Composite Tissue Transplantation

TISSUE ENGINEERING I N T E L L I G E N C E U N I T 2 Charles W. Hewitt • Kirby S. Black Composite Tissue Transplantat...

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TISSUE ENGINEERING I N T E L L I G E N C E U N I T

2

Charles W. Hewitt • Kirby S. Black

Composite Tissue Transplantation

R.G. LANDES C O M P A N Y

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TISSUE ENGINEERING INTELLIGENCE UNIT 2

Composite Tissue Transplantation Charles W. Hewitt, Ph.D. Robert Wood Johnson Medical School Cooper Health System Camden, New Jersey

Kirby S. Black, Ph.D. CryoLife, Inc. Kennesaw, Georgia

R.G. LANDES COMPANY AUSTIN, TEXAS U.S.A.

TISSUE ENGINEERING INTELLIGENCE UNIT 2 Composite Tissue Transplantation R.G. LANDES COMPANY Austin, Texas, U.S.A. Copyright © 1999 R.G. Landes Company All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the U.S.A. Please address all inquiries to the Publishers: R.G. Landes Company, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081

ISBN: 1- 57059-554-2

While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data

Composite Tissue Transplantation / edited by Charles W. Hewitt, Kirby S. Black. p. cm. -- (Tissue engineering intelligence unit) ISBN 1-57059-554-2 (alk. paper) 1. Transplanting of organs, tissues, etc. I. Hewitt, Charles W. II. Black, Kirby S. III. Series. [DNLM: 1. Tissue Transplantation WO 660 C738 1998] Q89. C66 1998 617.9'5--dc21 98-8851 DNLM/DLC CIP for Library of Congress

TISSUE ENGINEERING INTELLIGENCE UNIT 2 PUBLISHER’S NOTE


R.G. Landes Company produces books in six Intelligence Unit series: Medical, Molecular Biology, Neuroscience, Tissue Engineering, Biotechnology and Environmental. The authors of our books are acknowledged leaders in their fields. Topics are unique; almost without exception, no similar books exist on these topics. Our goal is to publish books in important and rapidly changing areas of bioscience for sophisticated researchers and clinicians. To achieve this goal, we have accelerated our publishing program to conform to the fast pace at which information grows in bioscience. Most of ourRobert books are published within 90 to 120 days of receipt of Wood Johnson Medical School the manuscript. WeCooper would Health like to thank our readers for their System continuing interest and welcome any comments or suggestions they Camden, New Jersey may have for future books.

Charles W. Hewitt, Ph.D.

Kirby S. Black, Ph.D.

Stephanie Stewart

CryoLife, Inc. Production Manager Kennesaw, Georgia R.G. Landes Company

R.G. LANDES COMPANY AUSTIN, TEXAS U.S.A.

DEDICATION This book is dedicated to my family: my children Nicole, Ryan and Noah; my wife Michaele; my mother and father; my sister Nancy; my aunt Helen; my cousin Frank; and in–laws Dean, Dolores and Dave. They have supported my efforts with patience and encouragement during this effort. Charles W. Hewitt I would like to dedicate this book, in gratitude for their long time encouragement and support, to my wife Christine, my parents and my children Matthew, Colin and Meredith. Without their support and understanding, even the prospect of this effort would have been daunting. Kirby S. Black

CONTENTS Section I: Introduction 1. The First Limb Transplant Experiments with Cyclosporine .................. 3 Charles W. Hewitt and Kirby S. Black Introduction ............................................................................................. 3 The Questions .......................................................................................... 4 Materials and Methods ............................................................................ 4 Cyclosporine and Limb Transplants ...................................................... 4 The Return of Cosmas and Damian ....................................................... 5 Section II: Immunobiology of Composite Tissue Transplantation 2. Relative Antigenicity of Limb Allograft Components and Differential Rejection ......................................................................... 9 Mark A. Randolph and W.P. Andrew Lee Introduction ............................................................................................. 9 Transplant Immunology ....................................................................... 10 Humoral Response ................................................................................ 12 Immunogenic Components of Composite Tissue Skeletal Allografts 12 Relative Antigenicity of Limb Tissue .................................................... 14 Large Animal Data ................................................................................. 19 Conclusion ............................................................................................. 22 3. Induction of Transplantation Tolerance in Large Animal Models Without Long Term Immunosuppression: Strategies to Manipulate the Immune System of the Fetal and the Adult Recipient .......................................................................... 31 J. Peter Rubin, Sheldon Cober, Peter E. M. Butler and W. P. Andrew Lee Introduction ........................................................................................... 31 Manipulation of the Adult Immune System: Swine Model ................ 32 Manipulation of the Fetal Immune System: Swine Model .................. 34 4. Dendritic Cells and Alloimmune Chimerism in Limb Transplantation ......................................................................... 41 Mia Talmor, Ralph M. Steinman and Lloyd A. Hoffman Introduction ........................................................................................... 41 The Dendritic Cell System .................................................................... 41 Identification of Mature DCs ................................................................ 42 Maturation and Migration of DCs ....................................................... 44 The Dendritic Cell in Transplantation ................................................. 44 Dendritic Cells in Limb Transplantation ............................................. 45

Section III: Vascularized Bone Marrow Transplantation 5. Composite Tissue/Vascularized Bone Marrow Transplantation: Development of Donor-Host Immune Chimerism and Tolerance ..... 57 Charles W. Hewitt and Kirby S. Black Introduction ........................................................................................... 57 Initial Experiments on Immune Chimerism in Rat Limb Transplant Recipients ................................................... 57 Flow Cytometric Analysis for T Cell Chimerism ................................. 58 Cellular Kinetics of Chimerism and Mechanisms of Immune Nonresponsiveness ........................................................ 59 Discussion .............................................................................................. 60 6. Vascularized Bone Marrow Transplantation: Pathology of Composite Tissue Transplantation-Induced Graft Versus Host Disease ............... 65 Rajen Ramsamooj and Charles W. Hewitt Background ............................................................................................ 65 Gross Clinical Aspects of VBMT .......................................................... 66 Acute and Chronic GVHD .................................................................... 67 Histopathology ...................................................................................... 67 Conclusions ........................................................................................... 68 Section IV: New Composite Tissue Transplant Models 7. New Models of Vascularized Bone Marrow Transplantation Based on Composite Tissue Allografts ............................................................. 73 Martha S. Matthews and Charles W. Hewitt Introduction ........................................................................................... 73 Background ............................................................................................ 73 Models of Vascularized Bone Transfer ................................................. 75 Laboratory Investigations ..................................................................... 75 Conclusions ........................................................................................... 76 8. Composite Tissue Transplants in Rats: A Whole Limb/Hemipelvis Model ......................................................... 79 Kirby S. Black and Charles W. Hewitt Background ............................................................................................ 79 CTA Functionality ................................................................................. 79 Surgical Model ....................................................................................... 80 Conclusion ............................................................................................. 82 Section V: Individual Component Tissues of the Composite Tissue Transplant 9. Transplantation of the Peripheral Nerve Allograft ............................... 87 Vaishali B. Doolabh and Susan E. Mackinnon Introduction ........................................................................................... 87 Transplantation Immunology .............................................................. 88

The Nerve Allograft Response .............................................................. 90 Long Nerve Allograft Regeneration ...................................................... 93 Nerve Allograft Preservation and Storage ............................................ 93 Nonspecific Immunosuppressive Strategies ........................................ 94 Induction of Donor Specific Immunosuppression ............................. 97 UV-B Irradiation ................................................................................... 98 Immune Privilege ................................................................................ 100 Clinical Applications ........................................................................... 100 Conclusion ........................................................................................... 101 10. Peripheral Nerve Allotransplants Immunosuppressed with 15-Deoxyspergualin ...................................................................... 107 Keiichi Muramatsu and Kazuteru Doi Introduction ......................................................................................... 107 Peripheral Nerve Allo- and Xenotransplantation .............................. 107 Immunosuppressive Drugs Applied to Peripheral Nerve Allograft .. 108 Immunosuppressive Effects of 15-Deoxyspergualin ......................... 109 Peripheral Nerve Allotransplantation Using 15-Deoxyspergualin ... 112 Conclusions and Future Directions .................................................... 115 11. Therapeutic Uses of Muscle and Factors Controlling the Efficiency of Whole Muscle Graft Regeneration ........................... 121 Miranda D. Grounds and John K. McGeachie Therapeutic Benefits of Regeneration and Grafting .......................... 121 Factors Controlling the Regeneration of Whole Muscle Grafts ........ 123 Myofiber Survival and the Importance of the External Lamina ....... 126 Identification and Behavior of Myoblasts .......................................... 127 Inflammatory Cell Response and Revascularization ......................... 128 Denervation ......................................................................................... 130 Influence of the Host Environment .................................................... 132 Clinical Implications ........................................................................... 132 12. Myoblast Transfer as a Platform Technology of Gene Therapy and Tissue Engineering ......................................................................... 139 Peter K. Law Introduction ......................................................................................... 139 Vectors ................................................................................................. 139 MTT Technology ................................................................................. 142 Muscular Dystrophies—The Testing Ground ................................... 143 Animal Experiments ............................................................................ 143 Clinical Trials ....................................................................................... 145 Future Perspectives .............................................................................. 148 My Vision ............................................................................................. 149 Summary .............................................................................................. 150 Conclusion ........................................................................................... 151

13. Meniscal Allograft Transplantation ..................................................... 157 Thomas R. Carter Introduction ......................................................................................... 157 Functions of the Meniscus .................................................................. 157 Effects of Meniscectomy ...................................................................... 158 Processing of Meniscal Allografts ....................................................... 158 Animal Studies ..................................................................................... 160 Clinical Studies .................................................................................... 161 Indications ........................................................................................... 162 Surgical Techniques ............................................................................. 163 Author’s Experience ............................................................................ 164 Summary .............................................................................................. 166 Section VI: Immunosuppressants for Composite Tissue Transplantation 14. Potential New Immunosuppressants for Composite Tissue Transplantation ......................................................................... 173 Daniel Jung and Barry D. Kahan Introduction ......................................................................................... 173 Cyclosporine (CsA) Analogs ............................................................... 173 Other New Immunosuppressants ....................................................... 175 15. Rationale for Local Immunosuppression in Composite Tissue Allografting ......................................................... 197 Scott A. Gruber, Mansour V. Shirbacheh and Jon W. Jones Introduction ......................................................................................... 197 Rat CTA Models .................................................................................. 198 Canine and Primate CTA Models ....................................................... 199 Rejection of CTAs ................................................................................ 199 Local Immunosuppression ................................................................. 200 Conclusion ........................................................................................... 201 16. Long Term Limb and Nerve Allograft Survival with FK506 Immunosuppression ......................................................... 205 Neil F. Jones and Esther Voegelin Introduction ......................................................................................... 205 Mechanism of FK506 .......................................................................... 205 Limb Transplantation in Rats Immunosuppressed with FK506 ...... 206 Other Studies of Limb Transplantation with FK506 Immunosuppression ................................................... 209 Limb Transplantation with Other Immunosuppressive Agents ....... 215 Conclusions: Limb Transplantation and Immunosuppression ........ 216 Nerve Graft Transplantation in Rats Immunosuppressed with FK506 ....................................................................................... 217

Other Studies of Nerve Graft Transplantation Using FK506 and Cyclosporine Immunosuppression ......................................... 217 Conclusions: Nerve Graft Transplantation and Immunosuppression ................................................................ 219 17. Allogeneic Rat Hindlimb Transplants Immunosuppressed with Mycophenolate Mofetil (RS-61443) ............................................ 225 Stephen J. Mathes, Robert D. Foster and James P. Anthony Introduction ......................................................................................... 225 Mycophenolate Mofetil: Mechanism of Action and Clinical Efficacy ........................................................................ 225 Experimental Efficacy in Composite Tissue Transplantation ........... 226 Functional Outcomes: Nerve Regeneration ....................................... 234 Conclusion ........................................................................................... 236 18. Long Term Prevention of Rejection and Combination Drug Therapy .......................................................... 239 James P. Anthony, Robert D. Foster and Stephen J. Mathes Introduction ......................................................................................... 239 Long Term Prevention of Rejection ................................................... 239 Combination Drug Therapy ............................................................... 242 Conclusion ........................................................................................... 244 19. Efficacy of Rapamycin and FK506 in Prolonging Rat Hindlimb Allograft Survival .................................. 247 James Chang, Yvonne L. Karanas and Barry H.J. Press Introduction ......................................................................................... 247 History .................................................................................................. 247 Mechanism ........................................................................................... 248 Toxicity ................................................................................................ 248 Vital Organ Transplantation ............................................................... 248 Hindlimb Research .............................................................................. 248 Rapamycin ........................................................................................... 249 Mechanism ........................................................................................... 250 Toxicity ................................................................................................ 250 Vital Organ Transplantation ............................................................... 250 Hindlimb .............................................................................................. 250 Conclusions ......................................................................................... 252 Section VII: Clinical Composite Tissue Transplantation 20. Allogeneic Vascularized Transplantation of Human Knee Joints ........................................................................... 257 Gunther O. Hofmann Introduction ......................................................................................... 257 History .................................................................................................. 257 Indications ........................................................................................... 257

Trauma Management .......................................................................... 258 Transplantation ................................................................................... 260 Immunosuppression ........................................................................... 262 Follow-Up ............................................................................................ 262 Results .................................................................................................. 262 Discussion and Overview .................................................................... 262 21. Clinical Transplantation of Skin Using Immunosuppression ............ 267 Bruce M. Achauer and Victoria Vander Kam Introduction ......................................................................................... 267 Skin as an Immune Organ .................................................................. 268 Clinical Application ............................................................................. 269 Topical Immunosuppression .............................................................. 270 Skin Modification ................................................................................ 270 Conclusions ......................................................................................... 270 22. The Clinical Future of Composite Tissue Transplantation ................ 273 Robert D. Foster and James P. Anthony Introduction ......................................................................................... 273 Composite Tissue Transplantation: Past and Present ....................... 274 The First Significant Step Forward: Cyclosporine and Newer Immunosuppressive Agents ................. 275 Functional Recovery: The Application of Nerve Allografting from Animal Models to Humans ................................................... 277 Composite Tissue Transplantation: The Future—Towards More Complex Study Protocols: Canine Larynx Allotransplantation ... 278 Transplant Survival Without Immunosuppression: Allogeneic Tolerance Induction ..................................................... 282 Conclusion ........................................................................................... 282 Conclusion ....................................................................................................... 287 Index ................................................................................................................ 289

EDITORS Charles W. Hewitt, Ph.D. Robert Wood Johnson Medical School Cooper Health System Camden, New Jersey Chapters 1, 5, 6, 7, 8 Kirby S. Black, Ph.D. CryoLife, Inc. Kennesaw, Georgia Chapters 1, 5, 8

CONTRIBUTORS Bruce M. Achauer, M.D. UCI Burn Center University of California Irvine Medical Center Orange, California Chapter 21 James P. Anthony, M.D. University of California at San Francisco San Francisco, California Chapter17, 18, 22 Peter E. M. Butler, M.D. Massachusetts General Hospital Harvard Medical School Boston, Massachusettes Chapter 3 Thomas R. Carter, M.D. The Orthopedic Clinic Arizona State University Phoenix, Arizona Chapter 13 James Chang, M.D. Stanford University Medical Center Stanford, California Chapter 19

Sheldon Cober, M.D. Massachusetts General Hospital Harvard Medical School Boston, Massachusettes Chapter 3 Vaishali B. Doolabh, M.D. Washington University School of Medicine St. Louis, Missouri Chapter 9 Kazuteru Doi, M.D. Yamaguchi University School of Medicine Yamaguchi, Japan Chapter 10 Robert D. Foster, M.D. University of California at San Francisco San Francisco, California Chapter 17, 18, 22 Miranda D. Grounds, Ph.D. The University of Western Australia Australia Chapter 11

Scott A. Gruber, MD, Ph.D. University of Texas at Houston Health Center Houston, Texas Chapter 15 Gunther O. Hofmann, M.D., Ph.D. Trauma Center Murnau University of Munich Munich, Germany Chapter 20 Lloyd A. Hoffman, M.D., F.A.C.S. Rockefeller University and The New York Hospital Cornell Medical Center New York, New York Chapter 4 Daniel Jung, M.D. The University of Texas Medical School at Houston Houston, Texas Chapter 14 Jon W. Jones, M.D. University of Louisville School of Medicine Louisville, Kentucky Chapter 15 Neil F. Jones, M.D. UCLA Medical Center Los Angeles, California Chapter 16 Barry D. Kahan, Ph.D., M.D. The University of Texas Medical School at Houston Houston, Texas Chapter 14 Victoria Vander Kam, RN, BS, CPSN UCI Burn Center University of California Irvine Medical Center Orange, California Chapter 21

Yvonne L. Karanas, M.D. Stanford University Medical Center Stanford, California Chapter 19 Peter K. Law, Ph.D. Cell Therapy Research Foundation Memphis, Tennessee Chapter 12 W.P. Andrew Lee, M.D. Massachusetts General Hospital Harvard Medical School Boston, Massachusettes Chapter 2, 3 Susan E. Mackinnon, M.D. Washington University School of Medicine St. Louis, Missouri Chapter 9 Stephen J. Mathes, M.D. University of California at San Francisco San Francisco, California Chapter 17, 18 Martha S. Matthews, M.D. Cooper Health System/University Medical Center Camden, New Jersey Chapter 7 John K. McGeachie The University of Western Australia Australia Chapter 11 Keiichi Muramatsu, M.D., Mayo Clinic Rochester, Minnesota Chapter 10

Barry H.J. Press, M.D., F.A.C.S. Stanford University Medical Center Stanford, California Chapter 19 Rajen Ramsamooj, M.D. University of California, Davis Sacramento, California Chapter 6 Mark A. Randolph, M.A.S. Massachusetts General Hospital Harvard Medical School Boston, Massachusettes Chapter 2 J. Peter Rubin, M.D. Massachusetts General Hospital Harvard Medical School Boston, Massachusettes Chapter 3 Mansour V. Shirbacheh, M.D. University of Louisville School of Medicine Louisville, Kentucky Chapter 15 Ralph M. Steinman, M.D. Rockefeller University and The New York Hospital Cornell Medical Center New York, New York Chapter 4 Mia Talmor, M.D. Rockefeller University and The New York Hospital Cornell Medical Center New York, New York Chapter 4 Esther Voegelin, M.D. UCLA Medical Center Center for Health Sciences Los Angeles, California Chapter 16

PREFACE

U

pon entering a burn unit, it is absolutely clear how devastating a massive injury like a burn can be. Even though in many cases these individuals have not been injured fatally, the damage done can have a profound impact on their lives, resulting in what has been called social death. When reflecting on the marvelous work done by Nobel prize winner Jodi Williams, it is obvious that the individuals who have suffered from land mine injuries will forever bear tragic scars and missing limbs. Situations such as these have prompted investigation of composite tissue transplants. Modern transplantation initially focused on life threatening diseases. However, with advances in medical technology aside from transplantation, it can be postulated that kidney transplants are related more to quality of life than sparing of life. Similarly, we would argue from both a scientific and medical perspective that composite tissue transplantation should be a very viable part of the surgical armamentarium. Its use is warranted to repair such heinous integumentary/musculoskeletal injuries as those described above, as well as purely for quality of life considerations. The term “composite tissue transplant” was spawned by the observation that this particular branch of transplantation did not include one specific tissue or organ, but a unique combination of tissues. For example, if one were to reconstruct a lost limb, this would include a “composite” of skin, muscle, bone, joint, nerve, blood vessels and connective tissue. However, other repairs could involve simply two, three or four of the individual tissues or possibly even more. In addition, when studying a whole organ transplant such as kidney or liver, the function of that whole organ can usually be clearly identified by various physiologic and biochemical metabolic processes. However, when one examines this broad group of “composite” tissues and their interactions, function may be more difficult to define. Thus, there are several unique aspects to composite tissue transplantation that warrant very different approaches compared to organ transplantation. This book has been compiled to provide an overview of these important and related subjects. There has been an attempt to include the basic principles of immunosuppression and immunobiology as they relate to ongoing models of composite tissue transplantation, as well as a historical perspective on the subject, and to examine some of the first clinical applications in this emerging arena. Additional questions that are addressed herein include neuromuscular function, tolerance, potential for graft versus host disease, potential for bone marrow transplantation, muscle cell chimerism and many other subjects detailed by a diverse group of laboratories and investigators. The history of transplantation has revealed that this is a science driven by compelling individuals, scientists and surgeons. It is the editors’ hope that this book will inform, excite, and inspire those already working in the composite

tissue transplantation field, and serve as a basis for new individuals working in this area. The dream has been, and continues to be, restoration and repair of those individuals who have suffered life changing integumentary/ musculoskeletal injuries that have severely and dramatically affected their quality of life. Kirby S. Black Charles W. Hewitt

EDITORS' NOTE

T

he editor's chaired a workshop in September 1991 on the clinical use of Composite Tissue Allografts. This conference was sponsored by The Rehabilitation Research and Development Service of the Department of Veteran Affairs. Participants of the workshop evaluated the current state of composite tissue transplantation and the possibilities for clinical use. As noted in previous publications by the editors,1-4 it was the conclusion of the workshop attendees that in the relative near future, composite tissue transplantation would be a clinical reality. In fact it was concluded that “historic” first clinical applications of composite tissue transplants would occur in five years. In this regard, the editor’s feel compelled to comment on recent events reported by the popular news media regarding the first human hand transplant performed during the modern era of immunosuppression recently in France. For example, the Chicago Tribune published an article on Thursday, November 12, 1998, which reported that Dr. Erle Owen lead an international surgical team who performed the unprecedented transplant procedure. Although there is some controversy surrounding this particular historic feat (and whether this in fact even represents the first true clinical composite tissue transplant), these efforts do represent the closest analogy to our original limb transplant experiments performed in rats with cyclosporine 16 years ago.5,6 Thus, our conference attendees were in error by three years with respect to the first application of clinical composite tissue transplantation. However, most notable in the popular news reports is the fact that this first human hand transplant recipient has shown no evidence of rejection. In conclusion, as to clinical possibilities, it appears that we are now beginning to realize the foundation of studies performed by numorous investigators in the field, many of which are contributors to this book. It seems more than likely that the field of composite tissue transplantation will continue to evolve clinically and that significant new surgical treatments for integumentary/musculoskeletal disorders will be further developed. Charles W. Hewitt, Ph.D. Kirby S. Black, Ph.D. References: 1. Black KS, Hewitt CW. Report: Composite Tissue Workshop, Department of Veteran Affairs, Rehabilitation Research and Development Service, Washington, DC, 1991. 2. Hewitt CW, Puglisi RN, Black KS. Current state of composite tissue and limb allotransplatation: Does present data justify clinical application? Transplant Proc 1995; 27(1):1414-1415. 3. Hewitt CW. Update and outline of the experimental problems facing clinical composite tissue transplantation. Transplant Proc 1998; 30:2704-2707.

4. Llull R, Beko KR, Black KS et al. Composite tissue allotransplatation: Perspectives concerning eventual clinical exploitation. Transpl Rev 1992; 6(3):175-188. 5. Black KS, Hewitt CW, Fraser LA et al. Cosmas and Damian in the Laboratory. N Engl J Med 1982; 306:368-369. 6. Hewitt CW, Black KS, Fraser LA et al. Cyclosporin A (CyA) is superior to donorspecific blood (DSB) transfusion for the extensive prolongation of rat limb allograft survival. Transplant Proc 1983; 15:514-517.

ACKNOWLEDGMENTS I would like to acknowledge my good friend and colleague, Dr. Kirby S. Black, who shared a unique and unconventional vision with me 20 years ago of the possibilities for allotransplantation of composite tissues. That vision is still alive and successfully being pursued. I would also like to extend my appreciation to Mrs. Lisa Stressman, Administrative Coordinator, and Ms. Maria Perez, who served as editorial assistant on this book. I thank the contributors to this book, for their participation and for their interest in composite tissue allotransplantation. I would like to extend my appreciation to Chief of Surgery Anthony J. DelRossi, M.D., for encouraging an environment supportive of surgical research at Robert Wood Johnson Medical School, Camden/Cooper Health System. Lastly, I wish to thank various colleagues who have had a positive and beneficial impact upon my career: Edward Doolin, M.D., Jill Adler-Moore, Ph.D., and the late Edwin Howard, D.V.M., Ph.D. This work was supported in part by awards from the Orthopedic Research and Education Foundation, the American Heart Association, the Plastic Surgery Educational Foundation, the International Association of Fire Fighters Burn Foundation, the Foundation of UMDNJ, Edge Scientific, L.L.C., and by faculty practice grants from Robert Wood Johnson Medical School/Cooper Hospital/ University Medical Center. Address correspondence to Surgical Research, Cooper Hospital/University Medical Center, Three Cooper Plaza, Suite 411, Camden, New Jersey 08103. Charles W. Hewitt I would like to extend my appreciation to Christine Black, my wife of 22 years, for her assistance in reviewing and proof reading these manuscripts. Kirby S. Black

The First Limb Transplant Experiments with Cyclosporine

Section I Introduction

1

CHAPTER 1

The First Limb Transplant Experiments with Cyclosporine Charles W. Hewitt and Kirby S. Black

Introduction

I

n 1978, the Medical Science One Building was under construction at the University of California, Irvine, College of Medicine. At that time, Kirby Black was serving as Research Director for the Plastic Surgery Division within the Department of Surgery, and Charles W. Hewitt was serving as Director of Research for the Division of Urology within that same department. Following completion of the new Medical Science One Building and placement of Surgical Research within that building, serendipity brought Kirby Black and Charles Hewitt together; they found themselves next door to one another, each directing the research efforts of their respective divisions. This was next door in terms of both laboratories and offices. Kirby Black was interested at the time in developing models of ischemia reperfusion injury and flap studies in plastic surgery. Charles Hewitt was primarily interested in studying transplant rejection, as the Division of Urology was the division primarily responsible for kidney transplantation at the University of California, Irvine. He was interested in studying mechanisms of tolerance induction, and had some early successes in this area using a kidney transplant model. Kirby Black was interested in examining the effects of temperature on tissue survival in his ischemia reperfusion models. Yet, he did not have a refrigerator/ freezer available for this purpose. Thus, he borrowed one, an environmental chamber with a see-through glass door, in Hewitt’s lab. Now, this particular ischemia reperfusion model was an interesting one, in that it involved amputation and replantation of a rat hind limb. The Plastic Surgery Research Laboratory was looking to investigate mechanisms of ischemia reperfusion injury by reattachment of these preserved amputated limbs under various conditions. Each day, Hewitt would notice this preserved amputated limb in his refrigerated environmental chamber and after several days he became increasingly curious as to what experiments Plastic Surgery was undertaking. A discussion concerning these experiments ensued between Hewitt and Black, and ultimately an investigative partnership was formed, along with a very meaningful friendship. Each investigator became interested in the other investigator’s research, and further discussions ensued.

Composite Tissue Transplantation, edited by Charles W. Hewitt and Kirby S. Black. ©1999 R.G. Landes Company.

4


The Questions From the model developed for replantation of the amputated limbs, and the results of organ graft prolongation produced in Hewitt’s lab, came a mutual realization that it would be very interesting to test additional mechanisms related to transplantation using the leg model. Thus, a fairly unique question was eventually decided upon by the two investigators, namely, could this model be used to study limb transplantation and some of the developments found successful in prolonging organ transplantation? It was hypothesized that this new and unique type of transplant, an integumentary musculoskeletal transplants would be particularly useful in plastic and reconstructive surgery applications and indications. And so, this bond was formed, initially as an alliance between a transplantation immunology laboratory and a plastic surgery microvascular surgical laboratory, which eventually blended and integrated the two investigators’ interests into one focus of pursuit over the next 20 years, and which represented the pioneering efforts of these investigators in the field of composite tissue transplantation.

Materials and Methods However, all was not success in the early years. For a year and a half, these investigators used every proven technique that was successfully developed in the kidney transplant model and applied it to the rat hind limb composite tissue transplant model. There were only minor successes, with maybe a few days here and a few days there of prolonged graft survival.1,2 It readily became apparent that this particular transplant model was indeed a difficult one in which to achieve graft prolongation and success. The failures became frustrating, resulting in several discussions about dropping the whole idea of composite tissue transplantation, as it just did not seem feasible in view of the results that were obtained. Then, during this time, a novel new immunosuppressive compound came onto the scene. However, its reputation was rather uncertain. Cyclosporine’s promise was in debate, due to concerns about its reported various toxicities.3,4 In the Black/Hewitt laboratory, indeed, it was a drug that was initially viewed as not very promising. However, as the number of failures in prolonging limb transplant survival mounted, this attitude changed; any new promising intervention or drug that would achieve the desired objectives was considered.

Cyclosporine and Limb Transplants The introduction of cyclosporine into the laboratory was actually rather embarrassing. Although Kirby Black and Charles Hewitt were both quite aware of the new drug in development, a student who was working on the limb transplant project approached them one day about a new remarkable miracle immunosuppressant agent that he had read about in the literature. It turned out that this “literature” was a story put forth in the Los Angeles Times. The student, who at that time was a dedicated and motivated individual with good work habits in the laboratory, obviously found this journal more to his liking than Transplantation or the Lancet. Nevertheless, at this student’s urging, Hewitt and Black decided that it was time to try cyclosporine in the limb transplant model. It was decided that the student would write to the company that was developing the drug (Sandoz) to see if an experimental quantity could be obtained to study whether this “miracle drug” would prolong limb transplant survival. The student outlined a brief experiment that he wanted to try and wrote to Sandoz regarding his intentions. In the student’s haste, he neglected to have the principal investigators of the laboratory read the letter before it was sent. To their amazement and embarrassment, the student had mentioned how he had seen promising new data reviewed in the Los Angeles Times, as opposed to some prestigious medical journal. Yet, to their surprise, David Winter, a wonderful scientist and gentleman who was then the Director of Immunology at Sandoz, became intrigued with the possibilities of these proposed limb

The First Limb Transplant Experiments with Cyclosporine

5

Fig. 1.1. CTA limb transplant recipient (LBN to LEW) given cyclosporine A (CsA) at 25 mg/kg/d for 20 days. This particular recipient went on to tolerance and indefinite graft survival.

transplantation experiments. He sent the rather large quantity (as it turned out) of 1 g of cyclosporine to try in these rather unique experiments. Shortly after the drug arrived, it was mixed according to Sandoz’ directions and the laboratory initiated testing of the new compound for its ability to extend limb transplant survival. Almost immediately, within the first week of experimentation, it was noted that there were dramatic differences between these experiments and any previous ones that had been undertaken in the laboratory. By days 5 and 6 there was a notable stubble of hair growth forming on the limb. This had never been achieved before. And, after another week passed, luxuriant black hair growth completely covered the transplanted limb. The animals did quite well, and soon they were actually walking on these new limb transplants (Fig. 1.1). It was truly amazing, and the investigators in the laboratory became cyclosporine converts and even zealots. They were so impressed with the ability of this compound to prolong tissue transplant survival compared to any former drug therapy or treatment tried previously to manipulate the immune system that they were convinced this was a true 20th century miracle drug. The first experiments with cyclosporine were detailed in the literature,2,5-7 and the rest is history, so to speak.

The Return of Cosmas and Damian It should be noted that serendipity and coincidence were working to the advantage of the laboratory during this time. It turned out that there was a well known investigator who was also advancing his career in those days based on the development of this new miracle immunosuppressive drug. His name was Barry Kahan, another cyclosporine advocate. At about this same time, Dr. Kahan wrote a rather compelling editorial in the New England Journal of Medicine regarding analogies between the legend of Cosmas and Damian and


6

whether cyclosporine would usher in the 20th century equivalent of that legend.8 The title of the article was “Cosmas and Damian in the 20th Century.” The legend of Cosmas and Damian, twin saints, one a physician and one a surgeon, is well known to the transplant community. They have served as a symbol for the desired successes of transplantation, having themselves performed a miraculous transplantation back in the 4th century AD. The coincidence was that the type of tissue they actually transplanted was reported to be a limb. It had further similarities to the experiments performed in the Black-Hewitt laboratories. The limb was taken from an Ethiopian Moor and transplanted to a Roman caretaker of one of their shrines. As famous paintings depict, the black leg was successfully transplanted onto this white individual due to the miraculous healing powers of the sainted twins. However, the real miracle was that the sainted brothers had performed this procedure posthumously, since they were beheaded in the 3rd century AD as Christian martyrs.5,8,9 Due to the analogies drawn between the miraculous feats of Cosmas and Damian and the new compound cyclosporine, to in effect achieve the 20th century equivalent, some poetic license was granted in a short report published in the New England Journal of Medicine, in response to Dr. Kahan’s editorial appearing in a previous issue.5 The first reported results with limb transplants and cyclosporine were from small quantities of that initial cyclosporine granted by David Winter from Sandoz. The results were used to answer Dr. Kahan’s editorial in a most affirmative manner, again drawing the important analogies of the miraculous feat of the drug to prolong limb transplant survival, similar to what Cosmas and Damian had done in the 4th Century. There were no winged angels flying around the microsurgery operating table at Irvine; the real miracle was in this immunosuppressive compound. The other serendipitous analogy involved the genetic model that the investigators had chosen in Irvine due to the immunology and transplant barrier of the rat strains utilized: a black donor and white recipient were used. This further emphasized the parallels to the original Cosmas and Damian legend.

Acknowledgements This work was supported in part by awards from the Orthopedic Research and Education Foundation, the American Heart Association, the Plastic Surgery Educational Foundation, the International Association of Fire Fighters Burn Foundation, the Foundation of UMDNJ, BioFX Laboratories, L.L.C., Edge Scientific, L.L.C., and by faculty practice grants from Robert Wood Johnson Medical School/Cooper Hospital/University Medical Center.

References 1. Black KS, Hewitt CW, Woodard TL et al. Efforts to enhance survival of limb allografts by prior administration of whole blood in rats using a new survival end-point. J Microsurgery 1982; 3:162-167. 2. Hewitt CW, Black KS, Fraser LA et al. Cyclosporin A (CsA) is superior to prior donorspecific blood (DSB) transfusion for the extensive prolongation of rat limb allograft survival. Transplant Proc 1983; 15:514-517. 3. Thomson AW, Cameron ID. Immune suppression with cyclosporin A—optimism and caution. Scott Med J 1981; 26(2):139-144. 4. Kahan BD. Cyclosporine: The agent and its actions. Transplant Proc 1985; 17(4):5-18. 5. Black KS, Hewitt CW, Fraser LA et al. Cosmas and Damian in the laboratory. N Engl J Med 1982; 306:368-369. 6. Hewitt CW, Black KS, Fraser LA et al. Composite tissue (limb) allografts in rats: I. Dosedependent increase in survival with cyclosporine. Transplantation 1985; 39:360-364. 7. Black KS, Hewitt CW, Fraser LA et al. Composite tissue (limb) allografts in rats: II. Indefinite survival using low dose cyclosporine. Transplantation 1985; 39:365-368. 8. Kahan BD. Cosmas and Damian in the 20th century. N Engl J Med 1981; 305(5):280-281. 9. Kahan BD. Cosmas and Damian revisited. Transplant Proc 1983; 4(Suppl 1):2211.

Relative Antigenicity of Limb Allograft Components and Differential Rejection

Section II Immunobiology of Composite Tissue Transplantation

7

CHAPTER 2

Relative Antigenicity of Limb Allograft Components and Differential Rejection Mark A. Randolph and W.P. Andrew Lee

Introduction

T

he use of autologous skin grafts was studied intensely during the 19th century, and refinements in harvesting grafts and surgical technique made them common treatments for defect coverage. Limitations on donor sites, however, encouraged many clinicians and investigators to explore the use of allogeneic or xenogenic tissues, and skin allografts and nonvascularized bone allografts have been the subject of numerous investigations since the beginning of this century. Schone in 1912 and Lexer in 1914 demonstrated that allogeneic and xenogenic skin grafts to humans did not survive more than three weeks after transplantation. Two decades later, Padgett reported rejection of skin allografts within 35 days in a series of 40 patients; however, he described the indefinite survival of skin grafts transplanted between identical twins.1 Brown confirmed the observation that skin allografts between identical twins survived.2 A report by Brown and McDowell in 1942 described the use of skin allografts to cover massive burn injuries in humans, but this was followed by a report later in the year on the dissolution or, as we know today, rejection of the grafts.3,4 They also noted that placing a second set of skin allografts on the patients would inevitably lead to complete failure of the grafts.4 In 1943, Gibson, a plastic surgeon at the Glasgow Royal Infirmary, and Medawar reported on their use of skin allografts for treating burned pilots; they, too, confirmed the rejection of a second set of skin allografts.5 Medawar, in collaboration with Brent and Billingham, investigated this phenomenon of “second-set rejection” which laid the foundation for modern transplantation immunology.6 Since then, much has been learned about the cellular and molecular mechanisms of the rejection processes. In 1955, Murray et al reported on the successful transplantation of kidneys between identical twins.7 Subsequent advances in tissue typing, surgical technique, and immunosuppressive therapy in the last three decades have resulted in remarkable success in clinical allotransplantation of vital visceral organs. Kidney transplantation is now the preferred treatment for chronic renal failure, while the transplantation of heart, liver, and, more recently, heart-lung and pancreas are being achieved with improved results.8 Replantation of a child’s severed arm by Malt in 1962 opened the door for complex vascular reconstruction of limb tissues.9 Improved microsurgical techniques and tools and a reexamination of the vascular


10


anatomy supplying bones, muscles, and skin led to rapid expansion of free flap transfers for defect repair and coverage. Some investigators even sought to use vascularized allogeneic tissues, and Goldwyn reported on the homotransplantation of limbs in dogs in the late 1960s.10 Genetic matching and immunosuppressive therapy were rudimentary at that time and many experiments with allogeneic musculoskeletal tissues failed. Interest in allogenic musculoskeletal tissue transplantation resurged in the 1980s with the introduction of improved immunosuppressive agents such as cyclosporine, and of refined genetic matching, particularly in the rat.11 Large animal studies, primarily in dogs, have been confounded by the lack of genetic lines necessary for histocompatibility matching.12-18 Presently, the transplantation of vascularized limb tissue allografts can be achieved only with generalized host immunosuppression which results in significant systemic toxicity, thereby precluding its clinical use. Whereas the use of potentially morbid immunosuppressive agents can be justified for the transplantation of vital visceral organs, their prolonged use for transplanting musculoskeletal tissues cannot easily be rationalized. The transplantation of a vascularized limb allograft or its tissue components (skin, subcutaneous tissue, muscle, bone, blood vessels) has been made technically possible by the advent of microvascular surgery. The availability of limb tissue allografts would greatly expand the horizon of reconstructive surgery. Such allografts would be of virtually unlimited supply, while the problem of donor site morbidity would be obviated. Many researchers have demonstrated survival of various limb tissue allografts including skin,19-25 muscle,26-28 bone,29-33 nerve34-37 and whole limb38-47 in animals maintained continuously on cyclosporine or more recently introduced immunosuppressive agents. The adverse effects of chronic immunosuppression, however, preclude their use in non life-threatening situations. Clinical transplantation of limb tissue allografts, therefore, remains a theoretical proposition today. As the clinical feasibility of nonvital tissue transplantation depends on less toxic means of host treatment or the induction of tolerance, a better understanding of the immunogenic mechanisms of limb tissue allografts must be obtained. Such information may allow a more precise manipulation of the tissue transplanted, less toxic immunosuppression of the host, or the induction of tolerance in the host toward the allogeneic tissue. For example, skin has long been considered to be the most antigenic body tissue,48 presumably due to the epidermal Langerhans’ cells or skin-specific antigens.49-51 In the early years of allograft study, it was perceived that the results of skin allografting would accurately predict the fate of other tissue or organ allografts. As more and more information accrued, it became apparent that skin was very antigenic, and probably more so than many other tissues or organs. In 1973, Murray proposed a relative scale of antigenicity for tissue and organs (Table 2.1).48 The most antigenic on his proposed hierarchy were skin and lung, whereas the least antigenic were kidney and pancreas. The combination of organ allografts with skin allografts, however, resulted in two-fold prolongation of the skin grafts.52 Conceptually, a limb allograft devoid of skin could be better tolerated by the host. Another tissue component of particular significance is the bone marrow, which has been shown to be an early target of host immune response.53-55 Thus, if limb tissue allografts without antigenic marrow elements would be better accepted by the host, specific marrow suppressive therapy such as irradiation or cytotoxic agents may play a role in making such transplants feasible.56 These data supported the theory that not all tissues were immunologically identical and that some were more antigenic than others.

Transplant Immunology It is instructive to review the immunological aspects of allograft rejection in order to understand some of the differences in the host’s immune response to different organs and tissues, particularly the different tissues that comprise a composite tissue allograft. Rejec-


11

Table 2.1. Relative scale of antigenicity of tissues and organs Most antigenic

Skin Lung

Less antigenic

Liver Heart

Least antigenic

Kidney Pancreas

Source: Reprinted with permission from Murray JE. Organ transplantation (skin, kidney, heart) and the plastic surgeon. Plast Reconstr Surg 1971; 47:425.

tion of allogeneic tissues or organs occurs through cellular and humoral immunological responses by the graft recipient. The immunological response of the host is effected by certain alloantigens, in the form of glycoproteins, expressed on the cells of the donor tissue, and a second signal is provided by specialized antigen presenting cells (APC).57 In humans, these antigens are the products of six closely linked genes on the short arm of chromosome 6 referred to as major histocompatibility complex (MHC) antigens. The MHC antigens in humans are referred to as human leukocyte antigens (HLAs) and are divided into 4 two classes: Class I antigens coded by the genes known as HLA-A,-B, and -C, and Class II antigens coded by the genes HLA-DR, -DQ, and -DP. Class I MHC antigens are expressed on all nucleated cells of the organism and platelets, but may be sparsely expressed on some types of cells, including certain antigen presenting cells.58,59 Class I antigens are recognized by CD8+ cytotoxic cells and generally serve as the first signal to elicit an immune response by the host; however, they are poor immunogens by themselves. Class II MHC antigens are more selective in their distribution, being expressed on B lymphocytes, macrophages, dendritic cells, and activated T cells. Class II antigens may also be expressed on the vascular endothelium of humans and some large animal species such as swine and monkeys, but not rodents—an important distinction.60,61 Furthermore, the expression of Class II antigens on some tissues is not constant and varies according to several stimuli such as interferon gamma (INF-!) or interleukin 4 (IL-4).62 Effective activation of T cells requires stimulation by the specialized APCs which express Class II antigens and can trigger CD4+ helper T cells. Whereas the MHC antigens have a prominent role in graft rejection, they did not evolve in nature to prevent tissue grafting. The essential role of the MHC antigens is now believed to involve the presentation of peptides of foreign antigens to responding T cells from the host. The MHC antigens exhibit extraordinary polymorphism, which presumably provides an advantage to members of a species by ensuring a broad capacity to present and respond to a large number of foreign antigens. Because there are a large number of alleles encoded by each locus and there are at least six individual loci in the human MHC, the likelihood of unrelated humans having identical MHC antigens is extremely small. The immunologic rejection process begins when the MHC antigens from the foreign tissue are presented to the host’s immune system by the antigen presenting cells, which may be of donor origin or recipient origin. Dendritic cells, macrophages, and activated B cells all have antigen presenting capability.63-67 Kupffer cells in the liver and Langerhans cells in the skin are believed to be subpopulations of dendritic cells that have antigen presenting capability as well. When antigen is presented to the host by APCs of donor origin, it as referred to

12


as the “direct” antigen presentation pathway. If the APCs are of recipient origin, it is called the “indirect” pathway.68,69 APCs constitutively express Class II antigens, and the level of this expression can be increased by the addition of various lymphokines such as IFN-! and IL-4.70-72 There is also evidence that some APCs can express low levels of Class I antigens, which may protect these cells from destruction by the host’s activated immune response as they provide their full helper function.58 The presentation of alloantigen to the host’s immune system by APCs induces maturation and proliferation of immature T cells and release of interleukin-1 (IL-1).73 Presentation of Class II allogantigens causes sensitization and activation of the CD4+ subpopulation of T cells in the host and the production of IL-2. IL-2 stimulates lymphocytic subpopulations to proliferate, resulting in clonal expansion of both cellular (T cell) and humoral (B cell) responses by the host. The cellular response results in allograft infiltration of natural killer (NK) cells, cytotoxic T cells (CD8+), and macrophages. Allograft destruction is mediated through two direct effector pathways, a CD8+ cytotoxic T cell (Class I restricted) response and a CD4+ (Class II restricted) response.74 The intensity of allograft rejection is dependent on the degree of MHC antigen mismatch between the graft donor and the host, as well as the tissue being transplanted. The most vigorous rejection episode will occur when the donor and host are mismatched at all MHC loci. However, rejection can occur even when the donor and host are matched for MHC antigens because of multiple minor, non-MHC antigenic differences. This is possible through the direct stimulation of donor APCs or the indirect stimulation by reprocessing of donor alloantigen onto host APCs.75,76 The indirect route of antigen presentation can also occur in Class I mismatched and Class II matched combinations, whereas direct stimulation by donor APCs is more likely in Class I and II mismatched combinations.77,78 The cellmediated response to allografts is measured in vitro using either a cell-mediated lymphocytotoxicity assay for measuring the reactivity of cytotoxic T cells (primarily a Class I, CD8+ dependent assay) against alloantigen or a mixed lymphocyte reaction (primarily a Class II, CD4+ dependent assay).

Humoral Response Rejection of allogeneic tissues and organs can also be effected by antibodies produced in response to exposure to the donor graft. In most transplantation situations the B cell response for antibody production occurs simultaneously with the T cell-mediated response, and separating the two to demonstrate that an induced humoral response alone can cause graft rejection is difficult. Nonetheless, it has been demonstrated that there is generally a transient IgM antibody response in the early phase of antigen presentation involving B cells alone. Production of persistent IgG alloreactive antibodies, however, requires both B cells and CD4+ helper cells. There is some speculation that induced antibody responses are responsible for chronic forms of rejection because of the presence of antidonor antibodies found in biopsies of obliterative arteritis, which is commonly encountered with chronic rejection.79 However, the possible role of antibody-mediated rejection is not well understood. The humoral response is generally measured by a complement dependent, cytotoxic antibody assay to determine the antibody titer against allogeneic cells.

Immunogenic Components of Composite Tissue Skeletal Allografts Composite tissue skeletal allografts are comprised of tissues that potentially have different degrees of antigenicity. In order for clinical limb tissue transplantation to occur, one must know the relative degree of immunogenicity for each of the tissues involved. Whereas solid organ transplantation predominantly involves one or a few tissue types, composite limb tissue allografts include multiple tissue types. Analysis of the literature may be helpful in determining the relative antigenicity of organ allografts; however, the picture is not clear


13

for the tissues comprising a composite limb tissue allograft. Some portions of these grafts such as the skin or bone marrow may include highly antigenic cell populations, and other portions, like intact cartilage, may be rather benign from an immunological standpoint. Many investigators have explored the transplantation of whole limbs in rodent models, whereas others have explored the use of single tissues such as tendon, bone, or muscle. It is believed that the immunogenicity of allografts containing bone and bone marrow is directly related to the presence of bone marrow-derived immunogenic cells (APCs) that reside there and are capable of delivering the second signal necessary to activate the T lymphocyte system.53 These cells are known to express Class II MHC molecules, which play a role in their ability to trigger a host immune response. The other components of bone allografts probably contribute little to the immunogenicity of the bone grafts. For, example, osteoblasts and osteocytes express Class I, but not Class II, MHC antigens and do not stimulate lymphocytes in mixed cultures.80 The osteocytes are encased in matrix, which also isolates them from cell-cell contact by the immune system. The matrix components of bone, such as proteoglycan subunits and collagen, can be antigenic stimuli, but are inconsequential for alloreactivity and transplantation where an immune response is triggered by and directed at alloantigens on the donor cell surface. The scientific literature is replete with studies on the antigenicity of skin allografts, and readers are encouraged to seek other sources. Most of the investigations have been performed in precisely controlled genetic combinations of mice using conventional (nonvascularized) skin grafts. Few studies have explored the immune response of vascularized skin allografts. Nonetheless, the skin has a very heterogeneous population of cells, including specialized APCs, and it is now generally agreed that skin is highly alloantigenic. The nature of this antigenicity can be related to the antigen presenting cells that reside in the skin49-51 and possibly due to skin-specific antigens that have not been characterized.81 The latter may be responsible for skin rejection, which often proceeds even when animals are tolerized to solid allograft organs. Whereas rejection processes of heart (muscle) allografts in rodents have been studied intensely,82,83 the same is not true for skeletal muscle. The transplantation of vascularized skeletal muscle has received little systematic study, and studies of nonvascularized muscle have little clinical significance.40-42,84 It is useful to compare the histologic findings from vascularized skeletal muscle allografts performed in our laboratory to cardiac allografts.28 It is common to find a mononuclear infiltrate early in the rejection phase, followed by a predominant polymorphonuclear pattern by one week. These invading immune cells were most prevalent at the periphery of the graft despite a vascular network throughout the skeletal muscle. This finding is consistent with that noted in cardiac allografts.82,83,85 Immunologic assays confirmed the intensity of the muscle allograft rejection, and we demonstrated that continuous cyclosporine was effective in ameliorating the immune response. This experiment demonstrated that the muscle portion of a composite limb tissue allograft was a potent immunogenic stimulus, similar to cardiac allografts. Composite limb tissue allografts contain many additional components including vessels, nerves, tendons, ligaments, and cartilage. With the exception of vessels, little attention has been paid to vascularized transplantation of the remaining tissues. It is not clear what role tissues such as tendons or ligaments play in limb tissue allograft rejection, but since these tissues are not believed to harbor large numbers of APCs, their role in eliciting an immune response is probably minimized. Allograft nerves are being investigated for use in reinnervating injured limbs with sizable nerve involvement, but they are prepared as a cable graft and are not primarily vascularized (personal communication, Dr. Susan Mackinnon). It has been shown, however, that fresh allograft nerves can elicit an immune response by the host.37


14

Relative Antigenicity of Limb Tissue The only known attempt to dissect the relative antigenicity of the various tissues that comprise a composite limb tissue allograft was performed in our laboratory several years ago, a synopsis of which is presented here.86 Inbred and genetically pure adult Lewis (RT1l) and Buffalo (RT1b) rat strains differing strongly at the RT1 major histocompatibility locus were employed. Models for the microsurgical transplantation of individual vascularized limb tissues were developed: 1. Skin—a groin flap based on the superficial epigastric vessels was transplanted to an orthotopic position in the recipient rat. The flap artery and vein were anastomosed end to end to the host femoral vessels (Fig. 2.1). 2. Subcutaneous tissue—the groin flap without the overlying skin was transplanted and placed subcutaneously in the recipient groin. Vascular anastomoses were performed as for the skin flap (Fig. 2.2). 3. Muscle—the gastrocnemius muscle isolated on the femoral and popliteal vessels was transplanted heterotopically into a subcutaneous position in the recipient groin. End to end anastomoses between the donor and host femoral vessels were performed (Fig. 2.3).28 4. Bone—the knee joint, consisting of distal femur and proximal tibia, was transplanted on the femoral pedicle to host femoral artery and vein and placed subcutaneously in the anterior abdominal wall (Fig. 2.4).87 5. Blood vessels—1.5 cm segments of femoral artery and vein were transplanted as interposition grafts to the host femoral vessels (Fig. 2.5). 6. Whole limb—the entire rat hind limb was transplanted heterotopically on its femoral pedicle to the recipient’s flank. Host femoral vessels were used for vascular anastomoses (Fig. 2.6). Donor tissue was harvested from Lewis rats in all instances. The tissues were transplanted across a strong histocompatibility barrier to Buffalo rats and between genetically identical Lewis rats as isograft controls. For each tissue component, a subgroup of allografts was treated with cyclosporine at 10 mg/kg subcutaneously daily after transplant. Nonvascularized skin and bone allografts were also transplanted for comparison with their vascularized counterparts. Ten transplants (n = 10) were performed in each subgroup and sacrificed at one and two weeks postoperatively. After transplant, the external appearance of the allograft or isograft was noted daily where possible. At sacrifice, the graft was examined for gross appearance and submitted for histologic sectioning, and fluorochrome uptake in osteoid laid down by osteoblasts was also examined in the pertinent specimens.88 The cellular immune response generated by the host animals was measured by a cellmediated lymphocytotoxicity assay.85,89 The percent release of radioactive chromium 51 above the spontaneous release yielded a direct measure of the number of target cells lysed by the effector cells and was expressed as a relative cytotoxicity index (RCI), which provided a standardized measure (in percent) of the host’s cell-mediated cytotoxicity. The humoral immune response was determined from the host serum collected at sacrifice by a complement-dependent cytotoxic antibody assay. The percent release calculated for each serum dilution, according to the positive and negative controls and spontaneous release, provided


Skin Subcutaneous tissue

15

Fig. 2.1. Schematic diagram of the model for vascularized skin allograft. Reprinted with permission from Lee WPA et al. Relative antigenicity of components of a vascularized limb allograft. Plast Reconstr Surg 1991; 87(3):402.

Femoral a.&v.

Fig. 2.2. Schematic diagram of the model for vascularized subcutaneous tissue allograft. Reprinted with permission from Lee WPA et al. Relative antigenicity of components of a vascularized limb allograft. Plast Reconstr Surg 1991; 87(3):402. Subcutaneous tissue Femoral a.&v.

Gastrocnemius m.

Femoral a.&v.

Fig. 2.3. Schematic diagram of the model for vascularized muscle allograft. Reprinted with permission from Lee WPA et al. Relative antigenicity of components of a vascularized limb allograft. Plast Reconstr Surg 1991; 87(3):402.


16

Femur Tibia

Fig. 2.4.Schematic diagram of the model for vascularized bone allograft. Reprinted with permission from Lee WPA et al. Relative antigenicity of components of a vascularized limb allograft. Plast Reconstr Surg 1991; 87(3):402.

Femoral a.&v.

Fig. 2.5. Schematic diagram of the model for vascularized blood vessel allograft. Reprinted with permission from Lee WPA et al. Relative antigenicity of components of a vascularized limb allograft. Plast Reconstr Surg 1991; 87(3):402.

Donor Fem. a.&v.

Fig. 2.6. Schematic diagram of the model for vascularized limb allograft. Reprinted with permission from Lee WPA et al. Relative antigenicity of components of a vascularized limb allograft. Plast Reconstr Surg 1991; 87(3):402. Whole limb

Femoral a.&v.


A

17

Fig. 2.7. Cell–mediated responses in various control groups: un– operated animals, rats with isografts, and rats with allografts treated with cyclosporine (A). Humoral responses in various control groups (B). Reprinted with permission from Lee WPA et al. Relative antigenicity of components of a vascularized limb allograft. Plast Reconstr Surg 1991; 87(3):402.

B

a measure of cytotoxicity. Only serum dilutions where cytotoxicity was more than 25% of the positive control were considered indicative of a significant antibody response. Thus, the greatest dilution number at which this occurred yielded the humoral relative cytotoxicity index (H-RCI) and provided a standardized measure of the host antibody cytotoxicity. The isografts and cyclosporine-treated allografts demonstrated grossly and histologically normal-appearing tissues with only minimal inflammation in all groups. There was good fluorochrome uptake in the bone and limb allografts, indicating bone viability. In untreated allografts, there was progressive edema in the gross specimens beginning three to four days after transplant, with subsequent rejection and necrosis by two weeks. Histologically, there was an inflammatory infiltrate in these allografts at one week, consisting of predominantly mononuclear cells. At two weeks, the cellular infiltrate was more dense with the appearance of some neutrophils, accompanied by extensive tissue necrosis and microvascular thromboses. The whole limb allografts, however, demonstrated a more delayed pattern of rejection as compared to the individual limb tissue allografts. Here the cellular structures were mostly preserved at one week, with only a moderate amount of mononuclear infiltrate. The extent of rejection at two weeks as determined histologically resembled more closely that of the individual limb tissue allografts a week earlier.

18


A

Fig. 2.8. Cell–mediated responses in rats with various skin allografts: vascularized, nonvascularized, and vascularized with cyclosporine (A). Humoral responses in rats with various skin allografts (B). Reprinted with permission from Lee WPA et al. Relative antigenicity of components of a vascular– ized limb allo–graft. Plast Reconstr Surg 1991; 87(3):402.

B

In rats with isografts or cyclosporine-treated allografts, there were no significant immune responses at either time interval (Figs. 2.7A, B). In animals not treated with cyclosporine, both vascularized and nonvascularized skin allografts generated significant cell-mediated responses at one and two weeks (p290, 2/8 no signs of rejection days >280, >290

3/6 no reversal of rejection seen

12 months period, early deaths 2/8** days 4,7 3/8** days 2,5,7, 3/8 GVHD, days 87,97,97

3/10 GVHD 3/10 GVHD

Complications/Others

Long-Term Composite Tissue Allograft Survival with FK506 Immunosuppression 211

FK506 i.m.

Lew (RT1l) PVG (RT1c)



Kuroki 199165

JKuroki 199166

Kuroki 199367 -7 -7 Æ +6 -7

1ml blood 1 1ml gammairradiated blood 0 -7 Æ +6

-7 Æ +6

-7 ∀ +6

-7

-7 ∀ +6

0-14 0-14 0-14

7

6 6

7 6

6

6

13 14 14 10

Duration days # Rats

1

1 ml blood 1 0

DST¥ i.v. + FK-506

FK506 i.m. DST¥ i.v. + FK506 Radiated DST¥ i.v. + FK506

1

0.32 0.64 15 0

Dosage mg/kg/day

FK506 i.m.

CsA

Drug

Rat-strain

Authors

Table 16.4. Continued

11.0 ± 1.3

>57.3 ± 19.3 >60.0 ± 19.9

11.0 ± 1.3 >56.3 ± 22.3

>60.7 ± 19.1

>65.5 ± 27.0

31 (8/9 rats) 50 (8/12 rats) 34 (8/10 rats) 12 (8/8 rats)

Limb survival days

no statistical difference between FK506 alone and DST or radiated DST

no statistical difference between FK506 alone and DST

1/8 rats 4/12 rats ∀ 20% indefinite graft 2/10 rats survival i

Complications/Others

212 Composite Tissue Transplantation

* Animals sacrificed at days postoperatively ** Animals died at days postoperatively ¥ Donor-specific blood transfusion (DST)

0

orally

Lew (RT11)

6

i.p.

FK506

BN (RT1n)

Fealy 2,5,7,10,13 199569

0

6

6 6 10 2

4.5

FK506 orally

BN (RT1n) Lew (RT11)

Fealy 199468

Dosage mg/kg/day

Rapamycin

Drug

Rat-strain

Authors

1-14

1-14

1-90

1-14 1-14 until rejection resolved thereafter maintenance

3

3

4

3

3

3 5

Duration days # Rats Complications/Others

during 14 days Isografts, FK506 treated allografts Rapamycin treated + untreated allografts rejection not prevented

no rejection signs

IL-2, IL-1a, IFN-! increase (day 2-5), TGF-∃, PDGF-#, FGF and IL-6 peak (day 10-13)

IL-1, -2, -6 expression, PDGF-#, TGF-∃, bFGF and IFN-! low cytokine expression

Biopsies taken at day

28.0 ± 0.8 41-65 63*,65*,41** 2/5 >60 68, 85; 2nd rejection when 2 mg decreased to 1 mg >90 62,108,345 108*,62** no rejection at day 345 after discontinuation at day 255 4.3 ± 0.47

Limb survival days

Long-Term Composite Tissue Allograft Survival with FK506 Immunosuppression 213

214


prevented rejection for 104 days, 3 of 8 animals died within 1 week and 3 animals died between 87-97 days posttransplantation from GVHD (Table 16.4). Long term intermittent treatment with 3 mg/kg FK506 once a week after a single administration of 10 mg/kg of FK on the day of limb transplantation achieved indefinite limb allograft survival ( 200 days) although 3 of 8 rats died from Pneumocystis carinii pneumonia between 228-242 days and one of 8 rats died from an unspecific pneumonia. Two of the 8 rats recovered from these symptoms after discontinuation of FK506 and administration of steroids. Four weeks following discontinuation of FK506, signs of rejection could be reversed by a single injection of 10 mg/kg of FK506, followed by a maintenance dose of 1.5 mg/ kg per week. These two studies62,63 showed that indefinite graft acceptance can be achieved either by short term or long term intermittent treatment. However, unexpected infection with Pneumocystis carinii occurred in many of the recipients, suggesting chronic GVHD. This infection could have been secondary to nonspecific immunosuppression induced by excessive FK506 treatment because, except for the pneumonia, there were no other typical signs (alopecia, diarrhea) of GVHD in these animals (Table 16.4). Kuroki et al64 have demonstrated prolonged survival of limb allografts for 50 days across the Lewis ∀ PVG barrier after a 14 day course of 0.64 mg/kg FK506 compared to only 31 days in those animals receiving CsA (Table 16.4). In 4 of 12 FK506 treated animals, the transplanted donor skin survived between 20 and 36 weeks after transplantation, although alopecia and atrophy were present.65 In these tissues the degree of lymphocyte infiltration was limited, muscle bone and cartilage were maintained. However, once the skin of the transplanted limb was rejected, the entire limb was gradually rejected. In the same study Kuroki et al65 reported that functional recovery as assessed by weight-bearing gait and evoked potentials was similar in these FK506 treated animals to the function of long term isografts. It took almost one year to achieve useful recovery in motor as well as sensory function. In the same study,65 the cellular immune response using MLC (mixed lymphocyte cultures), cell mediated lympholysis (CML) and the humoral response using complement-dependent cytotoxicity showed unresponsiveness for donor alloantigens, suggesting that long term limb allograft survival was related to this immunologic tolerance. However, these results did not distinguish the precise mechanisms of long term limb allograft survival. Kuroki et al66 also reported the synergistic effect of donor-specific blood transfusion and FK506 immunosuppression in skin allografts, but not in limb allografts. They suggested that the effect of the donor-specific blood transfusion could be overridden by bone marrow cells contained within the limb allograft. In this protocol FK506, 1 mg/kg/d, was given one week before transplantation and continued for 6 days postoperatively with or without a donor-specific blood transfusion. In both groups the period of limb allograft survival was similar (Table 16.4). Furthermore, there was no synergistic effect of FK506 and heat or !-irradiated donor-specific blood transfusion on limb allograft survival67 (Table 16.4). A reduced sensitization, resulting in tolerance to the composite tissue allograft, could not be demonstrated with irradiated donor-specific blood and a short term peritransplant course of FK506 3-4 months after transplantation. Fealy et al68 demonstrated prolonged limb allograft survival for 28 days in 3 rats across the Brown Norway (RT1n) ∀ Lewis (RT11) barrier using 6.0 mg/kg FK506 orally for 14 days. Rejection after discontinuation of FK506 could be reversed by 10 mg/kg FK506 orally followed by a low dose maintenance of 2 mg/kg and graft survival was prolonged for 60 days. In 3 animals FK506 was given for 90 days in the same dosage and rejection was delayed for 62, 108 and 345 days respectively compared to 5 days in untreated allografted animals (Table 16.4). The cytokine mRNA expression in a limb allograft model across the Brown Norway ∀ Lewis barrier was studied during acute allograft rejection. Maximal cytokine expression correlated with peak graft rejection. However, a 14 day course of FK506 sup-

Long-Term Composite Tissue Allograft Survival with FK506 Immunosuppression

215

pressed cellular expression of various cytokines to below isograft levels, whereas rapamycin was ineffective in suppressing cytokine expression, and allograft rejection could not be prevented (Table 16.4). This downregulation of cytokine expression was associated with clinical allograft survival.69

Limb Transplantation with Other Immunosuppressive Agents Following discovery of cyclosporine (CsA) in 1976, several groups reported successful experimental limb transplantation with variable survival of the transplanted limbs and side effects across the Lewis x Brown Norway ∀ Lewis mismatch.8-13,20,34 Three studies achieved indefinite rejection-free survival, but only in a minority of rats receiving long term CsA therapy.10,12,13 Black et al15 produced long term survival of limb allografts in 6 rats receiving a tapered course of high dose CsA. Significant morbidity was noted at these CsA doses after 60 days of treatment. Other studies using high dose CsA (15-25 mg/kg/d subcutaneously) were able to delay rejection for 31-83 days posttransplantation.8,11,20,64 Some of these long term survivors developed lymphoid chimerism in that the lymphocytes from the peripheral blood and spleen of the Lewis recipients contained 20% Lewis x Brown Norway donorderived cells. Lymphoid chimerism was interpreted as a beneficial consequence of the development of tolerance.13 Hewitt et al14 confirmed the development of lymphoid chimerism in the fully allogeneic composite tissue allograft across the Lewis ∀ ACI mismatch after 100 days of immunosuppression with CsA. The development of donor-host lymphocyte chimerism in combination with a wasting syndrome in these long term composite tissue allograft survivors was suggestive of graft versus host disease (GVHD). Across semiallogeneic immunologic barriers such as Lewis x Brown Norway ∀ Lewis, however, this donor-host immune chimerism may not lead to a lethal GVHD, because the hybrid donor immune cells do not respond to parental self-immunogens. Several other authors have also shown the effectiveness of CsA in prolonging limb allograft survival using different strains, different dosage regimes and limited duration of immunosuppression between 14-60 days.7,16,18,19 Hotokebuchi et al20 demonstrated in 35 rats limb allograft survival for 45 days with short term immunosuppression and 56 days with long term immunosuppression, compared with rejection at 11 days in the nonimmunosuppressed controls. The articular cartilage of the CsA-treated animals maintained normal architecture and cell viability 52 weeks posttransplantation despite the gross appearance of skin rejection. Half of the CsA-treated animals, however, developed clinical signs of GVHD by 1 year, and histologic examination showed normal bone marrow with mild or moderate bone atrophy. The appearance of GVHD was seen in the use of fully allogeneic rat strains (Brown Norway RT1n and Fischer F344 RT11) compared to the studies with semiallogeneic strains.8-13,20,34 Inceoglu et al34 investigated the efficacy of combined immunosuppression with systemic low dose CsA and topical fluocinolone acetonide (FA) on the survival of rat hindlimb allografts. Across the Brown Norway ∀ Lewis mismatch, 10 rat limbs survived between 32-51 days compared to 3-5 days in the nonimmunosuppressd control group. This therapy was particularly effective for the prevention of skin rejection, but resulted in a higher incidence of infection and loss of body weight due to systemic synergistic effects. Fritz et al17 confirmed extended rat limb survival with CsA immunosuppression across the strong ACI ∀ Lewis mismatch. Five of seven animals treated continuously with CsA for up to 113 days showed no signs of rejection clinically, histologically, or immunologically, but 2 of 7 rats developed skin rejection within that time period. During this period, no immunosuppression-related complications were reported. Other studies of experimental limb transplantation have investigated 15-deoxyspergualin (DOS), rapamycin and RS-61443. A 10 or 20 day course of DOS improved survival of limb allografts to 18 and 24 days respectively across the Dark Agouti (RT1a) ∀ Lewis (RT11) barrier. The effect of this antitumor drug may be due to a suppression

216


of macrophage-dependent lymphocyte function.29 Rapamycin, a macrolide fungal fermentation product related in structure to FK506, prolonged survival of limb allografts for 60 days across the histocompatiblity barrier of Wistar (RT1u) ∀ Buffalo (RT1b). This study also confirmed the synergistic effect of subtherapeutic doses of rapamycin and CsA to prolong limb allograft survival.30 Fealy et al68 demonstrated limb allograft survival between 8-10 days across the Brown Norway ∀ Lewis barrier using rapamycin intraperitoneally in different doses for 14 days. In combination with CsA, rejection could be delayed for 20 days. The introduction of RS-61443, which is the morpholinoethyl ester prodrug of mycophenolic acid and inhibits lymphocyte proliferation by depletion of the purine cycle, seems to prevent acute and delayed rejection of limb allografts with minimal toxicity. Benhaim et al31- 33 have investigated the effectiveness of RS-61443 in rat limb transplantation. Five of six animals (Brown Norway RT1n ∀ Fischer F344 RT11) treated continuously with RS-61443 at 30 mg/kg/d showed no clinical or histologic evidence of rejection of the skin component of limb allografts when the animals were killed between 231 and 251 days after transplant. RS-61443 was significantly more effective than CsA.31 This is the first immunosuppressive agent that seems to be effective in preventing the delayed onset of acute rejection without significant toxicity, except moderate bone marrow suppression up to 32 weeks postoperatively. This anemia appears to be species-specific and is limited to the rat. RS-61443 also demonstrated a marked ability to reverse established moderate to severe acute rejection in rat limb allografts.32 Furthermore, combination therapy with low dose RS-61443 and CsA was efficacious in preventing rejection with minimal toxicity for more then 231 days in 11% (n =18) of rats posttransplantation.33

Conclusions: Limb Transplantation and Immunosuppression Our series of experimental limb transplantations is one of the largest ever reported to compare the efficacy of long term intermittent immunosuppression in preventing the rejection of a limb transplant across the strongest histocompatibility barrier (ACI ∀ Lewis) in rats, using the conventional immunosuppressive agent CsA (cyclosporine) and the newer immunosuppressive agents FK506 and RS-61443 (submitted for publication). All three immunosuppressive agents were able to prolong rejection of the skin component of a limb transplant compared with nonimmunosuppressed controls. There was no stastically significant difference between intermittent immunosuppression using CsA and RS-61443. All animals receiving 25 mg/kg CsA twice weekly showed signs of rejection while continuing to receive long term intermittent immunosuppression, with a mean rejection time (MRT) of 61.6 days. All animals immunosuppressed with 30 mg/kg RS-61443 twice weekly also showed signs of rejection while continuing to receive long term intermittent immunosuppression, with a mean rejection time of 43.6 days. Animals receiving 2mg/kg FK506 twice weekly showed no signs of rejection, but died of bacterial pneumonia between 273 and 334 days posttransplantation, with a mean survival time of 296 days. We therefore believe that long term intermittent immunosuppression with FK506 is significantly superior to CsA and RS-61443 in preventing rejection of all the component tissues of a limb transplant across this extremely strong histocompatibility barrier in rats. Unfortunately, however, just as in the few other studies of long term survival after limb transplantation,62,31 all of our long term survivors died without signs of rejection of the skin component of the limb transplant, just under 300 days postoperatively. This may have been due to either the development of graft versus host disease or to overwhelming infection due to their immunocompromised status.


217

Nerve Graft Transplantation in Rats Immunosuppressed with FK506 Peripheral nerve tissue is highly antigenic and without immunosuppression undergoes rejection after skin, but before or concurrently with muscle rejection in studies of composite tissue transplantation. Rejection can be successfully prevented temporarily using various immunosuppressive drugs, but rejection does occur again after cessation of immunosuppression. Functional results of peripheral nerve allografts after rejection remain controversial.70-74 In most of the published studies, cyclosporine has been used for immunosuppression.70-78 Our group (Buettemeyer et al79,98) has demonstrated that nerve allograft rejection can be successfully prevented by immunosuppression using FK506. Two centimeter nerve allografts were transplanted across the extremely strong histocompatibility barrier from donor ACI rats into a 0.5 cm gap in the sciatic nerve of recipient Lewis rats and immunosuppressed with FK506, 2 mg/kg per day intramuscularly for 3 months (n = 15). Five animals were sacrificed after evaluation by walking track analysis and somatosensory evoked potentials (SSEP), and histological examination. Five animals continued to receive intermittent immunosuppression with FK506, 2 mg/kg twice weekly for another 2 months, whereas the remaining 5 rats received no further immunosuppression, in order to determine whether rejection of nerve allografts can still occur after immunosuppression is withdrawn, even after the axons have regenerated through the nerve graft. The sciatic function index improved from -76.3 at 3 months to -46.6 at 5 months in those animals continuing to receive intermittent immunosuppression, but only improved to -66.5 at 5 months when immunosuppression was discontinued. Similarly, somatosensory evoked potentials demonstrated an improvement in relative latency from 2.3 msec at 3 months to 0.34 msec at 5 months in animals continuing to receive intermittent immunosuppression, but only improved to 1.29 msec at 5 months when immunosuppression was discontinued (Table 16.5). Nerve allografts continuing to receive intermittent immunosuppression showed no signs of rejection by light or electron microscopy and no significant difference compared with isografts, whereas nerve allografts whose immunosuppression had been stopped at 3 months showed mild signs of rejection, less regeneration, and a smaller number of nerve fibers (Table 16.5). Immunohistology revealed only a small number of Lewis-derived Schwann cells in the ACI nerve allografts in animals continuing to receive intermittent immunosuppression, but an increasing number of Lewis-derived Schwann cells in animals whose immunosuppression was discontinued. After continued immunosuppression for 3 and 5 months, histology confirmed that rejection could be successfully prevented with FK506, but after discontinuation of FK506 for 2 months there were histological signs of ongoing rejection compared with nerve allografts continuing to receive intermittent immunosuppression. Immunohistology demonstrated that, under continued immunosuppression, the Schwann cells, which are the main antigenic structures,83 remain donor (ACI)-derived. This confirmed the results of a previous study84 using cyclosporine. So, even if immunosuppression is withdrawn after the axons have regenerated across the distal nerve juncture, an ongoing rejection process continues to occur, which can be confirmed both histologically and by functional testing, and the Schwann cells then become recipient (Lewis)-derived. The electrophysiological and functional results in our study confirmed the results of other investigators using cyclosporine (CsA) immunosuppression.70-72,85,86

Other Studies of Nerve Graft Transplantation Using FK506 and Cyclosporine Immunosuppression

Kuroki et al87 have investigated regeneration in the sciatic nerve and the functional recovery in vascularized rat limb allografts after cessation of a short course of FK506 or CsA. In a series of 71 limb transplants from Lewis (RT11) to PVG (RT1-1c) rats, allografts were immunosuppressed with 0.32 mg/kg/d or 0.64 mg/kg/d FK506 or 15 mg/kg/d CsA

EM##: good myelination complete basal lamina

LM#: no signs of rejection

Histology

Lewis-derived

LM: severe signs of rejection, perineurial fibrosis, demyelinization.

4.87

-82.7

Allograft 3 months

LM: no signs of rejection, but mild perineurial fibrosis in 2/5 rats

2.36

-76.3

FK506 for 3 months

EM: some non- viable Schwann cells, more demyelinated fibers.

LM: mild signs of rejection, perineurial infiltration. Number of nerve fibers +

1.29

-66.8

FK506 discontinued after 3 months; results at 5 months

Schwann cells +++ Lewis-derived Lewis-derived Schwann cells + Schwann cells ++

EM: adjacent regenerating and degenerating cells with old myelin debris.

3.78

-65.8

Allograft 5 months

Lewis-derived Schwann cells +

LM: no signs of rejection, mild demyelinization, mild perineurial fibrosis. Number of nerve fibers ++ EM: viable Schwann cells, well-formed axons and myelin sheaths.

0.34

-46.6

FK506 continued twice a week after 3 months until 5 months

*Sciatic function index (SFI) after de Medinacelli.80 A SFI of -100 to -120 represents a complete paralysis of the sciatic nerve, whereas a functional nerve is defined as a SFI of 0 to -20. **Relative latency (msec) of nerve conduction velocity in the operated leg compared to the unoperated leg measured by somatosensory-evoked potentials (SSEP)81 ***OX antibody which stains only Lewis fibers, to determine the origin of the Schwann cells in the nerve allograft.82 # Light microscopy (LM) ## Electron microscopy (EM)

ImmunoLewis-derived Schwann histology*** cells +++

0.56

0.78

SSEP**

-38.2

-60.1

Isograft 5 months

SFI*

Isograft 3 months

Table 16.5. Functional and histological results in nerve isografts, nerve allografts without immunosuppression, and nerve allografts after discontinuation or with continuous immunosuppression using FK506

218 Composite Tissue Transplantation


219

intramuscularly from the day of transplantation for 14 days. Histological examination of the nerve allografts was evaluated between 2 and 14 weeks after transplantation and showed satisfactory nerve regeneration and reinnervation of target organs under this short term course of FK506. Nerve regeneration was found to begin 4 weeks after limb transplantation and gradually matured, demonstrating host nerve fibers regenerating throughout the distal donor nerve. However, long term survival of the limb allografts in 7/31 rats treated by this short term course of immunosuppression with FK506 in two different dosages or CsA again raises the question of the strength of the antigenic disparity between the recipient and donor animals. One would expect that the components of the composite limb allograft would be rejected after withdrawal of immunosuppression including the donor-origin Schwann cells in the transplanted limb. Furthermore, the regenerating host-origin axons would be expected to be adversely affected by the surrounding rejecting donor tissue in the allografted limb. In contrast, the short segmental nerve allograft model would be expected to show better functional recovery, because all the surrounding tissue is of host origin.88-90 Weinzweig et al93 reported satisfactory nerve regeneration across peripheral nerve allografts at 7 months by nerve conduction velocities after a 14 day short term course of immunosuppression with 1 mg/kg/d FK506 between histoincompatible Lewis and ACI rats. Conduction velocities were faster than in rats receiving a short term course of immunosuppression with CsA, and not significantly different from rats receiving long term immunosuppression with either CsA or FK506, or the isograft control. Their results suggested that a two week course of FK506 immunosuppression would produce the same nerve regeneration as a 7 month course of intermittent immunosuppression; this does not correlate with our findings,79 and we are extremely skeptical of the validity of this study. Other results of short term immunosuppression on nerve allograft transplantation are extremely variable, from complete loss of function to good recovery of neuromuscular function.71,89,93-97 Regenerating nerve fibers may be rejected after cessation of immunosuppression, but remnants of the endoneurial tube may still act as a conduit for regenerating axons. Midha et al91 demonstrated that donor Schwann cells are rejected and replaced by host Schwann cells after cessation of immunosuppression. Demyelinated host axons seemed to remyelinate and to regenerate across the allograft into the distal host nerve, resulting in functional nerve recovery. Lassner et al92 described the process of rejection by elimination of the Schwann cells but leaving the acellular nerve allograft as a structural scaffold through which axons could regenerate.

Conclusions: Nerve Graft Transplantation and Immunosuppression Our experience in nerve allograft transplantation has demonstrated that long term intermittent immunosuppression using both FK506 and CsA is superior to short term immunosuppression. After discontinuation of immunosuppression, the nerve allograft demonstrates histological evidence of rejection and compromised regeneration; this is reflected in less improvement in functional recovery as assessed by SSEPs and walking track analysis in animals that have their immunosuppression discontinued, compared with those animals that continue to receive intermittent immunosuppression. Problems in assessing peripheral nerve allograft rejection include the use of different ill-defined donor/recipient histocompatibility barriers and different electrophysiological and functional methods of assessing nerve function. Rather than simply using nerve conduction studies across the nerve allograft, the only truly objective electrophysiological parameter of nerve conduction across a nerve allograft is to compare it with the opposite leg by recording in the cerebral cortex, to provide a relative distal latency between the nerve allograft and normal sciatic nerve.98 In a recent study,99 various parameters including the sciatic functional index, nerve conduction velocity, muscle contraction and

220


axon morphometry were compared to determine the best measurement of nerve regeneration. The poor correlation between sciatic functional index and other parameters of nerve function led to the conclusion that the best measure of nerve function in the experimental animal still remains unproved or undiscovered. This explains the tremendous disparity of results between different studies of nerve allograft transplantation.

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45. Collier DS, Thiuru S, Calne R. Kidney transplantation in the dog receiving FK506. Transplant Proc 1987; 19(suppl 6):62. 46. Calne R, Collier DS, Thiuru S. Observation about FK506 in primates. Transplant Proc 1987; 19(suppl 6):63. 47. Todo S, Demetris A, Ueda Y, et al. Renal transplantation in baboons under FK506. Surgery 1989; 106:444-451. 48. O’Hara K, Billington R, James RW et al. Toxicologic evaluation of FK506. Transplant Proc 1990; 22(suppl 1):83-86. 49. Brazelton TR, Morris R. Molecular mechanisms of action of new xenobiotic immunosuppressive drugs: Tacrolimus (FK506), sirolimus (rapamycin), mycophenolate mofetil and leflunomide. Curr Opin Immunol 1996;8;710-720. 50. Lipson RA, Kawano H, Halloran PF et al. Vascularized limb transplantation in the rat. Transplantation 1983; 35:293-299. 51. Gill TJ, Kunz HW, Misra DN et al. The major histocompatibility complex of the rat. Transplantation 1987; 43:773-784. 52. Min Z, Jones NF. Limb transplantation in rats: Immunosuppression with FK506. J Hand Surg 1995; 20A:77-87. 53. Buettemeyer R, Jones NF, Min Z et al. Rejection of the component tissues of limb allografts in rats immunosuppressed with FK506 and cyclosporine. Plast Reconstr Surg 1996; 97:139-148. 54. Billingham ME, Cary NRB, Hammond ME et al. A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: heart rejection study group. J Heart Transplant 1990; 9:587-593. 55. Daniel RK, Egerszegi EP, Samulack DD et al. Tissue transplants in primates for upper extremity reconstruction: A preliminary report. J Hand Surg 1986; 11A:1-8. 56. Siliski JM, Simkin S, Green CJ. Vascularized whole knee joint allografts in rabbits immunosuppressed with cyclosporin A. Arch Orthop Trauma Surg 1984; 1j03:26-35. 57. Press BHJ, Sibley RK, Shons AR. Early histopathologic changes in experimentally replanted extremities. J Hand Surg 1983; 8:549-552. 58. Press BHJ, Sibley RK, Shons AR. Limb allotransplantation in the rat: Extended survival and return of nerve function with continuous cyclosporin/prednisone immunosuppression. Ann Plast Surg 1986; 16:313-321. 59. Arai K, Hotokebuchi T, Miyahara H et al. Limb allografts in rats immunosuppressed with FK506. Transplantation 1989; 48:782-786. 60. Weiland AJ. Fate of vascularized bone graft. In: Aebi M, Regazzoni P, eds. Bone Transplantation. Berlin: Springer, 1989:29-50. 61. Horowitz MC, Friedlander GE. The immune response to bone graft. In: Friedlander GE, Goldberg VM, eds. Bone and Cartilage Allografts. Park Ridge: American Academy of Orthopedic Surgeons, 1991:85-101. 62. Arai K, Hotokebuchi T, Miyahara H et al. Limb allografts in rats immunosuppressed with FK506. I. Reversal of rejection and indefinite survival. Transplantation 1989; 48:782-786. 63. Arai K, Hotokebuchi T, Miyahara C et al. Prolonged limb allograft survival with shortterm treatment with FK506 in rats. Transplant Proc 1989; 21:3191-3193. 64. Kuroki H, Ikuta Y, Akiyama M. Experimental studies of vascularized allogeneic limb transplantation in the rat using a new immunosuppressive agent, FK506: Morphological and immunological analysis. Transplant Proc 1989; 21:3187-3190. 65. Kuroki H, Ishida O, Daisaku H et al. Morphological and immunological analysis of rats with long-term-surviving limb allografts induced by a short course of FK506 or cyclosporine. Transplant Proc 1991; 23:516-520. 66. Kuroki H, Bean MA, Ikuta Y et al. Synergistic effect of FK 506 and donor-specific blood transfusion on rat skin but not on composite tissue (limb) allograft survival. Transplant Proc 1991; 23:3282-3284. 67. Kuroki H, Bean MA, Ikuta Y et al. Effect of FK506 and donor-specific blood transfusion on the rat composite tissue limb allograft and the mechanism of long-term graft survival. Transplant Proc 1993; 25:658-661.


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68. Fealy MJ, Umansky WS, Bickel KD et al. Efficacy of rapamycin and FK506 in prolonging rat hindlimb allograft survival. Ann Surg 1994; 219:88-93. 69. Fealy MJ, Most D, Huie P et al. Association of down-regulation of cytokine activity with rat hindlimb allograft survival. Transplantation 1995; 59:1475-1480. 70. Ishida O, Tsai T-M, Breidenbach WC et al. Peripheral nerve allografts: Functional and histologic assessment after withdrawal of cyclosporine. J Reconstr Microsurg 1992; 8:240. 71. Midha R, Mackinnon SE, Evans PJ et al. Comparison of regeneration across nerve allografts with temporary or continuous cyclosporin A immunosuppression. J Neurosurg 1993; 78:90-100. 72. Yu LT, Hickey WF, Sumner A et al. Survival and function of peripheral nerve allografts after cessation of long term cyclosporin immunosuppression in rats. Transplant Proc 1989; 21:3178-3180. 73. Zalewski AA, Gulati AK. Failure of cyclosporin-A to induce immunological unresponsiveness to nerve allograft. Exp Neurol 1984; 83:659-663. 74. Ansselin AD, Westland K, Pollard JD. Low dose, short term cyclosporin A does not protect the Schwann cells of allogeneic nerve grafts. Neurosci Lett 1990; 119:219-222. 75. Zalewski AA, Gulati AK. Survival of nerve and Schwann cells in allografts after cyclosporin A treatment. Exp Neurol 1980; 70:219-225. 76. Mackinnon SE, Hudson AR, Falk RE et al. The nerve allograft response—an experimental model in the rat. Ann Plast Surg 1985; 14:334-339. 77. Bain JR, Mackinnon SE, Hudson AR et al. The peripheral nerve allograft: A dose-response curve in the rat immunosuppressed with cyclosporin A. Plast Reconstr Surg 1988; 82:447-457. 78. Lassner F, Schaller E, Steinhoff et al. Cellular mechanisms of rejection and regeneration in peripheral nerve allografts. Transplantation 1989; 48:386-392. 79. Buettemeyer R, Rao U, Jones NF. Peripheral nerve allograft transplantation with FK506: Functional, histological, and immunological results before and after discontinuation of immunosuppression. Ann Plast Surg 1995; 35:396-401. 80. de Medinacelli L, Freed WJ, Wyatt RJ. An index of the functional condition of rat sciatic nerve based on measurements made from walking tracks. Exp Neurol 1982; 77:634-643. 81. Sen C, Moller AR. Comparison of somatosensory evoked potentials recorded from the scalp and dorsal column nuclei to upper and lower limb stimulation. Electroencephalogr Clin Neurophysiol 1991; 80:378-383. 82. Barclay AN. The localization of populations of lymphocytes defined by monoclonal antibodies in rat lymphoid tissues. Immunology 1981; 42:593-601. 83. Grochowicz P, Romaniuk A, Jedrzejewska A et al. Rejection pattern of nerve allografts: Changes in graft and host cell determinants. Transplant Proc 1987; 19:1131-1132. 84. Ishida O, Martin A, Firrell JC. Origin of Schwann cells in peripheral nerve allografts in the rat after withdrawal of cyclosporine. J Reconstr Microsurg 1993; 9:234-236. 85. Mackinnon SE, Hudson AR. Clinical application of peripheral nerve transplantation. Plast Reconstr Surg 1992; 90:695-699. 86. Schaller E, Lassner F, Becker M et al. Regeneration of autologous and allogenic nerve grafts in a rat genetic model: Preliminary report. J Reconstr Microsurg 1991; 7:9-12. 87. Kuroki H, Ikuta Y. Nerve regeneration of vascularized rat limb allograft and functional recovery of long-term graft survivals treated by short course of FK506 and cyclosporine. Transplant Proc 1995; 27(1):348-50. 88. Zalewski AA, Gulati AK. Survival of nerve allografts in sensitized rats treated with cyclosporine A. J Neurosurg 1984; 60:828-834. 89. Mackinnon SE, Hudson AR, Bain JR et al. The peripheral nerve allograft. An assessment of regeneration in the immunosuppressed host. Plast Reconstr Surg 1987; 79:436-444. 90. Ishida O, Daves J, Tsai TM et al. Regeneration following rejection of peripheral nerve allografts of rats on withdrawal of cyclosporine. Plast Reconstr Surg 1993; 92:916-926. 91. Midha R, Mackinnon SE, Becker LE. The fate of Schwann cells in peripheral nerve allografts. J Neuropath Exp Neurol 1994; 53:316-322.

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92. Lassner F, Schaller E, Steinhoff G et al. Cellular mechanisms of rejection and regeneration in the peripheral nerve allografts. Transplantation 1989; 48:386-392. 93. Weinzweig N, Grindel S, Gonzalez M et al. Peripheral-nerve allotransplantation in rats immunosuppressed with transient or long-term FK506. J Reconstr Microsurg 1996; 12:451-459. 94. Bain JR, Mackinnon SE, Hudson AR et al. The peripheral nerve allograft: An assessment of regeneration across nerve allografts in rats immunosuppressed with cyclosporine A. Plast Reconstr Surg 1988; 82:1052-1064. 95. Yu LT, England J, Hickey WF et al. Survival and function of peripheral nerve allografts after cessation of long-term cyclosporin immunosuppression in rats. Transplant Proc 1989; 21:3178-3180. 96. Frazier J, Yu LT, Rhee E et al. Extended survival and function of peripheral nerve allografts after cessation of long-term cyclosporin administration in rats. J Hand Surg 1993; 18A:100-106. 97. Mackinnon SE, Midha R, Bain J et al. An assessment of regeneration across nerve allografts in rats receiving a short course of cyclosporine A immunosuppression. Neurosci 1992; 46:85-93. 98. Buettemeyer R, Jones NF, Rao U. Peripheral nerve allotransplant immunosuppressed with FK 506: Preliminary results. Transplant Proc 1995; 27(2):1877-1888. 99. Kanaya F, Firrell JC, Breidenbach WC. Sciatic function index, nerve conduction tests, muscle contraction, and axon morphometry as indicators of regeneration. Plast Reconstr Surg 1996; 98:1264-1271.

CHAPTER 17

Allogeneic Rat Hindlimb Transplants Immunosuppressed with Mycophenolate Mofetil (RS-61443) Stephen J. Mathes, Robert D. Foster and James P. Anthony

Introduction

H

uman limb allotransplantation has been technically possible for more than 25 years, since microsurgeons first began performing digital and partial limb replantations following traumatic amputation.1-4 Despite this, clinical limb transplants have never been performed in the United States, primarily due to an inability to prevent rejection. To date, the most successful experimental models for limb or other composite tissue allografts have relied upon the use of clinically available immunosuppressants (e.g., cyclosporine, FK506) to prevent rejection. While these agents are effective in preventing rejection in human organ transplants, they have not provided reliable long term, rejection-free limb allograft survival. Several components of limb tissues, particularly the skin and muscle, are highly antigenic in comparison to solid organ tissues.5-8 Typically, then, with prolonged treatment of experimental limb transplants, some component of the allograft (most commonly the skin) is rejected and/or a form of chronic rejection ultimately develops.9,10 These observations prompted the use of a novel immunosuppressant for composite tissue allograft studies: mycophenolate mofetil, a drug with a unique mechanism of action and clinically shown to be useful in preventing renal allograft rejection.

Mycophenolate Mofetil: Mechanism of Action and Clinical Efficacy Mycophenolate mofetil (MMF), previously known as RS-61443 (Syntex, Palo Alto, CA), is derived from a Penicillium species, and when administered is hydrolyzed to its active moiety, mycophenolic acid (MPA).11 MPA is a potent, noncompetitive, and reversible inhibitor of inosine monophosphate dehydrogenase, a key enzyme in purine de novo synthesis.12 Because lymphocytes are unable to use salvage pathways for purine biosynthesis, MMF decreases DNA production, preferentially inhibiting T and B cell proliferation.13,14 Unlike other commonly used immunosuppressants such as cyclosporine (CsA) and FK506, antibody formation is also inhibited15 and, therefore, MMF prevents rejection by suppressing both cellmediated and humoral immune responses. Clinically, MMF has been applied to both renal and cardiac transplantation. Several large, randomized, double blind studies have evaluated MMF’s efficacy in preventing acute


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rejection after cadaveric renal transplantation. The results show a significant (60-70%) reduction in the number of biopsy-proven rejection episodes when MMF is combined with CsA and steroids compared to those patients treated with CsA and steroids alone.16 The most recent report of the International Mycophenolate Mofetil Renal Transplant Study Group, a pooled analysis of 3 randomized, double blind studies involving a total of 1493 patients, showed that MMF was more effective than azathioprine (AZA) when combined with CsA and corticosteroids posttransplant. MMF-treated groups exhibited a reduced incidence (16.5% vs. 40.8%) and severity of rejection, similar graft survival (90.4% vs. 87.6%), and better graft function over 12 months.17 MMF has also been used as rescue therapy for acute kidney rejection in multicenter, uncontrolled studies.18,19 MMF was compared to high dose intravenous methylprednisolone ± AZA. Treatment was standardized for the initial 7 days of the study, after which steroids or antilymphocyte therapy were given at the discretion of each center. Overall, graft loss was decreased at 6 months in the MMF group (46%) compared with the steroid group (60%). In addition, the need for anti-lymphocytic therapy was decreased in the MMF group. Controlled clinical trials using MMF in cardiac transplantation have yet to be published, although preliminary open label trials for treating both recurrent and persistent cardiac rejection have been promising.20 Adverse reactions to the clinical use of MMF are generally limited to gastrointestinal symptoms, including nausea, diarrhea, and cramping, which usually taper off with long term use. In contrast to CsA,21 drug-related nephrotoxicity or hepatotoxicity has not been reported. Currently MMF is recommended for use with CsA and steroids in patients receiving allogeneic renal transplants. The use of this agent in other organ transplants awaits the results of clinical trials. In addition to its use in transplantation, MMF appears promising for the treatment of rheumatoid arthritis by inhibiting lymphocyte proliferation, decreasing immunoglobulin levels, and reducing the number of painful and swollen joints,22,23 and there is continued interest in its use as an antineoplastic drug to treat leukemia, lymphoma, and a variety of solid tumors.24

Experimental Efficacy in Composite Tissue Transplantation Experimentally, MMF has proven to be useful to the field of composite tissue allotransplantation by: 1. Preventing rejection of allografts long term: 2. Reversing established rejection; and 3. As adjunctive therapy to reduce the required dosages of more toxic immunosuppressants.

Long Term Prevention of Rejection For immunosuppressive agents to play a role in composite tissue allotransplantation, effective long term regimens with minimal toxicity must be established. Relative to organ transplants (kidney, liver, heart, and pancreas),5-8 composite tissue allografts (CTAs) are more antigenic and, therefore, require much higher immunosuppressant dosages to prevent rejection. The skin and muscle, in particular, are especially antigenic, requiring immunosuppressant doses as much as 2-3 times higher than for solid organ allografts in animal models.25,26 Extrapolated to humans, this increased dosage requirement would be unacceptably high given the toxicity profile of CsA and FK506, particularly the renal and hepatic toxicity. In 1992, MMF was compared to CsA in a long term study as a single agent therapy to prevent acute rejection of hindlimb transplants across two inbred rat strains with a strong

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TABLE 17.1. Cutaneous histopathologic grading classification for rat hindlimb allograft rejection Grade 0 Grade 1 Grade 2 Grade 3 Grade 4

Normal epidermal appearance without evidence of rejection Focal basal cell layer vacuolization (focal or diffuse) Dyskeratosis of squamous cells in the epidermis or hair follicle epithelium Subepidermal clefting or microvesiculation Complete separation at the epidermal-dermal junction

Table 17.2. Histopathologic grading of skin biopsies Group

Description

1 2 3

Autografts Untreated allografts Allografts, CsA therapy

4

Allografts, RS-61443 therapy

Time of Biopsy (days post-op)

n

Histopathologic grade 0 1 2 3 4

375-386 13 116-140 223-244 69-76 231-251

4 6 6 6 5 6

4 2 1 4 5

3 1 1

1 3 -

2 -

6 -

antigenic mismatch at the major histocompatibility complex (MHC).27 Limbs from 12 Brown Norway donor rats were microsurgically transplanted onto 12 Fisher (F344) recipient rats. Six transplant recipients received immunosuppression with MMF (30 mg/kg/d) and six received CsA (10 mg/kg/d for 2 weeks, then 10 mg/kg twice weekly). The CsA dose was chosen to conform to the most frequently used dosing schedule cited in the literature for this model. The degree of rejection was determined by visual inspection and periodic skin biopsy. Untreated rat limb autograft and allograft recipients served as controls. Biopsies were graded 0 (normal) to 4 (severe rejection) based on a standard histopathologic grading scale (Table 17.1). As expected, untreated allografted rats developed rejection in 12-13 days, while untreated autografts remained rejection-free. Among the immunosuppressed allograft recipients, both CsA and MMF were initially effective in preventing limb rejection. However, CsA-treated rats developed mild to moderate rejection at 120-150 days after surgery, which persisted until sacrifice at postoperative day 223-224 (mean histologic rejection grade = 2.0). In contrast, rats treated with MMF demonstrated indefinite (>8 months) limb allograft survival with virtually no evidence of rejection (mean histologic rejection grade = 0.17). The difference between CsA and MMF with regard to the grade of rejection was significant (p200 days in eight rats. Six of these eight animals, however, developed evidence of Pneumocystis carinii pneumonia. More recent studies have demonstrated reliable skin survival for 300 days posttransplant using FK506 (2 mg/kg for 14 days followed by 2 mg/kg twice weekly). However, at greater

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than 300 days, skin rejection prevailed and all of the animals also died of a bacterial pneumonia.2 Therefore, FK506 does seem to be more effective at low doses than CsA, although again, if used as the sole immunosuppressant, indefinite, rejection-free survival is not reliably achievable. Mycophenolate mofetil (MMF) (Syntex Corp., Palo Alto, CA), derived from a Penicillium species, has a mechanism of action distinctly different from CsA or FK506. It is a potent, noncompetitive, and reversible inhibitor of inosine monophosphate dehydrogenase, a key enzyme in purine de novo synthesis.34 Because lymphocytes are unable to use salvage pathways for purine biosynthesis, MMF preferentially inhibits T and B cell proliferation, therefore suppressing both cell-mediated and humoral immune responses.35,36 MMF, administered at 30 mg/kg/d in hindlimb transplants, performed across two inbred rat strains with a strong antigenic mismatch at the major histocompatibility complex (MHC), has been shown to prevent rejection of all components of the allografts indefinitely (duration of the study: 231-252 days) in five of six animals. The results were confirmed in all of the animals histologically. The sixth rat displayed only slight skin rejection.26 Other immunosuppressive drugs have been tested in CTA models, including 15-deoxyspergualin and rapamycin, with mixed results.37,38 The most successful protocols for preventing rejection long term seem to involve the continued use of immunosuppressive drugs. However, even in cases of graft survival, the recipient animals often succumb to complications of the immunosuppression itself. In addition to general complications such as the increased risk of infection, all immuno-suppressive drugs produce specific side effects long term. Hepatotoxicity and nephrotoxicity are two well recognized side effects of CsA use in humans. The predominant toxicities associated with FK506 in humans also include nephrotoxicity as well as neurotoxicity, and MMF, although relatively nontoxic, can produce gastrointestinal symptoms including nausea, diarrhea, and cramping. Few experimental studies have attempted to document the incidence of these significant side effects in hindlimb allotransplantation. One study comparing the long term efficacy of MMF to CsA detected a moderate bone marrow suppression due to MMF toxicity, which appears to be species-specific and limited to the rat.39 This MMF-induced marrow suppression is manifested principally as a macrocytic anemia with moderate anisocytosis, polychromasia, and occasional poikilocytosis. The mean hematocrit was significantly lower in the MMF-treated group (27.0 ± 9.0) compared to CsA-treated animals (44.2 ± 3.8), rats with autografts (49.5 ± 1.7), or preoperative baseline values (42.7 ± 3.2) (p=0.001). Leukocyte counts of MMF-treated animals were significantly lower than baseline (5.1 vs. 9.1 cells/ml x 1000, p225 days), although no immunologic studies were done to characterize it.35 Further experiments by this same group attempted to delineate a dosing schedule that would yield maximal survival (using CsA at 0, 2, 4, 8, and 25 mg/kg/d for 20 days) and found 8 mg/kg/d to be the preferred dose.36 Such studies illustrate clearly that CsA was a significant improvement over prior regimens based on azathioprine/anti-lymphocyte serum/steroids. Despite the promising results using CsA to prevent rejection, no study using CsA immunosuppression has demonstrated reliable long term rejection-free survival to all of the components of a CTA. Most studies show indefinite rejection-free survival in only a handful

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of animals. Furnas et al37,38 used long term CsA therapy postoperatively (8 mg/kg/d for the first 20 days followed by a maintenance dose of 8 mg/kg twice a week) and had one out of five rats survive indefinitely (>400 days). Three out of the five rats in that study developed skin rejection beginning day 66-238 postoperatively. Similarly, CsA administered at 10 mg/ kg/d s.c. for 20 days, followed by twice a week thereafter, was unable to induce indefinite rejection-free survival, with five of six rats developing mild to moderate rejection when sacrificed at 32 to 35 weeks posttransplantation.39 Significantly, increasing the CsA dose has also not increased its effectiveness in preventing long term rejection. Using CsA at 15-25 mg/kg/d s.c., Hotokebuchi et al40 and Kuroki et al 41 observed early rejection of limb allografts at an average of 31-59 days posttransplantation. The only published reports of long term survival with high-dose CsA therapy have included small numbers of animals. In the first, Black et al42 produced rejection-free survival of limb allografts in six rats receiving a tapered dose of CsA up to 189 days as follows: 25 mg/kg/d on days 0 to 2, 15 mg/kg/d on days 3-19, and 10 mg/kg/d thereafter. However, unspecified animal morbidity was noted at these doses. Hewitt et al43 described eight long term limb allograft survivors without the need for continued immunosuppression. These animals were exposed to variable CsA regimens and represented the long term survivors from previous studies. However, the total number of animals transplanted to attain these eight animals was not specified, making estimation of the likelihood of long term CsA efficacy and interpretation of these findings difficult. The need for a more effective immunosuppressant than CsA for limb allografting is underscored by three recent studies examining CTAs in primates. Hovius et al44 performed radial partial hand allotransplantation in rhesus monkeys who received prednisone plus high dose CsA (25 mg/kg/d s.c.). Despite therapeutic blood CsA levels and supplemental attempts to reverse rejection, 10 of 12 monkeys developed rejection by postoperative day 21-144 (mean: 66 days). Gold et al45 studied the feasibility of performing vascularized composite mandibular allografts in monkeys using CsA at 15 mg/kg/d with additional methylprednisone reserved for rejection episodes. Three of the four recipients displayed clinical rejection within 27-65 days; the fourth monkey died prematurely on postoperative day 13 without allograft rejection. In a study of allogeneic neurovascular skin flaps and hand transplants in baboons, Skanes et al24 observed that the mean serum CsA concentration necessary to prevent rejection was 1100 ng/ml, a level that is two to three times higher than recommended clinically in humans. To achieve this minimal CsA concentration, the required CsA doses were 25-29 mg/kg/d s.c. and 35-48 mg/kg/d i.v.; these doses produced significant CsA-related toxicity. The fact that rather high doses of CsA were inadequate to ensure long term CTA survival in these primate studies is troubling, especially within the context of the well-recognized hepatotoxicity and nephrotoxicity associated with much lower doses of CsA in humans. For the conditions for which CTAs would likely be used (none of which is life threatening), the toxicity of high dose CsA would be unacceptable. Future work in composite tissue allotransplantation may rely on several promising immunosuppressants more recently introduced. Mycophenolate mofetil (MMF) (Syntex Corp., Palo Alto, CA), an inhibitor of de novo purine synthesis, preferentially inhibits both T and B cell proliferation. Used clinically to prevent and reverse renal allograft rejection, MMF has been shown experimentally to prevent rejection of CTAs long term, reverse established CTA rejection, and, along with CsA, serve as an adjunct therapy in reducing the required dosages of more toxic immunosuppressants.39,46,47 Probably most significantly, MMF has been shown long term to reliably prevent rejection of all of the components of a CTA, including the skin.39 FK506 (Fujisawa Pharmaceutical, Osaka, Japan), similar in action to CsA, but much more potent, has been used experimentally in limb allografting both as primary therapy

The Clinical Future of Composite Tissue Transplantation

277

and in combination with other immunosuppressant drugs. The most recent report using FK506 as primary immunosuppression,48 grafting between rats with a major antigenic mismatch, demonstrated rejection-free survival to 300 days posttransplant using a dose of 2 mg/kg/d for 14 days followed by 2 mg/kg twice weekly. Unfortunately, in animals surviving more than 300 days, a mild rejection was evident in the skin and muscle of all animals studied and all of them died of a bacterial pneumonia soon after that. For several weeks prior to their deaths, all of the animals lost significant amounts of body weight and, although not specifically tested for, the presumption was that graft versus host disease may have played a role in the animals’ demise. The development of graft versus host disease (GVHD) could become a significant complication if limb allografting is applied clinically, particularly because limb allografts contain a large load of immunocompetent cells within the donor bone marrow and lymph nodes. Therefore, future research must address this issue specifically. GVHD is a potentially fatal condition characterized in animals by ear erythema, footpad hyperkeratosis, dermatitis, weight loss, diarrhea, and alopecia, as well as liver and spleen involvement.49-51 The incidence of GVHD following experimental limb allotransplantation has never been documented. Only a handful of the numerous published rat limb allograft studies noted above even mention GVHD. Hotokebuchi et al40 noted the development of lethal GVHD in half of their recipient animals receiving long term CsA therapy, whereas researchers using a short term course of FK506 (14 days) observed nonlethal chronic GVHD in 30% of animals.52 Arai et al53 similarly noted chronic GVHD 8 to 10 months posttransplantation in three of five rats receiving only a single 50 mg/kg dose of FK506, but did not observe any GVHD in rats receiving long term FK506 therapy. Ferreira et al,54 in a long term study (189 days) using mycophenolate mofetil (MMF), also demonstrated no sign of GVHD which was confirmed by histopathologic analysis. If, as suggested by Hotokebuchi40 and Arai,53 GVHD requires many months to develop, many of the other studies were of insufficient duration to allow GVHD to develop. Alternatively, single dose or short term immunosuppressive therapy may predispose allograft recipients to GVHD through insufficient long term immuno-suppression of donor cells that survive in the host as chimeras. Clearly, further research is required to determine the incidence of GVHD and to develop therapies aimed at its treatment and prevention.

Functional Recovery: The Application of Nerve Allografting from Animal Models to Humans Even after rejection is prevented, CTAs cannot be applied clinically until a significant level of functional recovery is proven. For most CTAs, this will require a high degree of neural regeneration. Nerve allografts, now performed in humans, have greatly expanded our understanding of neural regeneration in CTAs. Over the past decade, our knowledge has progressed from understanding the nature of nerve allograft rejection to demonstrating function following nerve allografting in rats, monkeys, and now humans. Specifically, Mackinnon et al55,56 have shown that nerve allografts can restore function to muscles across a peripheral nerve gap in both rodents and primates using CsA immunosuppression. If immunosuppression is continued long enough for the nerve to regenerate across the nerve allograft, then the CsA treatment can be discontinued. Although the allografted Schwann cells will then be rejected, neural function resulting from the nerve regeneration will persist because the return in neural function is due to the host axons, not the allograft axons, which simply serve as a conduit for nerve growth. Thus, prolonged immunosuppression is not required for nerve allografting, probably as a result of the low antigenicity of neural tissue. Based on these experimental studies, in 1992 Mackinnon and Hudson reported the reconstruction of a 23 cm nerve gap in the sciatic nerve of a boy using a fresh nerve allograft,57

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with recovery of protective sensibility in the foot. Further experimental work demonstrated the feasibility of cold preservation for nerve allografts, stored using University of Wisconsin solution at 5° C for periods up to 5 weeks. As a result of this preservation, the immunogenicity of the allograft was decreased, which facilitated regeneration across the nerve graft.58,59 The Schwann cell basal lamina remained intact to provide an effective conduit for regenerating nerves.60 As with immediate nerve allografting, the foreign nerve allograft was eventually replaced by the host and indefinite immunosuppressive therapy was not necessary. The ability to effectively preserve nerve allografts allows the grafting procedure to be scheduled as an elective procedure, allows careful preoperative viral testing of the donor to be done, and allows time for the host to be adequately immunosuppressed prior to the surgery.61 This experimental protocol provided the basis for another clinical case demonstrating successful recovery of sensibility across a long peripheral nerve allograft in a 12 year old boy who sustained a severe posterior tibial nerve injury.62 Future directions for nerve allo-transplantation involve the use of monoclonal antibodies to block antigen recognition selectively, providing an antigen-specific immune tolerance.63,64 As with the experimental work on rat hindlimb allotransplantation, tolerance induction offers an attractive alternative to generalized host immunosuppression, especially for nonlife-threatening transplantation procedures in which the risks of immunosuppression must be carefully weighed against the benefits of anticipated recovered function.

Composite Tissue Transplantation: The Future—Towards More Complex Study Protocols: Canine Larynx Allotransplantation As long term rejection-free allograft survival becomes more reliably achievable, increasingly refined and comprehensive CTA study protocols will be possible and more complex reconstructions will be attempted. For example, in 1995 Anthony et al65 developed the first successful heterotopic model for studying laryngeal allotransplantation in dogs (Figs. 22.1 and 22.2), involving microvascular reattachment of the laryngeal transplant adjacent to the animal’s native larynx. The transplanted larynx was unilaterally revascularized through anastomoses to the native common carotid artery and external jugular vein, and unilaterally reinnervated by microsurgically attaching the superior and recurrent laryngeal nerves of the transplant to those of the recipient. Animals were evaluated via histologic and immunologic studies to monitor for signs of rejection, and nerve regeneration studies that included EMGs and fiberoptic laryngeal endoscopy. Three heterotopic laryngeal allotransplant recipients were immunosuppressed with a combination of CsA (5 mg/kg/d for the first 7 days postoperatively, followed by 15-20 mg/kg/d for the duration of the study), methylprednisolone (1 mg/kg/d), and MMF (20 mg/kg/d). All three transplants remained rejection-free for greater than 100 days posttransplant. All of the animals fed normally by postoperative day 2 and none required a tracheostomy. Since the transplant was exteriorized through the skin at both its upper and lower ends, the vocal cords could be examined daily under direct vision. Since no tracheostomy was needed, changes associated with a chronic tracheostomy, such as progressive cord adduction, were avoided. Since the native trachea remained intact, it could be manipulated (via airway obstruction, SLN stimulation, etc.) and EMGs and the resultant cord movements in the reinnervated side of the transplant could be easily measured and compared to both the intact side of the native larynx and the noninnervated side of the transplant. Early reinnervation was documented via histologic sections of the superior and recurrent laryngeal nerves (Fig. 22.3). Further work is needed to demonstrate the degree of neural recovery and improve the specificity of axonal regrowth so that muscle synkinesis due to crossedfiber reinnervation is avoided.


Fig. 22.1. Canine anatomy relevant to laryngeal transplantation.

279

280


Fig. 22.2. An anterior view of the pertinent anatomy in heterotopic laryngeal transplantation. The transplanted larynx is unilaterally revascularized and reinnervated. The unilaterally deinnervated native trachea remains in situ. Both ends of the heterotopic transplant are exteriorized, providing cord visualization (and functional testing) from both above and below.


281

A

Fig. 22.3. Histologic evaluation of the recurrent (A,B) and superior (C,D) laryngeal nerves 90 days posttransplantation. Both low and high power views of the regenerating nerves just distal to the operative repairs (toluidine blue stain). Although the axons in both nerves are irregularly shaped, their density within the nerves indicates that a high degree of axonal ingrowth (regeneration) has occurred.

B

C

D

282


Transplant Survival Without Immunosuppression: Allogeneic Tolerance Induction Despite the advances in immunosuppression therapy outlined above, the development of safe methods for achieving donor-specific tolerance across MHC barriers remains the ultimate goal in composite tissue allotransplantation. These transplant recipients would be tolerant to the antigens within their transplant but would otherwise remain fully immunocompetent. The only reports in the literature of tolerant CTAs have been limited to sporadic accounts in rat hindlimb allografts receiving long term immunosuppression.43 In these studies, however, tolerance was not reliably reproducible and a significant number of animals developed lethal graft versus host disease (GVHD). Recent models specifically designed to address the issue of tolerance induction in CTAs have thus far provided inconsistent results.66,67 The eventual solution may lie in the vast literature on experimental tolerance induction applied to organ allotransplantation. Although the mechanism of experimental tolerance induction is not well understood, it is readily achievable between mice with a major antigenic mismatch.68-70 In contrast, similar protocols applied to rats have proven inadequate in producing indefinite allotransplant survival. The reasons for this disparity are currently unknown. Unfortunately, the technical aspects of transplanting a hindlimb in a mouse have, thus far, precluded the usefulness of most tolerance induction protocols for CTAs. However, a newly developed model for mouse hindlimb transplantation71 may provide a solution. The applications and widespread acceptance of this model await further study. Mixed allogeneic chimerism is one approach to transplantation tolerance induction which has been successfully applied in both mice72 and rats.73 Irradiated animals, reconstituted with a mixture of host and donor T cell depleted bone marrow, develop normal immunocompetence and host-restricted responses. Mixed allogeneic chimerism offers several theoretical and practical advantages over other forms of tolerance induction. Since this technique provides (or leaves intact) a source of autologous marrow, there is minimal risk of potentially fatal aplasia should alloengraftment fail (one of the major risks of allogeneic marrow transplantation). Hematopoietic cells bearing host MHC molecules provide a source of accessory cells that can interact effectively with T cells educated in the host thymus. Such interactions probably account for the improved immunocompetence observed in such animals compared with fully allogeneic chimeras.74 The autologous or syngeneic bone marrow component of the graft also appears to prevent GVHD. In addition, when these chimeric rats are then challenged with skin allografts, complete acceptance is attained.73

Conclusion Composite tissue allotransplantation is not yet ready to be adopted into clinical use. Additional work needs to be done to improve the efficacy of the immunosuppressive drugs while minimizing their toxicity, determining the incidence and treatment of GVHD, and assessing and improving long term functional recovery. Our thoughts on the future of CTA are aptly expressed in Joseph Murray’s report in 196375 of the first successful allogeneic kidney transplants in humans. At the conclusion of the article he commented that, “...this report permits a note of cautious optimism in a problem that ten years ago was considered almost insoluble.” The editorial in the New England Journal of Medicine following Murray’s report76 recognized then that if transplantation could be successfully accomplished in man, “...the possible clinical applications are almost unlimited.” Our hope is that the many authors contributing to this text will continue in their efforts to substantiate this sentiment.


283

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73. Colson YL, Zadach K, Nalesnik M et al. Mixed allogeneic chimerism in the rat. donorspecific transplantation tolerance without chronic rejection for primarily vascularized cardiac allografts. Transplantation 1995; 60:971-980. 74. Deeg HJ, Tsoi MS, Storb R. Mechanisms of tolerance in marrow transplantation. Transplant Proc 1984; 16:933-937. 75. Murray JE, Merrill JP, Harrison JH et al. Prolonged survival of human-kidney homografts by immunosuppressive drug therapy. N Engl J Med 1963; 268:1315-1323. 76. Garland J. Uniqueness of the individual. N Engl J Med 1963; 268:1362-1363.

CONCLUSION

W

ith an increasing understanding of the immune response directed against allotransplants, there is a better awareness of how to effectively reduce or eliminate this response, with minimal untoward effects to the host. In addition, with the tremendous wealth of knowledge that has been gained over the years regarding organ transplantation, management of the organ transplant patient has become more predictable. Within the chapters of this book, we have had a chance to explore the immuno-suppressive basic science, physiology, and pathology of the various tissues that make up composite tissue transplantation. Although each reader must draw their own conclusions from these works, there are facts that remain evident. Composite tissue transplantation is most certainly surgically feasible. The techniques to transplant blocks of composite tissues for reconstruction and repair exist today. Secondly, the immunosuppressive regimens necessary to provide long term inhibition of the immune response exist today. Sophisticated drug therapies that are in routine use in many transplant centers have been examined within the composite tissue transplant experimental arena. Additionally, many more immunosuppressive drug therapies are currently in the pipeline. Many of the authors in this book have noted various and tremendous needs for this kind of reconstructive opportunity. The next steps in composite tissue transplantation will be bold ones. Pioneering teams of surgeons and patients will embark on one of the greatest adventures in transplantation. It is with this excitement and hope for the future that we submit this collection of works for the readers’ consideration.


272


Index

289

INDEX A

C

Allogeneic 9, 10, 12, 20-22, 42, 44, 45, 51, 74, 75, 79, 90, 91, 97, 98, 107, 144, 198, 215, 225, 226, 237, 238, 241, 257, 261, 262, 264, 270, 274, 276, 282 Allogeneic knee joints indication 257, 260, 262 trauma management 258 Allograft 9, 10, 12-14, 17-24, 31-34, 44, 45, 50, 51, 57, 65, 66, 68, 73, 79, 82, 87-94, 96-101, 107-109, 113, 115, 123, 149, 157-166, 173-182, 184-188, 192-195, 197-202, 205-207, 209, 214-217, 219, 220, 225-243, 247, 250-252, 259, 268-270, 274-278, 282 Allograft component 9 Alloimmune chimerism 41 Analog 173, 183, 241 Animal 5, 10, 11, 13, 14, 18-22, 24, 31-39, 50, 51, 58, 59, 123, 129, 143-145, 160, 174, 175, 177-179, 181-186, 188, 199, 201, 202, 205, 207, 209, 214-217, 219, 220, 226-230, 233-236, 239-244, 247, 249-252, 257, 268-270, 275-278, 282 Antigenicity 9, 10, 12-14, 19, 22, 23, 32, 159, 238, 239, 249, 277

Canine 23, 75, 108, 111, 143, 158, 160, 173, 175, 178, 183, 184, 188, 198-202, 228, 243, 247, 278 Chimerism 200, 215, 282 alloimmune 41 cellular kinetics 59 T cell chimerism 58, 65, 75 Clinical application 4, 38, 39, 73, 76, 79, 87, 100, 101, 108, 112, 121, 140, 148, 149, 151, 179, 188, 197, 201, 202, 228, 234, 236, 244, 267, 269, 274, 275, 277, 282 future 35, 79, 89, 93, 101, 115, 148, 151, 185, 197, 201, 236, 242, 244, 252, 264, 270, 273, 276-278, 282 implications 97, 132 past 101, 148, 157, 162, 197, 198, 247, 248, 274, 277 studies 3, 10, 13, 22, 23, 32, 34, 41, 44, 45, 51, 57, 59, 61, 65-68, 75, 76, 80, 90, 91, 93, 94, 96, 97, 100, 107, 108, 113, 126-129, 131, 132, 140, 143-146, 148, 157-163, 165, 166, 173-186, 188, 197-201, 209, 214-217, 219, 220, 225, 226, 228, 234, 236, 237, 240-244, 249, 250, 252, 268, 269, 274-278, 282 trials 123, 139, 140, 145, 148, 151, 173, 174, 176-179, 181, 183-188, 206, 226, 228, 237, 242, 248, 250 Combination drug therapy 239, 242, 243 Composite tissue 4, 10, 12, 33, 57, 65, 68, 71, 73, 76, 79, 82, 171, 173, 175, 178, 188, 197, 198, 201, 205, 214, 215, 217, 225, 226, 228, 234, 236-239, 241, 242, 244, 247, 273-276, 278, 282

B Bone marrow 10, 12, 13, 31, 32, 35, 37-39, 41, 42, 44, 57, 65-68, 73-76, 79, 89, 111, 129, 178, 182, 184, 186, 187, 197, 199, 214-216, 227, 228, 242, 262, 268, 274, 275, 277, 282

290

Clusterin in Normal Brain Functions and During Neurodegeneration

Cosmas and Damian 5, 6, 274 CsA (cyclosporine A) 21, 33, 34, 45, 50, 57, 79, 91, 92, 94, 96, 101, 108-110, 112, 113, 115, 144, 145, 173-178, 180-188, 198, 199-202, 205, 207, 209, 214-217, 219, 225-228, 234-236, -243, 269, 270, 275-278 CTA (composite tissue allograft) 10, 57-59, 61, 65-68, 73, 79, 82, 197-202, 226, 239-242, 244, 273-278, 282 Cyclosporine 3-6, 10, 13, 14, 17, 18, 21, 22, 24, 32-34, 45, 57, 91, 94, 108, 109, 112, 144, 145, 147, 148, 173, 174, 198, 205-207, 215-217, 225, 228, 237-239, 247-251, 262, 269, 275

F

D

Hemipelvis model 79 Hindlimb 51, 57, 59, 65-68, 73, 75, 239, 242, 243, 247-252, 275, 278, 282 Humoral response 12, 18, 19, 24, 31, 32, 61, 107, 175, 182, 185, 200, 214

Dendritic cells 11, 41, 42, 44-46, 50, 89, 94, 98, 268 maturation and migration 44 Denervation 121, 122, 126, 130-132 Deoxyspergualin 107, 109, 110, 112, 115, 173, 186, 206, 215, 242, 252 Differential rejection 9

E Efficacy 97, 100, 140, 143, 145, 146, 160, 162, 166, 179-182, 185, 200, 201, 206, 215, 216, 225, 226, 234, 237-239, 242, 243, 247, 248, 251, 252, 274-276, 282 Experiments 3, 5, 6, 10, 22, 35, 39, 46, 57, 100, 111, 113, 121, 123, 130, 131, 143, 144, 158, 173-175, 178, 179, 184-186, 197, 200, 205, 206, 215, 216, 220, 225, 226, 228, 236-239, 242, 243, 247, 248, 250-252, 257, 268, 273-278, 282

FK506 50, 90, 91, 109, 175, 180, 198-200, 202, 205-207, 209, 214, 216, 217, 219, 225, 226, 238-242, 247-252, 270, 276, 277

G Gene therapy 122, 139, 140, 142, 143, 145, 146, 148, 151 Graft versus host disease (GVHD) 57, 59, 60, 65-68, 73-76, 111, 181, 182, 185, 186, 200, 207, 209, 214-216, 236, 249, 277, 282

H

I Immune privilege 51, 100 Immune system 5, 11-13, 21, 23, 24, 31, 32, 35, 74, 96, 100, 101, 140, 188, 200, 250, 268, 270 adult 32 fetal 32, 34, 35 Immunosuppressants 4, 50, 108-111, 115, 142, 144, 145, 148, 171, 173, 175, 180, 182, 188, 198, 201, 225, 226, 228, 238, 239, 242, 249, 269, 270, 274, 276, 277 Immunosuppression 10, 21, 22, 23, 31-34, 39, 50, 75, 76, 87, 88, 90-92, 94, 96-98, 101, 109, 110, 115, 142, 145, 148, 150, 173, 176, 178, 184, 188, 197-202, 205-207, 209, 214-217, 219, 227, 228, 234, 239-244, 247-252, 262, 264, 267-270, 274-278, 282

Index

Immunosuppression (cont.) donor-specific 22, 50, 96 local 197, 200-202 topical 270 Immunosuppressive agents 10, 22, 35, 91, 173, 197, 198, 200, 205, 206, 209, 215, 216, 226, 238, 247, 249, 275 Inflammatory cell 96, 121, 128, 129, 249 Irradiation 10, 32, 46, 50, 73, 93, 97, 98, 108, 112, 198, 200, 205, 270

L Lamina 90, 121, 122, 125-127, 132, 278 Large animal 10, 11, 19, 20, 31, 32, 34, 35, 39, 111, 201, 202 Larynx 273, 274, 278 Limb 3-6, 9, 10, 12-14, 17-20, 22-24, 33, 41, 45, 46, 50, 57, 65-68, 74- 76, 79, 80, 87, 93, 112, 123, 144, 148, 165, 176, 181, 197-201, 205-207, 209, 214-217, 219, 225-228, 232-234, 236-241, 243, 247, 249-252, 274-277 limb transplant(ation) 3-6, 23, 41, 45, 50, 57, 74-76, 112, 176, 181, 197-199, 201, 205-207, 209, 214-217, 219, 225, 226, 234, 238, 242, 247-249, 251, 252, 275, 282

M Meniscus 157, 158, 160-163, 165, 166 function 157, 160, 163, 166 meniscectomy effects 157, 158, 160 Model 65-68, 71, 73-76, 79, 80, 82, 89-91, 93, 94, 96, 97, 99-101, 111, 112, 122, 123, 143, 145, 157, 160, 173, 175, 177-179, 181-184, 186, 188, 197-202, 205, 206, 214, 219, 225-228, 234, 236-239, 242, 243, 247-252, 269, 270, 274, 277, 278, 282 canine 160, 247 primate 237, 250

291

MTT Technology 142 Muscle 10, 13, 14, 18, 19, 22, 23, 33, 34, 46, 75, 79-82, 94, 121-124, 126-132, 142-151, 178, 197-199, 201, 205, 207, 209, 214, 217, 219, 225, 226, 236, 239, 249-251, 261, 273, 274, 277, 278 regeneration 121, 122, 127-132, 142, 150 Muscular dystrophies 122, 127, 131, 143, 144, 151 Mycophenolate mofetil 173, 177, 199, 225, 226, 236-238, 242, 276, 277 clinical efficacy 225 mechanism of action 174, 175, 177, 179, 180, 182-184, 186, 187, 225, 241, 242, 247, 250 Myoblast 121-123, 125-132, 139, 140, 142, 143-151, 175, 182 Myofiber 121-127, 130-132, 140, 142-146, 149

N Nerve 10, 13, 22, 75, 79-82, 87-91, 93, 94, 96-101, 107-109, 112, 113, 115, 127, 130, 146, 176, 186, 197, 198, 205, 206, 217, 219, 220, 234, 236, 238, 251, 274, 277, 278 Nerve allograft 13, 22, 87-91, 93, 94, 96-101, 107-109, 113, 115, 277, 278 peripheral 22, 87, 90, 91, 97, 100, 108, 217, 219, 278 preservation 88, 93, 100, 178, 186, 278 response 12, 88, 90, 93 storage 88, 93, 94, 100, 101 Nerve graft 87, 91, 93, 115, 217, 219, 278 FK506 immunosuppression 90

292

Clusterin in Normal Brain Functions and During Neurodegeneration

P

T

Pathology 65, 67, 68, 130 Peripheral nerve 22, 87, 90, 91, 93, 97, 98, 100, 101, 107, 108, 112, 115, 176, 186, 197, 198, 217, 219, 277, 278

Tissue engineering 139, 140, 142 Tolerance 3, 10, 20-22, 31-35, 37, 39, 41, 42, 44, 50, 57, 60, 61, 65, 66, 68, 73, 75, 76, 87-90, 96-101, 109, 111, 112, 115, 177, 180, 183, 188, 214, 215, 241, 244, 275, 278, 282 Toxicity 10, 12, 14, 17, 24, 32, 108, 110, 112, 140, 173-176, 178-180, 183, 184, 186-188, 198, 199, 201, 205, 206, 214, 216, 226-228, 234, 241-243, 248, 250-252, 270, 274-276, 282 Transfer 65, 74-76, 121, 122, 127, 139, 140, 142, 143, 145, 148, 151, 162, 178, 201, 209, 237

R Rapamycin 173, 180, 198, 200, 205, 215, 216, 238, 242, 247, 249-252 efficacy of 247, 248, 251 history 247, 249, 257 toxicity 248 Regeneration 22, 87, 91-94, 96-98, 100, 101, 107-109, 112, 113, 115, 121-123, 126-132, 142, 150, 234, 236, 238, 277, 278 Rejection 3, 9-13, 17, 21-24, 31-34, 41, 42, 44, 45, 50, 123, 142, 149, 173-185, 187, 188, 197-202, 205-207, 209, 214-217, 219, 225-244, 248-252, 257, 261, 262, 264, 268-270, 274-278

S Skeletal 4, 10, 12, 13, 20, 31-34, 65, 75, 109, 112, 121-123, 126-128, 130, 132, 140, 144, 198, 207 Skin 9-14, 18, 19, 21-24, 33-35, 37-39, 42, 44-46, 50, 66, 67, 75, 76, 79-82, 110, 140, 148, 174, 175, 181, 182, 185, 186, 188, 197-201, 205-207, 209, 214-217, 225-230, 232-234, 237, 239-243, 249-251, 267-270, 273-278, 282 modification 270 Surgical model 80

U UV-B 98-100

V VBMT (vascularized bone marrow transplantation) 57-59, 61, 65-68, 73-76 Vector 139, 140, 142

X Xenotransplantation 107, 108, 112, 115

Composite Tissue Transplantation

Transplantation of Composite Tissue Allografts

The Scientific Basis of Tissue Transplantation (Advances in Tissue Banking)

Lung Transplantation

Hand transplantation

Cardiac Transplantation

Hand Transplantation

Lung Transplantation

Organ Transplantation

Lung Transplantation

Scar Tissue

Scar Tissue

Scar Tissue

Tissue Engineering

Tissue Engineering

Composite Construction

Composite fermions

Composite Sheet Forming (Composite Materials Series)

Tissue Mechanics

Tissue Mechanics

Tissue Engineering

Tissue Engineering

Organ Transplantation stuart

Hair Transplantation (Cambridge Medicine)

Cardiac Transplantation (Contemporary Cardiology)

Bone Marrow Transplantation (Vademecum)

Imaging in Transplantation

Hematopoietic Stem Cell Transplantation

Living Donor Transplantation

Liver Transplantation - Vademecum

Hair Transplantation (Cambridge Medicine)

Composite Tissue Transplantation

Transplantation of Composite Tissue Allografts

The Scientific Basis of Tissue Transplantation (Advances in Tissue Banking)

Lung Transplantation

Hand transplantation

Cardiac Transplantation

Hand Transplantation

Lung Transplantation

Organ Transplantation

Lung Transplantation

Scar Tissue

Scar Tissue

Scar Tissue

Tissue Engineering

Tissue Engineering

Composite Construction

Composite fermions

Composite Sheet Forming (Composite Materials Series)

Tissue Mechanics

Tissue Mechanics

Tissue Engineering

Tissue Engineering

Organ Transplantation stuart

Hair Transplantation (Cambridge Medicine)

Cardiac Transplantation (Contemporary Cardiology)

Bone Marrow Transplantation (Vademecum)

Imaging in Transplantation

Hematopoietic Stem Cell Transplantation

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