt AR. Urine metabolite profiles associated with acute rejection are identified in this cohort. Before being applied clinically, validation with a larger independent sample is needed.

P-18 – ASSESSMENT OF KIDNEY ORGAN QUALITY AND OUTCOME USING THE TRANSCRIPTOME OF THE IMPLANT BIOPSY Thomas Mueller1, Motaz Obeidat1, Gordon Broderick1, Wenjie Wang2, Phillip Halloran1, Valerie Luyckx1. 1Division of Nephrology and Immunology, Department of Medicine, University of Alberta, Edmonton, AB, Canada; 2Division of Nephrology, Department of Medicine, University of Calgary, Calgary, AB, Canada. Robust prediction of early and late kidney transplant function using clinical and/or pathology based markers available at time of transplantation has not been achieved. Transcriptome studies are promising but the identification of predictive genes needs reliable reference markers. Our goal is to utilize objective measures of transplant function to refine transcriptome analysis and identify gene sets that characterize organ quality and predict early and long-term kidney function. Unsupervised microarray analysis was performed on implantation biopsies taken post-reperfusion in deceased (DD) and living donor (LD) kidneys (test set: 42 DD, 45 LD; validation set: 25 DD, 39 LD). A ‘livingness’ vs. ‘deceasedness’ gene set, reflecting an individual kidney’s similarity to best functioning LD kidneys or worst functioning DD kidneys, was derived from genes overlapping between those differentially expressed among LD vs. DD and low vs. high risk for delayed graft function,

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as scores of organ quality. Early function was measured by isotope scans on post-operative days 1 or 2. In the test set, 3718 genes differentiated LD vs DD, 1051 differentiated low vs. high risk kidneys (adj p<0.01, 676 genes overlapped between these two sets. Kidneys were plotted according to individual expression of the 15 highest, ‘livingness’, and 15 lowest, ‘deceasedness’, expressed genes within this set. Kidneys were strongly separated into those with best and worst early function. When applied to our validation set the same genes again demonstrated a continuum from best functioning to worst functioning kidneys (Figure 1). This data demonstrates that the closer a DD is to an LD kidney the better its function, and conversely the closer an LD is to a DD kidney, the worse its function. Prospectively the coordinates of an individual kidney plotted on this graph may reflect organ quality and permit prediction of utility of an organ for transplantation.

P-19 – ExPERIMENTAL DESIGN AND QUALITY ASSURANCE OF HIGH-THROUGHPUT CLINICAL TRANSPLANTOMICS STUDIES: A CRUCIAL STEP TOWARD ROBUST BIOMARKER DISCOVERY Zhong Gao1, Adam Asare1, Vicki Seyfert-Margolis1, Deborah Phippard1, Vincent Carey2. 1Immune Tolerance Network / UCSF, Tolerance Assays and Data Analysis, Bethesda; 2Harvard Medical School, Channing Laboratory, Boston; MA, USA. Background: Validation of biomarkers derived from smaller pilot studies requires careful consideration of technical variability associated with multi-site, high-throughput clinical trial settings. Microarray processing on peripheral whole blood samples is characterized by heterogeneous cell types that are subject to drastic technical fluctuations, lower signal/noise ratio, and higher risk of systemic batch bias. These issues hamper accuracy and robustness of biomarker discovery and validation. To tackle these challenges requires sound experimental design and standardized quality assurance methods. We address both of these aspects through: 1) Statistical design and planning for controlling batch effect; and 2) an automated microarray QA program based on statistical modeling. Methods: We surveyed the impact of batch effect under different circumstances including study design (cross-sectional and longitudinal studies), and processing (retrospective and incremental processing). A parametric multivariate outlier detection algorithm (PMVO) was developed within the Bioconductor/R statistical framework. We conducted a negative control test of the system using the MAQC Affymetrix data and applied the procedure to 507 Affymetrix HG-U133 2.0 Plus GeneChips© processed with human peripheral blood RNA from five Immune Tolerance Network (ITN) trials. Results: For retrospective studies, statistical modeling that includes batch effect as a parameter controlled systemic bias. For longitudinal studies with incremental sample processing, two RNA references with known differential expression were used as a normalization factor to adjust expression estimates on the array. For detection of technical artifacts using PMVO, we found 18/507 trial samples to be problematic based on poor microarray quality. Statistical power analysis indicated that exclusion of these outlier arrays substantially enhanced inferential power for detecting differential gene expression. No outliers were found using MAQC samples as the negative control. Conclusion: Integration of modeling for batch effects and PMVO in a statistical pipeline constitutes a novel approach to improving robustness of biomarker discovery.

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P-20 – PROTEOMICS IN ExPERIMENTAL AND CLINICAL LIVER ALLOGRAFT TOLERANCE Shigeru Goto, Li-Wen Hsu, Toshiaki Nakano, Chia-Yun Lai, Yu-Fan Cheng, Chao-Long Chen. Center for Translational Research in Biomedical Sciences, Liver Transplantation Program, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Kaohsiung, Taiwan. We have performed proteomic studies to investigate the mechanisms of immunological status of drug-free tolerance in experimental and clinical liver transplantation. In experimental setting of liver allograft tolerance, tolerogenic OLT (DA liver into PVG) rats were applied to proteome studies. On the other hand, as a clinical drug-free tolerance case, one OLT patient, who has not required immunosuppressive drugs for the last 9 years following post-transplant lymphoproliferative disease (PTLD) was applied to proteomic analysis using liquid chromatography-mass spectrometry. In rat OLT, the results differentiated the varying protein expressions in sera extracted from tolerogeneic OLT rats as compared with naïve rats. Proteomic assay also demonstrated 12 differentiated spots exclusive to a drug-free OLT patient. Among these proteins, haptoglobin (Hp), which is related to inhibition of T-cell proliferation, was found to be up-regulated following clinical and experimental OLT, may play an important role in the maintenance of experimental and clinical drugfree OLT tolerance as a natural immunological suppressor. Further identification of common proteins specific to experimental and clinical drug-free OLT is currently been carried out. Proteomic analysis will allow us to develop biomarkers to establish a novel weaning protocol for patients on long-term immunosuppression to avoid major immunosuppressant-related complications.

P-21 – PROTEIN MICROARRAYS IDENTIFY NOVEL NHLA ANTIBODIES SPECIFIC TO CHRONIC RENAL ALLOGRAFT INJURY Tara Sigdel, Li Li, Hong Dai, Poonam Sansanwal, Szu-Chuan Hsieh, Minnie Sarwal. Department of Pediatrics, Stanford University, Stanford, CA, USA. Chronic allograft injury (CAI) is mostly a humoral response driven by Ab against HLA and non-HLA antigens. Method: To study novel nHLA targets of CAI injury, we analyzed 60 serum samples at 0, 6, and 24 mo from 20 renal txp patients, at the time of paired protocol biopsies. Protein arrays (8300 antigens) targets were used to measure reactivities of non-HLA Abs. Histological CAI lesions were characterized by a semi-quantitative score for CAI applied to each biopsy based on the Banff, CADI and CNIT scores. Seven kidney compartment specific gene expression analysis was performed on microarray data (GEO-GSE:3931) by ian ntegrated informatics approach (Li et al PNAS 2009) and CAI specific non-HLA targets were mapped to 7 kidney regions (inner and outer cortex, inner and outer medulla, papillary tips, renal pelvis and glomeruli). Data handling, normalization and hypergeometric enrichment analysis was performed using customized algorithms. Results: We observed an increase of reactivity of a total of 577 abs against non-HLA antigens in response of graft injury. When mapped to kidney specific compartments, specific nHLA abs specificites were found maximally against the renal pelvis, followed by the renal cortex. Fewer nHLA Ab were also found against the glomerulus and medulla specific renal antigens. Specific nHLA Ab are mounted at the time of established CAI to different renal compartments (P= 6.00E-5), and a defined subset of these are also idenitified as critical for CAI progression, as their signal intensity is detected at significant levels in sera, prior to established injury on subsequent graft biopsies. Among the significant antigens, some are previously

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identified in matrix remodelling injury pathways and cell cycle progression but a number of them suggest novel pathways of tissue injury. Integrated statistical analysis of delta creatnine clearance, proteinuria and semiquantitive biopsy compartment scores for tubular atrophy, interstitial fibrosis, mesangial matrix expansion, glomerulosclerosis and intimal thickening are currently underway. Conclusion: We have identified and extensivelt mapped kidney compartment specific non-HLA abs specific to CAI that can not only detect graft injury but also can track evolution of graft injury in real time. Further correlation of the impact of these nHLA Ab with specific triggers of graft injury are underway. This immune atlas of the kidney is an exciting opportunity to discover and refine our understanding and monitoring of CAI.

P-22 – NOVEL SHOTGUN PROTEOMICS APPROACH IDENTIFIES PROTEINS SPECIFIC FOR ACUTE RENAL TRANSPLANT REJECTION Tara Sigdel1, Amit Kaushal1, Angela Norbeck2, Wei-Jun Qian2, Wenzhong xiao1, David Camp2, Richard Smith2, Minnie Sarwal1. 1Department of Pediatrics, Stanford University, Stanford, CA; 2Pacific Northwest National Laboratory, Richland, WA; USA. Background: Proteomic analysis using high-throughput proteomic analysis of urine is a unique approach to identify disease specific urine protein biomarkers to diagnose, predict and improve renal transplantation. Methods: We used high throughput shotgun proteomics using cutting edge LC-MS/MS and ELISA to analyze a set of 92 urine samples, from patients with AR, stable grafts (STA), proteinuria (NS), and healthy controls (HC). Urine proteins >10 kDa were were subjected for strong cation exchange (SCx) fractionation. The data was acquired by using ThermoScientific LTQ linear ion trap mass spectrometer and the proteins were identified with SEQUESTTM. ELISA validation assays was performed on UMOD, SERPINF1, and CD44 so as to test the validity of the method and utility of identified proteins. Results: A total of 1446 urinary proteins were identified along with a number of NS specific, renal transplantation specific and AR specific proteins. We identified alterations in a number of specific urinary proteins in AR, primarily relating to MHC antigens, the complement cascade and extra-cellular matrix proteins. Nine proteins were identified only in AR urine that included HLA class II histocompatibility antigen, DP(W4) beta chain (HLA-DBP), HLA class II histocompatibility antigen, DRB1-8 beta chain (IgHM), C4b-binding protein alpha chain (C4BPA), MHC class II antigen (HLA-DR), Myosin light chain 1 (MYL6B), HLA class II histocompatibility antigen DQ(3) beta chain (HLA-DQB1). A subset of proteins (UMOD, SERPINF1 and CD44), have been further cross-validated by ELISA for significant differences in the abundance of these urinary proteins in AR. The area under the curve for AR classification by the ROC analysis was calculated as 97.3% for CD44, 93.2% for SERPINF, and 84.6% for UMOD. (Figure 1).

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P-23 – INTEGRATIVE URINARY PEPTIDOMICS IN RENAL TRANSPLANTATION IDENTIFIES NOVEL BIOMARKERS FOR ACUTE REJECTION Tara Sigdel, Bruce Ling, Ken Lau, Lihua Ying, Irwin Lau, James Schilling, Minnie Sarwal. 1Stanford University Medical School, Stanford University, Stanford, CA, USA. Background: Non-invasive, peptidomic analysis combined with microarray and Q-PCR analysis is a unique approach to identify disease specific urine peptide biomarkers to predict and improve present status of renal transplantation. Methods: A total of 70 archived urine samples from 50 renal transplant patients including biopsy proven AR, stable graft (STA), BK virus nephropathy (BKV), and 20 control including non-specific proteinurea (NS) or healthy controls (HC). We used MALDI-TOF mass spectrometry in the discovery step. A quantitative multiple reaction monitoring (MRM) assay was used to verify two potential candidate peptides of uromodulin (UMOD). Available Affymetirx GeneChips data on matched kidney transplant biopsies (20 AR and 20 STA) (NCBI GEO: GSE14328) was used for transcriptomic analysis. We extracted total RNA from kidney biopsy samples using TRIzol reagent. cDNA was synthesized from total RNA and Q-PCR were performed on 5 ng of cDNA. Results: Urine peptidomic analysis of 70 unique samples, from renal transplant patients (n=50) and controls (n=20), identified a specific panel of 53 peptides for acute rejection (AR). Peptide sequencing revealed underlying mechanisms of graft injury with a pivotal role for proteolytic degradation of uromodulin (UMOD) and a number of collagens including COL1A2 and COL3A1. The 40 peptide panel discriminated AR in both training (n=46) and test (n=24) sets (ROC, AUC>0.96). Integrative analysis of transcriptional data from paired renal biopsies revealed coordinated transcriptional changes for the corresponding genes, in addition to dysregulation of extracellular matrix proteins in AR (MMP7, SERPING1 and TIMP1). Q-PCR on an independent set of 34 transplant biopsies, validated microarry data and verified a 6 gene biomarker panel (COL1A2, COL3A1, UMOD, MMP7, SERPING1, TIMP1) that can classify AR with high specificity and sensitivity (ROC, AUC 0.98). Conclusion: Integrated urine peptidomic and biopsy transcriptional analyses identified potential AR specific marker peptides and revealed that key collagen remodeling pathways are modulated in AR tissue.

P-24 – AUTOANTIGEN BIOMARKER DISCOVERY THROUGH IMMUNOLOGICAL PROFILING WITH FUNCTIONAL PROTEIN MICROARRAYS Dawn Mattoon1, Mary Brodey1, Gengxin Chen1, Barry Schweitzer1, Thien Dinh1, Dhavel Patel2. 1Life Technologies, Cupertino, CA; 2Novartis; USA. The diagnostic value of serum autoantibodies for many diseases, including cancer, diabetes, and autoimmune disorders is well established. Identifying the antigens that elicit an autoimmune response can yield panels of biomarkers that can be used as classifiers for particular diseases, disease stages, or as predictors of patient outcomes. The present study utilized high content protein microarrays comprised of more than 5,000 purified full-length human proteins, including a panel of 25 known autoantigens, to evaluate immunological profiles across panels of serum samples derived from healthy donors and Systemic Lupus Erythemasosus (SLE) patients. Three statistical algorithms were applied to analyze data from individual microarrays, to compare data between populations and identify candidate biomarkers. This line of investigation identified a panel of 18 novel biomarkers which could differentiate SLE patients from healthy individuals more accurately than a panel of 10 established SLE biomarkers. Studies utilizing the Luminex platform and a custom-printed protein microarray were used to validate the results obtained from the ProtoArray . Taken together, our results suggest that functional protein microarray technology is a powerful new tool for the rapid discovery of autoantigen biomarkers.

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P-25 – THE ROLE OF JUN IN APOPTOTIC DEATH OF INSULIN-PRODUCING CELLS FOLLOWING MYCOPHENOLIC ACID TREATMENT Yuri Cho1, Yun-Jong Park1, Dong Jin Joo1,2, Hyung Joon Ahn1,3, Yu Seun Kim1,2. 1The Research Institute for Transplantation, Yonsei University College of Medicine; 2Department of Surgery, Yonsei University College of Medicine; 3Department of Surgery, Kyunghee University School of Medicine, Seoul, South Korea. Mycophenolic acid (MPA) is widely used as an immunosuppressive drug after organ transplantations including pancreatic islet cell transplantation. However, MPA has cellular toxicity, and causes apoptotic cell death in several insulinoma cell lines and primary isolated pancreatic β-cells. However, the signal transduction mechanisms underlying this process have not been fully explored yet. In this study, we have used diverse technologies including illumina-microarray to examine genes that are regulated time-dependently following MPA treatment. We found that thousands of genes were altered during MPA-induced apoptosis. Among them, jun gene expression pattern was significantly altered, and cyclin D1 and fas-L also were directly affected by MPA treatment. Pancreatic β-cell line INS-1E cells were treated with MPA for 12hr, 24hr and 36hr. Functional screening was determined by using small interference RNA (siRNA)-mediated knockdown and over-expression of jun gene in INS-1E cell line. Expression levels of mRNA and protein in target genes in cell line and primary cells were determined by quantitative real-time PCR (qRT-PCR) and western blot analysis. We found that MPA significantly increased cell death by Caspase-3, Caspase-8 and p-JNK activation. Fas-L expression levels were increased following MPA treatment. Over-expressed Jun decreased cell viability, and increased activation of p38 MAPK, phosphorylation of JNK, Caspase-3, -8 and Fas-L expression after MPA treatment. However, knockdown of jun by siRNA decreased MPA-induced cell death and p-JNK and Fas-L activation. In conclusion, MPA induced apoptosis in insulinsecreting cell via up-regulation of jun gene that were closely linked with MAPK pathway. In this process, activated JNK leads Fas-L long-term expression and accumulation of activated caspase-8. This accumulation of caspase-8 initiates apoptosis through activation of caspase-3. JNK/c-Jun/FasL/caspase-dependent apoptotic pathway will play a critical role in mediating MPA-induced apoptosis of pancreatic β-cell line.

P-26 – ANTI-NUCLEAR AUTOANTIBODIES AS BIOMARKERS IN LIVER TRANSPLANTATION TOLERANCE Toshiaki Nakano1,2, Chia-Yun Lai1, Shigeru Goto1,6, Li-Wen Hsu1, Kuei-Chen Chiang3, Kazuhisa Ono4, Hideo Kawarasaki5, Chao-Long Chen1. 1Center for Translational Research in Biomedical Sciences, Liver Transplantation Program, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Kaohsiung, Taiwan; 2Graduate Institute of Clinical Medical Sciences, Chang Gung University College of Medicine, Kaohsiung, Taiwan; 3Kazusa Institute for Drug Discovery, Josai International University, Chiba, Japan; 4Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Japan; 5Department of Transplant Surgery, Jichi Medical University, Tochigi, Japan; 6 Iwao Hospital, Oita, Japan. Unlike other organ transplantation, orthotopic liver transplantation (OLT) is unique in terms of tolerance where recipients can survive without immunosuppressive drugs after experimental and clinical OLT. Recent proteomic approach allows us to investigate the diagnostic and therapeutic potentials of various biomarkers specific to liver allograft tolerance. Especially, a new insight has been given into our study since we found that post-transplant autoimmune responses with high titer of antinuclear antibodies against histone H1 and high mobility group box 1 (HMGB1) play an important role in induction of liver allograft tolerance in OLT rats and clinical drug-free OLT patients. Our previous studies showed that either treatment of recipient rats with commercially available anti-histone H1 polyclonal Ab or immunization with calf thymus histone H1 could

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prolong allograft survival in heterotopic heart transplantation. We have also reported that the blockade of histone H1 modulated dendritic cells toward tolerogenic status, decreased the cytotoxicity of lymphokine activated killer and natural killer cells, and induced CD4+CD25+ T cells. For further analysis of this mechanism, we generated an immunosuppressive anti-histone H1 monoclonal Ab (16G9 mAb). From phage display peptide library, we have selected one peptide (designated SSV) that binds directly to 16G9 mAb. The binding of SSV to 16G9 mAb was inhibited by histone H1. Immunization of mice with SSV induced immunosuppression in serum, suggesting that SSV was an epitope responsible for the immunosuppressive activity of 16G9 mAb. Furthermore, SSV binds to serum of both tolerogeneic OLT rats and clinical drug-free OLT patients, and the binding to SSV was also inhibited by histone H1. Further studies of biomarkers related to anti-nuclear antibodies including peptide SSV will allow us to establish a novel diagnostic and therapeutic therapy in liver transplantation.

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Anglicheau, Dany Bendall, Sean Blydt-Hansen, Tom Butte, Atul Chapman, Jeremy Cho, Yuri Contag, Christopher Dando, Caroline Davis, Mark Davis, Ronald W. Dinh, Thien Fan, Alice Gao, Zhong Ghanekar, Smita Goodsaid, Federico Goto, Shigeru Halloran, Philip F. Heeger, Peter Israni, Ajay Jacobs, Sharoni Kay, Thomas Kelly, Kathleen Khatri, Purvesh Kirk, Allan D. Lee, Da Hye Li, Li Lord, Graham Maecker, Holden Maluf, Daniel Mannon, Roslyn B. Marincola, Francesco M. Marsh, Steven G.E. page 28 page 13 Poster-17 page 20 page 16 Poster-25 page 25 page 13 page 21 page 16 Poster-24 page 13 Poster-19 page 13 page 38 Poster-20 page 17 page 43 page 27 page 13 Poster-10 page 50 Poster-15, 16 page 36 Poster-13 Posters-04, 05 page 26 page 13 Poster-01 page 29 page 39 page 47 Mas, Valeria McGlynn, Liane McManus, Bruce Miqueu, Patrick Mohanakumar, Thalachallour Mueller, Thomas Naesens, Maarten Nakano, Toshiaki Park, Yun-Jong Radu, Caius Reed, Elaine Reeve, Jeff Reich, Michael Rödder, Silke Rush, David Sanchez-Fueyo, Alberto Sarwal, Minnie Sellarés, Joana Shen, Zhenya Sigdel, Tara Smith, Richard Suthanthiran, Manikkam Tibshirani, Robert Vivi, Antonio Wishart, David page 24 Poster-06 page 32 Poster-14 page 41 Poster-18 page 22, Poster-09 Poster-26 Poster-07 page 49 page 42 Poster-03 page 37 Poster-08 page 46 page 31 page 18 Poster-02 Poster-11 Posters-21, 22, 23 page 44 page 18 page 48 Poster-12 page 45

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