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

Composite Tissue Transplantation

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

Composite Tissue Transplantation

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

Composite Tissue Transplantation

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

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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

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

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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-

Relative Antigenicity of Limb Allograft Components and Differential Rejection

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

Composite Tissue Transplantation

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

Relative Antigenicity of Limb Allograft Components and Differential Rejection

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

Composite Tissue Transplantation

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

Relative Antigenicity of Limb Allograft Components and Differential Rejection

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.

Composite Tissue Transplantation

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.

Relative Antigenicity of Limb Allograft Components and Differential Rejection

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

Composite Tissue Transplantation

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 (p<0.001 for vascularized versus control, p<0.01 for nonvascularized versus control) (Fig. 2.8A). The pattern of humoral responses was different in that there was no detectable antibody production in nonvascularized skin allografts until two weeks (p<0.001 for vascularized versus control, no significance for nonvascularized versus control (Fig. 2.8B). Similarly, in bone allografts, vascularization led to more prompt and intense cell-mediated and humoral responses (Figs. 2.9A,B). When the cell-mediated responses were compared for different tissues, muscle allografts generated a significantly higher response at one week (p<0.001 versus limb/vessels, p<0.01 versus bone, p<0.05 versus skin/subcutaneous) (Fig. 2.10A). Skin, subcutaneous tissue, and bone allografts were about equally antigenic as evidenced by statistically indistinguishable immune responses. The limb allografts, consisting of all tissue components, had a lower response (p<0.001 versus muscle, p<0.01 versus bone, p<0.05 versus skin/subcutaneous). Finally, vessel allografts led to the lowest responses (p<0.001 versus muscle/skin/subcutaneous/bone, p<0.05 versus limb).

Relative Antigenicity of Limb Allograft Components and Differential Rejection

A

19

Fig. 2.9. Cell–mediated responses in rats with various bone allografts: vascularized, nonvascularized, and vascularized with cyclosporine (A). Humoral responses in rats with various bone allografts (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

For humoral responses, the muscle allografts elicited no early antibody production (p<0.001 versus skin/bone) (Fig. 2.10B). There were no significant differences in humoral responses among skin, subcutaneous tissue, and bone allografts. Limb allografts generated lower responses (p<0.01 versus skin, p<0.05 versus bone) at one week. Again, vessel allografts were least antigenic (p<0.001 versus skin, p<0.01 versus bone).

Large Animal Data Although no large animal study has examined the relative antigenicity of limb tissue allografts, it is informative to review some of the data on organ transplantation in the only genetically defined large animal model—miniature swine. In particular, the availability of MHC homozygous and intra-MHC recombinant haplotypes make miniature swine the only large animal model in which one can reproducibly study the effects of selective matching for Class I and/or Class II loci on parameters of transplantation immunity.90-92 The Transplantation Biology Research Center at the Massachusetts General Hospital has developed miniswine with three homozygous swine leukocyte antigen (SLA) haplotypes, SLAa, SLAc, SLAd, and five lines bearing intra-SLA recombinant haplotypes. All of these lines differ by minor histocompatibility loci, thus providing a model in which most of the transplantation combinations relevant to human transplantation can be mimicked. For example, transplants

20

Composite Tissue Transplantation

A

B

Fig. 2.10. A systematic comparison of cell–mediated response in animals with various vascularized limb–tissue allografts: muscle, skin, subcutaneous tissue, bone, whole limb, and blood vessels (A). A systematic comparison of the humoral responses in animals with various vascularized limb–tissue allografts (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.

within an MHC homozygous herd simulate transplants between HLA identical siblings. Transplants between herds resemble cadaveric or nonmatched sibling transplants, and transplants between pairs of heterozygotes can be chosen to resemble parent into offspring, or one haplotype mismatched, sibling transplants. An additional advantage of this model is that swine, like humans, exhibit constitutive expression of Class II MHC antigens on vascular endothelium.61,93 In contrast, Class II antigens are not constitutively expressed on vascular endothelium in rodents, although they may be induced by treatment with IFN-!.94-96 Since endothelium is the first site at which host immune cell populations interact with a vascularized graft, it is possible that the ease with which tolerance can be induced to primarily vascularized allografts in rodents may be related to this difference in Class II expression on the vascular endothelium.97 Indeed, as described below, some of the techniques which have previously been shown to produce tolerance in rodents but not in other large animal models have been found to induce tolerance in miniature swine if, and only if, donor and recipient are matched for Class II.98,99 Although no systematic assessment of allogeneic limb tissues has been attempted in miniswine, there are valuable data on allogeneic kidney, heart, and musculoskeletal transplants in these herds to draw some inferences about relative antigenicities of various tissues.

Relative Antigenicity of Limb Allograft Components and Differential Rejection

21

Kidney Allografts Renal allografts between any miniature swine strain mismatched for Class II antigens (e.g., SLAcc ∀ SLAaa) always reject the graft acutely.98 When the swine are matched for Class I and II MHC antigens (e.g. SLAaa ∀ SLAaa) they develop specific and long term (>100 days) transplantation tolerance in approximately two-thirds of the cases without the use of any immunosuppressive therapy.98,100,101 When the recipients receive a short course of cyclosporine therapy consisting of 10 mg/kg for 12 days beginning on the operative day, these matched grafts are accepted in all animals. In contrast, when transplants are matched for Class II and differ for one haplotype at Class I (e.g., SLAag ∀ SLAad), approximately 30% of such allografts are accepted long term (>100 days).98,101 Spontaneous acceptors of Class II matched, one haplotype Class I mismatched, allografts demonstrate a significant rejection crisis during postoperative weeks 2-4, which subsides spontaneously; normal renal function is maintained long term. 98 Animals that accept Class I mismatched renal allografts do not ignore the MHC antigens on the graft, but rather become actively tolerized to these antigens. Thus, during the rejection crises, cytotoxic antibodies directed against the donor Class I antigens are detected in the serum of acceptor animals, but these antibodies are almost entirely of the IgM Class, suggesting failure of the usual IgM to IgG switch which characteristically occurs in animals that reject an organ allograft.98,101 Since the switch from IgM to IgG is generally thought to depend on T cell help,102 these results suggested that in the absence of sufficient T cell help, as might occur for single haplotype Class I only mismatched kidney allografts, tolerance rather than rejection had been induced. The acceptance of renal grafts across Class I plus minor differences does not indicate a failure of the immune system to recognize Class I, but results rather from an active immunologic process leading to tolerance. Consistent with this hypothesis, skin grafts from the original renal donor showed markedly prolonged survival (31.7 ± 20.4 d) over similar Class I mismatched skin grafts placed on control animals (11.67 ± 2.6 d). Although skin grafts were prolonged, they were eventually rejected, probably due to skin-specific antigens rather than to a break in tolerance, since there was no concomitant impairment of renal function, nor anti-Class I antibody formation.98 Since the development of spontaneous, long term tolerance to one haplotype, Class II matched, Class I mismatched, kidneys appeared to depend on a relative deficit of T cell help at the time of the host’s first exposure to antigen,97-99 pharmacological means of limiting T cell help is employed, using a short course of treatment with cyclosporine. Transplants across a selective two haplotype Class I mismatched, Class II matched, barrier without immunosuppression uniformly reject within three weeks. This treatment regimen induced long term, specific tolerance in eight of eight two haplotype Class II matched, Class I mismatched recipients.99 This result has subsequently been substantiated in 30 additional CsA-treated animals, all of which demonstrated long term tolerance, confirming a 100% rate of tolerance induction across this disparity.

Heart Allografts Comparable data between the various genetic lines of miniature swine have been generated for allogeneic heart transplantation as well. Heart allografts transplanted across a full MHC mismatch are rejected acutely in 7-8 days, similarly to kidney allografts. Whereas recipients of allogeneic kidney grafts matched for Class I and II, matched for Class II and mismatched for Class I, and a single haplotype Class I and II can survive with or without cyclosporine, heart allografts undergo rejection in all instances. Although cyclosporine administered for 12 days can prolong the heart allografts by several days, they are eventually rejected. Furthermore, in animals matched for Class II and mismatched for Class I, the hearts undergo cardiac allograft vasculopathy (CAV).103 These data suggest that the recipient’s

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Composite Tissue Transplantation

response to these two organs are at opposite ends of a spectrum of antigenicity. Whether the recipient’s response is a function of the organ or the tissues and cells that comprise these organs cannot be conclusively determined at this time.

Vascularized Limb Tissue Allografts Ongoing studies in our laboratory using the miniature swine indicate that vascularized limb tissue allografts fall somewhere in between kidney grafts and heart grafts on the spectrum of allogenicity. Using a composite vascularized limb tissue allograft isolated on the femoral artery and vein consisting of the tibia, fibula, and distal fragment of the femur along with a muscle cuff, we performed transplants among various matched and mismatched animals. Like kidney and heart grafts transplanted across a complete MHC mismatch, the limb tissue grafts were rejected within two weeks of stopping the cyclosporine therapy (given only for 12 days). When we matched for both Class I and II and gave 12 days of cyclosporine, the limb tissue allografts survived as long as 260 days—the end of the experiment. Donor skin grafts applied to these animals at day 100 also were prolonged and, in two swine, the donor skin grafts lived until the experiment was terminated (160 days for skin and 260 days for limb tissue graft). The grafts were rejected in these MHC matched recipients if cyclosporine was withheld—unlike the spontaneous acceptance noted in kidney allografts. Grafts across a Class I mismatch and a Class II match, as well as across a single haplotype Class I and II, were rejected, even when cyclosporine was given for 12 days. Whereas kidney allografts are least likely to stimulate a host immune rejection response in this model, and hearts are rejected in all matches, vascularized limb tissue allografts are intermediate in their ability to evoke an immune response.

Conclusion Despite the increasing clinical success of visceral organ transplantation, the era of limb tissue transplantation has not arrived in reconstructive surgery. The clinical need is obvious for limb tissue allografts consisting of any combination of skin, subcutaneous tissue, muscle, bone, blood vessels, or an entire limb, and the advent of microvascular surgery has made such allografts technically possible. With the introduction of cyclosporine, various researchers have demonstrated survival of skin,19-25 muscle,26-28 bone,29-33 nerve,34-37 and whole limb38-47 allografts in animals treated with this immunosuppressive agent.104,105 However, continuous or maintenance dosage of immunosuppressive therapy is necessary for prolonged survival of limb tissue allografts.33 Reports on animals tolerant to allogeneic limb tissues without the use of ongoing immunosuppression have been rare: Achauer reported a single spontaneous indefinite survivor in a rat with limb allograft which had received an initial 20 day course of cyclosporine,105 and Mackinnon noted regeneration in peripheral nerve allografts without apparent rejection following cessation of immunosuppression.37 Reasons for the observed tolerance were not clear. In the vast majority of experiments, however, graft rejection inevitably ensued once cyclosporine therapy was halted. Attempts at donorspecific immunosuppression or other immunosuppressive regimens have been largely unsuccessful.40,106-110 (Donor-specific immunosuppression refers to suppressing host immune response to graft antigens without compromising the host’s overall immune capabilities). As the adverse effects of chronic immunosuppression outweigh the possible benefits of nonvital organ transplants, transplantation of limb tissue allografts cannot be justified today. The basic strategy for dealing with host rejection in clinical organ transplantation consists of two parts, namely graft selection and host treatment. The options for host treatment are limited by the immunosuppressive means available and current state of knowledge in transplant immunology. With regard to graft selection, genetic matching has been shown to

Relative Antigenicity of Limb Allograft Components and Differential Rejection

23

Table 2.2. Relative antigenicity of limb tissues Cellular response

Humoral response

Most antigenic

Muscle

Least antigenic

Skin Subcutaneous tissue Bone Limb Vessel

Skin Subcutaneous tissue Bone Limb Muscle Bone

Note: Based on immune responses measured for each limb tissue at 1 week after transplant. Reprinted with permission from Lee WPA et al. Relative antigenicity of components of a vascularized limb allograft. Plast Reconstr Surg. 1991; 87(3):409.

delay rejection and may be a means to temper the immune response clinically.86 Decreasing the antigenicity of a limb tissue allograft by removing or suppressing its more antigenic portions, in accordance with Russell’s concept of “selective transplantation”, is another potential way of making such allografts clinically feasible.111 Knowledge of a hierarchy of antigenicity for limb tissues may allow selective removal or manipulation of components of limb tissue allografts to ameliorate host rejection. Our rodent study is the only known systematic immunologic assessment with regard to the relative antigenicities of individual limb tissues. The results yielded a scale more complex than the one proposed by Murray for skin and visceral organs (Table 2.2). The various limb tissues interacted with the host immune system in an intricate but predictable pattern with differing timing and intensity. The relative scale of antigenicity for limb tissues, therefore, varies according to the type of immune response measured (cellular versus humoral) and, to a lesser extent, the time at which it was measured. Our findings brought into question two long held assumptions regarding skin antigenicity. Skin has been thought to be the most antigenic body tissue and has therefore been used as a litmus for various immunologic studies.112,113 Furthermore, the Langerhans’ cells residing in the epidermis have been considered a primary source of antigenicity for skin grafts.50- 52 In that study, vascularized muscle allografts elicited even greater cell-mediated responses than skin. This finding, in fact, corroborates the observation made by Goldwyn in 1966 in canine limb transplant that skin and muscles were prime targets of immune destruction.10 With regard to the Langerhans’ cells, vascularized subcutaneous tissue allografts elicited immune responses very similar to those of skin flaps, even though the epidermaldermal layer was removed. This result does not refute the significance of Langerhans’ cells, but rather underscores the potent antigenicity of the remainder of skin flap. The antigenicity of venous allografts, even with cryopreservation, was documented by Axthelm.114 The vascular endothelium is thought to be the antigenic stimulus, as vascular allografts with endothelium removed encountered less rejection and remained patent without immunosuppression.115 Our findings confirmed the antigenicity of vascular allografts, but the immune responses they elicited were late and low compared to those of other limb tissues. Since the endothelium in rodent vessels does not express Class II MHC antigens, this type of response can be expected. Thus, the hypothesis that vascular endothelial damages from rejection was the cause of early failure of vascularized skin and visceral organ transplants is unlikely to be valid.116,117

Composite Tissue Transplantation

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An intriguing finding was that the limb allografts, which consisted of all these tissue components, were rejected more slowly and generated lower immune responses. This could represent a “consumption” phenomenon as the immune system was overwhelmed with the tremendous antigen load. This would be consistent with our observation at sacrifice that the animal’s spleen was at least twice as large as that with other allografts, whereas the number of spleen cells recovered was considerably lower. This explanation is supported by a pilot experiment where a rat received both a limb and a bone allograft. The resultant cellmediated and humoral responses were similar to those of a limb allograft alone. Other possible explanations for the relative survival of limb allografts include antigen competition, induction of enhancing antibodies, and an activation of suppressor T cells. The testing of these hypotheses, however, went beyond the scope of the experiment. The relative success of whole limb allograft was duplicated by Black, who found its long term survival remarkable and speculated that various tissue-specific antigens helped contribute to immune nonresponsiveness when combined with cyclosporine.42 Our results also demonstrated that primary vascularization of a limb tissue allograft significantly changed its rejection pattern. Vascularized skin allografts encountered considerable humoral responses at one and two weeks after transplant, whereas nonvascularized skin flaps did not elicit antibody production until two weeks. Similarly, vascularized bone allografts encountered more prompt and intense immune responses than nonvascularized bone allografts. This suggests that vascularization affects the way graft antigens are presented or handled and changes the rejection mechanisms of the allograft. Therefore, information for nonvascularized limb tissue allografts presently available may not be applicable for their vascularized counterparts. The immune assays employed in the experiment were consistent in their measurement of individual tissue antigenicities. For instance, the humoral responses were expressed on a logarithmic scale such that a difference of three units in the humoral relative cytotoxicity index (H-RCI) is equivalent to a eight-fold difference in host antibody concentration. Furthermore, the quantity of a particular tissue did not appear to alter significantly the immune responses generated. Vascularized and nonvascularized limb tissue allografts elicited immune responses of different timing and intensity, suggesting different mechanisms of rejection. When transplanted as a whole, the vascularized limb allograft encountered less rejection than did allografts of its individual components. No single limb tissue provided the dominant stimulus for rejection. Rather, the various tissues interacted with the host immune system in a complex, but predictable pattern. These data may also serve as a foundation for further elucidation of the rejection process so that a more precise manipulation of the host immune responses could be achieved.

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8. Transplantation Summary. Washington, D.C. Office of Health Technology Assessment & National Center for Health Services Research, U.S. Public Health Service. May, 1987. 9. Malt RA, McKhann C. Replantation of severed arms. JAMA 1964; 189:716-722. 10. Goldwyn RM, Beach PM, Feldman D et al. Canine limb homotransplantation. Plast Reconstr Surg 1966; 37:184-195. 11. Borel JF, Feurer C, Gubler HU, Stahelin H. Biological effects of cyclosporine A: A new antilymphocytic agent. Agents-Actions 1976; 6(4):468-475. 12. Yaremchuk MJ, Sedacca T, Schiller A, May JW Jr. Vascular knee allograft transplantation in a rabbit model. Plast Reconstr Surg 1983; 71(4):461-472. 13. Moore JR, Phillips TW, Weiland AJ, Randolph MA. Allogenic transplants of bone revascularized by microvascular anastomoses: A preliminary study. J Orthop Res 1984; 1(4):352-60. 14. Weiland AJ, Phillips TW, Randolph MA. Bone grafts—A radiologic, histologic, and biomechanical model comparing autografts, allografts, and free vascularized bone grafts. Plast Reconstr Surg 1984; 74(3):368-379. 15. Aebi M, Regazzoni P, Schwarzenbach O. Segmental bone grafting. Comparison of different types of graft in dogs. Int Orthop 1989; 13(2):101-111. 16. Welter JF, Shaffer JW, Stevenson S et al. Cyclosporin A and tissue antigen matching in bone transplantation. Fibular allografts studied in the dog. Acta Orthop Scand 1990; 61(6):517-527. 17. Stevenson S, Li XQ, Martin B. The fate of cancellous and cortical bone after transplantation of fresh and frozen tissue-antigen-matched and mismatched osteochondral allografts in dogs. J Bone Joint Surg, American Volume 1991; 73(8):1143-1156. 18. Bailleux A, Brennwald J, Rosso R et al. Bone and joint transplantation. Alternatives to bridging segmental bone defects and a model for allogenic bone transplantation. [German] Helvetica Chirurgica Acta 1990; 57(1):79-82. 19. Cosimi AB, Shield CF, Peters C et al. Prolongation of allograft survival by cyclosporine A. Surg Forum 1979; 30:287. 20. Borel JF, Meszaros J. Skin transplantation in mice and dogs. Transplantation 1981; 31:136. 21. Gratwohl A, Forster I, Speck B. Skin grafts in rabbits with cyclosporine A. Transplantation 1981; 31:136. 22. Achauer BM, Hewitt CW, Black KS et al. Cyclosporine prolongs skin allografts in rat burn model. Transplant Proc 1983; 15:3073-3076. 23. Egerszegi EP, Samulack DD, Daniel RK. Experimental models in primates for reconstructive surgery utilizing tissue transplants. Ann Plast Surg 1984 13:423. 24. Towpik E, Kupiec-Weglinski JW, Schneider TM et al. Cyclosporine and experimental skin allografts: II. Indefinite survival and development of specific immunologic unresponsiveness. Transplantation 1985; 40(6):714-718. 25. Towpik E, Kupiec-Weglinski JW, Tyler DS et al. Cyclosporine and experimental skin allografts: Long term survival in rats treated with low maintenance doses. Plast Reconstr Surg 1986; 77(2):268-276. 26. Watt DJ, Partridge TA, Sloper JC. Cyclosporine A as a means of preventing rejection of skeletal muscle allografts in mice. Transplantation 1981; 31:266-271. 27. Gulati AK, Zalewski AA. Muscle allograft survival after cyclosporine A immunosuppression. Exp Neurol 1982; 77:378-385. 28. Tan C, Lee WPA, Randolph MA, Yaremchuk MJ, Burdick JF, Weiland AJ. Vascularized muscle allografts and the role of cyclosporine. Plast Reconstr Surg 1991; 87(3):412-418. 29. Siliski JM, Green CJ. More on Cosmas and Damian. N Engl J Med. 1982; 307:1642. 30. Halloran PF, Bushuk M, Stewart JA. Effects of cyclosporine on the healing of vascularized and nonvascularized bone allografts in rodents. Transplant Proc 1983; 15:3053-3056. 31. Paskert JP, Yaremchuk MJ, Randolph MA et al. The role of cyclosporine in prolonging survival in vascularized bone allografts. Plast Reconstr Surg 1987; 80(2):240-247. 32. Sakai K, Hickey MJ, Hurley JV et al. Vascularized osteochondral allografts in an immunosuppressed rat model: Graft modulation and host immune tolerance. Plast Reconstr Surg, 1993; 91:597-605.

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33. Lee WPA, Pan YC, Kesmarky S et al. Experimental orthotopic transplantation of vascularized skeletal allografts: Functional assessment and long-term survival. Plast Reconstr Surg 1995; 95(2):336-349. 34. Zalewski AA, Gulati AK. Failure of cyclosporine A to induce immunological un-responsiveness to nerve allografts. Exp Neurol 1984; 83:659-663. 35. Zalewski AA, Gulati AK. Rejection of nerve allografts after cessation of immunosuppression with cyclosporine A. Transplantation 1981; 31:88-89. 36. Zalewski AA, Gulati AK. Survival of nerve and Schwann cells in allografts after cyclosporine A treatment. Exp Neurol 1980; 70:219-225. 37. Mackinnon SE, Hudson AR, Bain JR, Hunter DA. The peripheral nerve allograft: An assessment of regeneration in the immunosuppressed host. Plast Reconstr Surg 1987; 79:436-446. 38. Press BHJ, Sibley RK, Shons AR. Modification of experimental limb allograft rejection with cyclosporine and prednisone: A preliminary report. Transplant Proc 1983; 15:3057-3062. 39. Fritz WD, Swartz WM, Rose S et al. Limb allografts in rats immunosuppressed with cyclosporine A. Ann Surg 1984; 199(2):211-215. 40. Kim SK, Aziz S, Oyer P, Hentz VR. Use of cyclosporine A in allotransplantation of rat limbs. Ann Plast Surg 1984; 12(3):249-255. 41. Hewitt CW, Black KS, Fraser LA et al. Composite tissue (limb) allografts in rats: I. Dosedependent increase in survival with cyclosporine. Transplantation 1985; 39(4):360-364. 42. Black KS, Hewitt CW, Fraser LA et al. Composite tissue (limb) allografts in rats: II. Indefinite survival using low-dose cyclosporine. Transplantation 1985; 39(4):365-368. 43. Kniha H, Randzio J, Gold ME et al. Growth of forelimb allografts in young rabbits immunosuppressed with cyclosporine. Ann Plast Surg 1989; 22(2):135-141. 44. Muramatsu K, Doi K, Akino T et al. Results of vascularized joint allograft under immunosuppression with cyclosporine. Microsurgery 1993; 14:527-536. 45. Min Z, Jones NF. Limb transplantation in rats: Immunosuppression with FK506. J Hand Surg 1995; 20A(1):77-87. 46. Buttemeyer R, Jones NF, Min Z, Rao U. Rejection of the component tissues of limb allografts in rats immunosuppressed with FK506 and cyclosporine. Plast Reconstr Surg 1996; 97:139-148. 47. Benheim P, Anthony JP, Ferreira L et al. Use of combination of low-dose cyclosporine and RS-61443 in a rat hind limb model of composite tissue allotransplantation. Transplantation 1996; 61(4):527-536. 48. Murray JE. Organ transplantation (skin, kidney, heart) and the plastic surgeon. Plast Reconstr Surg 1971; 47(5):425-431. 49. Braathen LR, Thorsby E. Studies on human epidermal Langerhans’ cells: I. Allo-activating and antigen presenting capacity. Scand J Immunol 1980; 11:401-408. 50. Stingl G, Gazze-Stingl LA, Aberer W et al. Antigen presentation by murine epidermal Langerhans’ cells and its alteration by ultraviolet B light. J Immunol 1981; 127:1707-1713. 51. Alsbjorn B. A depression technique of the epidermal Langerhans’ cells in cadaver split skin. Scand Plast Reconstr Surg 1984; 18:61-64. 52. Moseley RV, Sheil AGR, Mitchell R et al. Immunological relationships between skin and kidney homografts in dogs on immunosuppressive therapy. Transplantation 1966; 4:678. 53. Czitrom AA, Axelrod T, Fernandes B. Antigen presenting cells and bone allotransplantation. Clin Orth Rel Res 1985; 197:27-31. 54. Gotfried Y, Yaremchuk MJ, Randolph MA, Weiland AJ. Histological characteristics of acute rejection in vascularized allografts of bone. J Bone Joint Surg 1987; 69-A(3):410-425. 55. Gornet MF, Schofield BH, Randolph MA et al. Immunologic and ultrastructural changes during early rejection of composite tissue allografts. Plast Reconstr Surg 1984; 88(5):860-868. 56. Lee WPA, Randolph MA, Weiland AJ, Yaremchuk MJ. Prolonged survival of vascularized limb tissue allografts by donor irradiation. J Surg Res 1995; 59(5):578-588. 57. Lafferty KJ, Andrus L, Prowse SJ. Role of lymphokine and antigen in the control of specific T cell responses. Immunol Rev 1980; 51:279-314.

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58. Caughman SW, Sharrow SO, Shimada S et al. Ia+ murine epidermal Langerhans cells are deficient in surface expression of Class I MHC. Proc Natl Acad Sci 1986; 83:7438-7442. 59. Harris HW, Gill TJ. Expression of Class I transplantation antigens. Transplantation 1986; 42:109-117. 60. Daar AS, Fuggle SV, Fabre JW et al. The detailed distribution of MHC Class II antigens in normal human organs. Transplantation 1984; 38:293-298. 61. Pescovitz MD, Sachs DH, Lunney JK, Hsu S-M. Localization of Class II MHC antigens on porcine renal vascular endothelium. Transplantation 1984; 37:627-630. 62. Glimcher LH, Kara CJ. Sequences and factors: A guide to MHC Class-II transcription. Annu Rev Immunol 1992; 10:13-49. 63. Steinman RM. Dendritic cells. Transplantation 1981; 31:151-155. 64. Faustman D, Steinman R, Gebel H et al. Prevention of rejection of murine islet allografts by pretreatment with anti-dendritic cell antibody. Proc Natl Acad Sci 1984; 81:3864-3868. 65. Glimcher LH, Kim KJ, Green I, Paul WE. Ia antigen-bearing B cell tumor lines can present protein antigen and alloantigen in a major histocompatibility complex-restricted fashion to antigen reactive T cells. J Exp Med 1982; 155:445-459. 66. Janeway CA, Ron J, Katz ME. The B cell is the initiating antigen presenting cell in peripheral lymph nodes. J Immunol 1987; 138:1051-1055. 67. Ron Y, Sprent J. T cell priming in vivo: A major role for B cells in presenting antigen to T cells in lymph nodes. J Immunol 1987; 138:2848-2856. 68. La Rosa FG, Talmage DW. Synergism between minor and major histocompatibility antigens in the rejection of cultured allografts. Transplantation 1985; 39:480-485. 69. Parker KE, Dalchau R, Fowler VJ et al. Stimulation of CD4+ T lymphocytes by allogenic MHC peptides presented on autologous antigen presenting cells. Transplantation 1992; 53:918-924. 70. Skoskievievicz MJ, Colvin RB, Schneeberger EE et al. Widespread and selective induction of MHC-determined antigens in vivo by interferon gamma. J Exp Med 1985; 162:1645-1664. 71. Benson EM, Colvin RB, Russell PS. Induction of Ia antigens in murine renal transplants. J Immunol 1985; 134:7-9. 72. Pober JS, Collins T, Gimbrone MA Jr et al. Inducible expression of Class II major histocompatibility complex antigens and the immunogenicity of vascular endothelium. Transplantation 1986; 41:141-146. 73. Cosimi AB, Burton RC, Colvin RB et al. Treatment of acute renal allograft rejection with OKT3 monoclonal antibody. Transplantation 1981; 32:535-539. 74. Hayry P. Intragraft events in allograft destruction. Transplantation 1984; 38:1-6. 75. Czitrom AA, Gascoigne NRJ, Edwards S et al. Induction of minor alloantigen-specific T cell subsets in vivo: Recognition of processed antigen by helper but not by cytotoxic T cell precursors. J Immunol 1984; 133:33-39. 76. Owens T, Czitrom AA, Gasciogne NRJ et al. The presentation of cell surface alloantigens to T cells. Immunobiology 1984; 168:189-201. 77. Sherwood RA, Brent L, Rayfield LS. Presentation of alloantigens by host cells. Eur J Immunol 1986; 16:569-574. 78. Lechler RI, Batchelor JR. Restoration of immunogenicity to passenger cell-depleted kidney allografts by the addition of donor strain dendritic cells. J Exp Med 1982; 155:31-41. 79. Jeannet M, Pinn V, Flax M et al. Homoral antibodies in renal allotransplantation inman. N Engl J Med 1970; 282:111-117. 80. Muscolo DL, Kawai S, Ray RD. In vitro studies of transplantation antigens present on bone cells in the rat. J Bone Joint Surg 1977; 59B:342-348. 81. Steinmuller D, Wachtel SS. Transplantation biology and immunogenetics of murine skinspecific (Sk) alloantigens. Transpl Proc 1980; 12:100-106. 82. Tilney NL, Strom TB, Macpherson SG et al. Populations of infiltrating cells removed from rejecting rat cardiac allografts. Surg Forum 1974; 25:289-292. 83. Tilney NL, Strom TB, Macpherson SG et al. Studies on infiltrating host cells harvested from acutely rejecting rat cardiac allografts. Surgery 1976; 79:209-217.

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84. Black KS, Hewitt CW, Grisham GR et al. Two new composite tissue allograft models in rats to study neuromuscular functional return. Transplant Proc 1987; 19:1118-1119. 85. Cerottini JC, Brunner KT. Cell-mediated cytotoxicity, allograft rejection, and tumor immunity. Adv Immunol 1974; 18:67-132. 86. Lee WPA, Yaremchuk MJ, Pan YC et al. Relative antigenicity of components of a vascularized limb allograft. Plast Reconstr Surg 1991;87(3):401-411. 87. Yaremchuk MJ, Nettelblad H, Randolph MA et al. Vascularized bone allograft trans-plantation in a genetically defined rat model. Plast Reconstr Surg 1985; 75(3):355-362. 88. Jowsey J, Kelly PJ, Riggs BL et al. Quantitative microradiographic studies of normal and osteoporotic bone. J Bone Joint Surg 1965; 47-A:785. 89. Burdick JF, Clow LW. Rejection of murine cardiac allografts. Transplantation 1987; 43(4):509-514. 90. Sachs DH, Leight G, Cone J et al. Transplantation in miniature swine. I. Fixation of the major histocompatibility complex. Transplantation 1976; 22:559-567. 91. Sachs, DH MHC homozygous miniature swine. In: Swine as Models in Biomedical Research. Swindle MM, Moody DC, Phillips LD, eds. Iowa: Iowa State University Press 1992;2. 92. Pennington LR, Lunney JK, Sachs DH. Transplantation in miniature swine. VIII. Recombination within the major histocompatibility complex of miniature swine. Transplantation 1981; 31:66-71. 93. Fabrega AJ, Pollak R. Survival of nationally shared, HLA-matched kidney transplants. N Engl J Med 1993; 328:212. 94. Benson EM, Colvin RB, Russell PS. Induction of IA antigens in murine renal transplants. J Immunol. 1985; 134:7-9. 95. Skoskiewicz, MJ, Colvin RB, Schneeberger EE et al. Widespread and selective induction of major histocompatibility complex-determined antigens in vivo by gamma interferon. J Exp Med 1985; 162:1645-1664. 96. Gaspari AA, Katz SI. Induction and functional characterization of Class II MHC (Ia) antigens on murine keratinocytes. J Immunol 1988; 140:2956-2963. 97. Gianello P, Fishbein JM, Sachs DH. Tolerance to primarily vascularized allografts in miniature swine. Immunol Rev 1993; 133:19-44. 98. Pescovitz MD, Thistlethwaite JR Jr, Auchincloss H Jr et al. Effect of Class II antigen matching on renal allograft survival in miniature swine. J Exp Med 1984; 160:1495-1508. 99. Rosengard BR, Ojikutu CA, Guzzetta PC et al. Induction of specific tolerance to Class I disparate renal allografts in miniature swine with cyclosporine. Transplantation 1992; 54:490-497. 100. Kirkman RL, Colvin RB, Flye MW et al. Transplantation in miniature swine. VI. Factors influencing survival of renal allografts. Transplantation 1979; 28:18-23. 101. Rosengard BR, Ojikutu CA, Fishbein J et al. Selective breeding of miniature swine leads to an increased rate of acceptance of MHC-identical, but not of Class-I disparate, renal allografts. J Immunol 1992; 149:1099-1103. 102. Davie JM, Paul WE. Role of T lymphocytes in the humoral response. I. Proliferation of B lymphocytes in thymus-deprived mice. J Immunol 1974; 113:1438-1445. 103. Madsen JC, Sachs DH, Fallon JT, Weissman NJ. Cardiac allograft vasculopathy in partially inbred miniature swine. I. Time course, pathology, and dependence on immune mechanisms. J Thoracic Cardiovascular Surg 1996; 111:1230-1239. 104. Towpik E, Kupiec-Weglinski JW, Tilney NL. The potential use of cyclosporine in reconstructive surgery. Plast Reconstr Surg 1985; 76(2):312-322. 105. Achauer BM, Black KS, Hewitt CW et al. Immunosurgery. Clinics Plast Surg 1985; 12(2):293. 106. Poole M, Bowen JE, Batchelor JR. Prolonged survival of rat leg allografts due to immunological enhancement. Transplantation 1976; 22:108-111. 107. Doi K. Homotransplantation of limbs in rats: A preliminary report on an experimental study with nonspecific immunosuppressive drugs. Plast Reconstr Surg 1979; 64:613-621. 108. Black KS, Hewitt CW, Woodard TL et al. Effects to enhance survival of limb allografts by prior administration of whole blood in rats using a new survival end point. J Microsurg 1982; 3:162-167.

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109. Hewitt CW, Black KS, Fraser LA et al. Cyclosporine A is superior to prior donor-specific blood (DSB) transfusion for the extensive prolongation of rat limb allograft survival. Transplant Proc 1983; 15:514-517. 110. Paskert JP, Yaremchuk MJ, Randolph MA et al. Prolonging survival in vascularized bone allograft transplantation: developing specific immune unresponsiveness. J Reconstr Microsurg 1987; 3(3):253-263. 111. Russell PS. Selective transplantation: An emerging concept. Ann Surg 1985; 201(3):255. 112. White E, Hildemann WH. Allografts in genetically defined rats: Difference in survival in kidney and skin. Science 1968; 162:1293-1295. 113. Gratwohl A, Forster I, Speck B. Activity of cyclosporin-A in skin graft rejection and in graft-versus-host disease in rabbits. Transplant Proc 1983; 15(1):497-499. 114. Axthelm SC, Porter JM, Strickland S et al. Antigenicity of venous allografts. Ann Surg 1979; 189(3):290-293. 115. Galumbeck MA, Sanfilippo FP, Hagen PO et al. Inhibition of vessel allograft rejection by endothelial removal. Ann Surg 1987; 206(6):757-764. 116. Patel R, Teraski PI. Significance of the positive crossmatch test in kidney transplantation. N Engl J Med 1969; 280:735-739. 117. Dvorak HF, Mihm MC Jr, Dvorak AM et al. The microvasculature is the critical target of the immune response in vascularized skin allograft rejection. J Invest Dermatol 1980; 74:280-284.

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Transplantation Tolerance in Large Animal Models

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CHAPTER 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 J. Peter Rubin, Sheldon Cober, Peter E. M. Butler and W. P. Andrew Lee

Introduction

T

he use of vascularized skeletal tissue allografts in humans would profoundly expand the domain of reconstructive surgery. There would be a potentially unlimited supply of tissue with no donor site morbidity. Although there has been sporadic occurrence of tolerance to vascularized skeletal tissue allografts, the responsible immunologic parameters were not defined.1 As with other types of vascularized tissue grafts, continuous immunosuppression of the host has been found to be necessary for graft survival. This requirement for depression of the recipient immune response stands as a major obstacle to the use of skeletal tissue allograft in humans. Immunosuppressive drugs carry potentially life threatening side effects, and cannot be justified for the treatment of non life threatening skeletal tissue defects. Thus the clinical feasibility of skeletal tissue transplantation depends upon inducing host tolerance to the allograft while preserving an immune response to all other foreign antigens. The host response to skeletal tissue allografts has been characterized in a rat model by our laboratory. To summarize the findings, a vascularized knee, including bones and surrounding soft tissues, was transplanted heterotopically across both a strong and a weak histocompatibility barrier, demonstrating that vascularized skeletal tissues were as susceptible to rejection as visceral organ allografts and, similarly, that genetic disparity determined the intensity and timing of rejection.2 Histologic analyses by light and electron microscopy showed that the bone marrow was an early target of the rejection process and that the growth plate was exquisitely sensitive to this process.3,4 In vitro immunologic data demonstrated the host cell-mediated and humoral responses to be specific and predictable.5 Having obtained this basic information, the roles of nonspecific and specific means Composite Tissue Transplantation, edited by Charles W. Hewitt and Kirby S. Black. ©1999 R.G. Landes Company.

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of immunosuppression to prolong allograft survival were determined. Using cyclosporine, the nonspecific agent which has revolutionized clinical visceral organ transplantation, it was found that vascularized skeletal tissue allografts would survive6 and bone junctures would heal7 while recipients were treated with cyclosporine. However, once cyclosporine was stopped, the grafts would eventually be rejected. Other means of immunosuppression, such as donor-specific blood transfusion, were also employed but were unsuccessful in prolonging allograft survival.8 More recent studies in the rat model have examined the relative antigenicity of the specific components of these allografts.9 Selective alteration by preoperative donor or graft gamma irradiation or donor bone marrow transplant was found to ameliorate rejection.10,11 Long term survival of functional skeletal allografts was achieved in an orthotopic model without significant host toxicity from sustained immunosuppression.12 Such allografts healed to the recipient bone and supported a mechanical load without fracture or resorption. Although experimental transplants in the rat have provided valuable data, fundamental differences between the rodent and human immune systems underscores the need for large animal models with a closer analogy to the human immune system. The NIH miniature swine represents the only large animal species with a genetically defined major histocompatibility complex (MHC). Several herds of partially inbred miniature swine have been developed, allowing investigators to experimentally simulate human transplantation between siblings with an identical MHC match, between family members with a one haplotype MHC mismatch, or between unrelated donor and recipient.13 Moreover, recombinant haplotypes may be used to investigate the influence of specific MHC Class I or Class II mismatches in allogenic transplantation.14 We have attempted to induce tolerance to vascularized skeletal allografts in the swine without long term immunosuppression. Our efforts have been directed along two primary lines of research with different challenges. In one area of investigation, we have manipulated the fetal immune system so that specific tolerance to donor tissue would be imparted to the host prior to maturation of the immune response. Our second area of investigation has focused on modification of the rejection response in the adult animal. The experimental details of each model follow.

Manipulation of the Adult Immune System: Swine Model Background and Specific Aim It is obvious that imparting specific tolerance to donor tissue in the adult immune system without long term immunosuppression will be a highly practical goal for transplantation research. The examination of allograft rejection in closely matched animals has led to the hypothesis that critical steps in donor tissue recognition can be experimentally altered. A comprehensive study of the effects of MHC matching on renal allografting in NIH miniature swine revealed that about 30% of kidneys transplanted with an MHC Class II match and a single haplotype Class I mismatch were permanently accepted without immunosuppression. However, grafts with a double Class I haplotype mismatch or Class II mismatch were rejected.15 It was later demonstrated that Class II matched, Class I single haplotype mismatched, animals which spontaneously accepted renal allografts showed a humoral response consisting of IgM anti-Class I antibodies with failure to convert to IgG. This led to the hypothesis that a lack of T cell help and inadequate cytokine release during early graft exposure contributed to tolerance. This theory was tested by administering a short term regimen of cyclosporine (12 days) to miniature swine with a Class II match and a Class I double haplotype mismatch. All animals treated with cyclosporine enjoyed renal graft acceptance, while all untreated animals suffered graft rejection. Furthermore, systemic tolerance in the experimental group animals was verified by prolonged survival of a skin graft

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from respective kidney donors.16 In a separate experiment, it was noted that exogenous IL-2 administration could block the induction of tolerance by cyclosporine during early graft exposure.17 Based on the encouraging results with short term cyclosporine in renal allografts, we have developed a skeletal tissue allograft model with the goal of inducing specific tolerance in the adult animal. A vascularized composite tissue graft including bone and muscle was transplanted in adult miniature swine, followed by treatment with 12 days of cyclosporine. We have started by transplanting across a minor antigen mismatch (equivalent to MHC matched siblings). These minor antigen differences will eventually lead to graft rejection in a majority of swine, although at a slower pace than in MHC mismatched animals.

Methods Harvest of Skeletal Tissue Grafts and Skin Grafts Under isoflurane anesthesia, a longitudinal incision was made on the medial thigh of the donor swine. The femoral vessels were isolated on a pedicle from the inguinal ligament to the suprageniculate popliteal region. After removing the skin overlying the limb, the ankle was disarticulated and the distal femur and thigh muscles were transected. This resulted in a graft consisting of tibia and fibula, knee joint, a portion of femur, and surrounding muscle. Two skeletal tissue grafts were harvested from each donor swine prior to sacrifice. Skin grafts were harvested from the lateral thorax with a Brown dermatome and frozen in cryoprotective medium (Whittaker products, Walkersville, MD) and stored at -70°C. Recipient Graft Placement Recipient animals were anesthetized with isofluorane anesthesia. An oblique incision centered over the groin allowed exposure and isolation of the femoral vessels. The allograft was then placed in a subcutaneous pocket made in the ipsilateral lower abdominal wall and secured in place with nonabsorbable sutures. Microsurgical anastomoses were performed under magnification using 9-0 nylon suture. The arterial anastomosis was completed in an end to side fashion, while the venous anastomosis was sewn end to end. Cefazolin was used for pre- and postoperative antimicrobial prophylaxis. Immunosuppression In selected animals, an intravenous preparation of cyclosporine (CsA) (Sandimmune, i.v.) was given daily through an indwelling central venous catheter at an initial dose of 10 mg/ kg. The first dose was given as anesthesia was being administered prior to the transplant procedure. Trough levels were checked on the fifth postoperative day, and the dose adjusted to keep levels between 500-800 ng/ml. The duration of therapy in all cases was 12 days. Perioperative and Rejection Monitoring Rejection was monitored grossly and histologically by performing open biopsies under isoflurane general anesthesia at two, three, six, eight, and twelve weeks postoperatively. The biopsies were obtained in successively more proximal locations on the graft to avoid devascularized regions of the allograft. Muscular and bony specimens were taken at each biopsy. Skin Grafting All animals with grafts surviving longer than 100 days received split thickness skin grafts from the donor swine, along with a self control, a donor matched control, and a third party. Grafts measured approximately 4 cm2 and were applied to a split thickness bed on the

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back of the recipient. Rejection was monitored by daily inspection, and considered to be complete when greater than 90% of the graft lost viability. Experimental Groups There were three experimental groups. Animals in Group I (n = 4) received grafts from a complete MHC mismatched donor, followed by 12 days of cyclosporine. Animals in Group II (n = 2) received grafts from an MHC matched/minor antigen mismatched donor, but no cyclosporine. Animals in Group III (n = 4) received grafts from an MHC matched/minor antigen mismatched donor, followed by twelve days of cyclosporine.

Results Group I (n = 4): Miniature swine receiving grafts across a complete MHC mismatch and twelve days of CsA showed gross and histologic evidence of allograft rejection by 42 days posttransplant (Table 3.1). Histologic section of muscle showed diffuse lymphocytic infiltration, while bone section showed empty lacunae devoid of osteocytes. Group II (n = 2): Allografts with MHC match and only minor antigen differences not treated with cyclosporine showed rejection at 63 and 84 days, respectively. Group III (n = 4): Allografts with MHC match and only minor antigen differences treated with 12 days of CsA were harvested between 178 days and 280 days posttransplant. All of these grafts demonstrated patent vessels, healthy bleeding from marrow cavities, and viable bone and soft tissue on microscopic examination at harvest. Histologic sections showed few lymphocytes in muscle and viable osteocytes in bony lacunae. All four animals received further skin grafts. Skin autografts survived indefinitely, and third party skin allografts were rejected in the usual time frame of 8-12 days. Skin grafts from the skeletal allograft donor survived 48 and 96 days in two animals, and were still viable at the time of sacrifice in the remaining two animals (>100 days).

Discussion Our study has shown that tolerance to vascularized skeletal tissue allografts could be induced in genetically defined miniature swine with only a 12 day course of cyclosporine. The graft recipient accepted the donor skin grafts (48 days to indefinitely) and rejected third party control skin grafts, thus confirming specific tolerance to the skeletal donor while maintaining immune competence. The transplant barrier in this study is equivalent to that of MHC matched human siblings (not identical twins). Studies are also underway in our animal model on transplants across greater genetic barriers of double haplotype Class I mismatch and single haplotype Class MHC mismatch (equivalent to a parent to sibling transplant). Demonstration of skeletal tissue allograft survival in a large animal model without long term immunosuppression represents an important step toward transplantation of skeletal tissue allografts in humans. The clinical implication is that these principles could be applied to human donors and recipients with similar MHC matches.

Manipulation of the Fetal Immune System: Swine Model Background and Specific Aim The fetal immune system must learn to distinguish self antigens from foreign antigens. This period of immunologic development provides a unique window for altering the recipient immune system. The development of tolerance to foreign antigens presented to the immature immune system is a concept born out of Owen’s observation in 1945 that fraternal cattle twins retained circulating red cells that had been exchanged through a fused placental circulation.18 Because each twin had a different genetic profile and should have rejected

Transplantation Tolerance in Large Animal Models

35

Table 3.1. Swine receiving grafts accross a complete MHC mismatch Group

n

Transplant Barrier

12 days CsA

Vascularized Graft Survival (days)

I

4

MHC Mismatch

Yes

Rejected <42 days

II

2

Minor Antigen

No

Rejected 63–84 days

III

4

Minor Antigen

Yes

All grafts alive at harvest 178–280 days

Donor Skin Graft Survival (days)

48, 96, >100, >100

each other’s hemopoietic cells, the findings suggested that the fetal immune system developed tolerance to these foreign antigens at the same time that tolerance was established to self antigens. It was later demonstrated that these fraternal cattle twins possessed a mutual and specific tolerance for skin grafts transplanted between them, yet retained immunocompetence to third party grafts.19 The landmark work of Sir Peter Medawar’s group in 1953 adapted the natural exchange of antigens in cattle twins to a set of controlled laboratory experiments using inbred mice. Allogenic cells from one murine strain were injected into fetal or newborn animals of another strain. The injected animals then demonstrated specific tolerance for transplanted donor tissue through adulthood.20 Shortly after, Burnet speculated that clones reactive to self antigens were selected during ontogeny.21 There is evidence that the persistence of chimerism is necessary to maintain tolerance induced by in utero inoculation, as demonstrated in the elegant experiments by Lubaroff and Silvers.22 They eliminated the donor cell population in an adult mouse that had been tolerized in the neonatal period by introducing third party lymphoid cells that were sensitized to the donor strain. The third party cells attacked the donor cells, and were later eradicated themselves by the host immune system. After disappearance of both donor cells and third party cells, the host animal rejected a donor skin graft in a primary response. Engraftment of cells by in utero injection has been accomplished previously in a large animal model by Flake.23 He transplanted hematopoietic stem cells into fetal sheep, but did not perform functional testing for tolerance. Moreover, the use of fetal donor tissue precluded any future organ transplants from the same donor animal. In a later study by the same group, adult T cell depleted bone marrow was used to inoculate the fetuses.24 Despite engraftment of donor cells, kidneys transplanted to the fetal recipients after they matured to adulthood were rejected. Cowan25 injected fetal Rhesus monkeys at gestational days 41 and 64 with T cell depleted marrow harvested from one parent. The overall engraftment rate in this one haplotype match was 73% as evaluated by polymerase chain reaction (PCR). Donor cells were detected in blood, bone marrow, spleen, and liver. Kidneys were transplanted from parental marrow donors to two chimeric animals. One kidney rejected in 5 weeks, while the other survived after rescue with a limited course of immunosuppressive agents. In our in utero model, we attempted to induce tolerance across a complete MHC mismatch. We used the NIH miniature swine as a source of donor marrow. This allowed for a consistent genetic profile of the donor tissue in each specific experiment. Moreover, the development of monoclonal antibodies to the donor MHC provided a useful tool for detecting donor cells in the recipient animals.

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Methods Selection of Yorkshire Breeding Pair Peripheral blood lymphocytes (PBLs) from outbred Yorkshire sows and boars were screened by flow cytometry for binding with a murine antibody directed against the Class I region of the donor MHC. Animals negative for antibody binding were bred. Prior to transplantation, immune responsiveness of both sow and boar to the donor was confirmed in vitro by a mixed lymphocyte reaction. Isolation of Peripheral Blood Lymphocytes (PBLs) by Gradient Centrifugation Heparinized blood obtained from experimental animals was diluted 2:1 with Hank’s buffered saline solution (HBSS) (Gibco/BRL, Grand Island, NY) in conical centrifuge tubes. Lymphocyte separation medium (LSM) (Organon, Teknika, Durham, NC) was added to the bottom of each tube prior to centrifuging at 1800 rpm, 36°C, for 30 minutes. The mononuclear cell layer was removed and washed once in HBSS. Remaining red cells were lysed with ACK buffer (B&B Research Laboratories, Fiskeville, RI) and the lymphocytes washed a second time in HBSS. Flow Cytometry Isolated PBLs were stained with murine monoclonal antibodies directed against cell surface antigens. Purified pig Ig was used to block FcR nonspecific binding. Murine antibody 2.12.3A (anti-swine SLAxd MHC Class I) was used to screen for chimerism. Murine antibodies 2.27.3A (anti-swine MHC Class I) and 36.7.5 (anti-mouse MHC Class I) served as positive and negative controls, respectively. To label T cells, murine antibodies 2.6.15 (anti-swine CD3), 74.12.4 (anti-swine CD4), and 76.2.11 (anti-swine CD8) were used. A goat anti-mouse fluoresceinated Ab was used as a secondary stain. In addition, a goat antirat fluoresceinated Ab was used to detect the presence of T cells labeled with the magnetic bead antibodies used for depletion. Cell staining and washing was performed at 4°C in HBSS containing 0.5% bovine serum albumin (BSA) and 0.5% sodium azide. Flow cytometry (FCM) was performed using a Becton Dickinson FACScan II (Sunnyvale, CA). Dead cells were gated out with propidium iodide. Mixed Lymphocyte Reaction (MLR) Stimulator and responder lymphocytes were cocultured in 96 well flat bottomed plates (Costar, Cambridge, MA) in a one way mixed lymphocyte reaction assay. Each well contained 4 x 105 responders and 4 x 105 irradiated (25 Gy) stimulators in 0.2 ml of tissue culture medium. All assays were run in triplicate. Medium consisted of RPMI 1640 (Gibco, Grand Island, NY) with 6% fetal pig serum, 100 U/l penicillin, 135 mg/ml streptomycin, 50 mg/ml gentamicin, 2 mM L-glutamine, 10 nM HEPES, 5 x 10-5 M beta 2-mercaptoethanol (2-ME), and 1 mM sodium pyruvate. Cultures were incubated for 5 days at 37°C in 7% CO2 nd 100% humidity. On the 5th day of culture, each well was pulsed with 1 mC of [3H] thymidine and harvested six hours later with the betaplate system (Tomtec; Wallac, Gaithersburg, MD). Proliferation of responder cells was assessed by the uptake of [3H] thymidine and expressed both as raw counts and as a stimulation index (SI), calculated as the ratio of average counts per minute (cpm) in the experimental wells to average cpm in the negative control wells (responders cocultured with irradiated self stimulaters). Donor Bone Marrow Harvest Under isofluorane anesthesia, a longitudinal incision was made over each proximal humerus of a 3 month old SLAad miniature swine donor (#12107). The humeral head was

Transplantation Tolerance in Large Animal Models

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exposed and a 1 cm2 window cut in the cortex. Bone marrow was then removed from each humeral head with a curette and placed in RPMI 1640 with 1% DNAse I (Sigma, St. Louis, MO). Bone fragments were cut into small pieces with scissors and then removed from the marrow by filtering through sterile coarse nylon mesh. The cavities in the humeral heads were filled with methyl methacrylate cement and skin wounds closed with nylon sutures. The animal was kept in a nonweight bearing sling for 8 hours postoperatively. Bone marrow was stored in a preservation medium consisting of RPMI 1640 with 5% donor serum, 5% DNAse I, 100 U/l penicillin, 135 mg/ml streptomycin, 50 mg/ml gentamicin, and 2 mM L-glutamine. T cell Depletion of Bone Marrow Bone marrow cells (BMCs) were stained with the murine antibody 2.6.15 (anti-swine CD3) and washed twice in HBSS with 0.5% BSA and 5% DNAse I. The BMCs were then counterstained with rat anti-mouse IgG2a/b magnetic bead antibodies (Miltenyi Biotec, Sunnyvale, CA) and washed again. The cells were resuspended in buffer and passed through a type CS separation column in the MACS magnetic cell sorting system (Miltenyi Biotec). The marrow T cell fraction was reduced to less than 0.5%, as confirmed by FCM. In Utero Transplantation A midline laparotomy was performed under isofluorane anesthesia on each sow at 50 days gestation. The gravid uterus was exposed and fetuses identified by transuterine ultrasound. 1-2 x 108 T cell depleted BMCs were injected into the portal veins of fetuses with a 25 gauge spinal needle by a transuterine ultrasound guided approach. Detection of Chimerism and In Vitro Tolerance of Offspring All offspring of the treated sow were tested for peripheral chimerism and in vitro immune responsiveness at 12, 16, and 20 weeks of age by FCM and MLR, respectively. Postmortem analysis of chimerism was also performed. Skin Grafting As an in vivo assay for tolerance, all offspring received split thickness skin grafts from the bone marrow donor along with a self control, a donor matched control, and a third party. Grafts were harvested from the lateral thorax with a Brown dermatome and frozen in cryoprotective medium (Whittaker products, Walkersville, MD) and stored at -70°C. Grafts measured approximately 4 cm2 and were applied to a split thickness bed on the back of the recipient. Rejection was monitored by daily inspection, and considered to be complete when greater than 90% of the graft lost viability.

Results Two parent animals, sow #979 and boar #169, both showed strong reactivity to the donor antigens in the mixed lymphocyte reaction and no binding to the antibody directed against the donor MHC Class I antigen. Following bone marrow transplantation, the sow delivered seven live piglets at 113 days gestation. Analysis of PBLs by FCM revealed reproducible chimerism in one piglet, with up to 0.95% donor cells detected (Fig. 3.1). All other animals in the litter showed no evidence of chimerism. MLR assays revealed persistent hyporesponsiveness of the chimeric piglet to donor cells, while maintaining a strong reaction to third party controls (Fig. 3.2). The other offspring all reacted strongly to both donor and third party antigens in MLR tests (Fig. 3.3). Donor and third party skin grafts were rejected between 6 and 7 days in the nonchimeric piglets (average 6.4 days). The chimeric piglet rejected a third party graft in 8 days, but the donor graft and donor MHC matched

38

Composite Tissue Transplantation Fig. 3.1. Chimeric Piglet PBL's at 16 weeks of age. Number of gated cells per run=20,000. 36.7.5 is negative control antibody, 2.12.3A is the experimental positive antibody.

Fig. 3.2. MLR of chimeric pig at 16 weeks of age. CPM=counts per minute, AD/AA/CC=miniature swine haplotype, and Yuc=Yucatan 3rd party allogeneic mismatch.

Fig. 3.3. MLR of nonchim– eric sibling of chimeric pig at 16 weeks of age. CPM=counts per minute, AD/AA/CC=miniature swine haplotype, Yuc= Yucatan 3rd party allogeneic mismatch.

control remained viable for 65 days. At 65 days after application of the skin graft, the chimeric animal died of respiratory failure. Necropsy revealed a mediastinal lymphoma compressing the trachea. The tumor was demonstrated not to be of donor origin by FCM. Additional FCM analysis of tissue demonstrated 1.1% chimerism in the liver and 0.7% in the spleen. No chimerism was detected in the bone marrow or thymus.

Discussion In our model, we achieved tolerance to donor skin grafts across a complete MHC mismatch with no immunosuppression. As in Cowan’s primate study, we detected donor cells in the spleen and liver, suggesting a prominent role in hematopoiesis for these organs dur-

Transplantation Tolerance in Large Animal Models

39

ing fetal life. Our inability to detect donor cells in bone marrow may be related to either sampling error or the decreased sensitivity of flow cytometry as compared with PCR. Use of this swine model has clear advantages over other large animal models. Swine carry a large number of fetuses and thus provide a greater sample size for each sow. In addition, the inbred miniature swine herd enables the genetic profile of the donor marrow cells to be held constant across a number of experiments. Furthermore, the differential tolerance to Class I, Class II, and single haplotype differences can be assessed in experimental animals. For example, the animal inoculated with SLAAD cells showed hyporesponsiveness to SLAAD cells in MLR assays, but reacted to SLAAA cells in the same test. This reaction to single haplotype mismatched cells may represent an incomplete deletion of reactive clones during ontogeny. The induction of tolerance by in utero injection has potential applications for disorders that can be detected in the prenatal period. In 1959, Woodruff 26 provided evidence that allogenic tolerance can be induced in fetal humans. He performed reciprocal skin grafts between human fraternal twins (male and female) previously identified as blood group chimeras. One of the twins accepted the graft, as demonstrated by clinical observation and chromatin sexing at one year postoperatively. There are also reports of successful ultrasound guided allogenic stem cell transplantation in human fetuses for treatment of thalassemia and severe combined immunodeficiency disease.27 These reports indicate that in utero injection could have a potential role in inducing tolerance for vascularized tissue grafts in humans.

References 1. Achauer BM, Black KS, Hewitt CW et al. Immunosurgery. Clin Plast Surg 1985; 12:293. 2. Yaremchuk MJ, Nettelblad H, Randolph MA et al. Vascularized bone allograft trans-plantation in a genetically defined rat model. Plast Reconstr Surg 1985; 75: 355. 3. Gotfried Y, Yaremchuk MJ, Randolph MA et al. Histological characteristics of acute rejection in vascularized allografts of bone. J Bone Joint Surg 1987; 69A:410. 4. Gornet MF, Randolph MA, Schofield BH et al. Immunologic and ultrastructural changes during early rejection of vascularized bone allografts. Plastic Reconstr Surg 1991; 88:860. 5. Innis PC, Randolph MA, Paskert JP et al. Vascularized bone allografts: In vitro assessment of cell-mediated and humoral responses. Plastic Reconstr Surg 1991; 87:315. 6. Paskert JP, Yaremchuk MJ, Randolph MA et al. The role of cyclosporine in prolonging survival in vascularized bone allografts. Plastic Reconstr Surg 1987; 80:240. 7. Kesmarky S, Randolph MA, Yaremchuk MJ et al. Vascularized bone allografting: The effect of cyclosporine in an orthotopic rat model. Orthop Transact 1987; 11:293. 8. Paskert JP, Yaremchuk MJ, Randolph MA et al. Prolonging survival in vascularized bone allograft transplantation: Developing specific immune unresponsiveness. J Reconstr Microsurg 1987: 3:253. 9. Lee WPA, Yaremchuk MJ, Pan YC et al. Relative antigenicity of components of a vascularized limb allograft. Plastic Reconstr Surg 1991; 87:401. 10. Lee WPA, Randolph MA, Weiland AJ et al. Prolonged survival of vascularized limb tissue allografts after donor irradiation. J Surg Res 1995; 59:578. 11. Lee WPA, Yaremchuk MJ, Manfrini M et al. Prolonged survival of vascularized limb allografts from chimera donors. Plast Surg Forum 1988; 11:51. 12. Lee WPA, Pan YC, Kesmarky S et al. Experimental orthotopic transplantation of vascularized skeletal allografts: Functional assessment and long-term survival. Plast Reconstr Surg 1995; 95:336. 13. Sachs D, Leight G, Cone J et al. Transplantation in miniature swine. I. Fixation of the major histocompatibility complex. Transplantation 1976; 22(6):559. 14. Pennington L, Lunney J, Sachs D. Transplantation in miniature swine. VIII. Recombination within the major histocompatibility complex of miniature swine. Transplantation 1981; 31:66.

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15. Pescovitz M, Thistlethwaite J Jr, Auchincloss H Jr et al. Effect of Class II antigen matching on renal allograft survival in miniature swine. J Exp Med 1984; 160:1495. 16. Rosengard B, Ojikutu C, Guzetta P et al. Induction of specific tolerance to Class I disparate renal allografts in miniature swine with cyclosporine. Transplantation 1992; 4(3):490. 17. Gianello P, Blancho G, Fishbein J et al. Mechanism of cyclosporine induced tolerance to primarily vascularized allografts in miniature swine: Effect of administration of exogenous IL-2. J Immunol 153:4788, 1994. 18. Owen RD. Immunogenetic consequences of vascular anastomoses between bovine twins. Science 1945; 102:400. 19. Anderson D, Billingham RE, Lampkin GH et al. The use of skin grafting to distinguish between monozygotic and dizogotic twins in cattle. Heredity 1951; 5:379. 20. Billingham RE, Brent L, Medawar PB. Actively acquired tolerance of foreign cells. Nature 1953; 172:603. 21. Burnet FM. The clonal selection theory of acquired immunity. Cambridge: Cambridge University Press 1959. 22. Lubaroff DM, Silvers WK. The importance of chimerism in maintaining tolerance of skin allografts in mice. J Immunol 1973; 111:65. 23. Flake AW, Harrison MR, Adzick NS et al. Transplantation of fetal hematopoietic stem cells in utero: The creation of hematopoietic chimeras. Science 1986; 233:776. 24. Hedrick MH, Rice HE, MacGillivray TE et al. Hematopoietic chimerism achieved by in utero hematopoietic stem cell injection does not induce donor specific tolerance for renal allografts in sheep. Transplantation 1984; 10. 25. Cowan MJ, Tarantal AF, Capper J et al. Long term engraftment following in utero T cell depleted parental marrow transplantation into fetal rhesus monkeys. Bone Marrow Transplant 1996; 17:1157. 26. Woodruff MFA. Reciprocal skin grafts in a pair of twins showing blood chimerism. Lancet 1959; 100:476. 27. Raudrant D, Touraine JL, Rebaud A. In utero transplantation of stem cells in humans: Technical aspects and clinical experience during pregnancy. Bone Marrow Transplant 1992; 9(supp):98.

CHAPTER 4

Dendritic Cells and Alloimmune Chimerism in Limb Transplantation Mia Talmor, Ralph M. Steinman and Lloyd A. Hoffman

Introduction

D

endritic cells are bone marrow-derived leukocytes that act as potent antigen-presenting cells during the afferent phase of rejection. Both lymphoid and nonlymphoid DCs exhibit a similar constellation of functional properties that are not readily observed with other APCs. Mature DCs grown from cultures supplemented with cytokines exhibit striking dendritic processes, as well as high expression of MHC products and a number of costimulatory molecules necessary for T cell activation, including ICAM-I, B7, and CD40. Graft-derived dendritic cells have been shown to migrate from a transplanted limb to the host’s lymphoid tissues, where they prime alloreactive T cells. Examination of lymphoid tissue from immunosuppressed, nonrejecting rat recipients of microvascularized limb transplants reveals the presence of a considerable number of donor-derived cells with features of dendritic cells. These cells are bone marrow-derived, and largely confined to the peripheral lymphoid tissues (lymph node cortex, and splenic white pulp) unless further immunomodulation is performed. It is known that tolerance to donor antigens develops when recipients are made chimeric with donor marrow. We describe a more restricted form of chimerism after limb transplantation, involving dendritic cells, primarily in host lymphoid tissues. It is possible that under certain conditions, this dendritic cell chimerism may lead to tolerance, rather than alloreactivity. In vitro studies have shown that it is only upon exposure to cytokines (GM-CSF, IL-4) that DCs mature and develop their characteristic phenotype and immunostimulatory potential. Immature dendritic cells that do not express costimulatory molecules (B7) have been shown to prolong survival in vivo models of vascularized organ transplantation. Another potential approach to tolerance utilizing limb transplants focuses on expression of Fas ligand by a subset of graft-derived DCs that may migrate to T cell regions and bind to Fas-producing CD4+ T cells, resulting in their death. Manipulation of DC expression of Fas ligand may promote tolerance rather than rejection. We are currently testing these possibilities using the microvascularized rat limb model.

The Dendritic Cell System Specialization of Form and Function The dendritic cell system is comprised of a phenotypically and functionally diverse group of lymphoid and nonlymphoid leukocytes specialized in many ways for inducing T cell mediated immunity. Dendritic cells (DC)s act as antigen presenting cells (APC)s during Composite Tissue Transplantation, edited by Charles W. Hewitt and Kirby S. Black. ©1999 R.G. Landes Company.

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the afferent phase of acute rejection after transplantation of both skin and vascularized organs.1 DCs exhibit four functional features that are not readily observed in other types of APCs (Reviewed in Inaba).2 First, DCs are extremely potent. Very small numbers of DCs stimulate strong T-dependent responses when they are cultured with allogeneic T cells. For example, when mature rat DCs are grown from bone marrow progenitors in enriched culture media, and graded doses are tested against a constant number of 300,000 T cells (as is standard in assays for T cell responses) a DC:T cell ratio of 1:1000 gives a strong MLR. (Fig. 4.1) (Unpublished data). DCs are typically 100 times more potent than unfractionated populations of leukocytes such as mouse spleen suspensions or human mononuclear cells. In addition, alloreactive CTL are generated in the MLR, even in the absence of CD4+ T cells, which suggests that DCs can activate both CD4+ and at least some CD8+ T cells directly.1 It is important to note that in the primary MLR, DCs are able to initiate the response. Once the T cell has been activated, it responds vigorously to antigens presented by other APCs like B cells and macrophages.3,4 The second specialization of DCs is their capacity to stimulate quiescent and naive T cells. One line of evidence for this developed when DCs were found to stimulate the naive T cells that predominate in neonatal cord blood.5 Third, DCs that bear antigen are able to sensitize T cells in vivo in rodent models in the absence of any other adjuvant. This was shown first in transplantation models. Lechler and Batchelor were able to induce tolerance of parental strain rats to kidneys from an F1 strain. Tolerance could be broken by the injection of small numbers of F1 dendritic cells, whereas much larger numbers of macrophages and lymphocytes were without effect.6 Similar observations were made after pancreatic islet and thyroid transplants in rodent models.7,8 Fourth, in vivo, DCs are the main cells that capture injected antigens for presentation to T cells, in contrast to macrophages, which are known to be the main cell types that capture antigens for the purpose of clearance and destruction.

Identification of Mature DCs While DCs are a trace cell type, rising to levels of only 1-3% in a few tissues such as the epidermis or afferent lymph, large numbers of these cells can be generated from progenitors in both blood and bone marrow. The study of phenotypic and functional features of DCs at the cellular level is thus facilitated. Enriched DCs grown from cultures of progenitors supplemented with a number of cytokines, including GM-CSF, TNF-#, and IL-4 exhibit the characteristic DC morphology and phenotypic markings of mature lymphoid DCs. (Fig. 4.2). Most striking are the large lamellipodia, or dendritic processes, that extend from the cell body of these irregularly shaped cells. Phenotypically, DCs are characterized by extremely high levels of antigen-presenting MHC products.9 The levels of Class II on DCs are 10 or more times higher than for other leukocytes. In addition, DCs exhibit high levels of T cell adhesive and costimulatory molecules that are known to facilitate the APC-T cell interaction. Especially abundant are ICAM-I (CD54), B7-2(CD86), and CD40. These markers are not unique to dendritic cells, but they are not usually expressed at such high levels on other leukocytes. While B cell surface markers are typically absent, markers more typical of T cells or macrophages can be expressed, though usually at much lower levels. CD4, CD2 and CD8 (aa homodimer) are expressed by enriched rat DCs cultured from bone marrow progenitors, though at low levels. The expression of CD8 by some dendritic cells, first shown by Shortman and colleagues, has peaked great interest.10 Recent data indicate that this subset expresses Fas ligand and can delete Fas-expressing, responding T cells.11 While a number of DC-restricted markers have been identified (CD11c, DEC-205 in mice,12-16 OX-62 in some rat DCs,17 and S100, CD83, and p55 in humans18-20), none is fully characterized functionally, and none is entirely DC specific.

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Fig. 4.1. Allostimulatory data for rat bone marrow–derived dendritic cells. After !–irradiation, various numbers of cells were used to stimulate a constant number of responder T lymphocytes (300,000/well) separated from syngeneic (WF) and allogeneic (ACI) spleen. Cells are labeled with [3H] thymidine 6–8 h before harvesting.

Fig. 4.2. Photomicrograph of a cytospin preparation of rat bone marrow derived dendritic cells stained with OX–6, a monoclonal antibody directed against MHC Class II molecules.

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Maturation and Migration of DCs While dendritic cells have been identified in both lymphoid and nonlymphoid tissues, there exist important phenotypic and functional differences between the two. Among the nonlymphoid dendritic cells, epidermal Langerhan’s cells (LCs) have been the most widely studied.9 Functionally, freshly isolated LCs are unable to stimulate the MLR, despite their expression of the relevant alloantigens. These cells have the ability to capture and process antigen, but lack immunostimulatory activity. However, when LCs are cultured in the presence of cytokines, they acquire the typical features of lymphoid DCs.21 After this maturation period in culture, LCs become large dendritic shaped cells that express very high levels of antigen presenting and accessory molecules, and are powerfully immunostimulatory for resting T cells. Immature DC progenitors from blood and bone marrow follow a similar course of maturation following exposure to cytokines. After transplantation of both vascularized and nonvascularized allografts, there is a considerable flux of DCs from nonlymphoid to lymphoid tissues via afferent lymphatics. The dendritic cell system is designed to allow immunogens to be captured and presented (likely as foreign peptide-allogeneic MHC complexes in the case of transplantation) at sites of antigen deposition. DCs then migrate and simultaneously mature, developing antigen presenting and accessory molecules that enable them to stimulate reactive T cells in lymphoid organs. While direct sensitization of the host against donor DCs is thought to provide the major stimulus for rejection, the “indirect” pathway, whereby graft antigens are presented on host APCs, has been shown to contribute to rejection of nonvascularized skin22 and vascularized kidney allografts,23 though the principal cell type that mediates this response is unknown.

The Dendritic Cell in Transplantation

The “passenger leukocyte” theory first described by Snell24 over 30 years ago proposed the concept that leukocytes within an allograft act as the critical stimulus for the sensitization of recipient alloreactive T cells. Early studies with both vascularized and nonvascularized allografts provided direct evidence that dendritic cells were the leukocytes that function as APCs during the afferent phase of rejection.25,26 Investigations into the mechanisms of rejection after transplantation of immunoincompatible skin revealed that LCs from the graft increased in both size and upregulated expression of MHC markers before migrating into the dermal lymphatics, and ultimately into draining lymph nodes, to initiate an immune response. A rapid loss of interstitial DCs from rat heart allografts was reported by Larsen and colleagues,27 who subsequently detected these donor-derived cells within recipient lymphoid tissues in close association with CD4+ T cells. Similar findings were observed after rat kidney, spleen and pancreas transplantation.28,29 Given the potent immunostimulatory potential of DCs, several early studies strove to inhibit rejection by ablating dendritic cells in the transplant. As previously mentioned, such depletion facilitates the acceptance of small endocrine grafts,7,8 and the severance of lymphatics does likewise for skin allografts, presumably by blocking the access of lymph-trafficking dendritic cells to host lymphoid tissues. Depletion of graft-derived DCs would block the direct pathway of allosensitization, but the indirect pathway, whereby the host’s own DCs acquire graft antigens, would not be blocked, and this pathway has been shown to participate in the rejection schema. Therefore, instead of ablating APCs in the transplant, several groups have suggested that DCs might possibly be used to establish chimerism, and then tolerance, to donor antigens.30,31 The tolerogenic properties of DCs have thus become a major focus for investigators interested in the role of the DC in the immune response after transplantation.

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Dendritic Cells in Limb Transplantation Early Studies In light of previous studies which implicated DCs as the major antigen presenting cell after vascularized organ transplantation, we began to look for donor-derived leukocytes in rat recipients of allografted limbs. In preliminary studies, we found an acute influx of presumptive dendritic cells in lymphoid tissues of nonimmunosuppressed hosts by staining sections with monoclonal antibodies that were specific for donor MHC Class I or II antigens.32,33 The cells were suggestive of dendritic cells because of their large size, dendritic shape, very strong staining with antibodies to MHC products and localization. It was assumed that the majority of donor cells had migrated from donor skin, where they had resided as LCs. In order to test this hypothesis, and in hopes of rendering the transplant tissue less immunogenic, all allogeneic skin was removed from a subset of limbs. However, it was found that removal of skin did not significantly alter the flux of donor cells from the transplant, or the onset of rejection.

Dendritic Cell Chimerism in Cyclosporine Treated Rats Receiving Limb Transplants In order to gain a better understanding of the role of the DC in the immune response after limb transplantation, further studies were performed on cyclosporine (CsA)-treated rats who had received limb transplants.13 The effectiveness of CsA in preventing rejection of allografted limbs had previously been established.34,35 While it was known at the time that CsA exerted its major effect on T cells via an inhibition of various cytokines, including IL-2 and IFN-!, the effect on APCs was less clear. We therefore performed transplants between two mismatched rat strains (WF,RT1u; ACI, RT1a), and investigated DC trafficking in CsA-treated rats (15 mg/kg/d s.c.). In contrast to what we had expected, we found enhanced donor-derived chimerism in CsA-treated recipients. Without CsA, the limb was rejected at 7-9 days, and dendritic cell trafficking followed a similar pattern to that previously described, resulting in an absence of donor-derived profiles by day 5, which corresponded with the histologic onset of rejection. In contrast, with CsA, donor cells were noted for up to 56 days. Significant differences in numbers of donor cells in recipient spleen and lymph node were noted between the treated and untreated groups at all time points after day 4 (Fig. 4.3). Several lines of evidence led us to the conclusion that the donor-derived cells were in fact dendritic cells. The OX-3+ (donor specific MHC II) donor cells had a similar shape and intensity of MHC II stain as the DCs in donor spleen and nodes, where OX3 stained large profiles in the T cell areas, and less intensely, small round cells in the B areas (Fig. 4.4). In transplant recipients (Fig. 4.5) large, irregularly shaped, strongly OX-3 positive profiles were noted in both T and B cell regions. The distribution of donor cells was verified by staining adjacent sections with antibodies to B cells, T cells, or macrophages or with two color immunolabeling on the same section (Fig. 4.6). While it is known that the T cell regions are the major location for dendritic cells in the steady state,15,36-39 we hypothesize that the DCs we identified in B cell areas are in the process of migration to T cell regions, where they subsequently die. It is known that dendritic cells in spleen and in afferent lymph turn over at a substantial rate (half time of about 1 week),40,41 and that dendritic cells are hard to find in efferent lymph. Presumably, migratory dendritic cells only remain in the T cell areas are if a productive interaction with a T cell takes place. Since this is blocked by CsA, those dendritic cells that access the T cell area simply die; otherwise, the sizable numbers of migratory cells that are present in afferent, but not efferent, lymph would swell the lymph node. Given the fact that donor cells did not have the appearance of B lymphocytes (and in

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Fig. 4.3. Number of donor–derived cells in the spleen and draining inguinal lymph nodes of limb transplant recipients. Limbs from OX–3+ donors (OX–3 selectively stains MHC Class II of the donor and not the recipient) were grafted onto OX–3– recipients that were or were not treated with CsA. At the indicated times, recipient tissues were cryopreserved, sectioned, and stained for OX–3 profiles. The data given are the average number (± SEM) of OX–3+ profiles per high power field (hpf) and are the means for 2–5 animals. Differences between CsA and nonCsA recipients are significant at the p<0.05 level for all time points after day 4. Reprinted with permission from Talmor M, Steinman RM, Codner MA et al. Bone marrow–derived chimerism in nonirradiated, cyclosporine–treated rats receiving microvascularized limb transplants: Evidence for donor derived dendritic cells in recipient lymphoid tissues. Immunol 1995; 86:448–455. Copyright 1995, Blackwell Scientific Ltd.

lymph node were at least as abundant in the T cell area as the B cell area), and did not localize to the macrophage-rich regions of the lymphoid organ, we propose that donorderived cells are dendritic cells that are participating in the normal turnover of this lineage.

Donor-derived Chimerism Is Primarily Bone-marrow Derived As limbs are composite organs, donor cells might derive from skin, muscle, bone or marrow. Because donor cells were as abundant in mesenteric nodes (which did not drain the graft) as in draining nodes, we suspected that the donor cells were not accessing the node simply by lymphatics that drain the graft, but instead by a systemic route from the marrow. To pursue this issue, two experiments were performed. First, the limb graft was irradiated with 1000 rads to block proliferation of marrow progenitors. The irradiation totally ablated the influx of donor cells into the recipient spleen and lymph node at all time points. Second, grafts of skin and muscle flaps (latissimus dorsi) without bone did not engraft donor cells in recipient spleen and lymph node.

Vascularized Bone Mediates Chimerism Better Than Marrow Suspensions Marrow suspensions are typically used to establish chimerism. We compared a graft of 108 marrow cells (two femur equivalents) with an intact limb. Some donor cells were evident at day 4 in CsA-treated recipients of marrow suspensions, but none was detected by day 7,

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Fig. 4.4. Morphology of MHC Class II positive cells in the lymphoid tissues of the OX–3+ donor strain (left) and in the recipients of limbs from the donor strain (right). (a,b) Low and high power views respectively of donor spleen stained for MHC Class II. At low power, the MHC Class II+ dendritic cells in the peripheral (arrow) T cell regions are larger and stain more intensely than the B cells in follicles (B) and marginal zone (MZ). The few dark profiles in the red pulp are granulocytes with endogenous peroxidase activity. (c,d) Higher power views of the T cell and B cell areas of donor lymph node showing the difference in size, shape and intensity of Class II stain between dendritic cells (c) and B cells (d). An arrow marks MHC Class II–rich macrophages in the subcapsular sinus region. (e,f) Sections of recipient spleen at low power stained for donor MHC Class II antigens (e) and the B cell antigen (f). Note that the brown OX–3+ donor–derived cells are localized primarily to regions with B cells, with only a few profiles in the periarterial (arrows) T cell areas. (g,h) Higher powers of recipient spleen (g) and lymph node (h) showing the donor–derived, OX–3+ cells, and their irregular shape and strong MHC Class II stain. Figure reprinted with permission from Talmor M, Steinman RM, Codner MA et al. Bone marrow– derived chimerism in nonirradiated, cyclosporine–treated rats receiving microvascularized limb transplants: Evidence for donor derived dendritic cells in recipient lymphoid tissues. Immunol 1995; 86:448–455. Copyright 1995, Blackwell Scientific Ltd.

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Fig. 4.5. Immunolabeling of donor–derived cells in the lymph node cortex (left) and the spleen (right) of recipients treated with CsA. The localization of donor–derived cells, which are the orange/brown profiles, is representative of all time points beyond day 4. Sections were stained (brown reaction product) for donor MHC Class II (OX–3 monoclonal; top row) and counterstained with hematoxylin. Adjacent sections were stained to identify leukocyte subsets, i.e., OX-33 anti–B cell (middle row) and W3/35 anti–CD4 T cell (lower row; W3/25 also stains CD4 on macrophages in the splenic red pulp). Arrows indicate periarterial areas. Reprinted with permission from Talmor M, Steinman RM, Codner MA et al. Bone marrow–derived chimerism in nonirradiated, cyclosporine–treated rats receiving microvascularized limb transplants: Evidence for donor derived dendritic cells in recipient lymphoid tissues. Immunol 1995; 86:448–455. Copyright 1995, Blackwell Scientific Ltd.

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Fig. 4.6. Irregular shape and strong MHC Class II expression of the donor–derived cells in recipient lymphoid organs. Spleen (top): Adjacent sections were stained with W3/25, to identify the CD4+ T cells (left) that constitute the periarterial sheath of lymphocytes, and with OX–3 to show the donor dendritic cells (right) at the junction of B and T cell areas. The central artery of the white pulp is shown with a large arrow, and some OX–3+ donor cells with the short arrow. Lymph node (Middle, bottom): Two color immunolabeling of donor–derived cells in the T cell regions of the lymph node cortex of recipients treated with CsA. The donor–derived cells are stained with OX–3 and alkaline phosphatase (purple reaction product), while recipient cells are stained with different anti–leukocyte monoclonals and peroxidase (brown reaction product). The latter mAbs were OX–33 anti–B cell, W3/25 anti–CD4+ T cell, ED–2 anti–macrophage, and no primary mAb. A few of the donor cells are arrowed to illustrate the strong MHC Class II staining and irregular shape that is characteristic of dendritic cells. Reprinted with permission from Talmor M, Steinman RM, Codner MA et al. Bone marrow–derived chimerism in nonirradiated, cyclosporine–treated rats receiving microvascularized limb transplants: Evidence for donor derived dendritic cells in recipient lymphoid tissues. Immunol 1995; 86:448–455. Copyright 1995, Blackwell Scientific Ltd.

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in contrast to vascularized bone grafts. When the recipients were irradiated (600 rads), an injection of marrow yielded large numbers of donor cells in spleen, as expected.

Dendritic Cells and the Induction of Tolerance While dendritic cells have been studied primarily in the context of immunogenicity, tolerogenic properties of DCs have been described both in vitro42,43 and in vivo.44,45 DCs have been shown to mediate tolerance at the level of the thymus. By allowing dendritic cells to migrate into the thymus,43 or by reaggregating DCs with thymic epithelial cells,42,46 a role in central tolerance, presumably by negative selection, has become evident in thymic organ cultures. In our experience, low level thymic chimerism is seen in long term rats treated with a variety of immunosuppressant agents (CsA, Neoral, FK506)(Unpublished data). Thymic chimerism with DCs is increased if the recipient is treated with a donor-specific transfusion (DST) one day prior to transplant. While the functional relevance of this thymic chimerism is unknown, rejection is delayed in recipients treated with CsA and a single pretransplant DST that exhibit high levels of thymic chimerism. It is known that transplantation tolerance is induced when foreign hematopoietic cells are administered into fetal and neonatal recipients. To establish marrow chimeras in immunologically mature adults, ablative therapies are applied. Utilizing limb transplants, we describe a more restricted form of chimerism that seems to involve primarily dendritic cells in host lymphoid tissues, but does not require irradiation of the recipient. We propose that graft-derived dendritic cell chimerism might be used to establish tolerance to donor antigens. This concept was first championed by Starzl and colleagues, who noted donor-derived “microchimerism” in recipients 25 years after organ transplantation.30,47-52 They hypothesized that the small numbers of donor-derived cells included DCs which could tolerize the recipient and eventually allow a cessation of immunosuppression. Despite high levels of peripheral lymphoid chimerism in immunosuppressed rats, we have been unable to tolerize recipients. Continuous immunosuppression (CsA or Neoral daily or FK506 thrice weekly) is required to maintain the rat in a nonresponsive state, and donor-derived lymphoid chimerism is maintained only as long as the animal remains in this state. Whether the DC chimerism is a result of immunosuppressant-induced nonresponsiveness, or a precondition for it, is unknown, but several lines of evidence suggest that under certain conditions DCs will predominantly tolerize rather than sensitize host T cells. One theory of DC-induced tolerance relates to the fact that dendritic cells may need to undergo a process of maturation before strong T cell stimulatory function develops; immature dendritic cells may induce tolerance rather than immunity. Immature DCs found in mouse skin and spleen,9,21,53 human blood,54 and rat lung55 are poor simulators of the MLR. It is only when they are exposed to cytokines (GM-CSF being a major example) that they develop the characteristic immunostimulatory phenotype. It has been shown that exposure to cytokines increases DC expression of B7 and other costimulators. The B7 system is a major costimulator for IL-2 production by T cells.56-58 Boussiotis et al have shown that transfection of ICAM-1 generates an APC that induces tolerance in peripheral, alloreactive helper T cell clones, whereas transfection of B7 leads to memory.59 Fu et al have shown that costimulatory molecule-deficient dendritic cell progenitors prolong cardiac allograft survival in immunosuppressed recipients in vivo.60 It is possible that transplantation of dendritic cells under conditions that block their full maturation might lead to tolerance rather than immunity. A new approach to this issue has become apparent based on the finding by Suss and Shortman that a subclass of dendritic cells expresses Fas ligand, and can kill CD4+ Fasexpressing T cells via Fas/Fas ligand-induced apoptosis.11 In subsequent in vivo studies,

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Bellgrau and colleagues found that testis grafts which were derived from mice that could express functional Fas ligand survived indefinitely when transplanted under the kidney capsule of allogeneic animals, whereas testis grafts derived from mutant gld mice which express nonfunctional Fas ligand were rejected. Further analysis of testis showed that Sertoli cells constitutively expressed Fas ligand, which explains its status as an immune privileged site.61 If one were able to enhance graft-derived DC expression of Fas ligand, the alloreactive Fas positive T cells responding to them would undergo apoptotic cell death, and the transplant would therefore theoretically be accepted indefinitely. We are currently testing these possibilities, using the rat hindlimb allograft model.

References 1. Austyn JM. Dendritic cells in transplantation. Adv Exp Med Biol 1993; 329:489-494. 2. Inaba K, Steinman RM. Dendritic cells. In: Lung Biology in Health and Disease. Lipscomb MF, Russell SW, eds. 1996; 102:87-107. 3. Inaba K, Steinman RM. Resting and sensitized T lymphocytes exhibit distinct stimulatory (antigen-presenting cell) requirements for growth and lymphokine release. J Exp Med 1984; 160:1717-1735. 4. Inaba K, Steinman RM. Protein-specific helper T lymphocyte formation initiated by dendritic cells. Science 1985; 229:475-479. 5. Sallusto F, Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor a. J Exp Med 1994; 179:1109-1118. 6. Lechler RI, Batchelor JR. Restoration of immunogenicity to passenger cell-depleted kidney allografts by the addition of donor strain dendritic cells. J Exp Med 1982; 155:31-41. 7. Faustman DL, Steinman RM, Gebel HM et al. Prevention of rejection of murine islet allografts by pretreatment with anti-dendritic cell antibody. Proc Natl Acad Sci USA 1984; 81:3864-3868. 8. Iwai H, Kuma S-I, Inaba MM et al. Acceptance of murine thyroid allografts by pretreatment of anti-Ia antibody or anti-dendritic cell antibody in vitro. Transplantation 1989; 47:45-49. 9. Schuler G, Steinman RM. Murine epidermal Langerhans cells mature into potent immunostimulatory dendritic cells in vitro. J Exp Med 1985; 161:426-546. 10. Vremec D, Zorbas M, Scollay R et al. The surface phenotype of dendritic cells purified from mouse thymus and spleen: Investigation of the CD8 expression by a subpopulation of dendritic cells. J Exp Med 1992; 176:47-58. 11. Suss G, Shortman K. A subclass of dendritic cells kills CD4 T cells via Fas/Fas ligand induced apoptosis. J Exp Med 1996; 183:1789-1796. 12. Metlay JP, Witmer-Pack MD, Agger R et al. The distinct leukocyte integrins of mouse spleen dendritic cells as identified with new hamster monoclonal antibodies. J Exp Med 1990; 171:1753-1771. 13. Witmer-Pack MD, Crowley MT, Inaba K et al. Macrophages, but not dendritic cells, accumulate colloidal carbon following administration in situ. J Cell Sci 1993; 105:965-973. 14. Jiang W, Swiggard WJ, Heufler C et al. The receptor DEC-205 expressed by dendritic cells and thymic epithelial cells is involved in antigen processing. Nature 1995; 375:151-155. 15. Kraal G, Breel M, Janse M et al. Langerhans cells, veiled cells and interdigitating cells in the mouse recognized by a monoclonal antibody. J Exp Med 1986; 163:981-997. 16. Witmer-Pack MD, Swiggard WJ, Mirza A et al. Tissue distribution of the DEC-205 protein that is detected by the monoclonal antibody NLDC-145. II. Expression in situ in lymphoid and nonlymphoid tissues. Cell Immunol 1995; 163:157-162. 17. Brenan M, Puklavec M. The MRC OX-62 antigen: A useful marker in the purification of rat veiled cells with the biochemical properties of an integrin. J Exp Med 1992; 175:1457-1465.

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18. Takahashi K, Isobe T, Ohtsuki Y et al. Immunohistochemical localization and distribution of S-100 proteins in the human lymphoreticular system. Am J Pathol 1984; 116:497-503. 19. Zhou LJ, Tedder TF. Human blood dendritic cells selectively express CD83, a member of the immunoglobulin superfamily. J Immunol 1995; 154:3821-3835. 20. Mosialos G, Birkenbach M, Ayehunie S et al. Circulating human dendritic cells differentially express high levels of a 55-Kd actin bundling protein. Am J Pathol; 1996; 148:593-600. 21. Witmer- Pack MD, Olivier W, Valinsky J et al. Granulocyte/ macrophage colony-stimulating factor is essential for the viability and function of cultured murine epidermal Langerhans cells. J Exp Med 1987; 166:1484-1498. 22. Fangman J, Dalchau R, Fabre JW. Rejection of skin allografts by indirect allorecognition of donor Class 1 major histocompatibility complex peptides. J Exp Med 1992; 175:1521. 23. Benham AM, Sawyer GJ, Fabre JW. Indirect T cell allorecognition of donor antigens contributes to the rejection of vascularized kidney allografts. Transplantation 1995; 59:1028-1032. 24. Snell GD. The homograft reaction. Annu Rev Microbiol 1957; 11:439. 25. Steinman RM, Kaplan G, Witmer MD et al. Identification of a novel cell type in peripheral lymphoid organs of mice: V. Purification of splenic dendritic cells, new surface markers and maintenance in vitro. J Exp Med 1979; 149:1. 26. Austyn JM, Steinman RM. The passenger leukocyte: A fresh look. Transplant Rev 1988; 2:139. 27. Larsen CP, Morris PJ, Austyn JM. Migration of dendritic leukocytes from cardiac allografts into host spleens: A novel pathway for the initiation of rejection. J Exp Med 1990; 171:307. 28. Tilney NL, Gowans JL. The sensitization of rats by allografts transplanted to alymphatic pedicles of skin. J Exp Med 1971; 133:951. 29. Steiniger B, Klempnauer J. Donor-type MHC-positive cells in the host spleen after rat organ transplantation: Differences between pancreas and heart allograft recipients. Transplantation 1986; 41:787. 30. Starzl TE, Demetris AJ, Trucco M et al. Cell migration and chimerism after whole organ transplantation: The basis of graft acceptance. Hepatology 1993; 17:1127. 31. Talmor M, Steinman RM, Codner MA et al. Bone marrow-derived chimerism in nonirradiated, cyclosporine-treated rats receiving microvascularized limb transplants: Evidence for donor-derived dendritic cells in recipient lymphoid tissues. Immunology 1995; 86:448-455. 32. Codner MA, Shuster BA, Steinman RM et al. Migration of donor leukocytes from limb allografts into host lymphoid tissues. Ann Plast Surg 1990; 25:353. 33. Hoffman LA, Codner MA, Shuster BA et al. Donor leukocyte migration following extremity transplantation in an experimental model. Plast Reconstr Surg 1992; 90:999. 34. Fritz WD, Schwartz WMR. Limb allografts in rats immunosuppressed with cyclosporine A. Ann Surg 1984; 39:211. 35. Kim SK, Aziz S, Oyer P. Use of cyclosporine A in allotransplantation of rat limbs. Ann Plast Surg 1984; 12:249. 36. Agger RM, Witmer-Pack MD, Romani H et al. Two populations of splenic dendritic cells detected with M342, a new monoclonal to an intracellular antigen of interdigitating dendritic cells and some B lymphocytes. J Leukoc Biol 1992; 52:34-42. 37. Fossum S. Lymph-borne dendritic leukocytes do not recirculate, but enter the lymph node paracortex to become interdigitating cells. Scand J Immunol 1989; 27:97-105. 38. Witmer MD, Steinman RM. The anatomy of peripheral lymphoid organs with emphasis on accessory cells: Light microscopic, immunocytochemical studies of mouse spleen, lymph node and Peyer’s patch. Am J Anat 1984; 170:465-481. 39. Dijkstra CD. Characterization of nonlymphoid cells in rat spleen, with special reference to strongly Ia-positive branched cells in T cell areas. J Reticuloendothel Soc 1982; 32:167-178. 40. Steinman RM, Lustig DS, Cohn ZA. Identification of a novel cell type in peripheral lymphoid organs of mice III. Functional properties in vivo. J Exp Med 1974; 139:1431-1445. 41. Pugh CW, MacPherson GG, Steer HW. Characterization of nonlymphoid cells derived from rat peripheral lymph. J Exp Med 1983; 157:1758-1779.

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42. Jenkinson EJ, Jhittay P, Kingston R et al. Studies of the role of the thymic environment in the induction of tolerance to MHC antigens. Transplantation 1985; 39:331. 43. Matzinger P, Guerder S. Does T cell tolerance require a dedicated antigen-presenting cell? Nature 1989; 338:74. 44. Clare-Salzler MJ, Brooke J, Chai A et al. Prevention of diabetes in nonobese diabetic mice by dendritic cell transfer. J Clin Invest 1992; 90:741. 45. Khoury SJ, Gallon L, Chen W et al. Mechanisms of acquired thymic tolerance in experimental autoimmune encephalomyelitis: Thymic dendritic-enriched cells induce specific peripheral T cell unresponsiveness in vivo. J Exp Med 1995; 182:357. 46. Moore NC, Anderson DEJ, McLoughlin JJT et al. Differential expression of Mtv loci in MHC Class II-positive thymic stromal cells. J Immunol 1994; 152:4826-4831. 47. Starzl TE, Demetris AJ, Trucco M et al. Chimerism and donor specific nonreactivity 27 to 29 years after kidney allotransplantation. Transplantation 1993; 55:1272-1277. 48. Demetris AJ, Murase N, Fujisaki JJ et al. Hematolymphoid cell trafficking, microchimerism, and GVHD reactions after liver, bone marrow, and heart transplantation. Transplant Proc 1993; 25:3337-3344. 49. Starzl TE, Demetris AJ, Murase N et al. Cell migration, chimerism and graft acceptance. Lancet 1992; 339:1579-1582. 50. Qian S, Demetris AJ, Murase N et al. Murine liver allograft transplantation: Tolerance and donor cell chimerism. Lancet 1994; 19:916-924. 51. Starzl TE, Demetris AJ, Trucco M et al. Chimerism after liver transplantation for type IV glycogen storage disease and type I Gaucher’s disease. N Engl J Med 1993; 14:326-332. 52. Starzl TE, Demetris AJ, Murase N et al. Cell chimerism permitted by immunosuppressive drugs is the basis of organ transplant acceptance and tolerance. Immunol Today 1993; 14:326-332. 53. Crowley MT, Inaba K, Witmer-Pack MD et al. Use of the fluorescence activated cell sorter to enrich dendritic cells from mouse spleen. J Immunol Meth 1990; 133:55-66. 54. O’Doherty U, Steinman RM, Peng M et al. Dendritic cells freshly isolated from human blood express CD4 and mature into typical immunostimulatory dendritic cells after culture in monocyte-conditioned medium. J Exp Med 1993; 178:1067-1078. 55. Holt PGJ, Oliver N, Bilyk C et al. Downregulation of the antigen presenting cell function(s) of pulmonary dendritic cells in vivo by resident alveolar macrophages. J Exp Med 1993; 177:397-407. 56. Linsley PS, Brady W, Grosmaire A et al. Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation. J Exp Med 1991; 173:721-730. 57. Freeman GJ, Borriello RJ, Hodes RJ et al. Murine B7-2, an alternative CTLA4 counterreceptor that costimulates T cell proliferation and interleukin 2 production. 1993; 178:2185-2192. 58. Freeman GJ, Gribben JG, Boussiotis JW et al. Cloning of B7-2: A CTLA-4 counter-receptor that costimulates human T cell proliferation. Science 1993; 262:909-911. 59. Boussiotis A, Freeman GJ, Gray G et al. B7 but not intercellular adhesion molecule-1 costimulation prevents the induction of human alloantigen-specific tolerance. J Exp Med 1993; 178:1753-1763. 60. Fu F, Li Y, Qian S et al. Costimulatory molecule-deficient dendritic cell progenitors (MHC Class II, CD80dim, CD86-) prolong cardiac allograft survival in nonimmunosuppressed recipients. Transplantation 1996; 62:659-665. 61. Bellgrau D, Gold D, Selawry H et al. A role for CD95 ligand in preventing graft rejection. Nature 1995; 377:630-632.

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Donor–Host Immune Chimerism and Tolerance in CTA/VBMT

Section III Vascularized Bone Marrow Transplantation

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CHAPTER 5

Composite Tissue/Vascularized Bone Marrow Transplantation: Development of Donor-Host Immune Chimerism and Tolerance Charles W. Hewitt and Kirby S. Black

Introduction

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he induction of stable mixed immune chimerism is related to alloimmune tolerance development in vascularized bone marrow transplant (VBMT) models.1-4 A VBMT can be represented by a composite tissue allograft (CTA).1-9 The most widely studied model of CTAs is the rat hindlimb transplant. A CTA/VBMT is unique because it provides immediate engraftment of the bone marrow by surgical means. A CTA/VBMT undergoes increased recipient hemopoietic repopulation compared to conventional bone marrow transplantation.5 With a parental to hybrid genetic combination (LEW to LBN; RT1l to RT1n), the majority of these VBMT recipients fail to express graft versus host disease (GVHD) and instead develop tolerance (Fig.5.1). In clinical and experimental models of cellular bone marrow transplantation, it has been shown that donor T cell subsets present in the graft are responsible for inciting GVHD. Yet, tolerant CTA/VBMT recipients in our experiments demonstrated stable donor mixed immune chimerism without GVHD.2-4,9 Conversely, GVHD positive CTA/VBMT positive recipients develop unstable aggressive allogenic mixed immune chimerism.2,6-8

Initial Experiments on Immune Chimerism in Rat Limb Transplant Recipients In initial studies, long term surviving LEW rat recipients of LBN vascularized hindlimb transplants or composite tissue allografts (CTAs) were tested for donor-host lymphoid chimerism.1 These recipients underwent various cyclosporine (CsA) regimens to induce long term acceptance. Splenic or peripheral lymphocytes from long term CTA hosts demonstrated a mean of 19.7 % (± 9.7 %) donor LBN chimerism. Thus, this was the first demonstration that lymphoid cells originated from the donor bone marrow of the limb grafts, resulting in chimeric repopulation of hemopoietic tissues.1 It was suggested that the presence of donor immunocytes in these limb allograft recipients may have been beneficial and contributed to the long term CTA survival. Composite Tissue Transplantation, edited by Charles W. Hewitt and Kirby S. Black. ©1999 R.G. Landes Company.

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Composite Tissue Transplantation Fig. 5.1. Percentage of survival of CTA animals (LEW ∀ LBN) vs. days posttransplantation. The majority (62.5%) of the recipients showed antigen–specific tolerance with absence of GVHD. The minority fraction (32.5%) underwent acute and chronic (mostly the latter) lethal GVHD. Reprinted from ref. 2 by permission of Williams and Wilkins.

Flow Cytometric Analysis for T Cell Chimerism Assays were performed with mononuclear lymphocytes from experimental CTA/VBMT recipients, normal BN and normal LEW animals. Cells were pooled, purified with FicollHistopaque, and Ig depleted by means of magnetic particles coated with sheep antibody against rat IgG. Lymphocytes were concentrated to 1.5 x 106 cells/ml in Hank’s balanced salt solution with bovine serum albumin. Cell suspensions were incubated with sheep anti-rat IgG-coated dynabeads (Dynabeads M-450, Dynal Robbins Scientific, Mountain View, CA). The resulting mixtures were incubated on a Dynabead Rotator. Thereafter, each individual sample was washed thrice in the magnetic particle concentrator and supernatants were then centrifuged and resuspended in DPBS/Az. Mixed proportions of normal LEW and normal BN lymphocytes totaling 1 x 106 cells/tube were stained separately with antisera against LBN determinants, LEW determinants, and normal LEW serum as previously described,4 in order to yield chimeric standard curves. Five different values were chosen: 100% LEW, 0% LBN; 75% LEW, 25% LBN; 50% LEW, 50% LBN; 25% LEW, 75% LBN and 0% LEW, 100% LBN. These standard mixtures of normal lymphocytes and experimental lymphocyte suspensions were indirectly stained by either LEW allosera against LBN determinants (positive control) or normal LEW serum (negative control), and a FITC conjugated rabbit antirat IgG antibody (Cappel, Rogers, AK) as the secondary reagent. The stained lymphocytes were protected from light and fixed in 1.0% buffered paraformaldehyde. Fluorescence was assessed on a Coulter Epics V Fluorescent Activated Cell Sorter (Coulter Electronics, Hialeah, FL). A minimum of 5,000 cells were counted. The cells were analyzed by light green fluorescent intensity and 90° forward light scatter (FALS). FALS was used to isolate the viable lymphocytes for analysis. The gate was set such that contaminant erythrocytes and monocytes were excluded in the analysis. Data analysis was performed using Coulter Easy 2 Immuno software. The Immuno program allows for the quantitation of true positive labeling. It discards autofluorescence and background by graphically subtracting the positive antisera from the normal or naive antisera along a window previously set with a common channel number, then performs statistical analysis on the resulting curve, determining frequency of events and intensity of fluorescence. After obtaining true positive labeling, the percentage of LBN lymphocytes was calculated. Linear regression statistics were run (Human Systems Dynamics, Northridge,

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Fig. 5.2. Stable mixed allogeneic T cell chimerism in tolerant VBMT chimeras. Chimerism was determined by flow cytometry, regression analysis and inverse prediction. Reprinted from ref. 3 by permission of Williams & Wilkins.

CA). It was then determined whether there was a significant linear relationship between the percent of positively stained BN cells and the percentage of BN cells present.

Cellular Kinetics of Chimerism and Mechanisms of Immune Nonresponsiveness We tested whether the level of donor chimerism was associated with alloimmune reactivity. Flow cytometric measurements of chimerism were correlated with in vitro cellular immune responses in both tolerant and GVHD positive CTA/VBMT recipients. The CTA/ VBMT model consisted of transplantation of a hindlimb from parental LEW rats onto hybrid LBN recipients. Slow delayed development of donor immune chimerism resulted in the peripheral blood of tolerant CTA/VBMT animals during the first 100 days following surgery (Fig. 5.2). The tolerant chimeras presented stable donor lymphocyte populations averaging 18.3% ± 3.9% (Table 5.1). Proliferative cellular immune responses of donor LEW lymphocytes from the tolerant CTA/VBMT recipients demonstrated antigen-specific anti-BN immune reactivity early during the first 30 days posttransplantation (Fig. 5.3). Cellular immune assays also showed increased responses against self LEW determinants during this time (Fig. 5.4). Subtle but limited clinical signs of GVHD were present at this time, but were short-lived. Following this time, the tolerant CTA/VBMT chimeras appeared normal clinically, and remained otherwise healthy throughout their course. Then, following long term posttransplantation (>200 days), cellular mixed lymphocyte responses demonstrated antigen-specific suppression (Fig. 5.5). Cytotoxic studies confirmed that antigen-specific reactivity was suppressed to host BN determinants, but not to third party allodeterminants (Fig. 5.6). In summary, different sequential mechanisms of immune regulation were shown in tolerant CTA/VBMT chimeras associated with stable mixed immune chimerism. In unreported results, we have recently found that this immune regulation is associated with nonspecific suppressor cells.

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Table 5.1. Levels of donor lymphoid chimerism in T-enriched cell populations obtained from VBMT recipients Cell sample/ animal No.

Recipient (LBN) % BN+ (LBN)

Donor (LEW) % LEW+ /BN–

73.8 71.6 61.9 85.5 91.2 92.2 97.8 74.2 87.3

26.2 28.4 38.1 14.5 8.8 7.8 2.2 25.8 12.7

Tolerant VBMT recipients: 278 279 288 289 297 298 299 300 301

x = 18.3 ± 3.9% GVHD positive VBMT recipients: 282 285 304

36.8 16.3 66.4

63.2 83.7 33.6 x = 60.2 ± 14.5%

Indefinite surviving VBMT animals demonstrate low–level stable mixed lymphoid chimerism with donor chimerism averaging 18.3 ± 3.9%. However, GVHD animals demonstrate a greater level donor chimerism, averaging 60.2 ± 14.5%. Specific staining represented theamount of BN+ staining by LEW anti-BN antibodies minus background staining by LEW normal serum and FITC-conjugated rabbit anti–rat IgG: i.e., (exppos—expneg). In control cell samples that were 100% LBN, the specific staining was 85.7%, while in cell samples containing 100% LEW there was 0.0% specific staining. The values for % recipient BN+ (LBN) cells in the table above were derived from the following equation: ([exppos—expneg/LBNpos—LBNneg] x 100). The values for % LEW+/BN– (LEW) cells in the table above were derived from the following equation: 100—([exppos—expneg/ LBNpos—LBNneg ] x 100). Reprinted from ref. 2 by permission from Williams & Wilkins.

Discussion There are two major theoretical mechanisms by which the induction of specific transplantation tolerance associated with mixed chimerism is generally possible: 1. Clonal deletion or clonal inactivation of immune cells destined to react against the graft; and 2. Continuous inhibition of the alloreactive cells by antigen-specific regulatory cells. A third possibility may combine some aspects of both of these mechanisms and include the elimination of activated cells by a nonspecific mechanism. This may involve evolution of antigen-specific mechanisms via nonspecific actions. One such example includes nonspecific suppressor cells, which are large granular lymphocytes. Natural suppressor (NS) cells bear the null phenotype and have been implicated in several mechanisms of tolerance induction. NS cell development is well known to occur in environments of chimerism, hematopoiesis, GVHD, and tolerance. The induction of NS cells has been implicated as one of the underlying mechanisms of tolerance development in many of these models.10-12 The

Donor–Host Immune Chimerism and Tolerance in CTA/VBMT

61 Fig. 5.3. Cellular immune regulation to host-BN allo– determinants in tolerant VBMT chimeras. Allo-BN (stimulator) antigen–acti– vated mixed lymphocyte responses were determined over time with immunocytes from tolerant VBMT recipients (responder). Allo-BN antigen (target)– activated immunocytolytic responses were determined over time with immunocytes from tolerant VBMT recipients (effector) at an effector/target ratio of 100:1 (▲ Alloimmune prolifera– tion, ■ direct immuno– cytolysis). Reprinted from ref. 3 by permission of Springer–Verlag.

Fig. 5.4. Cellular immune regulation to self–LEW de– terminants in tolerant VBMT chimeras. Self–LEW (stimul– ator) antigen–activated mixed lymphocyte responses were determined over time with immunocytes from tolerant VBMT recipients (respond– er). Self–LEW antigen (target)–activated immuno–cytolytic responses were determined over time with effector/target immunocytes from tolerant VBMT recipi–ents (effector) at an effector/target ratio of 100:1 (▲ immune proliferation, ■ direct immunocytolysis). Reprinted from ref. 3 by permission of Springer– Verlag.

findings of tolerance and nonspecific suppressor cells in our CTA/VBMT chimeras share striking similarities with other studies on NS cells. These similarities include the following: NS cells reside in the spleen, are transferable, inhibit polyclonal and humoral responses, are associated with inducing antigen-specific suppressor cells and play a role in clonal inactivation; and their presence is directly related to tolerance induction.

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Composite Tissue Transplantation Fig. 5.5. (A) In vitro lymphocyte cultures of tolerant VBMT animals demonstrate antigen– specific nonresponsiveness against host BN de– terminants while responses against third party alloantigen (ACI) were normal.

Fig. 5.5. (B) Mitogen responses to both con– canavalin A (CON-A) and phytohemagglutinin P (PHA-P) show normal polyclonal activation in tolerant VBMT animals. Reprinted from ref. 2 by permission of Williams & Wilkins.

Acknowledgments 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 faculty practice grants from Robert Wood Johnson Medical School/Cooper Hospital/University Medical Center.

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A

B

C

63 Fig. 5.6. Direct cell–mediated lympholysis effector assays in tolerant VBMT animals. The ex– perimental animals’ (A) normal third party responses toward ACI targets; (B) antigen–specific unresponsiveness against host BN targets; responses com– parable to normal LBN effectors against self-BN targets; and (C) expected unresponsiveness against self-LEW targets. Reprinted from ref. 2 by permission of Williams & Wilkins.

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References 1. Hewitt CW, Black KS, Dowdy SF et al. Composite tissue (limb) allografts in rats: III. Development of donor-host lymphoid chimeras in long-term survivors. Transplantation 1986; 41:39-43. 2. Hewitt CW, Ramsamooj R, Patel M et al. Development of stable mixed T-cell chimerism and transplantation tolerance without immune modulation in recipients of vascularized bonemarrow allografts. Transplantation 1990; 50:766-772. 3. Llull R, Ramsamooj R, Black KS et al. Cellular mechanisms of alloimmune nonresponsiveness in tolerant mixed lymphocyte chimeras induced by vascularized bone marrow transplants. Transpl Int 1994; 7(1):S453-S456. 4. Ramsamooj R, Patel MP, Llull R et al. Use of regression analysis and flow cytometry for determining levels of mixed semiallogeneic immune chimerism. J Invest Surg 1996; 9:273-281. 5. Lukomska B, Durlik M, Cybulska E et al. Comparative analysis of immunological reconstitution induced by vascularized bone marrow versus bone marrow cell transplantation. Transpl Int 1996; 9(Suppl 1):S492-S496. 6. Woolley DS, Hou A, Strande L et al. Vascularized bone marrow transplantation and graft versus host disease: Morphometric analysis of muscle. Transplant Proc 1994; 26(6):3321-3322. 7. Hewitt CW, Englesbe MJ, Tatem L et al. Graft-versus-host-disease in extremity transplantation: Digital image analysis of bone marrow in situ. Ann Plast Surg 1995; 35(1):108-112. 8. Ramsamooj R, Llull R, Tatem LD et al. Graft-versus-host-disease in limb transplantation: Digital image analysis of bone marrow and TGF-beta expression in situ using a novel 3-D microscope. Transplant Proc 1996; 28(4):2029-2031. 9. Tatem LD, Hirpara S, Dalsey RM et al. Role of in situ IL-2r and TGF-beta expression in tolerant vascularized bone marrow (limb) transplant chimeras. Transplant Proc 1997; 29(4):2194-2197. 10. Schwadron RB, Gandour DM, Strober S. Cloned natural suppressor cell lines derived from the spleens of neonatal mice. J Exp Med 1985; 162(1):297-310. 11. Tilkin AF, Begue B, Gomard E et al. Natural suppressor cell inhibiting T killer responses against retro viruses: A model for self tolerance. J Immunol 1985; 134(4):2279-2782. 12. Maier T, Holda JH, Claman HN. Synergism between T and non-T cells in the in vivo and in vitro expression of graft-versus-host-disease-induced natural suppressor cells. J Exp Med 1985; 162(3):979-992.

CHAPTER 6

Vascularized Bone Marrow Transplantation: Pathology of Composite Tissue TransplantationInduced Graft Versus Host Disease Rajen Ramsamooj and Charles W. Hewitt

Background

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omposite tissue allografts (CTA) represent the transplantation of several tissue types, including integumentary, musculoskeletal, cutaneous and hematopoietic elements. The rat hindlimb CTA using a parental limb to an F1 hybrid host actually represents a vascularized bone marrow transplant model.1 The hindlimb CTA provides transplantation of precursor hematolymphoid (bone marrow) and mature (blood and lymph nodes) elements by a surgical approach, along with transfer of their syngeneic/supportive microenvironments. Immediate engraftment with and without immune modulation has been previously shown.2-7 By comparison, other bone marrow transplant models have a finite rate of engraftment failures, of which none have been reported for the hindlimb CTA/vascularized bone marrow transplant. There are significant advantages to the transplantation of composite tissues when considering bone marrow transplantation. In contrast to other methods of bone marrow transplantation,8-10 CTAs allow immediate engraftment of donor lymphoid cells, with development of donor-specific lymphoid chimerism.1,4,5,7 Chimerism produces two profound effects in the hybrid recipient of a parental limb. These are the development of donor-specific immune tolerance and graft versus host disease. The tolerant animals showed significantly lower levels of donor-specific T cell chimerism.1 Mechanisms of host specific tolerance are associated with the fate of the chimeric T cell populations. A curious phenomenon occurs during the first 30 days post-vascularized bone marrow transplantation (VBMT). These animals become polyclonal, self- and host-specifically unresponsive in vitro studies.11 These results are similar to the immune reactivity associated with GVHD in other models.12-14 This initial dysregulated immune response is later replaced by polyclonal unresponsiveness at 100 days and host specific unresponsiveness at 200 days.11 Thus, this initial immune dysregulation in the tolerant animals is associated with a stable low level mixed chimerism. Secondly, it appears as though the chimeric environment supports the development of suppressor circuits and thus host specific tolerance. Composite Tissue Transplantation, edited by Charles W. Hewitt and Kirby S. Black. ©1999 R.G. Landes Company.

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Fig. 6.1. Graft versus host disease in a LEW to LBN CTA/VBMT recipient. LEW to LBN composite tissue allograft/vascularized bone marrow transplant undergoing one way donor antihost graft versus host disease on day 41 post-transplant.

Unfortunately, the converse of tolerance, when considering vascularized bone marrow transplantation, is graft versus host disease. This undesired outcome is associated with unstable higher levels of donor chimerism. In the recipient animal, these chimeric T cells become effector cells that lead to the development of GVHD. In murine studies, evidence of GVHD correlates with the presence of increased levels of donor CD8+ lymphocytes.15 Thus, it follows that the depletion of the CD8+ cells would decrease the chance of developing GVHD,15 but there is an increased risk of graft failure.15-17

Gross Clinical Aspects of VBMT The majority of recipients (60-70%) of parental to F1 hybrid rat hindlimbs are tolerant. They thrive after allograft transplantation. Weight gain is at the usual rate and they remain in excellent health.18 The minority develops GVHD. Graft versus host disease is best described as a cachectic wasting syndrome of dermatitis, enteritis and hepatitis. The dermatitis takes the form of a macular erythematous rash that can involve any part of the body. In the rat hindlimb model, though, the most affected areas include the ears, nose and genitalia (Fig. 6.1). In the initial stages of the disease, there is an erythematous appearance to the skin. There is concomitant alopecia. In the later stages, the rash changes to a lichen planuslike rash that eventually becomes sclerotic. The enteritis is manifested clinically as profound diarrhea and weight loss with loss of appetite. In the rat hindlimb CTA/VBMT, average weight loss ranges from 25-40% of original weight at the time of transplantation.18 The hepatitis component is usually detected by abnormal bilirubin levels and/or the presence of jaundice, which is not readily apparent in the rat hind limb CTA. In human

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bone marrow transplantation, these parameters are easily determined and/or observed with serum liver enzyme tests and as bilirubin, etc. Yet in animal models, this component is typically not clinically relevant.

Acute and Chronic GVHD All animals that develop GVHD in the hindlimb CTA/VBMT model eventually die of the disease within an average of 163 days post-transplantation.18 A curious phenomenon occurs in the development of the disease. Two distinct groups, designated acute and chronic, develop. In general, the acute group shows a significantly greater weight loss over a shorter period of time compared to the chronic group.18 In addition, the onset of the precipitous weight loss occurs earlier in the course of the disease. Acute and chronic GVHD is not unique to the rat hind limb CTA/VBMT model. In fact, these two groups have been described in other animal models, as well as in human GVHD associated with bone marrow transplantation. It is generally accepted that acute and chronic GVHD represent two distinct disease/immunologic processes. The mechanisms of this dichotomy are beyond the scope of this chapter. Nevertheless, numerous studies in mouse models have shown that there are two separate pathways, at least from an immune response aspect, and that acute GVHD does not beget chronic GVHD. In human GVHD, acute and chronic GVHD are usually arbitrarily defined as a GVHD crisis occurring before 100 days for the former and after 100 days for the latter.19 In general, acute GVHD is regarded as an abrupt onset of dermatitis, enteritis and/or hepatitis that is immediately life threatening. Conversely, chronic GVHD is typically thought to be a more sclerosing process.

Histopathology Skin Graft versus host disease in the skin and epidermal surfaces is characterized by a lichenoid inflammation at the dermal-epidermal junction. The inflammatory infiltrate is composed almost exclusively of mature lymphocytes. This inflammatory infiltrate is most pronounced in the early stages of the disease. There is concomitant dyskeratosis, and individually necrotic keratinocytes are associated with this inflammation.18 There is usually inflammation and destruction of the adnexal structures. As the disease progresses, there is replacement of the inflammation by dense sclerosis at the dermal-epidermal junction.

Liver When considering graft versus host disease, there is typically a prominent infiltrate that appears almost identical to a viral hepatitis. That is, there is a prominent portal inflammatory infiltrate of mature lymphocytes. The inflammation can extend beyond the limiting plate (active hepatitis). The inflammation usually surrounds the vessels within the portal tracts and may show endothelialitis. But, the more diagnostic histopathologic feature is bile duct damage.18 The changes can range from mild ductulitis, which is composed of intraepithelial lymphocytes, to complete bile duct damage. The epithelial damage consists of irregularly shaped bile ducts with vacuolization of the nuclei and nuclear dropout.

Gastrointestinal Tract Unlike the liver and skin, lymphocytic inflammation is usually not a prominent feature. The sine qua non of GVHD in the GI tract is crypt cell apoptosis. Histologically, apoptosis is best characterized as a ballooned crypt cell containing a pyknotic, karyorrhectic nucleus.18 The apoptosis can range from single cells involving individual crypts

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to complete necrosis of the bowel wall with ulceration and granulation tissue. Fibrosis of the bowel wall may or may not be present.

Other Tissues The bone marrow of recipients of rat hind limb CTA/VBMT show several interesting findings. Typically, there is increased cellularity in those animals developing GVHD, compared to the tolerant animals in both the transplanted and contralateral limbs.20 By immunohistochemistry, in situ TGF-∃ expression is significantly increased in the CTA/VBMT chimeras who develop GVHD. This increased expression is present in both the transplanted and contralateral limbs. When considering the transplanted vs. contralateral limb, there is an increased level of TGF-∃ in the contralateral versus transplanted limb marrow in the animals that are tolerant. Although in situ TGF-∃ expression was present in the tolerant animals, the level of immunostaining is less than that in the GVHD animals.20,21 These findings suggest an important mechanism involving the immunomodulator TGF-∃. The differences in the GVHD and tolerant CTA chimeras, with regard to TGF-∃ expression, suggest an auto/alloimmune dysregulation that occurs in GVHD CTA/VBMT chimeras. The lower level of TGF-∃ expression in the tolerant versus GVHD animals may also support the maintenance of tolerance in these chimeras.

Conclusions F1 hybrid recipients of parental rat hindlimb composite tissue allograft (CTA), and therefore vascularized bone marrow transplants develop one of several outcomes. All of these CTA/VBMT develop donor-specific lymphoid chimerism. The level of this chimerism, to some extent, determines whether or not these animals develop tolerance or graft versus host disease (GVHD). Studies of this model have shown that stable low levels of mixed donor chimerism are associated with tolerance. Conversely, GVHD is linked to higher, unstable levels of donor chimerism. The pathology associated with CTA/VBMT-induced graft versus host disease is best described as a syndrome of dermatitis, hepatitis and enteritis. The CTA/VBMT recipients developed characteristic clinical and histopathological findings of GVHD. With respect to the immunomodulator TGF-∃, GVHD was associated with high levels of in situ expression in the bone marrow compared to the tolerant animals. Thus, TGF-∃ may represent a mechanism of tolerance and immune dysregulation associated with CTA/VBMT and GVHD.

References 1. Hewitt CW, Ramsamooj R, Patel M et al. Development of stable mixed T cell chimerism and transplantation tolerance without immune modulation in recipients of vascularized bone marrow allografts. Transplantation 1990; 50:766-772. 2. 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. 3. Hewitt CW, Black KS, Fraser LA et al. Composite tissue (limb) allografts in rats: I. Dose dependent increase in survival with cyclosporine. Transplantation 1985; 39:360. 4. Hewitt CW, Black KS, Dowdy DF et al. Composite tissue (limb) allografts in rats: III. Development of donor-host lymphoid chimeras in long-term survivors. Transplantation 1986; 41:39. 5. Hewitt CW, Black KS, Henson LE et al. Lymphocyte chimerism in a full allogeneic composite tissue (rat limb) allograft model prolonged with cyclosporine. Transplant Proc 1988; 20:272. 6. Henson LE, Hewitt CW, Black KS. Use of regression analysis and the complement-dependent cytotoxicity typing assay for predicting lymphoid chimerism. J Immunol Methods 1988; 114:139.

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7. Hewitt CW, Black KS, Ramsamooj R et al. Lymphoid chimerism and graft-versus-host disease (GVHD) in rat-limb composite tissue allograft recipients. FASEB J 1989; 3:5233. 8. Thomas ED, Buckner CD, Cliff RA et al. Marrow transplantation for acute non lymphoblastic leukemia in first remission period. N Engl J Med 1979; 301:597. 9. Santos GW. Bone marrow transplantation. Adv Inter Med 1979; 24:157. 10. Beschorner WE, Tutschka PJ, Santos GW. Chronic graft-versus-host-disease in the rat radiation chimera. Transplantation 1982; 33:393. 11. Llull R, Patel MP, Ramsamooj R et al. Mechanisms of alloimmune tolerance associated with mixed chimerism induced by vascularized bone marrow transplants. Submitted. 12. Rolink AG, Radaszkiewicz T, Melchers F. The autoantigen-binding B cell repertoire of normal and of chronically graft versus host disease mice. J Exp Med 1987; 165:1675-1687. 13. Luzuy S, Merino J, Engers H et al. Autoimmunity after induction of neonatal to alloantigens: Role of B cell chimerism and F1 donor B cell activation. J Immunol 1986; 146:4420-4426. 14. Wilson DB. Idiotypic regulation of T cells in graft-versus-host-disease and autoimmunity. Immunol Rev 1989; 107:159-177. 15. Korngold R, Sprent J. Variable capacity of L3T4+ T cells to cause lethal graft-versus-hostdisease across minor histocompatibility barriers in mice. J Exp Med 1987; 165:1552-1564. 16. Hamilton BL. L3T4-positive T cells participate in the induction of graft-versus-host-disease in response to minor histocompatibility antigens. J Immunol 1987; 139:2511-2515. 17. Truitt Rl, Atasoylu AA. Contribution of CD4+ and CD8+ T cells to graft-versus-host-disease and graft-versus-host leukemia reactivity after transplantation of MHC-compatible bone marrow. Bone Marrow Transplant 1991; 8:51-58. 18. Ramsamooj R, Llull R, Black KS et al. Composite tissue allografts in rats: IV. Pathological manifestations of graft-versus-host-disease (GVHD) in recipients of vascularized bone marrow allografts. Submitted. 19. Wick MR, Moore SB, Gastineau DA et al. Immunologic, clinical and pathologic aspects of human graft-versus-host-disease. Mayo Clin Proc 1983; 58:603. 20. Hewitt CW, Englese MJ, Tatem LD et al. Graft-versus-host-disease in extremity transplantation: Digital image analysis of bone marrow in situ. Ann Plastic Surg 1995; 35:108-112. 21. Ramsamooj R, Llull R, Tatem LD et al. Graft versus host disease in extremity transplantation: Digital image analysis of bone marrow and TGF-∃ expression in situ using a novel 3-D microscope. Transplant Proc 1996; 28:2029-2031.

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New Models of VBMT Based on CTA

Section IV New Composite Tissue Transplant Models

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CHAPTER 7

New Models of Vascularized Bone Marrow Transplantation Based on Composite Tissue Allografts Martha S. Matthews and Charles W. Hewitt

Introduction

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omposite tissue allografts containing bone with significant amounts of marrow have been shown to maintain functioning donor marrow, as evidenced by the development of a chimeric state and by GVHD.1,2 This fact leads to interesting speculation in the area of bone marrow transplantation. Although a moderately successful modality, conventional bone marrow transplantation is fraught with difficulties in engraftment, infectious complications, and GVHD. Transplantation of viable bone marrow, within its protective milieu and retaining its normal architecture, may provide some advantages.3 Development of long term chimerism might also prove advantageous for inducing immune tolerance in conjunction with organ transplantation.

Background Conventional Bone Marrow Transplantation The early history of bone marrow transplantation is linked to the study of the effects of radiation on the hematopoietic system. As early as 1922, Fabricious-Moeller noted that shielding of a hindlimb in guinea pigs that received total body irradiation (TBI) protected them from depression of platelet counts and bleeding.4 The opening of the atomic age in the mid-40s stimulated government interest and funding of radiation effects and their treatment. The 1950s saw elucidation of the ability of the body to repopulate the marrow from protected spleen or bone marrow sites, or from donors.5-8 The term “chimera” was introduced, first as a manifestation of the postirradiated, transplanted state, and then expanded to include those from other causes.9 In the following decade, GVHD was discovered and studied.10 Clinical application of bone marrow transplantation in man began with end stage acute leukemia. Throughout the late 1960s and the 1970s, preparatory regimens were refined, and the clinical courses of successful and unsuccessful engraftment were studied. The natural history of GVHD was charted. As success with transplantation increased, there developed a willingness to treat patients who were not in end stage disease. This also improved results. Composite Tissue Transplantation, edited by Charles W. Hewitt and Kirby S. Black. ©1999 R.G. Landes Company.

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Allogeneic bone marrow transplantation is now an accepted modality of treatment under certain circumstances for various hematologic malignancies such as ALL and CML, hematologic disorders such as aplastic anemia, immunodeficiencies such as severe combined immunodeficiency, and metabolic bone disorders such as Gaucher’s disease. The goal of the transplant procedure is to transfer pluripotent hematopoietic progenitor cells from donor marrow to recipient marrow. Bone marrow is harvested from the iliac crests of the donor, anticoagulated, and centrifuged to remove erythrocytes, mature WBCs, and plasma. The remaining cell mass may be treated in various ways to remove or disable functioning T cells. The recipient is prepared, or conditioned, to receive the transplant. The transplant material is infused into the recipient vascular system, where the donor cells proceed to find their “homes” in recipient marrow. Satisfactory engraftment is indicated by hematopoietic recovery within 21 days.11 Numerous obstacles lie in the path of a successful transplant. The method of preparation is imprecise, as there is no direct test of the viability of the processed cells. Engraftment requires the transfused cells to find a niche in the donor marrow space in which to settle, reproduce, and differentiate. The cells must find the donor marrow space, and then have room in the space. The host must remain immunologically unresponsive to the graft, yet retain the ability to fight infection. The graft itself must also stay immunologically unresponsive to the host. Recipient conditioning is an important aspect of successful transplant as well as a cause of considerable morbidity. Although methods vary, the end desired result is ablation of the recipient’s cellular marrow. This accomplishes three goals: the ablation of malignant cells, if present; the destruction of the recipient immune system, so that the donor graft will be accepted; and production of “space” for the new cells. As a consequence, the recipient experiences anemia, thrombocytopenia, neutropenia and loss of immune function, with their resultant ill effects. During the period between marrow ablation and graft reconstitution, infectious complications are common and severe. Transfusions of both red cells and platelets are required.12 As a requirement of successful engraftment, pluripotential progenitor cells must migrate to the marrow space and physically take up residence there. This process, known as homing, is complex and not well defined. It appears that the local milieu of the marrow space is essential both to successful engraftment and to the eventual ability of the pluripotential stem cells to replenish themselves as well as differentiate into dedicated cell lines.13 After establishment of the donor cells, GVHD becomes an issue. Donor lymphoid stem cells repopulate the peripheral blood. New T cells are initially naive and must be “schooled” to recognize the host as self.13 GVHD represents a failure of this schooling. Acute GVHD is reported to occur in 30-70% of bone marrow transplant patients, and chronic GVHD in 15-40% of patients. GVHD is more common with greater degrees of HLA mismatch, and with increasing age. T cell depletion of the graft is possible and leads to a diminished incidence of GVHD; however, the incidence of graft failure is increased.11

Vascularized Bone Marrow Transplant—A New Horizon? Transplantation of vascularized bone marrow could offer advantages over conventional bone marrow transplant. Immediate engraftment would occur, with the donor stem cells remaining within their original microenvironment. This has been demonstrated in the whole limb transplant model, with more than 50% of stem cells engrafted.15 Because the cells would come complete with their supporting microenvironment, the need for marrow “space” would be obviated. Indeed, the quality and quantity of recipient marrow space would be inconsequential. In some cases, recipient marrow ablation might be avoided. Hematopoietic recovery might be accelerated by the transfer of already functioning bone marrow.

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It has been shown in rats that vascularized bone marrow transplant within a whole limb model produces stable mixed T cell chimerism, both in a semiallogeneic and a fully allogeneic model.16,17 Sixty to 70% of animals develop stable chimerism without GVHD. It is speculated that this macrochimerism might lead to lower levels of GVHD and induction of tolerance. This might have advantages in conjunction with organ transplantation as well.18,19 Whole hindlimb transplants have been the mainstay of studies in this area. The hindlimb model in rats has been used for many years, mostly in the investigation of the potential for limb transplantation. Only incidentally was the potential for the limb as a carrier for bone marrow recognized. The whole limb model has some potential difficulties when used primarily for bone marrow transplantation studies. Skin, muscle, nerve, bone and mature lymph nodes are also transferred with the bone marrow. It is well known that the immunogenicity of these tissues varies, with skin being highly antigenic, and bone being least antigenic.20 Long term survival of fully allogeneic limb transplants, even in established chimeras, seems to depend on continuing immunosuppression.15 It is unknown if this would be the case in a bone plus marrow only transplant. The transplantation of mature lymph nodes might also affect the immunologic responses of the host. Clearly, a vascularized bone marrow transplant model, consisting of marrow-containing bone with a minimum of other tissue, would be of great interest.

Models of Vascularized Bone Transfer There has been considerable work in the area of vascularized bone transfer for replacement of skeletal elements lost to trauma or tumor. One well established model is the canine knee joint graft.21-23 The vascularized knee joint model in the rabbit has also been described.24 Rat knee joint transplants and intercalary bone transplants in dogs have been reported.25 These bone plus marrow only models have not had the immunologic scrutiny that has occurred with whole limb transplants, so it is not known if they produce the same levels of chimerism. Evaluation of radiographs of many of these models shows extensive fixation hardware both within the marrow cavity and through the cavity, as well as relatively small amounts of marrow-containing bone being transferred.24-26 As used, they would not be ideal models for vascularized bone marrow transplant. The search for an animal model for bone marrow transplant begins with examination of potential bone donor sites in man. Autologous vascularized bone transfer is a clinical reality, with multiple available donor sites including the rib, scapula, fibula, radius, and iliac crest.27 The iliac crest is an appealing donor site for vascularized bone marrow, as it is already used for conventional bone marrow transplantation. Although certainly not as trouble free as a conventional bone marrow donation, a large piece of vascularized iliac crest could be taken from a living donor without long term deformity or disability. Even larger pieces could be taken from cadaver donors, where cosmesis and hernia are not concerns.

Laboratory Investigations Work was begun in our laboratory to identify potential vascularized bone marrow transplant models in the rat. Attention was directed to bones having a large marrow space and large feeding vessels. Numerous dissections revealed the pelvic bones to be suitable. Dissection revealed that the rat pelvis receives periosteal branches to the iliac crest from the iliolumbar arteries. The major blood supply to the remainder of the pelvis, however, comes via numerous periosteal branches from the external iliac artery. Additional periosteal branches also come from the obturator artery and the hypogastric system. Various bone flaps were dissected. The iliac crest was isolated on the iliolumbar vessels. This proved to be a difficult dissection with a tenuous blood supply. A hemipelvis flap was

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designed with both the iliolumbar and external iliac periosteal supplies, both transferred on the aorta, vena cava, and ipsilateral external iliac artery and vein. This was even more difficult, and often led to the intraoperative death of the donor prior to completion of flap harvest. Finally, a model based only on the iliac system was developed. This model transfers the entire ischium, with the exception of the tuberosity, based on perfusion from periosteal branches of the external iliac and hypogastric arteries. The bone is transected through the pubic ramus anteriorly, the obturator foramen inferiorly and the sciatic notch posteriorly. This encompasses a large marrow cavity. The nonessential branches of the hypogastric artery are tied off, and the flap can be transferred on the common iliac vessels. The femoral vessels can be used for a flow through flap. Dye injection studies show perfusion of the marrow space. Successful isogeneic transfers have been accomplished short term in four animals, indicating that this has potential application as a purer model for vascularized bone marrow transplants. Our laboratory plans studies of engraftment and development of chimerism, tolerance, and GVHD, with comparisons to whole limb transplant models. This model has several drawbacks. The dissection is difficult and tedious, has significant blood loss, and is undoubtedly metabolically stressful for the donor. We have tried to maintain viability of the donor until the time of transfer in order to minimize ischemia time. Although bone is relatively resistant to ischemia, it is not known what effect this has on the marrow. The blood flow through the flap is slow. With systemic dye injection in a living donor, it takes from five to ten minutes for visible staining of the marrow. This leads to technical difficulty with vessel patency, and has led us to explore a flow through model. As the isolated vascularized bone marrow transplant model is defined, there are many interesting areas of speculation. How does the transplantation of highly antigenic tissue (skin), affect the transplantation of bone marrow? Will the need for long term immunosuppression in tolerant bone marrow transplant recipients be different with composite tissue transplants as opposed to bone only transplants? Will vascularized bone marrow transplant be a viable alternative to those who fail conventional bone marrow transplant? Could vascular bone marrow transplant serve as an adjunct to conventional cellular marrow transplantation to facilitate engraftment and tolerance induction, or serve as a “fail safe” backup system to ensure engraftment?

Conclusions Conventional bone marrow transplant, while effective in most cases, has a significant failure rate of engraftment. There are also known problems related to the logistics of the procedure, including hematopoietic disarrangements and infectious complications. The immediate engraftment that could be provided by a vascularized transfer of bone marrow and its supporting milieu might prove to be advantageous. Vascularized bone marrow transplant, with its attendant production of a tolerant state, might also be helpful in organ transplantation. New models of vascularized bone marrow transplant that do not carry the excess baggage of nonessential tissues need to be studied with respect to these issues. The vascularized pelvis in the rat is one such potential model.

References 1. Hewitt CW, Black KS, Fraser LA et al. Composite tissue (limb) allografts in rats II. Indefinite survival using low-dose cyclosporine. Transplantation 1985; 39(4):365-368. 2. Hewitt CW, Black KS, Fraser LA et al. Composite tissue (limb) allografts in rats III. Development of donor-host lymphoid chimeras in long-term survivors. Transplantation 1986; 41(1):39-43. 3. Lukomska B, Durlik M, Cybulska E et al. Comparative analysis of immunological reconstitution induced by vascularized bone marrow versus bone marrow cell transplantation. Transplant Int 1996; 9(1):492-496.

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4. Santos GW. Historical background. In: Atikinson K, ed. Clinical Bone Marrow Transplantation. Cambridge: Cambridge University Press, 1994:1-9. 5. Jacobson LO, Simmins EL, Marks EK et al. Recovery from radiation injury. Science 1951; 113:510-511. 6. Lorenz E, Uphoff DE, Reid TR et al. Modification of acute irradiation injury in mice and guinea pigs by bone marrow injection. Radiology 1951; 58:863-877. 7. Lindsley DL, Odell TT, Tausche FG. Implantation of functional erythropoietic elements following total-body irradiation. Proc Soc Exp Biol Med 1955; 90:512-515. 8. Mitchison NA. The colonization of irradiated tissue by transplanted spleen cells. Br J Exp Pathol 1956; 37:239-247. 9. Ford CE, Hamerton JL, Barnes DWH et al. Cytological identification of radiation chimeras. Nature 1956; 177:239-247. 10. Billingham RE. The biology of graft-versus-host reactions. Harvey Lect 1966-1967; 62:21-78. 11. Meagher RC, Herzig RH. Techniques of harvesting and cryopreservation of stem cells. Hematol Oncol Clinic North Am 1993; 7(3):501-506. 12. Atkinson K. Hemopoietic reconstruction posttransplant. In: Atkinson KM ed. Clinical Bone Marrow Transplantation. Cambridge: Cambridge University Press, 1994:31-41. 13. Clark BR, Dexter TM. Bone marrow homing and early engraftment. In: Atkinson KM ed, Clinical Bone Marrow Transplantation. Cambridge: Cambridge University Press, 1994; 19-25. 14. Ross DW. The immune system following bone marrow transplantation. Arch Pathol Lab Med 1996; 120:885-886. 15. Hewitt CW, Black KS, Henson LE et al. Lymphocytic chimerism in a full allogeneic composite tissue (rat-limb) allograft model prolonged with cyclosporine. Transplant Proc 1988; 20:272. 16. Hewitt CW, Ramsamooj R, Patel MP et al. Development of stable mixed T cell chimerism and transplantation tolerance without immune modulation in recipients of vascularized bone marrow allografts. Transplantation 1990; 50(5):766-772. 17. Yazdi B, Patel MP, Ramsamooj R et al. Vascularized bone marrow transplantation (VBMT): Induction of stable mixed T cell chimerism and transplantation tolerance in unmodified recipients. Transplant Proc 1991; 23(1):739-740. 18. Llull R, Ramsamooj R, Black KS et al. Cellular mechanisms of alloimmune non-responsiveness in tolerant mixed lymphocyte chimeras induced by vascularized bone marrow transplants. Transpl Int 1994; 7 (suppl 1):S453-S456. 19. Llull R, Murase Q, Ye R et al. Vascularized bone marrow transplantation in rats: Evidence for amplification of hematolymphoid chimerism and freedom from graft-versus-host reaction. Transplant Proc 1995; 27(1):164-165. 20. Llull R, Murase N, Demetris Q et al. Multilineage amplification of graft-vs-host diseaseresistant chimerism following rat vascularized bone marrow allotransplantation. Transplant Proc 1995; 27(4):2363-2364. 21. Buttemeyer 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(1):139-148. 22. Slome D, Reeves B. Experimental homotransplantation of the knee joint. Lancet 1966; 2:205. 23. Reeves B. Studies of vascularized homotransplants of the knee joint. J Bone Joint Surg 1968; 50B:226. 24. Porter BB, Lance EM. Limb and joint transplantation. Clin Orthop 1974; 104:249-274. 25. Yaremchuk MJ, Sedacca T, Schiller AL et al. Vascular knee allograft transplantation in a rabbit model. Plast Reconstr Surg 1983; 71(4):461-471. 26. Doi K, Akino T, Shigetomi M et al. Vascularized bone allografts: Review of current concepts. Microsurgery 1994; 15(12): 831-841. 27. O’Brian, BM, Morrison WA. Vascularized bone grafts. In: O’Brian BM, Morrison WA, eds. Reconstructive Microsurgery. New York: Churchill Livinston, 1987:315-326.

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Composite Tissue Transplantation in Rats: A Whole Limb/Hemipelvis Model

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Composite Tissue Transplants in Rats: A Whole Limb/Hemipelvis Model Kirby S. Black and Charles W. Hewitt

Background

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t has been observed that donor-specific skin or macrophage pretreatment can actually have an adverse effect upon limb allograft survival,1 while donor-specific blood transfusions have no beneficial effect.2 Thus the composite tissue allograft (CTA) does not always respond to immunological manipulation in the same way that other organ transplants have been shown to. CTAs demonstrate a CsA dose-dependent increase in survival with short term administration,3 as well as indefinite survival with long term administration.4 Histological examination of long term surviving CTAs have revealed normal appearing bone marrow,5 and lymphoid chimerism has been demonstrated utilizing mononuclear cells isolated from both host spleen and/or blood of various long term survivors.5-8 It was established that all of the long term CTA recipients tested possessed lymphoid chimerism of both donor and host origin. Some important questions remain in consideration of future possible clinical CTA applications. What amount of allogeneic tissue can be tolerated by the recipient? If very large defects are to be treated, the resulting allografted tissue, by way of massive antigen and marrow infusion, might be detrimental to the host through a graft versus host response. The model presented herein includes a large amount of bone marrow in the pelvis, which conceivably could illicit such a response. The possibility of antigen overload from a large transplant must be investigated, since the best functional possibilities in massive tissue defects would include large portions of muscle, skin, bone, connective tissue, bone marrow and epithelium. Many of these tissues have been cited as being extremely antigenic.

CTA Functionality An issue paramount to the eventual usefulness of CTAs is the functional capabilities of these tissues. Previously, we took the approach of utilizing an isolated, vascularized neuromuscular allograft model (Fig. 8.1).9-11 This model worked well in a system where only a few muscles needed to be transplanted. However, in a situation where a complex set of muscles, bone, tendons, vessels, and nerves must be transplanted, a more complex experimental model is required. The model presented in this chapter was designed for this purpose. In the limb/hemipelvis allograft model, all insertions and origins, as well as the nerve supply, are kept intact. The femur, tibia, and pelvic region are transplanted, as well as

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Fig. 8.1

Medical illustration of the isolated muscle allograft model typically used in refs. 9-11.

the ankle joint. In this case, immediate revascularization of the bone and cartilage is an important advantage over studies where a lack of vascularization has led to a collapse of articular surface and bone. In addition, the model was designed such that the leg is semifunctional immediately postoperation. This model can be utilized to examine the intricacies of functional return in a complex system, as well as the immunological impact that a large group of tissues might present.

Surgical Model Donor Limb Preparation A circumferential incision is made slightly above the thigh in the donor, and the knee exposed. The skin is cleared of the underlying muscles until the attachment to the spine is visible. An incision running parallel to the spine is made through the superficial muscle groups, m. biceps femoris and m. gluteus superficial, until the femur is visible. Various muscle groups are then separated from mid-thigh to the knee. This exposes the length of the sciatic nerve and the tendon attached to the femur. This tendon is then bisected. The membranous muscle, m. gluteus superficial, is then separated from the underlying quadricep and gluteal muscles. The insertion of the gluteal muscles is dissected off the greater trochanter. The

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ilium is scraped with an osteotome. The ventral fat pad is then cleared and the epigastric vessels cauterized. Next, the m. adductor et brevis and m. gracilis are separated to expose the psoas tendon which wraps around the lesser trochanter. The lesser trochanter is exposed by incising the psoas and clearing with an osteotome. Two holes are drilled parallel to the long axis of the femur using a 21 gauge needle in the area of the psoas. On the dorsal side, the sciatic nerve is bisected just proximal to the greater trochanter after using xylocaine without epinephrine as a local anesthetic. The sciatic nerve is reflected and a metal plate slid under the ileum to protect the underlying tissue. One hole is drilled in the ileum close to the distal end of the exposed area. An osteotomy is performed on the ileum just proximal to the hole using a hobby circular saw blade. The greater trochanter is scraped with a scalpel until a flat edge is exposed. The fat near the pelvis is then cleared using blunt dissection. The edge of the pelvis is isolated and cut medially up to the pubic symphysis. This area is highly vascularized, so care must be taken when dissecting. The pubic symphysis is then cleared. The femoral vessels and nerve are isolated and branches cauterized. The donor is set aside.

Recipient Preparation A circumferential incision is made just below the knee of the recipient. Skin is retracted until the knee is exposed. Medially, both the ventral and the dorsal fat pads are cleared and epigastric vessels cauterized. The femoral vessels are isolated and the branches cauterized. The femoral nerve is tagged with 6-0 silk suture and cut distal to the tag after local anesthesia with xylocaine without epinephrine. Femoral vessels, artery first, are ligated twice near the epigastric branch and cut between ligations. The dorsal skin is retracted until the spine is exposed. The superficial muscles (m. biceps femoris and m. gluteus superficial) are cut parallel to the spine to the junction of the gluteus superficial, leaving some muscle attached to the spine for later attachment to the donor muscles. An incision is made along the facial plane of the gluteus superficial to the knee, exposing the underlying sciatic nerve and the tendon attached to the femur. This tendon is cut close to the femur. The membranous muscle is then separated from the underlying quadricep and gluteal muscles. A trigonal flap is made by cutting the membranous muscle at the knee. Once the quadriceps and gluteals are well exposed, the greater trochanter is removed using a scalpel at a 45° angle to the plane of the femur, leaving a small piece attached to the gluteal. The cut is parallel to the insertion of the gluteal muscles on the greater trochanter. Next, the quadricep muscles are excised and discarded. The ileum, lying beneath the gluteal muscles, is then cleared with an osteotome. The sciatic nerve is locally anesthetized with xylocaine and cut proximally to the point where it wraps around the greater trochanter. A metal protection plate is slid underneath the ileum and one hole drilled toward the proximal end of the exposed area. An osteotomy is done on the ileum distal to the drilled hole. The tibia is then separated from the femur using a scalpel. The femur is likewise disarticulated. The femur is then cleared of everything except the psoas tendon and muscle. Next, fat is cleared off the back of the pelvis using blunt dissection. Muscles are then cut off the pelvis to the pubic symphysis. The pubic symphysis is gently cleared off with the osteotome and cut, leaving most with the recipient. The leg is removed by carefully cutting through the remaining muscle and connective tissue. The portion of the femur not involved in the psoas insertion is removed (by burring), thus leaving a half cylinder of bone attached to the psoas. The femur is then cut to include only the psoas attachment. Two 21 gauge holes initiated from the top of femur (psoas side), angled so as not to hit the psoas, are drilled in the femur. Three-0 proline on a double straight needle is fed through the holes in the femur from the outer to the inner edge. Both needles of a double armed 3-0 proline suture are then carefully placed through the cartilaginous portion of the pubic symphysis ventral side to dorsal side.

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Transplantation Xylocaine without epinephrine is applied locally to the femoral nerve in the donor. The nerve is tagged with 6-0 silk just distal to the inguinal ligament and cut just distal to the tag. Femoral vessels, artery first, are ligated twice just distal to the profundus branches and cut between ligations. The underside of pelvis at the pubic symphysis is gently cleared off. The pubic symphysis is cut, taking most with the leg. The back of the pelvis is cleared off and the leg removed as described for the recipient above. Three-0 proline suture is threaded through holes in donor and recipient ileums and a 21 gauge needle is inserted for use as an intermedullary rod. Three-0 proline already threaded through the recipient pubic symphysis is likewise threaded through donor pubic symphysis. Ends of the ileum are then pushed together and the suture tied. The suture in the pubic symphysis is tied also. Anastomoses of femoral vessels are done with 10-0 nylon microsuture on a 70 m needle (Ethicon, Somerville, NJ) using microsurgical techniques. Ischemic times are an average of one hour. Femoral and sciatic nerves are then repaired with 10-0 nylon microsuture. Three-0 proline already threaded through the recipient at mid-femur is fed through two holes in the donor lesser trochanter, and the bones approximated and secured. Two 27 gauge holes are then drilled by hand in both the recipient and donor greater trochanters, leaving the needles in following drilling. Four-0 silk suture is threaded through the needles and the recipient and donor greater trochanters are sutured together in this manner. Four-0 vicryl on a taper needle is used to close muscle on the lateral aspect with a horizontal mattress stitch. Three-0 vicryl on a cutting needle is used to close the skin.

Conclusion In this paper we have detailed a massive composite tissue allograft model. We have achieved long term survival in these animals without detrimental effects due to the massive tissue allograft. The model presented in this study should be ideal for studying functional properties of composite tissue transplants during chronic rejection. A significant mortality rate is related to the complexity of the surgery and its postoperative effects on the animals, rather than to immune phenomena.

Acknowledgment This work was supported in part by the National Institutes of Health (GM 31974 and AM 32263), International Association of Fire Fighters Burn Foundation, the Orthopedic Research and Education Foundation, the Plastic Surgery Educational Foundation, the Foundation of UMDNJ, and faculty practice grants from Robert Wood Johnson Medical School/Cooper Hospital/University Medical Center.

References 1. Grisham GR, Black KS, Hewitt CW et al. Prior administration of donor-strain epidermal cells or macrophages to enhance survival of rat hind limb allografts. Transplantation 1987; 44:572-574. 2. Black KS, Hewitt CW, Woodard TL et al. Efforts to enhance rat limb allografts by prior administration of blood using a new survival end-point. J Microsurg 1982; 3:162-167. 3. Hewitt CW, Black KS, Fraser LA et al. Composite tissue (limb) allografts in rats: I. Cyclosporine prolongs survival in a dose-dependent manner. Transplantation 1985; 39:360-364. 4. 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. 5. Hewitt CW, Black KS, Dowdy SF et al. Composite tissue (limb) allografts in rats: III. Development of donor-host lymphoid chimeras in long-term survivors. Transplantation 1986; 41:39-43.

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6. Hewitt CW, Ramsamooj R, Patel M et al. Development of stable mixed T cell chimerism and transplantation tolerance without immune modulation in recipients of vascularized bone marrow allografts. Transplantation 1990; 50:766-772. 7. Hewitt CW, Black KS, Henson LE et al. Lymphocyte chimerism in a full allogeneic composite tissue (rat limb) allograft model prolonged with cyclosporine. Transplant Proc 1988; 20:272. 8. Llull R, Ramsamooj R, Black KS et al. Cellular mechanisms of alloimmune non-responsiveness in tolerant mixed lymphocyte chimeras induced by vascularized bone marrow transplants. Transpl Int 1994; 7(1):S453-S456. 9. Black KS, Hewitt CW, Grisham GR et al. Two new composite tissue allograft models in rats to study neuromuscular functional return. Transplant Proc 1987; 19:1118-1119 10. Black KS, Hewitt CW, Caiozzo VJ et al. Neuromuscular capabilities in long term composite tissue allografts. Transplant Proc 1988; 20(2):269-271. 11. Caiozzo VJ, Black KS, Hewitt CW et al. Histochemical properties of muscle allografts enhanced via cyclosporine. Transplantation 1989; 48:840-844.

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Transplantation of the Peripheral Nerve Allograft

Section V Individual Component Tissues of the Composite Tissue Transplant

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Transplantation of the Peripheral Nerve Allograft Vaishali B. Doolabh and Susan E. Mackinnon

Introduction

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he restoration of meaningful nerve function and sensation following peripheral nerve injury continues to pose a considerable challenge to the reconstructive surgeon. The impact of human peripheral nerve injuries secondary to extensive trauma, burns, or neoplastic processes is defined by the devastating morbidity of a nonfunctional limb. Surgical techniques of bridging the peripheral nerve gap, including direct apposition and conduit entubulation of nerve stumps, frequently yield satisfactory clinical results. The merits of nerve grafting have established it as a viable, therapeutic option to reconstruct long gap lengths. Early attempts at nerve grafting utilized autografts, allografts and xenografts to bridge the peripheral nerve gap (for review, see ref. 1). Historically, the wide spectrum of results from all three methods made interpretation difficult and placed routine clinical applications in disrepute until recently. Of the notable advances in the 19th century, Philipeaux and Vulpian are credited with the first descriptions of experimental nerve autografting.2 In 1878, Albert described the first clinical use of a nerve allograft to repair a 3 cm gap in the median nerve of his patient; however, the clinical outcome of this repair was never reported.3 Shortly thereafter, the first successful clinical nerve allograft report was made by Mayo-Robson who repaired a 2.5 cm median nerve defect.4 Within the last two decades, Millesi has brought credibility to autografting techniques by emphasizing the importance of using microsurgical instruments, sutures and magnification, and of completing tension free repairs in order to recover eventual functional outcome.5,6 Currently, grafts of small caliber, expendable, autologous sensory nerve, such as the sural nerve, lateral antebrachial cutaneous nerve and anterior division of the medial antebrachial cutaneous nerve, are the standard methods of repair of significant defects. However, the management of multiple, complex, long gaps is restricted by the limited supply and sizes of these nerves. Nerve allograft reconstruction under immunosuppression is a recent experimental maneuver that offers a limitless supply of nerve material and obviates donor site sensory loss and unsightly scarring associated with autologous nerve harvest. The peripheral nerve allograft differs significantly from solid organ transplants in that it functions primarily as a temporary scaffold. Host axons regenerate through the allograft to reinnervate host sensory and motor targets. Successful nerve regeneration through transplanted allografts depends upon the establishment of long term, donor-specific tolerance to the graft until regeneration is complete. With the advent of microneurosurgical techniques

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coupled with an evolving understanding of the immunology of transplantation, we believe nerve allografts now have an appropriate clinical role in the repair of extensive injuries that supersedes current autogenous nerve repair methods. This review briefly addresses the early experimental and clinical endeavors to understand the nerve allograft response and allograft replacement under immunosuppression, the proven methods of allograft preservation and storage, and select nonspecific immunosuppressive regimens. Next, recent developments in establishing donor-specific immunotolerance are presented. We conclude with a report of the few clinical cases in which these advances have been applied.

Transplantation Immunology Recent progress in nerve allografting has been made through an improved understanding of the general principles of transplantation immunology and naturally operative mechanisms of antigen specific nonreactivity, or tolerance. It is well known that the host’s effector response to a new allograft depends upon T lymphocyte recognition of donor peptide processed by host antigen presenting cells (APCs) and presented in the context of host Class I or Class II major histocompatibility (MHC) loci. T cell activation results when the MHC-antigen peptide complex binds the T cell receptor (TCR), and CD3 transmembrane proteins transduce signals to the cytoplasm of the T cell which lead to a rise in intracellular calcium and protein kinase activation. The TCR imparts specificity to the response. Full responsiveness and clonal expansion requires an accessory cell-delivered costimulatory signal in addition to receptor engagement (Fig. 9.1). TCR binding to the MHC-peptide complex is augmented by coreceptor crosslinking, such as the binding of CD4 or CD8 molecules on the T cell to Class II or Class I MHC molecules on APCs, respectively. Delivery of costimulatory signals is facilitated by cell surface molecules and their ligands (e.g., intercellular adhesion molecule-1 (ICAM-1) lymphocyte function antigen-1(LFA-1), CD28/B7-1/ 2) that secure T cell-APC interaction.7,8 The costimulatory molecule B7-1 and B7-2 on APCs is inducible, such that levels of expression correlate quantitatively with the costimulatory potency of the APC.9 Subsets of T cells are operationally defined by the profile of cytokines they produce in response to activation. T helper 1 (Th1) cells secrete lymphokines such as interleukin 2 (IL-2) and interferon gamma (IFN-!) that play a central role in the maturation of precursor cells to functional effector cells, in the migration of immunocompetent cells and in controlling levels of expression of Class I and II MHC molecules in the allograft and on other cell types.10,11 T helper 2 (Th2) cells have been associated with the production of interleukins 4, 5 and 6 (IL-4, IL-5, IL-6). At the time of antigen exposure, the profile of secreted cytokines regulates subsequent T cell activities. Hence, a rapid, tailored (i.e., antigen-specific) T cell proliferative response to foreign antigen relies upon appropriate antigen presentation, multiple auxiliary cues from coreceptor engagement and the appropriate cytokine milieu. Antigen-specific immunologic unresponsiveness may be achieved by T cell clonal deletion at the site of lymphocyte development and/or T cell suppression from “external” or “internal” suppressors that results in quantitative or qualitative changes in antigen presentation.12 These mechanisms may not be mutually exclusive in action. Central clonal deletion is illustrated by the deletion of self-reactive T cell clones within the thymus during embryonic development to provide tolerance to self antigen. Mechanisms of peripheral deletion of T cell clones also exist. The existence of “external” suppressors that recognize either the responsive clone or the target antigen itself, with subsequent downregulation of other lymphocytes, has been postulated.9 Preferential cytokine production may alter the preponderance of active T cell subsets (i.e., Th1 vs. Th2 ). Clonal anergy refers to “internal” changes within the mature T lymphocyte that prohibit TCR engagement from leading to

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Fig. 9.1. T cell activation requires both receptor engagement and the delivery of a second costimulatory signal from the antigen presenting cell (APC). Adhesion molecules initiate a nonspecific interaction. As the dialogue between additional T cell receptors and their respective ligands on the APC occurs, either activation or anergy results.

activation of the immune response. Unlike clonal deletion, both external suppression and anergy allow antigen-specific T lymphocytes to remain in the mature repertoire. Faulty antigen binding to the TCR complex is central to the induction of tolerance. Blockage of adhesion molecules or other coreceptors and their ligands will interfere with signal transduction to the cytoplasm of the T cell and/or the APC, thereby influencing activation, cell-cell proximity and overall immune surveillance. Anergy may result from ineffective antigen presentation secondary to binding the TCR with an appropriate ligand in the absence of secondary costimulatory signals, as well as the provision of the appropriate signals with subsequent inhibition of cell division. The T cell will fail to respond to antigen and “disengage” internal mechanisms, such that future receptor stimulation with appropriate costimulation also fails. Clonal anergy is characterized by a profound inability of the T cell to produce IL-2.13 Under normal conditions, TCR engagement and secondary auxiliary signals together induce the expression of nucleoregulatory proteins which bind to an enhancer of the IL-2 gene, thereby augmenting transcription and message stabilization.14 The downregulation of IL-2 production will lead to the induction of immunologic tolerance. Anergy may be reversed or prevented by the exogenous addition of IL-2 during TCR activation.13 This supports the idea that anergy is maintained by a defect in TCR-mediated IL-2 mRNA accumulation, not TCR, CD4 or IL-2 receptor modulation.9 Interestingly, in several experimental models, clonal anergy has been reversed in vivo once the specific antigen is removed.15,16 As discussed later, understanding this phenomenon is paramount in the nerve allograft model where donor Schwann cells (SCs) are replaced over time by recipient SCs. Transplantation provides the unique situation in which for a period of time there are two types of APCs present, donor APCs and host APCs. This realization has given rise to much speculation as to the role of “passenger leukocytes” or dendritic cells (DC) within transplanted grafts. Indeed, donor derived murine DCs have been found in the bone marrow

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of surviving murine liver allograft recipients, raising the question of whether their presence influences the induction and maintenance of tolerance.17 In related studies of murine liver transplantation, immature DCs that lack Class II MHC and/or B7 expression, but are avidly phagocytic, were shown to migrate to and reside in secondary lymphoid tissues (e.g., spleen, peripheral lymph nodes). In the face of allograft acceptance, their persistence suggests the establishment of tolerogenic progenitors of chimeric cells within the recipient.18 Allografts may then induce and maintain tolerance simply by their survival. Similar low level chimerism associated with allograft survival has been noted under a short course of systemic FK506 immunosuppression in a rat liver transplant model.19 Yu has postulated the existence of cells within peripheral nerve from the monocyte/dendritic cell family that can be stimulated to express MHC molecules.20 Hence, microchimerism constitutes another possible mechanism of tolerance to peripheral nerve allografts.

The Nerve Allograft Response The goal of nerve allografting is to direct regenerating host axons through a donor conduit so that they may successfully establish contact with distal host effectors. Understanding the structure of the allograft is essential to meeting this goal. In the uninjured setting, unmyelinated nerve fibers are composed of several axons wrapped by a single SC, and the axons of myelinated nerve fibers are enveloped individually by a single SC. The membrane of the SC wraps circumferentially around the axon to form a multilaminated myelin sheath. Individual myelinated nerve fibers and groups of unmyelinated fibers are surrounded by the double membrane of the basal lamella of the SC. This double membrane serves as the conduit through which proximal regenerating fibers elongate. Following nerve injury, Wallerian degeneration occurs distal to the level of the nerve injury. This is characterized by degeneration of both the axon and myelin, and a proliferation of columns of SCs. In the proximal nerve, traumatic degeneration occurs in relation to the extent and type of injury, increasing with avulsion injuries and the proximity of the injury to the cell body. This traumatic degeneration is histologically similar to Wallerian degeneration. Without immunosuppressive protection, surveys of the cellular infiltrate have revealed that the host’s reaction to a nerve allograft across maximal MHC barriers peaks 7-9 days postengraftment. It is characterized by Wallerian degeneration and an epineurial invasion of lymphocytes, plasma cells and macrophages.21-23 The ensuing severe inflammatory response occurs as macrophages enter the endoneurium and, along with SCs, phagocytose myelin and axon debris, leading to disruption of the entire nerve architecture and subsequent collapse of the SC basal lamina (SCBL) tubes. These obliterative changes frequently misdirect axonal growth down incorrect SCBL tubes or altogether prevent the advancement of regenerating axons through the allograft, which severely compromises ultimate outcome. Under immunosuppression, this cascade of events is subdued and the majority of SCBL structures are preserved. The high inherent potential for aberrant reinnervation underscores the importance of correct microsurgical alignment at the time of transplantation. Many studies have implicated donor SCs as the prime antigenic target in nerve allograft rejection.24,25 There is evidence of inducible and constitutive expression of Class I and Class II MHC expression on SCs in allografts.20,26,27 Yu et al have reported that rejecting allografts under MHC incompatibility demonstrated enhanced donor-derived Class I and Class II MHC antigen expression that peaked 8-15 days after transplantation and became undetectable by 30 days.20 Allogeneic donor MHC antigen can be presented by donor APCs and directly recognized by host T cells (i.e., without being presented in association with self MHC molecules).12 SCs can serve as APCs in the graft that initiate direct host allorecognition. Conversely, the progressive loss of donor SCs could promote allograft survival. Several investigators have demonstrated that the degree of major, but not minor, histocompatibility

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loci disparity is believed to determine nerve allograft rejection.28,29 We have shown that regeneration across an allograft with minor histocompatibility loci disparity was superior to that across major histocompatibility loci disparity.28 Indeed, regeneration across minor histocompatibility differences has been found to be similar to regeneration across autografts.22 The peripheral nerve allograft is a unique transplantation model in that it serves a structural, and not a functional, purpose for the host. As host axons regenerate, the survival of the peripheral nerve allograft is only temporarily dependent upon immunosuppression (see below). The classic teachings of Aguayo state that the nerve graft is static and that immunogenic SCs within the graft are of donor origin.30 As such, when immunosuppressive agents are withdrawn, the foreign graft cells should be rejected. Proponents of this theory report a modified rejection response with delayed degenerative changes, axonal loss and partial loss of functional recovery following discontinuation of the immunosuppression. Recently, Buttemeyer et al explored intermittent immunosuppressive strategies using FK506. After cessation of the FK506, fewer donor-derived SCs were seen compared to continuously immunosuppressed animals, presumably from ongoing, albeit retarded, rejection.31 Animals that received 3 months of immunosuppression followed by a tapered regimen showed inferior walking function recovery and poorer regeneration compared to isografts and continuously immunosuppressed animals. These studies argue in favor of continuous immunosuppression for peripheral nerve allograft recipients. In contrast, the replacement theory suggests that SCs migrate into the donor graft along with regenerating host axons and repopulate the graft, rendering it nonimmunogenic. The nerve allograft is viewed as progressively losing its target antigenic stimulus and passing through a chimeric state, ultimately to one composed of host tissue without donor allogeneic cells. In the Trembler mouse model, repopulation of host SCs was tracked by immunohistochemical staining and shown to occur in the presence of cyclosporine A (CsA), albeit at a slower pace.32 Ishida et al demonstrated that nerve allografts undergo a rejection phase after immunosuppression withdrawal with subsequent recovery and regeneration, suggesting that they can still serve as effective nerve conduits.33 We have consistently found recovery of function after immunosuppression withdrawal, possibly secondary to replacement of SCs by recipient cells, either progressively during the immunosuppressive period or rapidly following CsA withdrawal. Other explanations include the downregulation of expression of allodeterminants by donor cells and/or the reconstitution of a previously impervious blood-nerve barrier that would minimize the subsequent migration and response of immunocompetent cells.34 We believe that the host SCs gradually replace donor SCs under CsA immunosuppression and will remyelinate growing host axons (Fig. 9.2).32,35 Studies in a rat allograft model by Midha et al demonstrated that temporary CsA immunosuppression yields equivalent functional and morphologic results to long term immunosuppression, and that it may be safely discontinued once proximal axons traverse the graft and reinnervate distal end organs.36 Similar results were also noted in a primate nerve allotransplantation model.37,38 Thus, it is possible that once significant donor alloantigen replacement has occurred, the rejection response is extinguished, regeneration proceeds uninhibited, and immunosuppression is no longer required. In ongoing studies, we continue to investigate the replacement phenomenon and the need for only finite host immunosuppression. A group of animals receiving 10 weeks of CsA immunosuppression, followed by abrupt withdrawal, demonstrated that replacement had occurred sufficiently to allow allografts to survive when reimplanted into a second animal of the original recipient strain. In contrast, when reimplanted into an animal of the original donor strain, massive lymphocytic infiltration and rejection of the nerve allograft was seen (Fig. 9.3). Moreover, in vitro MLR, CTL and LDA assays suggest that the donor strain secondary recipients of

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Fig. 9.3. Electron micro– graph of a posterior tibial nerve allograft infiltrated by immunocompetent cells in a second recipient of the donor strain. The allograft was transferred from a rat receiving 10 weeks of cyclo– sporine A immunosup– pression, followed by abrupt withdrawal. This rejection is consistent with replacement by host SCs. Fibroblasts (F), macro– phages (M), and lympho– cytes (L) are seen in close proximity to the Schwann cell nuclei (SC) of myelinated (m) and un– myelinated (u) fibers (uranyl acetate–lead citrate stain; magnification x10,800).

these temporarily immunosuppressed allografts were mounting an immune response. Animals under 20 weeks continuous CsA immunosuppression demonstrated some degree of replacement, as good regeneration was seen when primary allografts were reimplanted into a second animal of either donor or recipient strain.39 In vitro assays suggest that both secondary recipients of continuously immunosuppressed allografts were less reactive. These results are consistent with the notion that progressive SC replacement (i.e., slow loss of graft antigenicity) occurs under continuous immunosuppression, and sufficient, rapid replacement follows immunosuppression withdrawal. To help further delineate the process of re-

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placement, we are attempting immunohistochemical staining for the H-Y antigen in male SCs in a model of nerve allograft transplantation from male into female rats. Our results will be confirmed by in situ hybridization using a DNA probe specific for the Y chromosome.

Long Nerve Allograft Regeneration Differences between results of short and long allograft transplantation have suggested that the nerve allograft response is influenced by total graft length in a way yet unknown. Early on, in a review of 375 clinical reconstruction cases, Delangeniere noted diminishing success with transplants as the graft length increased.40 Our recent experimental studies in a long nerve allograft transplantation model simulate the extensive reconstruction required in many human traumatic and neoplastic injuries to peripheral nerves. For long term studies of the immunological response and of nerve regeneration, the rodent model has proven inadequate. Over long periods of time, rats demonstrate a superior intrinsic regenerative capacity that frequently overcomes the immunological challenge of a short allograft.41 Additionally, rodent limb sizes restrict the total nerve graft length that may be studied. Our earlier investigations using the cynomolgus monkey demonstrated no significant histomorphometric or electrophysiologic differences between untreated allografts and autografts across a 3 cm graft.38 Furthermore, because the use of primates was wrought with ethical concerns, we have since established both the sheep and pig as reliable models to investigate regeneration across the long nerve allograft. These models should provide important information on the kinetics of the nerve allograft response that has direct extrapolation to the clinical situation. Both animals allow the creation of a gap length that adequately challenges their intrinsic regenerative capacity, and both demonstrate excellent regeneration across autografts, and complete rejection with no regeneration across allografts.42,43 Recently available inbred herds of miniature swine homozygous at the MHC promise to be particularly useful in our future long allograft studies. Transplantation in these swine may be completed across minor antigens only (MHC matched), or minor plus some combination of Class I or Class II mismatch.44,45 They will allow similar rigorous immunological testing and experimental manipulations as do the smaller rodent nerve allograft models.

Nerve Allograft Preservation and Storage Many early research efforts by other investigators were filled with enthusiasm for the use of grafts from cadavers, with an emphasis on methods of preservation. Multiple advantages to establishing a method of nerve allograft preservation have since been realized. Appropriate storage techniques ensure graft viability and flexible scheduling of surgery, which translates into nonemergent, less expensive procedures. Moreover, preoperative requirements for tissue typing and transportation of grafts between cities has lead to variable storage times between harvest and implantation that can be accommodated by these techniques. Ideally, defining optimal preservation conditions would facilitate the establishment of a nerve bank with readily available, abundant graft material of known antigenicity for allograft reconstruction. A variety of methods attempting to mitigate the host immune response to nerve allografts have relied on decreasing allograft immunogenicity to prevent rejection. Early on, we demonstrated that lyophilization and high dose irradiation (30,000 rads) render grafts nonimmunogenic.46 There continues to be considerable debate as to whether viable SCs are required for good regeneration, or simply the presence of intact SCBL tubes lined with laminin. In 1986, Hall established that when SC migration into acellular grafts is impeded, axonal elongation is significantly retarded.47 As such, the presence of viable SCs following

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storage is an important determinant of successful preservation protocols. Nerve allografts stored in Belzer/University of Wisconsin Cold Storage Solution (UWCSS), commonly used for flushing and for cold storage of solid organs prior to transplantation, have survived and demonstrated good nerve regeneration. Electron microscopy of these stored nerves reveals that the SCBL tubes remain structurally intact, and hence can serve as effective structural guides for the regenerating axons.1 In vitro assays of cell viability have demonstrated that numerous viable cells remain after one week storage in UWCSS, and that storage for lengthier time yields a conduit that is primarily acellular (Fig. 9.4).48 By identifying the specific surface proteins S100 and NGFr on rat and human SCs respectively, we have demonstrated that SCs are capable of surviving at least 3 weeks storage in UWCSS (Fig. 9.5). However, regeneration was inferior through grafts stored for 3 weeks in comparison to grafts stored for one week.49 From these studies, we propose that the donor SC is required for axonal elongation, and, following its replacement, the host SC proceeds to remyelinate the regenerated axon (Fig. 9.2). In other studies in the rat model, we have demonstrated good return of gastrocnemius muscle isometric contractile function after storage of nerve allografts at varying times and temperatures.50 Moreover, cold preservation of the nerve allograft at 5° C potentiated the immunosuppressive effect of cyclosporine A, thus allowing a decrease in the systemic CsA requirements necessary to support regeneration across a short nerve allograft.51 The host’s response to a preserved nerve allograft has also been examined. With the use of radioactively labeled lymphocytes, we have shown that cold preservation delays or prevents the biphasic increase in efferent lymphocyte output observed after fresh allograft implantation in an ovine model.52 In a separate study, there was no statistically significant difference in lymphocyte migration between grafts harvested from live or cadaveric donors after 5 days cold preservation. The associated proportional increase in CD4+ T helper lymphocytic infiltration and increase in MHC Class II expression on the nerve allograft was also delayed or prevented by cold preservation. Indeed, Yu et al has demonstrated no reduction in lymphocytic infiltration in rat sciatic nerve allografts after culture for 7 days at 37°C in vitro.20 This suggests that the reduced lymphocytic infiltration seen is an effect of the cold temperature and not an effect of in vitro culture alone. The effects of cold preservation may be attributed to possible loss or deactivation of passenger lymphocytes or APCs (e.g., Schwann cells, dendritic cells) and/or inhibition of antigen presentation, lymphokine production and adhesion or coreceptor molecule expression.53-55 Hence, preservation decreases the overall cost of donor nerve harvest and makes it feasible to prolong preoperative ischemia. It decreases graft immunogenicity, which translates into a lesser evoked immune response, and works synergistically with CsA. Furthermore, it allows time for identification of potential donor pathogens, to begin systemic preoperative immunosuppression or, as discussed later, to pretreat with donor antigen.

Nonspecific Immunosuppressive Strategies In other transplantation models, experimental immunosuppressive strategies have been directed at the various stages of early cell mediated response such as antigen processing and presentation with MHC by the APC. Immunotherapies have attempted to mask antigen recognition by monoclonal antibodies (mAbs) as discussed below, or manipulate MHC expression by altering preferential cytokine production. These therapies gain added benefit from their secondary effects of blocking clonal expansion, T cell differentiation and chemoattraction of immunocompetent cells. Current immunosuppressive regimens globally suppress the host effector response to all foreign antigens by nonspecific, systemic drugs. The disadvantages of this approach lie in the increased susceptibility to infection and higher incidence of lymphoproliferative disorders. In contrast to graft pretreatment methods, this approach does not alter the viability of al-

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95 Fig. 9.4. Rat peripheral nerve explants stored in UWCSS demonstrate a significant reduction in the total number of cells over 3 weeks (adapted with permission from: Levi ADO, Evans PJ, Mackinnon SE et al. Cold storage of peripheral nerves: An in vitro assay of cell viability and function. GLIA 1994; 10:121-131. © 1994 Wiley–Liss, Inc.)

Fig. 9.5. Electron micrograph of a rat posterior tibial nerve preserved for 3 weeks at 5°C in UWCSS solution. An intact Schwann cell basal lamina (BL), nuclei (N) and multilaminated axon (A) are visible. Viable donor Schwann cells are necessary for axonal elongation across long gaps (uranyl acetate–lead citrate stain; magnification x10,800).

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lograft constituents. Prior investigations into the use of azathioprine and antilymphocyte globulin in nerve allotransplantation have been met with limited success.22,56 In the last two decades, the promising results of CsA immunosuppression in other transplantation models promoted its use in clinical and experimental nerve allografting studies. Parenthetically, within the cell, CsA associates with cyclophilin and binds calcineurin, which subsequently reduces the expression of IL-2 and other cytokines. As detailed earlier, we have demonstrated equivalent histologic, electrophysiologic and functional regeneration across MHC-disparate nerve allografts in CsA-treated hosts compared to autografts.35,57 Nerves regenerating under CsA therapy were found to have less inflammatory cell infiltration than untreated controls. When combined with cold preservation techniques, lower doses of CsA were required to support nerve regeneration.51 Furthermore, we have demonstrated a synergistic effect of CsA and monoclonal antibody therapy against the adhesion molecules ICAM-1 and LFA-1 that allows for lower, nontoxic doses of CsA to be used (manuscript in preparation, see below). There is, however, considerable morbidity associated with global immunosuppression by continuous CsA therapy that must always be weighed against the benefits of transplanting a nonvital tissue. This remains a critical issue, and is the stimulus for ongoing research into devising methods for donor-specific immunosuppression. Monoclonal antibodies are homogenous immmunoglobulins of defined specificity that recognize specific epitopes on target molecules and, hence, can be used to probe specific events that regulate the immune system. Short term administration of mAbs directed against ICAM-1 (CD54) and LFA-1#,∃ (CD11#,CD18) have been shown to alter the adhesion of lymphocytes to a graft, resulting in decreased rejection and prolonged graft survival.58,59 ICAM-1 is a member of the immunoglobulin superfamily and is a cell surface glycoprotein expressed on a variety of APCs, B and T cells, fibroblasts, keratinocytes and endothelial cells. ICAM-1 is normally expressed in low quantities on the surface of these cells, but may be upregulated in association with inflammation and other immunological reactions.60,61 LFA-1, a member of the ∃2 integrin family, is a counterreceptor for ICAM-1. It is expressed constitutively on the surface of lymphocytes and undergoes a conformational change with inflammation.62 Both this conformational change and increased expression of ICAM during inflammatory stimuli leads to increased adherence of lymphocytes to the endothelial cells of affected tissues.63 The ICAM-1-LFA-1 adhesion also facilitates APC-T cell interaction and signal transduction resulting from TCR occupancy, thereby promoting alloantigen recognition, lymphokine production, lymphocyte migration and subsequent graft destruction during T cell mediated rejection. Blockage of ICAM-1 on APCs and LFA-1 on T helper cells may inhibit the auxiliary signals to result in nonalloantigen-specific tolerance. Hence, when administered at the time of first encounter with a foreign graft, anti-ICAM-1 and anti-LFA-1 mAbs alter the pattern of costimulation by changing the context of antigen presentation (Fig. 9.1). We have proposed that under the umbrella of mAb therapy directed against ICAM-1/ LFA-1, peripheral tolerance is induced sufficiently for SC replacement to occur and to alter the perception of the nerve allograft as foreign. In a rat model, the survival of nerve allografts was improved such that functional recovery and histomorphometric evidence of regeneration was comparable to nerve autografts.64-66 While the protective effect of these mAbs persisted for a limited time after the mAbs were cleared from the circulation, long term tolerance was not established. In a murine model of nerve allotransplantation, normal nerve architecture was preserved with a short course of anti-ICAM-1 and anti-LFA-1 therapy. In contrast, untreated allografts demonstrated severe structural disorganization and cellular infiltrate consistent with acute rejection.64,65 In our rat model, we have established an

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appropriate dosing regimen for nerve allograft survival, and doses above which toxic systemic side effects are seen.66 Recently, mAbs against the CD4 molecule have been developed which allow for specific immunomodulation of T cell interaction with antigen in the context of the MHC Class II molecule. They have the potential to determine whether engagement of the TCR by antigen results in activation or inactivation of the T helper cell. The purported mechanisms of action of these anti-CD4 mAbs include: 1. 2. 3. 4.

Elimination of newly maturing alloreactive CD4+ T cells or peripheral CD4+ T cells; Modulation of the CD4 molecule from its interaction with other proteins; Blocking of an activation signal; or Transmission of a negative signal to the T cell to alter its response.

Studies have shown that depleting anti-CD4 mAbs can prolong graft survival in proportion to the degree of T helper cell depletion. This has led, however, to a “profound, longterm elimination” and subsequent nonspecific immunosuppression.67 The potent in vivo immunosuppressive efficacy of the nondepleting anti-CD4 mAb RIB 5/2 has recently been demonstrated in other solid organ transplantation models to modulate recipient CD4+ T cell function, without causing T cell elimination. This is believed to occur through depression of IL-2 and IFN-! production by the subset of Th1 cells.68 Motoyama et al have reported donor-specific tolerance to cardiac transplants was achieved with the pretransplant administration of donor antigen in conjunction with RIB 5/2. The delivery of antigen must coincide with the T cell inactivation in order for tolerance to develop.69,70 Swain has demonstrated that the CD4 effectors and their cytokines, generated by antigen stimulation, are usually short lived in the absence of chronic stimulation.71 This will have important implications in the replacement phenomenon seen in nerve allotransplantation. We have ongoing studies to identify the efficacy of RIB 5/2 immunosuppression in the peripheral nerve allograft regeneration model.

Induction of Donor Specific Immunosuppression Portal Venous Tolerance The form and route of antigen exposure determine the APC that presents antigen to responding T cells and subsequently the character of the ensuing immune response. In other words, depending on its presentation, antigenic challenge may be perceived as immunogenic or tolerogenic by the body. This is well illustrated by the ability to tolerate absorbed antigen from the gastrointestinal tract without mounting an immune response. The phenomenon of oral tolerance has led to the idea that donor antigen administered directly into the portal vein would also be tolerated. In animal models, the intraportal administration of cellular alloantigen has been shown to lead to an abrogated cytolytic T cell response.72 Kamei et al have shown that when allogeneic splenocytes treated with ultraviolet B irradiation are provided to the hepatic environment, a donor-specific tolerogenic signal is generated.73 In contrast, the administration of untreated cells into the portal vein results in accelerated rejection.74 The same laboratory further demonstrated that impairment of phagocytosis and antigen presentation by the Kupffer cells, the APCs of the liver, abolished donor specific tolerance.75 This was attributed to passage of alloantigen through the liver to the systemic circulation, where it sensitized systemic lymphocytes rather than locally tolerizing intrahepatic T cells. Gorczonski detected a defect in vitro IL-2 production by hepatic, but not splenic APCs, in mice inoculated with donor antigen via the portal vein. He concluded

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that the hepatic, not the splenic, APCs were central to the development of donor specific anergy of IL-2-producing T cells following intraportal pretreatment with donor antigen.76

UV-B Irradiation Irradiation of a whole animal by photons of the UV-B region of the solar spectrum (280-320 nm) has been shown to result in a state of antigen-specific, T lymphocyte-mediated immunosuppression.77 In vivo, this is believed to result from damage to DNA, modification of cell surface antigens, and interference with intercellular interactions, antigen presentation and cytokine release. 77,78 UV-B irradiation has been found to increase Th2 proliferation and decrease Th1 cell proliferation by rendering them unable to respond to subsequent antigen stimulation (i.e., introduction of the allograft) due to a defect in autocrine IL-2 production. This change in the cytokine environment in which T cells recognize the peptide-MHC complex, without altering the “foreignness” of the antigen, is conducive to anergy induction.9 In vitro, decreased cellular proliferation has been shown to be correctable by the addition of exogenous IL-2. This suggests that there is a deficit in IL-2 production, not responsiveness to IL-2.9 Since supplementation with exogenous IL-2 restores function, one can conclude that unresponsiveness is due to functional inactivation, and not the release of soluble factors or induced cell death.79 The addition of nonirradiated allogeneic splenic APCs during the induction of unresponsiveness by portal vein inoculation of UV-B-irradiated APCs has been shown to prevent subsequent unresponsiveness, also suggesting that UV-B renders the APC a poor provider of the costimulatory signals normally provided by allogeneic unirradiated APCs.80 Furthermore, the alloantigen-specific CD4+ T cells rendered unresponsive with pretransplant exposure to donor MHC without appropriate costimulation are unable to provide helper signals to precursor CTLs, leading to their functional inactivation as well.9 The development of anergy is influenced by the initial presentation of antigen. One of two mechanisms is operative: either donor antigen (e.g., UV-B-treated splenic APCs) given intraportally is recognized directly by recipient T cells in the hepatic microenvironment, or the UV-B treated cells are processed by the hepatic APCs (e.g., Kupffer cells, dendritic cells) and indirectly presented to recipient T cells, the normal physiologic response. Processing of the UV-B modified antigen is believed to result in a less avid interaction between the altered peptide-MHC complex of the hepatic APC and the TCR, a loss of essential costimulatory signals and, hence, depressed T cell clonal expansion. The same relative T cell unresponsiveness will then result when donor antigen is reintroduced in association with the allograft. Flye has postulated that the induction of anergy may also be the result of uptake of UV-B-irradiated antigen by immature hepatic dendritic cells that, as discussed earlier, are unable to provide appropriate costimulatory signals to stimulate T cells. If true, the finding that inhibition of phagocytosis and antigen presentation by the Kupffer cell abolishes tolerance suggests that proper Kupffer cell function is necessary for maintenance of hepatic dendritic cells in their immature state (personal communication). We have shown that pretransplant intraportal administration of UV-B-treated donor splenocytes is capable of supporting peripheral nerve regeneration (Figs. 9.6A-D). Tolerance to the nerve allografts was further enhanced by the addition of a short course of antiICAM-1 and anti-LFA-1. These two therapies were synergistic, as shown by improvements in electrophysiologic testing (e.g., nerve conduction velocity) and morphometric analysis (e.g., nerve fiber density, percent neural regeneration, total axon number). In vitro cytotoxic T lymphocyte (CTL) and limiting dilution analysis (LDA) assays demonstrated a reduction in clonal T helper cell precursor frequency, with reduced cytolytic activity and IL-2 production. The rejection of third party MHC-disparate nerve allografts confirmed that the UV-B irradiation pretreatment conferred an allospecific tolerance.81

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C

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B

D

Fig. 9.6. Distal section of a rat posterior tibial nerve: (A) autograft, (B) untreated allograft, (C) 8 week allograft under immunosuppression from a single pretransplant, portal venous administration of UV-B–irradiated donor antigen, (D) 16 week allograft under the same UV-B immunosuppression regimen. Immunosuppressed allografts demonstrate good reconstitution of the perineurium and nerve architecture, as well as recovery of total fiber number and density when compared to unprotected allografts (toludine blue stain, magnification x580).

A common theme in peripheral tolerance is that persistence of antigen in the appropriate location, form and concentration are important factors for the induction of unresponsiveness. Continued antigen exposure acts as a reinforcing tolerogen that maintains tolerance. Kamei et al also demonstrated in a heterotopic rat cardiac allograft model that pretransplant administration of UV-B-modified donor antigen through the portal vein combined with continued portal vein drainage of the graft’s venous effluent synergistically permitted donor specific, long term acceptance of the allografts.73 The graft survival was significantly greater than that noted after a single dose of antigen. Flye has proposed that continued unresponsiveness also requires site-specific antigen persistence to continuously stimulate a Th2 response. In our rat model, we are currently exploring the importance of donor antigen presence in maintaining tolerance induced by the same method of pretransplant administration of UV-B-modified donor antigen. The replacement phenomenon seen in nerve allografts predicts that as donor antigen (e.g., Schwann cell) is replaced by host cells, UV-B-induced tolerance will extinguish after a finite period. Preliminary results suggest that this is indeed the case. Tolerance to nerve allografts is clearly seen 8 weeks postengraftment by electrophysiologic, functional and histomorphometric parameters. However, at 16 weeks, when a second donor allograft is introduced, tolerance appears to have faded. In vitro MLR, CTL and LDA demonstrate that the recipient is mounting an immune response after the introduction of a second donor MHC-disparate allograft (manu-

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script in preparation). This would not be expected if tolerance had persisted unabated by continued antigen exposure (i.e., no replacement of donor SCs). In contrast to vital organ transplants, reconstitution of the immune response after a limited time of tolerance is a reassuring finding that will be of benefit in clinical applications of nerve allotransplants. Our initial results in the rodent model have encouraged us to begin working in our long allograft swine and ovine models. We have established the appropriate protocols for in vitro MLR with sheep cells, and have demonstrated in vitro suppression of proliferation between outbred sheep. We have also successfully administered 180 million UV-B-irradiated splenocytes through fluoroscopic injection into the portal vein in sheep. However, this did not demonstrate tolerance in vivo (unpublished observations). It is not clear whether the quantity of antigen was excessive, spilling into the systemic circulation and causing a heightened immune response, or if the quantity was insufficient to induce tolerance. Experiments are underway to resolve this issue, as well as to explore the efficacy of this immunotherapy in our miniature swine model. Hence, the intrahepatic immune response that results in systemic tolerance is an important natural regulatory function that can be manipulated to generate the acceptance of allografts. Pretreatment with donor antigens can specifically promote nerve allograft survival and regeneration across maximal MHC barriers, provided an optimal source and amount of antigen is administered, and the appropriate interval between pretreatment and subsequent transplantation are utilized. Unlike in solid organ transplantation, nerve allograft preservation techniques make it feasible to downregulate the recipient’s immune system with donor antigen prior to transplantation.

Immune Privilege Apoptosis is an active cell death process that is critical to normal embryonic development and physiologic cell turnover. Recently, the interaction of the cell surface receptor Fas (CD95) with Fas ligand (Fas-L) has been found to result in apoptosis of Fas expressing cells.82,83 The Fas receptor is a cysteine-rich type I membrane protein belonging to the NGF receptor family. Upon contact with crosslinking antibodies or the natural ligand (Fas-L), a type II membrane protein, cells expressing Fas undergo apoptosis by a rapid intracellular signaling pathway. The Fas system is believed to play a crucial role in peripheral deletion of autoimmune cells. It has also been implicated in activation-induced T cell death, since Fas-L is an important effector molecule for CD8+ cytolytic T cells.84 Following studies in the testis and eye, Fas-L expression is purported to be a mechanism for protecting against Fas-bearing T lymphocyte-mediated immune damage.85,86 Indeed, in tissues with relatively low reparative capacities such as nervous tissue, inflammatory responses can lead to nonspecific injury to adjacent tissues, which may in turn lead to permanent consequences. This is crucial to the nerve allograft setting, where even minimal scarring may have profound effects. We have found Fas-L mRNA in normal and recently injured fresh human peripheral nerves undergoing Wallerian degeneration (unpublished observation). Further analysis of preserved nerve allografts is precluded by the destruction of mRNA by storage in UWCSS. Studies using immunohistochemical staining with an antibody to Fas-L are underway to determine the location and expression of Fas-L in peripheral nerve allografts.

Clinical Applications Nerve allograft transplantation is a surgical option that is still in its infancy, with only a few clearly defined indications, approaches and postoperative care recommendations. The senior author has reserved nerve allografting for injuries with no recovery pattern, such as

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Class V and VI Sunderland or Seddon neurotmesis injuries that clinically demonstrate a nonprogressing Tinel’s sign. In 1988, the senior author performed the first human nerve allograft transplant under temporary CsA immunosuppression in a boy with a sciatic nerve injury.87 A 23 cm gap was reconstructed with ten donor fresh nerve allografts. The patient was treated with CsA immunosuppression for 2 years until successful sensory regeneration across the nerve allograft occurred. He had an episode of soft tissue breakdown, but has maintained sensibility in his foot and continues to ambulate unassisted. In 1993, a posterior tibial nerve injury was reconstructed in a 12 year old boy using small caliber, cold preserved nerve allografts.88 These allografts were initially stored in UWCSS at 5°C for 7 days. The recipient was started on a course of CsA (50 mg/kg p.o.) immunosuppression 4 days prior to the planned transplantation. At the time of surgery, a gap distance of 20 cm in the main posterior tibial nerve was reconstructed with eight donor nerve allografts. Two years later, the patient demonstrated good sensory recovery and the CsA immunosuppression was stopped. He currently maintains this recovery. In three other patients, regeneration is proceeding across nerve allografts at a rate comparable to that which would be expected across a nerve autograft. In these patients, their own sural nerves were used, as well as significant lengths of nerve allograft material. In two patients, sensory and motor recovery has occurred and the immunosuppression has been stopped. In one patient, serum immunosuppression levels were subtherapeutic three weeks posttransplant, and the long allograft was rejected. In this patient, regeneration continues in appropriate fashion across the two sural nerve autografts which were also used in the reconstruction.

Conclusion Progress in nerve allotransplantation in the past resulted primarily from the advent of microsurgical techniques and from advances in understanding the factors that guide nerve regeneration. In this chapter, we have discussed the basic principle that specificity of a T cell response is confirmed by the interaction of the TCR with antigen bound to the appropriate MHC molecule of the APC. Adhesion molecules and other coreceptors facilitate this interaction and enhance signal transduction resulting from TCR occupancy. When this interaction occurs in the absence of a second costimulatory signal delivered by the APC, anergy of the responding T cell results. We have also presented the recent developments in nerve preservation and in understanding the nerve allograft replacement response. Furthermore, we emphasize that the induction of donor-specific tolerance to major histocompatibility antigens remains a major goal in the application of nerve allograft transplantation. While CsA is the current front line immunosuppressive regimen, improved graft survival sometimes pays the price of complications resulting from this systemic therapy. Current cold preservation methods have lowered immunosuppressive requirements by decreasing graft immunogenicity. We have also shown that blockade of adhesion molecules ICAM-1/ LFA-1 leads to immunosuppression; however, it is not alloantigen-specific. Suppression of the immune system by orally fed antigens is a natural form of tolerance that has been adapted to enhance nerve allograft survival. We have demonstrated, in a rat model, the induction of donor-specific tolerance and improved nerve allograft survival with the pretransplant, portal venous administration of donor antigen. Nerve allograft storage protocols make this immunosuppressive approach clinically feasible. Preliminary success in short and long allograft models holds promise for future, widespread applications of nerve allograft transplantation in the clinical management of peripheral nerve injuries.

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References 1. Evans PJ, Midha R, Mackinnon SE. The peripheral nerve allograft: A comprehensive review of regeneration and neuroimmunology. Prog Neurobiol 1994; 43:187-233. 2. Philipeaux J, Vulpian A. Note sur des essais de greffe d’un troncon du nerf lingual entre les deux bouts du nerf hypoglosse, apres excision d’un segment de ce dernier nerf. Arch de Physiol Norm et Path 1870; 618-620. 3. Albert E. Einige operationen an nerven. Wied Med Presse 1885; 26:1285-1288. 4. Mayo-Robson AW. Nerve grafting as a means of restoring function in limbs paralyzed by gunshot or other injuries. Br Med J 1917; 1:117. 5. Millesi H. Microsurgery of peripheral nerves. Hand 1973; 5:157-160. 6. Millesi H. Indication, technique and results of nerve grafting. Handchirurgie (suppl) 1977; 2:1-24. 7. Marlin SD, Springer TA. Purified intracellular adhesion molecule-1 (ICAM-1) is a ligand for lymphocyte function associated antigen-1 (LFA-1). Cell 1987; 51:813-819. 8. Linsley PA, Ledbetter JA. The role of the CD28 receptor during T cell responses to antigen. Annu Rev Immunol 1993; 11:191-211. 9. Jenkins MK. Antigen-presenting cell regulation of T cell activation and anergy induction. In: Bach FH, Auchincloss H Jr, eds. Transplantation Immunology. New York; Wiley-Liss, Inc., 1995:295-304. 10. Bach FH, Sachs DH. Transplantation immunology. N Engl J Med 1987; 317:489-492. 11. Hayry P, Renkinen R, Leszczynski D et al. Local events in graft rejection. Transplant Proc 1989; 21:3716-3720. 12. Auchincloss H. Strategies to induce tolerance. In: Bach FH, Auchincloss H Jr, eds. Transplantation Immunology. New York: Wiley-Liss, Inc., 1995:211-218. 13. Beverly B, Kang SM, Lenardo MJ et al. Reversal of in vitro T cell clonal anergy by IL-2 stimulation. Int Immunol 1992; 4:661-671. 14. Su B, Facinto E, Hibi M et al. JNK is involved in signal integration during costimulation of T lymphocytes. Cell 1994; 77:727-736. 15. Rocha B, Tanchot C, Von Boehmer H. Clonal anergy blocks in vivo growth of mature T cells and can be reversed in the absence of antigen. J Exp Med 1993; 177:1517-1521. 16. Rocha B, Von Boehmer H. Peripheral selection of the T cell repertoire. Science 1991; 251:1225-1228. 17. Lu L, McCaslin D, Starzl TE et al. Bone marrow-derived dendritic cell progenitors (NLDC 145+, MHC Class II+, B7-1dim, B7-2-) induce alloantigen-specific hyporesponsiveness in murine T lymphocytes. Transplantation 1995; 60(12):1539-1545. 18. Thomson AW, Lu L, Subbotin VM et al. In vitro propagation and homing of liver-derived dendritic cell progenitors to lymphoid tissues of allogeneic recipients. Transplantation 1995; 59(4):544-551. 19. Demetris AJ, Murase S, Fujisaki S et al. Hematolymphoid cell trafficking, microchimerism, and GVH reactions after liver, bone marrow, and heart transplantation. Transplant Proc 1993; 25(6):3337-3344. 20. Yu LT, Rostami A, Silvers WK et al. Expression of major histocompatibility complex antigens on inflammatory peripheral nerve lesions. J Neuroimmunol 1990; 30:121-128. 21. Mackinnon SE, Dellon AL, O’Brien JP et al. Selection of optimal axon ratio for nerve regeneration. Ann Plast Surg 1989; 23:129-134. 22. Mackinnon SE, Hudson AR, Bain JR et al. An assessment of nerve regeneration in the immunosuppressed host. Plast Reconstr Surg 1987; 79:436-444. 23. Mackinnon SE, Hudson AR, Falk RE et al. The nerve allograft response—a quantitative immunological assessment of pretreatment methods. Neurosurg 1982; 10:61-84. 24. Sanders FK. The preservation of nerve grafts. In: Wolstenholme GEW, Cameron MP. A CIBA Foundation Symposia: Preservation and Transplantation of Normal Tissues. London: Little, Brown and Company, 1954:175-189. 25. Aguayo AJ, Kasarjian J, Skamene E et al. Myelination of mouse axons by Schwann cells transplanted from normal and abnormal human nerves. Nature 1977; 268:753.

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26. Armati PJ, Pollard JD, Gatenby P. Rat and human Schwann cells in vitro can synthesize and express MHC molecules. Muscle Nerve 1990;113:106-116. 27. Wekerle H, Schwab M, Linington C et al. Antigen presentation in the peripheral nervous system: Schwann cells present endogenous myelin autoantigens to lymphocytes. Eur J Immunol 1986; 16:1551-1557. 28. 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. 29. Zalewski AA, Silvers WK. An evaluation of nerve repair with nerve allografts in normal and immunologically tolerant rats. J Neurosurg 1980; 52:557-563. 30. Romine JS, Aguayo AJ, Bray GM. Absence of Schwann cell migration along regenerating unmyelinated nerves. Brain Res 1975; 98(3):601-606. 31. Buttemeyer 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. 32. Midha R, Evans PJ, Mackinnon SE et al. Temporary immunosuppression for peripheral nerve allografts. Transplant Proc 1993; 25:532-536. 33. 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. 34. Zalewski AA, Kadota Y, Azzam NA et al. Observations on the blood and perineurial permeability barriers of surviving nerve allografts in immunodeficient and immunosuppressed rats. J Neurosurg 1993; 78:794-806. 35. Midha R, Mackinnon SE, Becker LE. The fate of Schwann cells in peripheral nerve allografts. Neuropath Exp Neurol 1994; 53:316-322. 36. Midha R, Evans PJ, Mackinnon SE et al. Comparison of regeneration across nerve allografts with temporary or continuous cyclosporine A immunosuppression. J Neurosurg 1992; 78:90-100. 37. Fish JS, Bain JR, McKee N et al. The peripheral nerve allograft in the primate immunosuppressed with cyclosporine A: II. Functional evaluation of reinnervated muscle. Plast Reconstr Surg 1992; 90:1047-1052. 38. Bain JR, Mackinnon SE, Hudson AR et al. The peripheral nerve allograft in the primate immunosuppressed with cyclosporine A: I. Histologic and electrophysiologic assessment. Plast Reconstr Surg 1992; 90:1036-1046. 39. Atchabahian A, Mackinnon SE, Doolabh VB et al. Donor tissue replacement by recipient cells in nerve allografts with cyclosporine A immunosuppression. J Neurosurg, submitted 1997. 40. Delageniere H. A contribution to the study of the surgical repair of peripheral nerves based on 375 cases. Surg Gynecol Obstet 1924; 39:543-553. 41. Mackinnon SE, Hudson AR, Hunter DA. Histologic assessment of nerve regeneration in the rat model. Plast Reconstr Surg 1985; 75:384-388. 42. Strasberg S, Mackinnon SE, Genden EM et al. Long segment nerve allograft regeneration in the sheep model: Experimental study and review of the literature. J Reconstr Microsurg 1996; 12(8):529-537. 43. Atchabahian A, Genden EM, Mackinnon SE et al. Regeneration through long nerve grafts in the swine model. J Reconstr Microsurg, submitted 1996. 44. Pennington LR, Lunney JH, Sachs DH. Transplantation in miniature swine-recombination within the major histocompatibility complex of miniature swine. Transplantation 1981; 31:66-71. 45. Sachs DH, Leight G, Schwarz CJ et al. Transplantation in miniature swine—fixation of the major histocompatibility complex. Transplantation 1976; 22:559-567. 46. Mackinnon SE, Hudson AR, Falk RE et al. Peripheral nerve allograft: An immunological assessment of pretreatment methods. Neurosurg 1984; 14(2):167-171. 47. Hall SM. Regeneration in cellular and acellular autografts in the peripheral nervous system. Neuropath appl Neurobiol 1986; 12:27-46. 48. Levi ADO, Evans PJ, Mackinnon SE et al. Cold storage of peripheral nerves: An in vitro assay of cell viability and function. Glia 1994; 10:121-131.

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49. Evans PJ, Mackinnon SE, Wade JA et al. Regeneration across preserved peripheral nerve grafts. Muscle Nerve 1995; 18:1128-1138. 50. Evans PJ, Awerbuck DC, Mackinnon SE et al. Isometric contractile function following nerve grafting: A study of graft storage. Muscle Nerve 1994; 17:1190-2000. 51. Strasberg SR, Hertl MC, Mackinnon SE et al. Peripheral nerve allograft preservation improves regeneration and decreases systemic cyclosporine A requirements. Exp Neurol 1996; 139:306-316. 52. Hare GMT, Evans PJ, Mackinnon SE et al. Effect of cold preservation on lymphocyte migration into peripheral nerve allografts in sheep. Transplantation 1993; 56(1):154-162. 53. Imagawa DK, Millis JM, Seu P. The role of tumor necrosis factor in allograft rejection. Transplantation 1991; 51:57. 54. Isobe M, Yagita H, Okumura K et al. Specific acceptance of cardiac allograft after treatment with antibodies to CD4, LFA-1 and ICAM-1. Transplant Proc 1993; 25:828-830. 55. Kupiec-Weglinski JW, Tilney NL. Lymphocyte migration patterns in organ allograft recipients. Immunol Rev 1989; 108:63-82. 56. Zalewski AA, Gulati AK, Silvers WK. Loss of host axons in nerve allografts after abolishing immunological tolerance in rats. Exp Neurol 1981; 72:502-506. 57. Mackinnon SE, Midha R Bain J et al. An assessment of regeneration across nerve allografts in rats receiving short courses of cyclosporine A immunosuppression. Neurosci 1992; 46(3):585-593. 58. Cosimi AB, Conti D, Delmonico FL et al. In vivo effects of monoclonal antibody to ICAM-1 (CD54) in nonhuman primates with renal allografts. J Immunol 1990; 144:4604-4612. 59. Heagy W, Waltenbaugh C, Martz E. Potent ability of anti-LFA-1 monoclonal antibody to prolong allograft survival. Transplantation 1984; 37:520-523. 60. Pober JS. Cytokine mediated activation of endothelium. Am J Pathol 1988; 133:426-433. 61. Buckle AM, Hogg N. Human memory T cells express intercellular adhesion molecule-1 which can be increased by interleukin-2 and interferon gamma. Eur J Immunol 1990; 20:337-341. 62. Dustin ML, Springer TA. T cell receptor crosslinking transiently stimulates adhesiveness through LFA-1. Nature 1989; 341:619-624. 63. Taylor PM, Rose ML, Yacoub MH et al. Induction of vascular adhesion molecules during rejection of human cardiac allografts. Transplantation 1992; 54(3):451-457. 64. Nakao Y, Mackinnon SE, Hertl MC et al. The immunosuppressive effect of monoclonal antibodies to ICAM-1 and LFA-1 (CD11a) on peripheral nerve allograft in mice. Microsurg 1995; 16:612-620. 65. Nakao Y, Mackinnon SE, Hertl MC et al. Monoclonal antibodies against ICAM-1 and LFA-1 prolong nerve allograft survival. Muscle Nerve 1995; 18:93-102. 66. Hertl MC, Strasberg S, Mackinnon SE et al. The dose-related effect of monoclonal antibodies against adhesion molecules ICAM-1 and LFA-1 on peripheral nerve allograft rejection in a rat model. Restor Neurol Neurosci 1996; 10:147-159. 67. Pearson TC, Madsen JC, Larsen CP. Induction of transplantation tolerance in adults using donor antigen and anti-CD4 monoclonal antibody. Transplantation 1992; 54:475-483. 68. Siegling A, Lehmann M, Riedel H et al. A nondepleting anti-rat CD4 monoclonal antibody that suppresses T helper 1-like but not T helper 2-like intragraft lymphokine secretion induces long-term survival of renal allografts. Transplantation 1994; 57:464-467. 69. Motoyama K, Arima T, Lehmann M et al. Tolerance to heart and kidney grafts induced by nondepleting anti-CD4 monoclonal antibody (RIB 5/2) versus depleting anti-CD4 mAb (OX-38) with donor antigen administration. Transplantation (In Press). 70. Arima T, Lehmann M, Flye MW. Induction of donor specific transplantation tolerance to cardiac allografts following treatment with nondepleting (RIB 5/2) or depleting (OX 38) anti-CD4 mAb plus intrathymic or intravenous donor alloantigen. Transplantation In Press. 71. Swain SL. Generation and in vivo persistence of polarized Th1 and Th2 memory cells. Immunity 1994; 1:543. 72. Qian JH, Kokudo S, Sato S et al. Tolerance induction of alloreactivity by portal venous inoculation with allogeneic cells followed by injection of cyclophosphamide. Transplantation 1987; 43:538-543.

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73. Kamei T, Callery M, Flye MW. Pretransplant portal venous administration of donor antigen and portal venous allograft drainage synergistically prolong rat cardiac allograft survival. Surgery 1990; 108:415-422. 74. Yu S, Roland CR, Goss JA et al. Different immune responses to UV-B treated or untreated donor spleen cells via portal venous administration. Transplant Proc 1992; 24(6):2908. 75. Callery MP, Kamei T, Flye MW. Kupffer cell blockade inhibits induction of tolerance by the portal venous route. Transplantation 1989; 47:1092-1094. 76. Gorczynski RM. Immunosuppression induced by hepatic portal venous immunization spares reactivity in IL-4 producing T lymphocytes. Immunol Lett 1992; 33:67. 77. Kripke ML. Immunologic unresponsiveness induced by ultraviolet radiation. Immunol Rev 1984; 80:87-102. 78. Kripke MI. Ultraviolet radiation and immunology—something new under the sun. Cancer Res 1994; 54:6102-6105. 79. Flye MW, Yu S. Restoration of lymphocyte proliferation and CTL generation by murine rIL-2 after treatment of allogeneic stimulator cells by ultraviolet B irradiation, heat or paraformaldehyde. Transplantation 1991; 51:1066-1071. 80. Simon JC, Tigelaar RE, Bergstresser R. Ultraviolet B irradiation converts Langerhans cells from immunogenic to tolerogenic antigen presenting cells: Induction of specific clonal anergy in CD4+ T helper 1 cells. J Immunol 1991; 146:485. 81. Genden EM, Mackinnon SE, Flye MW et al. Intraportal injection of donor alloantigens induces donor specific tolerance in a nerve allograft regeneration model. J Exp Med, submitted 1997. 82. Takayama H, Kojima H, Shinohara N. Cytotoxic T lymphocytes: The newly identified Fas (CD95)-mediated killing mechanism and a novel aspect of their biological functions. Adv Immunol 1995; 60:289-312. 83. Itoh NS, Yonehara A, Ishii M et al. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 1991; 66:233-243. 84. French LE, Hahn M, Viard I et al. Fas and Fas ligand in embryos and adult mice: Ligand expression in several immune-privileged tissues and coexpression in adult tissues characterized by apoptotic cell turnover. J Cell Biol 1996; 133 (2):335-343. 85. Griffith TS, Brunner T, Fletcher SM et al. Fas ligand-induced apoptosis as a mechanism of immune privilege. Science 1995; 270 (5239):1189-1192. 86. Bellgrau D, Gold D, Selawry H et al. A role for CD95 ligand in preventing graft rejection. Nature 1995; 377(6550):630-632. 87. Mackinnon SE. Nerve allotransplantation following severe tibial nerve injury-case report. J Neurosurg 1996; 84:671-676. 88. Mackinnon SE, Hudson AR. Clinical application of peripheral nerve transplantation. Plast Reconstr Surg 1992; 90:695-699.

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CHAPTER 10

Peripheral Nerve Allotransplants Immunosuppressed with 15-Deoxyspergualin Keiichi Muramatsu and Kazuteru Doi

Introduction

N

erve autografts have produced satisfactory clinical results for reconstruction of peripheral nerve defects and are now regarded as the gold standard.1 The nerve autograft has no immunological problem and the Schwann cells (SC) in a grafted nerve induce axonal regeneration. These concepts had been proven both experimentally and clinically.2 The significant shortcoming of a nerve autograft is the permanent functional loss of donor tissue. Another shortcoming of nerve autografts is that the donor nerve is a limited source, and there is insufficient donor tissue for reconstruction of a large nerve defect such as sciatic nerve injury. An option to resolve these problems is nerve allotransplantation or nerve xenotransplantation, both of which could serve as a rich donor source. The first clinical trial of nerve allotransplantation was reported in the 1880s3 and experimental studies soon followed.4 After more than a century since these first studies were reported, only now is nerve allotransplantation regarded as a reliable procedure for reconstruction of nerves and this is due to recent advances in transplantation medicine, especially immunosuppressive therapy.5 This chapter reviews nerve allo- and xenotransplantation, followed by a description of the immunosuppressive effects of 15-deoxyspergualin (DSG), and then presents some results on nerve allotransplantation using DSG.

Peripheral Nerve Allo- and Xenotransplantation History of Peripheral Nerve Allo- and Xenotransplantation

The first clinical nerve allotransplantation was reported by Albert.3 He operated on two cases using nerve allografts, but no satisfactory nerve regeneration was obtained. Mayo,6 Burk7 and Eden8 all reported similar unsatisfactory results. The cause of failure was necrosis of the allografted nerve induced by acute rejection. When a fresh allogeneic nerve is transplanted, acute rejection occurs similar to rejection of other transplanted allogenic organs such as kidney or liver. All viable donor cells in the transplanted nerve, especially SC, fibroblast, and endothelial cells, are recognized by the recipient’s lymphocytes, which induce cellular and humoral responses within several days postoperatively.

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The first clinical nerve xenotransplantation was reported in 1895 by Huber,9 and involved transplantation from the canine to the human. Following Huber’s work, some experimental studies10,11 were reported, but no satisfactory results were obtained. Sanders12 and Marmor13 concluded that nerve xenografts could not induce good axonal regeneration even with graft pretreatment. There are no recent reports that support the clinical application of nerve xenografting, even with immunosuppressive therapy. Recipients rapidly reject xenografts because the host normally has preexisting antibodies to xenoantigens.14 Recognition of xenoantigens strongly activates the complement system. This process is called hyperacute rejection; the grafted nerves necrose within several hours to a few days. Therapies to prevent rejection reactions can be classified into two categories: 1. To reduce the antigenicity of the donor nerve; and 2. To immunosuppress the recipient immune response. Immunology was poorly understood by the early investigators, which is why they had difficulty explaining why they failed to obtain nerve regeneration. If you apply our current understanding of immunology to their results, it is clear that all allo- and xenografts failed due to rejection, and the early investigators had developed no therapies to prevent rejection. Many authors have demonstrated the antigenicity of peripheral nerve in connection with autoimmune diseases. Lassner15 and Grochowicz16 demonstrated that SC presented MHC Class II antigens during ongoing rejection, and Annselin17 showed that SC presented both MHC Class I and II antigens 2 days posttransplantation, which is before the start of the rejection. Yu18,19 reported that SC, endothelial cells and perivascular macrophage-like cells can act as antigen presenting cells in the grafted nerve. The antigenicity of SC may change depending on the conditions of either the grafted nerve or graft bed and the progress of the rejection reaction. Viable cells are the first target of the rejection reaction and reducing the number of viable cells will reduce the antigenicity of nerve allografts.20,21 Various pretreatments of the nerve allograft have been attempted, but these treatments were concerned with preserving the donor nerve. Pretreatments include chemical treatment,22 predegeneration,23 deep freezing,24,25 freeze-drying,26,27 freezing and irradiation25 and freezethawing.28-30 Although these treatments reduce cell viability in the grafted nerve and reduce rejection, axonal regeneration is interrupted by intraneural scar formation and fibrosis. Therefore, pretreatment of the nerve allograft is qualitatively and quantitatively inferior to the autograft.31,32 The basement membrane of SC plays an important role in inducing axonal regeneration,33 but only a short length (<4 cm) of nerve can regenerate without the presence of viable SC.34-36 To our knowledge no reports demonstrate that a pretreated nerve allograft can induce nerve regeneration equal to that of the nerve autograft.

Immunosuppressive Drugs Applied to Peripheral Nerve Allograft In general, immunosuppressive regimes can be classified into pharmacological, immunological,37,38 physical, and surgical treatments. Until now, mainly pharmacological therapy has been used for nerve allotransplantation. The first clinical use of immuno-suppression was by Marmor.13 He used nerve allografts pretreated with freezing and irradiation, and azathioprine (AZA) was given postoperatively, but no satisfactory results were obtained. Gye39 used a combination of AZA and predonine postoperatively, but the results were poor. Microneural surgery40 and cyclosporine (CsA)41 may help to make clinical nerve allotransplantation a reality. The development of CsA was a great advance in transplantation medicine. CsA improved the survival rate of kidney allografts by 20-40% and reduced adverse effects such as infectious diseases or nephrotoxicity.42-44 But, because serious adverse effects are possibly caused by long term use of CsA and the nerve is not a life supporting organ, the general consensus is that administration of this immunosuppressant should be limited to the short term.45 This is a problem shared by all nonvital organ allotransplants.

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The first experimental use of CsA in nerve allotransplantation was by Zalewski.46 He demonstrated that rat nerve transplantation could be achieved across the MHC major barrier with CsA treatment for 4 weeks, but rejection occurred 13 weeks postoperatively. The results indicated that CsA could prevent acute rejection to the allografted nerve and induce axonal regeneration into the graft, but immunotolerance could not be induced with short term CsA treatment. Mackinnon and others47-49 reported that immunosuppression protected regenerated axons against rejection reactions. Midha50 demonstrated in the rat that when a sciatic nerve (2 cm) allotransplantation was performed with 12 weeks CsA treatment, regenerated axons grew into the grafted nerve, but the nerves were demyelinated after the withdrawal of CsA due to rejection. However, nerve conduction had recovered by the migration of recipient-derived SC, and re-myelination of surviving axons occurred 30 weeks postoperatively. Midha51 used mutant mice and demonstrated that the donor SC in the grafted nerves was replaced by recipient SC after rejection. There is evidence for and against present nerve conduction after rejection. Zalewski52-54 and Annselin55 reported that regenerated axons disappeared after rejection, but Ishida,56 Fraizer,57 Yu58 and de la Monte59 reported that nerve conduction could be maintained even after rejection and now this concept is almost universally accepted. We performed a similar experiment using CsA for short term administration, and investigated whether differences in donor nerve diameter influence the protection of regenerated axons against rejection. The results indicated that large diameter nerve allografts could induce better axonal regeneration than small diameter ones, which contrasts with nerve autografts.60 The first clinical use of CsA was reported by Mackinnon et al.61,62 They reported two cases using the nerve allograft, one of which was a sciatic nerve defect in an 8 year old and the other a tibial nerve defect in a 12 year old; CsA was administered 26 and 17 months respectively. Their clinical results were satisfactory, and a protective sensation in the foot could be obtained in both cases. The use of FK506 (FK) as an immunosuppressant was reported by Buttemeyer.63 FK was expected to induce immunotolerance in a nerve allograft, but Buttemeyer’s results showed that grafted nerve was rejected after withdrawal of short term FK treatment and a only small number of regenerated axons survived. Another immunosuppressive therapy consists of the administration of antibodies to block lymphocyte adhesion to endotherium. Nakao64,65 demonstrated the strong immunosuppressive effects of antibodies that blocked the function of intercellular adhesion molecule 1 (ICAM-1) and lymphocyte function associated molecule 1 (LFA-1) in nerve allografts. Various immunosuppressants have been attempted in nerve allotransplantation, but only CsA is used clinically. CsA causes a temporary decrease in nerve conduction; therefore, immunosuppressants without this deleterious effect are needed required to improve results. We wish to report a new immunosuppressant, 15-deoxyspergualin (DSG), which has unique immunosuppressive effects.

Immunosuppressive Effects of 15-Deoxyspergualin Progress of Immunosuppressive Drugs and DSG The advancement of transplantation medicine in the 1980s was mainly due to the development of new immunosuppressants. The most beneficial immunosuppressant was cyclosporine.41 CsA improved clinical transplantation results and made possible nonvital organ transplantations such as skeletal tissue61,62 and simultaneous multiple organ transplantations such as the heart and lung.66 Table 10.1 shows the progress of recent immunosuppressants.67 Normally immunosuppressive drugs have to be continuously administered to prevent rejection. However, rescue drugs are available which can treat on-going rejection,

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Table 10.1. Progress of immunosuppressive drugs and DSG

Preventive therapy Rescue therapy

1st generation (conventional era)

2nd generation (CsA era)

3rd generation (FK era)

Azathioprine Steroid ALG MP ALG

Cyclosporine Mizoribine ALG OKT3

FK506 15-deoxyspergualin

ALG: anti-lymphocyte globulin, MP: methylprednisolone

but, these drugs should be administered in large quantities and should only be administered temporarily. The representative immunosuppressant in the preCsA era was AZA, which was in clinical use in the early 1960s.68 AZA was the first drug reported which could prevent acute rejection, which made visceral organ transplantation possible for the first time. During the CsA era, CsA therapy improved inhibition of acute rejection, and the ability of CsA to prevent acute rejection also improved. CsA therapy was sometimes combined with a new drug, OKT3, which specifically acted on a T lymphocyte subset, which further improved graft survival.69 But the adverse effects of immunosuppression, such as nephrotoxicity70 and infectious disease,71 have not been resolved and delivery of the drug is still problematic, because of its irregular absorption from the gastrointestinal tract.72 The representative immunosuppressant in the postCsA era is FK.73 The mechanism of this drug is similar to that of CsA.74,75 In fact, the immunosuppressive effects of FK are stronger than CsA, even with low dose administration,76 and combination of FK with other immunosuppressants is more effective than CsA.77 The immunosuppressive mechanisms of FK are similar to those of CsA; however, FK has other immunosuppressive effects. In the postCsA era, the representative rescue immunosuppressant is DSG, which has a unique immunosuppressive action.

Immunosuppressive Mechanism of DSG

In 1981, Umezawa and Takeuchi78 derived a new antibiotic from a metabolite of Baccilus latetosporus which had an inhibitive effect on transformation of chick embryo fibroblasts due to Rous sarcoma virus; it was named spergualin. 15-deoxyspergualin79 (Fig. 10.1), the analogue of spergualin, exhibited a marked effect against mouse L-1210 leukemia cells and was proposed as an antitumor drug at first.80 DSG was developed for use as an immunosuppressant by Dicknite and coworkers.81 They demonstrated in rats that skin survival was significantly prolonged by DSG treatment at a dose of 0.5 or 2.0 mg/kg/d for 10 days. Nemoto and coworkers reported graft survival with rat Langerhans’ cell transplants to be dose-dependent.82-85 The immunosuppressive mechanism of DSG has not been fully characterized, but some authors reported that DSG inhibits macrophages, which recognize the antigen at the first step of the immune response,86 and others reported that DSG had no effect on macrophages.87 These conflicting results suggest further investigations into its mechanism are necessary. DSG does not act on the helper T cells (Th) and does not inhibit IL-2,87 both of which are the main targets of CsA and FK. DSG may inhibit the proliferation of cytotoxic T cells which have recognized the

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111

(±) H2N-C-NH-(CH2)6-CO-NH-CH-CO-NH-(CH2)4-NH-(CH2)3-NH2-3HCl || | NH OH Molecular Formula: C17H37N7O3•3HCl Molecular Weight: 496.91 Fig. 10.1. Structure of 15-deoxyspergualin

Table 10.2. Immunoresponse of 15-deoxyspergualin and cyclosporine 15-Deoxyspergualin

Cyclosporine

➔ ➔ ➔ ➔ ➔

➔ ➔ ➔ ➔ ➔

IL-1 production IL-2 production ConA Blastogenesis MLR CTL induction IL-2 dependent lymphocyte proliferation INF-r dependent lymphocyte proliferation T cell induction antibody formation Ts cell proliferation ConA: concanavalin A, MLR: mixed lymphocyte reaction ➔ = no inhibition; = inhibition

antigen on donor cells.88 The DSG mechanism for B cell inhibition is similar to the one in T cells. (Table 10.2, Fig. 10.2) Suzuki89 reported that DSG had no effects on suppressor T cells (Ts), and felt that relatively activated Ts (Ts > Tc, Th) might induce the immunotolerance. Recently, Nadler90 demonstrated that an intercellular binding protein to DSG, i.e., immunophilin, is heat shock cognate 70 (Hsc70), which is similar to heat shock protein 70 (Hsp70). The DSG-Hsc70 complex may facilitate translocation into the nucleus, where DSG may exert its immunosuppressive action.91

Immunosuppressive Effect of DSG in Animal Transplantation Models

DSG prolonged survival in rat experimental models of heart,92 pancreas,93 kidney94 and liver.95 The immunosuppressive effect varies in the different visceral organs and immunotolerance has been obtained in transplanted kidney, liver96 and heart.97 Interestingly, DSG could reverse ongoing rejection and induce immunotolerance in these tissues.97 This suggests that DSG inhibits the activated lymphocyte clone which recognizes the donor antigen. If this is true, then DSG is an ideal immunosuppressant for nonvital organ transplantations because it induces immunotolerance with short term administration. DSG was effective for treatment of graft versus host disease in bone marrow transplantation,98 and further investigation may reveal other immunosuppressive effects of DSG. Experiments using DSG in large animals, i.e., canine and monkey models, have been reported. In dogs, a sufficient dose to demonstrate the immunosuppressive effects also causes gastrointestinal symptoms such as diarrhea and appetite loss.99 The upper limit of the canine

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Fig. 10.2. Effector phase of DSG

dose is reported to be 0.6 mg/kg/d.100 The toxicity of DSG has been found to be species dependent. Long term treatment with DSG is possible in monkeys because the adverse effects are not as severe.101 Clinically, no serious adverse effects have been reported and humans may be the most suitable species for DSG administration.102 DSG may have an application in visceral xenotransplantation. Walter103 demonstrated DSG treatment could prolong the survival of the grafted hearts in the hamster to rat model as compared to CsA treatment. This result raises the possibility that DSG may be effective against hyperacute rejection, which can not be suppressed by CsA. Xenotransplantation results may also be improved by a combination of donor-specific transfusion, splenectomy and total lymphoid irradiation.104 The use of xenotransplants may resolve the problem of donor shortage. The first skeletal tissue allotransplantation was reported by Walter.105 They performed a rat limb transplant across a major MHC barrier and demonstrated that graft survival was significantly prolonged with DSG treatment at a dose of 2.5 mg/kg/d for 10 or 12 days relative to no immunosuppressive control. We achieved similar results using a limb transplant model (Muramatsu, personal communication). Although graft survival wasn’t prolonged with a combination of DSG and CsA, it was significantly prolonged with a combination of DSG and FK relative to each drug alone, suggesting a synergistic immunosuppressive effect (Muramatsu, personal communication). To our knowledge, the application of DSG for allotransplantation of other skeletal tissues has not been described.

Peripheral Nerve Allotransplantation Using 15-Deoxyspergualin Immunosuppressive Effect of DSG in Nerve Allotransplantation The primary purpose of this study was to investigate the immunosuppressive effect of DSG in rat nerve allotransplantation.106 Cyclosporine was compared to short term DSG to determine whether short term DSG therapy improves immunotolerance and axonal regeneration after withdrawal of the immunosuppressive therapy. Inbred rats whose MHC were

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113

well defined were used and the transplantation was from DA (RT1a) rats to Lewis rats (RT1l). The 20 mm long sciatic nerve dissected from the donor was interposed into a similarly sized (20 mm long) nerve defect in the recipient and both ends were microsurgically sutured. The animals were divided into the following 5 groups: Group 1: Autograft; Group 2: Allograft with no DSG treatment; Group 3: Allograft with continuous DSG treatment for 12 weeks; Group 4: Allograft with 6 weeks DSG treatment; and Group 5: Allograft with 6 weeks CsA treatment. DSG was administered at a dose of 2.5 mg/kg/d in accordance with Walter’s experiments104 and CsA was administered at a dose of 3 mg/kg/d, which maintained a physiologically active concentration in the blood. Rats were observed daily to check for any adverse effects of DSG, and nerve regeneration was assessed by sciatic function index (SFI). At the end of the 12 weeks, the wet weight ratio of the gastrocnemius was determined, and electrophysiological and histological studies were carried out (Table 10.3). The animal groups administered DSG showed severe diarrhea and lost body weight. DSG had more severe side effects than CsA at the doses used in this experiment. The nerve allografts that received continuous DSG treatment showed no histological evidence of rejection and had excellent axonal regeneration similar to that in the autograft. Grafted nerve conduction was temporarily lost after withdrawal of short term CsA treatment, and the grafted nerve showed severe histological evidence of rejection during this period. Nerve conduction loss was not observed after withdrawal of short term DSG treatment, and regenerated axon counts continued to increase after initiation of the rejection reaction (Fig. 10.3). Histological findings of rejection appeared 6 weeks after discontinuation of DSG immunosuppressive therapy. The results suggested that the decrease in regenerated axons in the short term CsA group is due to an acute, severe rejection and demyelination. However, the rejection reaction was delayed after short term DSG treatment and there was only a minimal decrease in the number of regenerated axons. The remaining incomplete immunosuppressive effect may cause these two results. Midha51 demonstrated that donor SC were replaced by recipient SC after the rejection reaction; therefore, an increase in the period of delay before the onset of the rejection reaction would benefit the early migration of recipient SC (Fig.10.4).

Table 10.3. Results of nerve allografts Group

IMS

BW

SFI

GWR

ES

1 Autograft 2 Allograft 3 Allograft 4 Allograft 5 Allograft

none none DSG 12 wk DSG 6 wk CsA 6 wk

% % ∋∋ ∋ %

∀ ∀ ∀ ∀ ∀

% ∋∋ % % ∀

%&% ∋&∀ %&% %&% ∋&%

IMS: immunosuppressant, BW: body weight, SFI: sciatic function index, GWR: gastrocnemius wet ratio, ES: electrophysiological study (after withdrawal of therapy & 12 weeks) Conduction of muscle action was detected from the gastrocnemius muscle. % = detected, ∋ = not detected, ∀ = partially detected.

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A

B

Fig. 10.4. Nerve regeneration using nerve allografts. (A) No immunosuppression; (B) Immunosuppression by CS or DSG.

Peripheral Nerve Allotransplants Immunosuppressed with DSG

115 Fig. 10.5. Comparison of sciatic nerve and saphenous nerve allografts. *p<0.05,sciatic versus saphenous group.

Nerve-Generating Effect of DSG The second purpose of the experiment was to clarify whether differences of donor nerve diameter influenced nerve regeneration after short term DSG treatment.107 The center of a large caliber nerve autograft was subject to necrosis as a result of delayed revascularization from the transplant bed. The scar formation due to necrosis interrupted axonal regeneration.11 This observation led to the successful use of cable grafts, which interposed several thin nerve grafts in a bundle. A large caliber nerve allograft has many antigen presenting cells and has poor nerve regeneration, possibly due to severe rejection.12 How does the difference in donor nerve caliber influence nerve regeneration and protection against rejection reaction after withdrawal of short term immunosuppressive treatment? Twenty millimeter sciatic nerves were orthotopically allografted as large caliber nerves (diameter = 800 mm) and 20 mm saphenous nerves were allotransplanted as small caliber nerves (200 mm).108 Nerve regeneration was evaluated by a histomorphometrical method based on regenerated axon count and density in the grafted nerve compared to the contralateral nerve. The regenerated axon count did not decrease in the sciatic nerve allografts after withdrawal of short term DSG therapy, but significantly decreased in the saphenous nerve allografts (Fig. 10.5). This result is opposed to the autograft data and suggests that a large caliber nerve allograft would be advantageous to protect the regenerated axons against rejection. Another interesting result was that the nerve allografts with short term treatment of DSG or CsA induced more regenerated axons than the autografts, suggesting that DSG and CsA may accelerate axonal regeneration. Therefore, the functional aspect of the grafted nerve should be further investigated.

Conclusions and Future Directions It may be possible to use peripheral nerve allotransplants as a reliable reconstructive procedure for nerve defects. DSG may be an important immunosuppressant, because it has an immunosuppressive action which is different from CsA and FK. Although DSG could not induce immunotolerance in rat nerve allotransplantation after short term treatment, it had demonstrated greater nerve regeneration compared to CsA, suggesting that it may improve nerve regeneration. The possibility of inducing immunotolerance by a combined use of CsA, FK and DSG should be investigated.

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Cryopreservation can keep SC viable and reduce the antigenicity of the grafted nerve109 and may lead to the establishment of a nerve bank. The immunosuppression effect of DSG in xenotransplantation should be investigated because of the unlimited source of xenodonors. Nerve growth factors can induce axonal regeneration, and the duration of immunosuppressant can be shortened if regenerated axons reach the end organs faster. The present operative indication of nerve allotransplantation should be considered carefully. Should nerve allografts be used instead of nerve autografts for reconstruction of digital nerve gaps? Should their use be limited to cases which cannot be reconstructed by autografts such as after sciatic nerve injury? The authors believe that an easy operative indication should never be contemplated until a safe and reliable immunosuppressive regimen has been established.

Acknowledgment We express our thanks to Nippon Kayaku Pharmaceuticals Co., Ltd., for providing the drug 15-deoxyspergualin, and to Daniel Crowe, Ph.D. for his help in preparing the manuscript.

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45. Muramatsu K, Doi K, Kawai S. Vascularized allogeneic joint, muscle and peripheral nerve transplantation. Clin Orthop 1995; 320:194-204. 46. Zalewski AA, Gulati AK. Rejection of nerve allograft after cessation of immunosuppression with cyclosporine A. Transplantation 1981; 31:88-89 47. Bain JR, Mackinnon SE, Hudson AR et al. The peripheral nerve allograft: A dose-response curve in the rat immunosuppressed with cyclosporine A. Plast Reconstr Surg 1988; 82:447-455. 48. 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 Recontr Surg 1988; 82:1052-1066. 49. Mackinnon SE, Midha R, Bain J et al. An assessment of regeneration across nerve allografts in rats receiving short courses of cyclosporine A immunosuppression. Neuroscience 1992; 46:585-593. 50. Midha R, Evans PJ, Mackinnon SE et al. Comparison of regeneration across nerve allografts with temporary or continuous cyclosporine A immunosuppression. J Neurosurg 1992: 78:90-100. 51. Midha R, Mackinnon SE, Becker LE. The fate of Schwann cells in peripheral nerve allografts. J Neuropath Exp Neuro 1994; 53:316-322. 52. Zalewski AA, Gulati AK, Silvers WK. Loss of host axons in nerve allografts after abolishing immunological tolerance in rats. Exp Neuro 1981; 72:502-506. 53. Zalewski AA, Slivers WK, Gulati AK. Failure of host axons to regenerate through a once successful but later rejected long nerve allograft. J Comp Neuro 1982; 209:347-351. 54. Zalewski AA, Gulati AK. Survival of nerve allografts in sensitized rats treated with cyclosporine A. J Neurosurg 1984; 60:828-834. 55. Anselinn AD, Pollard JD, Davey DF. Immunosuppression in nerve allografting; is it desirable? J Neuro Sci 1992; 112:160-169. 56. 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. 57. Fraizer J, Bronx NY, Yu L. Extended survival and function of peripheral nerve allografts after cessation of long-term cyclosporine immunosuppression in rats. J Hand Surg 1993; 18A:100-106. 58. Yu LT, England J, Hickey WF. Survival and function of peripheral nerve allografts after cessation of long-term cyclosporine immunosuppression in rats. Transplant Proc 1989; 21:3178-3180. 59. de la Monte SM, Bour C. Effects of cyclosporine A and predegeneration on survival and regeneration of peripheral nerve allografts in rabbits. Surg Neurol 1988; 29:95-100. 60. Muramatsu K, Doi K, Kawai S et al. Nerve regenerating effect of short-course administration of cyclosporine after fresh peripheral nerve allotransplantation in rat. Microsurgery 1995; 16:496-504. 61. Mackinnon SE, Hudson AR. Clinical application of peripheral nerve transplantation. Plast Reconstr Surg 1991; 90:695-699. 62. Mackinnon SE. Nerve allotransplantation following severe tibial nerve injury. J Neurosurg 1996; 84:671-676. 63. Buttemeyer 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. 64. Nakao Y, Mackinnon SE, Hertl MC et al. Monoclonal antibodies against ICAM-1 and LFA-1 prolong nerve allograft survival. Muscle Nerve 1995; 18:93-102. 65. Nakao Y, Mackinnon SE, Strasberg SR et al. The immunosuppressive effect of monoclonal antibodies to ICAM-1 and LFA-1 on peripheral nerve allograft in mice. Microsurgery 1995; 16:612-620. 66. Reitz BA, Wallwork JL, Hunt SA et al. Heart-lung transplantation, successful therapy for patients with pulmonary vascular disease. N Engl J Med 1982; 306:308.

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67. Amemiya H, Japan Collaborative Transplant Study Group for NKT-01. 15-deoxyspergualin: A new developed immunosuppressive agent and its mechanism of action and clinical effect. Artif Organs 1996; 20:832-835. 68. Murray JE, Merrill JP, Harrison JH et al. Prolonged survival of human kidney homografts by immuno-suppressive drug therapy. N Eng J Med 1963; 268:131569. Cosimi AB, Colvin RB, Burton RC et al. Use of monoclonal antibodies to T cell subsets for immunologic monitoring and treatment in recipients of renal allografts. N Eng J Med 1981; 305:308. 70. Farthing MG, Clark ML et al. Nature of the toxicity of cyclosporine A in the rat. Biochem Pharmacol 1981; 30:3311-3316. 71. Hardy AM. Infection in renal transplant recipients on cyclosporine: Pneumocystis carinii pneumonia. Transplant Proc 1983; 15:2773-2774. 72. Wassef R, Cohen Z, Langer B. Pharmacokinetic profiles of cyclosporine in rats. Transplantation 1985; 40:489-493. 73. Goto T, Kino T, Hatanaka H et al. Discovery of FK506, a novel immunosuppressant isolated from Streptomyces tsukubaensis. Transplant Proc 1987; 19:4-8. 74. Yoshimura N, Matsu S, Hamashima T et al. Effect of a new immunosuppressive agent, FK506, on human lymphocyte responses in vitro. II. Inhibition of the production of IL-2 and r-IFN, but not B cell-stimulating factor. Transplantation 1989; 47:35-39. 75. Thomson AW. FK506. Profile of an important new immunosuppressant. Transplant Rev 1990; 4:1-13. 76. Sawada S, Suzuki G, Kawase Y et al. Novel immunosuppressive agent, FK506. In vitro effects on the cloned T cell activation. J Immunol 1987; 139:1797-1803. 77. Nakajima K, Sakamoto K, Ochiai T et al. Prolongation of cardiac xenograft survival in rats treated with 15-deoxyspergualin alone and in combination with FK506. Transplantation 1988; 45:1146-1148. 78. Takeuchi T, Iinuma H, Kunimoto S et al. A new antitumor antibiotic, spergualin: Isolation and antitumor activity. J Antibiot 1981; 34:1619-1621. 79. Iwasawa H, Kondo S, Ikeda D et al. Synthesis of (-)-15-deoxyspergualin and (-)spergualin-15-phosphate. J Antibiot 1982; 35:1665-1669. 80. Umezawa H, Kondo S, Iinuma H. Structure of an antitumor antibiotic, spergualin. J Antibiot 1981; 76:312-322. 81. Dickneite G, Walter P, Schorlemmer HU et al. The immunosuppressive properties of 15-deoxyspergualin and its effects on experimental skin and islet cell transplantation. Recent Advances in Chemotherapy; Ishigami J ed. Univ of Tokyo Press, 1985; 949-950. 82. Umezawa H, Moriguchi M, Takeuchi T. Suppression of tissue graft rejection by spergualin. J Antibiot 1985; 38:283-284. 83. Nemoto K, Hayashi M, Abe H. Immunosuppressive activities of 15-deoxyspergualin in animals. J Antibiot 1987; 40:561-562. 84. Nemoto K, Hayashi M, Fuji H. Effect of 15-deoxyspergualin on graft-versus-host disease in mice. Transplant Proc 1987; 19:3985-3986. 85. Nemoto K, Hayashi M, Abe H. Inhibition by deoxyspergualin of allo-reactive cytotoxic activity in mouse graft-versus-host disease. Transplant Proc 1989; 21:3028-3030 86. Dickneite G, Scholemmer HU, Sedlacek HH et al. Suppression of macrophage function and prolongation of graft survival by the new guanidine-like structure, 15-deoxyspergualin. Transplant Proc 1987; 19:1301-1304. 87. Nemoto K, Abe F. Blastogenic responses and the release of interleukins 1 and 2 by spleen cells obtained from rat skin allograft recipients administered with 15-deoxyspergualin. J Antibiot 1987; 40:1062-1064. 88. Nishimura K, Tokunaga T. Effect of 15-deoxyspergualin on the induction of cytotoxic T lymphocytes and bone marrow suppression. Transplant Proc 1989; 21:1104-1107. 89. Suzuki S, Kanashiro M, Watanabe H et al. Therapeutic effect of 15-deoxyspergualin on acute graft rejection detected by 31P nuclear magnetic resonance spectrography, and its effect on rat heart transplantation. Transplantation 1988; 46:669-672.

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90. Nadler SG, Tepper MA, Schacter B et al. Interaction of the immunosuppressant deoxyspergualin with a member of the Hsc70 family of heat shock proteins. Science 1992; 258:484-486. 91. Muller WEG, Weissmann N, Maidhof A et al. Deoxyspergualin, a potent antitumor agent; further studies on the cytobiological mode of action. J Antibiot 1987; 40:1028-1035. 92. Walter P, Thies J, Harbauer G et al. Allogeneic heart transplantation in the rat with a new antitumor drug 15-deoxyspergualin. Transplant Proc 1986; 18:1293-1294. 93. Walter P, Dickneite G, Schorlemmer HU et al. Prolongation of graft survival in allogeneic islet transplantation by 15-deoxyspergualin in the rat. Diabetologia 1987; 30:38-40. 94. Walter P, Dickneite G, Feifel G et al. Deoxyspergualin induces tolerance in allogeneic kidney transplantation. Transplant Proc 1987; 19:3980-3981. 95. Thies JC, Walter P, Zimmermann FA et al. Prolongation of graft survival in allogeneic pancreas and liver transplantation. Eur Surg Res 1987; 19:4241-4243. 96. Engemann R, Gassel HZ, Laferenz E et al. Transplantation tolerance after short-term administration of 15-deoxyspergualin on orthotopic liver transplantation. Transplant Proc 1987; 19:4241-4243. 97. Suzuki S, Kanashiro M, Amemiya H. Effect of a new immunosuppressant, 15-deoxyspergualin on heterotopic rat heart transplantation, in comparison with cyclosporine. Transplantation 1987; 44:483-487. 98. Nemoto K, Ito J, Hayashi M et al. Effects of spergualin and 15-deoxyspergualin on the development of graft-versus-host disease in mice. Transplant Proc 1987; 19:3520-3521. 99. Collier DSJ, Calne R, Thiru S et al. 15-deoxyspergualin an immunosuppressive agent in dogs. Transplant Proc 1988; 20:240-241. 100. Amemiya H, Suziki S et al. A new immunosuppressive agent, 15-deoxyspergualin, in dog renal allografting. Transplant Proc 1989; 21:3468-3470. 101. Reichenspurner H, Hildebrandt A, Human PA et al. 15-deoxyspergualin for induction of graft nonreactivity after cardiac and renal allotransplantation in primates. Transplantation 1990; 50:181-185. 102. Amemiya et al. Clinical study of NKT-01 for refractory renal allograft rejection. Clin Rep 1991; 25:3501-3508. 103. Walter P, Bernhard U, Seitz G. Xenogeneic heart transplantation with 15-deoxyspergualin. Prolongation of graft survival. Transplant Proc 1987; 19:3993-3994. 104. Marchman W, Araneda D, DemMasi R et al. Prolongation of xenograft survival after combination therapy with 15-deoxyspergualin and total-lymphoid irradiation in the hamster to rat cardiac xenograft model. Transplantation 1992; 53:30-34. 105. Walter P, Menger MD, Thies J et al. Prolongation of graft survival in allogeneic limb transplantation by 15-deoxyspergualin. Transplant Proc 1989; 21:3186. 106. Muramatsu K, Doi K, Kawai S. Immunosuppressive effect of 15-deoxyspergualin applied to peripheral nerve allotransplantation in the rat. Exp Neuro 1995; 132: 82-90. 107. Muramatsu K, Doi K, Kawai S et al. Nerve-regenerating effect of 15-deoxyspergualin: Peripheral nerve allotransplants in the rat. Acta Orthop Scand 1996; 67:399-402. 108. Alpson D, Lal S. Combined light and electron microscopic study of the rat saphenous nerve. Acta Anat 1980; 106:141-149. 109. Karlsson JO, Toner M. Long-term storage of tissues by cryopreservation: Critical issue. Biomaterials 1996; 17:243-253.

CHAPTER 11

Therapeutic Uses of Muscle and Factors Controlling the Efficiency of Whole Muscle Graft Regeneration Miranda D. Grounds and John K. McGeachie

T

his review will consider mainly factors that control the cellular events during the regeneration of whole muscle grafts. The events that lead to the formation of new muscle in such grafts have been very well described and will only be reiterated briefly here. This review is essentially in two parts: it first outlines briefly the therapeutic applications for regeneration, myoblast transfer and whole muscle grafts, and secondly discusses in detail the factors controlling the early events of regeneration in whole muscle grafts, focusing in particular on experimental evidence related to the external lamina, the behavior of myoblasts, the importance of inflammatory cells and revascularization, the effects of denervation, and the influence of the host environment. The reinnervation, maturation and functional properties of such grafts are not addressed.

Therapeutic Benefits of Regeneration and Grafting Skeletal muscle can be transplanted as relatively intact tissue in the form of whole muscle grafts, where there is minimal physical damage or disruption of the myofiber structure.1 These grafts are the main subject of this review. The situation where whole muscles are chopped up into fragments before transplantation, widely referred to as “minced” muscle grafts,2-4 will not be discussed directly in this review. However, all of the cellular events during regeneration apply equally to minced muscle grafts, although the extensive disruption to the myofiber structure in these minced grafts generally results in extensive fibrosis and poor new muscle formation. The situation where muscle cells are isolated and cultured prior to transplantation is very topical and is discussed in some detail below. The fundamental steps of muscle regeneration are similar after any kind of damage or injury that results in necrosis and are described in detail elsewhere.1,5 In brief, they are: 1. The fragmentation of the sarcoplasm and pyknosis of myonuclei in the damaged muscle; 2. Infiltration of leukocytes from the blood stream, essential to remove the necrotic muscle—these cells also produce many growth factors, extracellular (ECM) molecules and enzymes essential for the regenerative process; 3. Activation and proliferation of mononucleated muscle precursor cells (myoblasts), widely considered to be derived from reserve myogenic cells called “satellite” cells,

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which in normal uninjured adult muscle are quiescent and located between the sarcoplasm and external lamina of myofibers; 4. Fusion of myoblasts with each other to form multinucleated new muscle cells (myotubes) which fuse with each other, with myoblasts and with surviving myofibers to form new muscle that replaces the damaged muscle; and 5. Functional innervation. Before proceeding to discuss in detail factors controlling these events in intact whole muscle grafts, we present some comments on the therapeutic aspects of the induced regeneration of muscles in situ, the transplantation of isolated cultured myoblasts and other types of muscle grafts.

Induced Regeneration of Resident Muscles The process of inducing a muscle to regenerate has been shown to have a novel effect in ablating mitochondrial myopathies.6 This unusual observation suggests that the process of muscle regeneration alone was able to reverse a genetic and biochemical defect in the patient, without using an external source of DNA. Mitochondrial myopathies are due to adverse “pathological” mutations in the extrachromosomal mitochondrial DNA (mtDNA). Both wild type and mutated mtDNA are present in the sarcoplasm of myofibers of affected patients. However, it appears that the amount of mutated mtDNA is very low or absent in the sparse cytoplasm of quiescent satellite cells (the reason for this apparent preferential lack of mutated mtDNA in these precursor cells is not clear). In response to injury induced by the local anesthetic, Bupivacaine, the satellite cells proliferate and fuse to repair the damaged myofibers. Consequently, the regenerated muscle then lacks the pathological mtDNA, thus allowing the presence of the endogenous wild type mtDNA to restore “normal” function to the muscles in these patients.6 A similar beneficial effect was demonstrated in muscles from such patients grown under tissue culture conditions.6 This correction of an inherited myopathy by the selected expression of endogenous (wild type) DNA is a most unexpected observation, and it is of interest to compare this with the approach of gene replacement by exogenous administration of DNA, either through gene therapy or myoblast transplantation (see below). Another situation where regeneration, or probably more accurately the denervation associated with whole muscle grafts, has been reported to have a beneficial effect on a myopathy is in muscular dystrophy in the laminin #2 chain (merosin)-defective dy/dy mouse model, which is discussed below under “Denervation.”

Myoblast Transplantation In the last 10 years, there has been considerable interest in the transplantation of isolated cultured myoblasts, for a diverse range of reasons: as a means of gene replacement by cell therapy in muscle (see below); to provide additional myoblasts to enhance muscle repair after damage;4,7 as a mechanism for delivery of genes into the blood stream8,9 or other specific sites such as the brain10 or joints;11 and as a replacement for cardiac muscle cells when implanted into acutely injured myocardium.12 The cell therapy approach is widely referred to as Myoblast Transfer Therapy (MTT). It is a promising cell strategy to replace the defective genes in various myopathies such as Duchenne’s muscular dystrophy (reviewed in refs. 13, 14). MTT depends on the fact that skeletal muscle is a multinucleated cell formed by fusion of mononucleated myoblasts. Thus, if the donor myoblast nucleus with the replacement “healthy” gene can be induced to fuse with defective myofibers, the donor gene product can diffuse within the shared cytoplasm of the syncytium of the mosaic muscle fiber to replace this missing gene product.15 Traditionally, such myoblast transplantation has been undertaken by culturing “normal” myoblasts to expand their numbers, followed by injection into the diseased muscles. Several

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clinical trials of MTT have already been carried out on boys with DMD with little or no success, and this has generated considerable controversy.16 The main reason for this failure appears to be that the donor myoblasts die soon after injection, due to an inflammatory and immune response by the host against the cultured myoblasts (see commentary in ref. 17, review in ref. 18). It has recently been demonstrated that cultured myoblasts can produce neoantigens which can provoke rejection after allotransplantation,19 and this probably contributes to the failure of clinical trials. However, in marked contrast to the rapid death of injected myoblasts,20 if the same donor muscle, instead of having the myoblasts isolated and cultured for injection, is implanted in the form of allografted whole muscles or sliced segments of relatively intact myofibers, then there is no immune rejection and the donor myoblasts survive for up to a year.21 Extensive movement of donor myoblasts, up to 2.3 mm away from the graft, and fusion with host myofibers can be demonstrated with such sliced muscle grafts,21,22 although there is no movement of donor cells out from intact grafts, presumably due to the connective tissue barrier of the epimysium. Furthermore, with the sliced muscle grafts, the survival and movement of donor myoblasts within the host muscle is enhanced when the host is immunodepleted with anti-CD4 and anti-CD8 antibodies prior to graft implantation, again indicating a role for immune cells in these processes.22 Thus, such grafts of muscle segments, where the replacement donor cells readily survive the transplantation event, may prove to be of considerable value as a strategy for MTT.

Whole Muscle Grafts Grafts of whole muscles, where the vasculature and neural supplies are completely disrupted and may or may not be reconnected by microsurgery, have been of considerable surgical interest for many years. Their use by Thompson in 197123 marked a resurgence of clinical interest, and subsequently they have been used clinically to treat disorders such as anal and urinary incontinence;24 facial paralysis;25,26 and the repair of injury in the upper limb27, 28 (see also refs. 29-31). Skeletal muscles, particularly the anterior latissimus dorsi (ALD), have also been used experimentally in dogs to construct skeletal muscle ventricles to assist cardiac function.32 The sternocleidomastoid muscle has also been used to repair cardiac ventricular defects in dogs.33 In humans, the ALD has also been used for cardiomyoplasty34 in order to reinforce myocardial defects to assist cardiac function when cardiac transplantation is not available. In some of these surgical procedures, the muscle is relocated but remains attached at one end (to sustain a viable blood supply) throughout the procedure, rather than being completely removed and transplanted. Unlike skeletal muscle, mature cardiac muscle lacks myogenic cells with the capacity to proliferate and it is therefore unable to regenerate. Thus, the skeletal muscle transplant with appropriate electrical stimulation is designed to replace (or at least augment) the cardiac muscle. Conventional grafts of intact whole muscles have been widely studied in experimental animal models, particularly rats1,31,35-40 and mice,41-47 where relatively small muscles such as the soleus or extensor digitorum longus (EDL) are usually transplanted; these are discussed in detail below.

Factors Controlling the Regeneration of Whole Muscle Grafts The experimental procedure used in our laboratory for orthotopically transplanted EDL muscle grafts in mice is illustrated in Figure 11.1. The cellular changes during the early stages of regeneration in whole muscle grafts are illustrated in Figure 11.2. The sequence of events throughout regeneration have been described extensively for both mice 45,47 and rats.1,35,36,38

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Fig. 11.1. Schematic diagram of the grafting and tissue sampling procedures. (A) The extensor digitorum longus (EDL) is isolated from its anatomical bed and neurovascular connections. (B) The EDL is placed over the underlying tibialis anterior (TA) muscle. (C) The proximal and distal tendons of the EDL are sutured to the tendon of quadriceps femoris and TA respectively. (D) The EDL and TA muscles are removed intact, usually after perfusion with fixative. (E) Fixed muscle samples are transected in the mid-belly region. (F) Each half is embedded, either in resin or paraffin wax and sectioned, as shown. Fig. 11.2. (opposite) Transverse sections through a whole extensor digitorum longus muscle graft and the underlying host tibialis anterior muscle (as illustrated in Fig. 11.1F) sampled at 4 and 5 days after transplantation. (A) This low power view of a graft removed after 4 days shows the relationship between the graft and the underlying host muscle. (B) A higher power view through the graft shows the main cellular events from the periphery (at the bottom) towards the center. Some surviving myofibers, identified by their darkly staining cytoplasm and peripheral myonuclei, are present at the edge of the graft (SM). Note the active

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cellular zone of the graft where there is evidence of the removal of necrotic sarcoplasm by macrophages (ND), new blood vessels (BV), myoblasts in the satellite cell position, “cuffing” within the surviving external lamina of necrotic myofibers (SC), and small multinucleated myotubes (MT). Persisting necrotic myofibers (NF) are seen towards the center of the graft; these will be progressively removed over time as the regenerative zone moves inwards.

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Myofiber Survival and the Importance of the External Lamina Survival of Myofibers Most of the myofibers in whole muscle grafts usually undergo necrosis except for a few that survive at the edge, presumably kept alive by diffusion of nutrients and oxygen from the extracellular fluid. Autolysis of the sarcoplasm is initially more pronounced at the periphery compared with the center of the graft, and studies from our laboratory indicate that this can occur to a limited extent in the absence of both revascularization and leukocyte infiltration (unpublished data). However, denervation of muscles prior to transplantation enhances the survival of myofibers in whole muscle grafts in rats,48 and more than 80% of the myofibers in 7 month denervated grafts survive the transplantation process.30 It is suggested that long-term denervation may produce changes in calcium metabolism and thereby reduce the calcium-induced necrosis that normally follows ischemia (see further discussion under “Denervation”, below).

Persistence of Components of the External Lamina in Necrotic Myofibers of Grafts The external lamina (often referred to as the basal lamina or basement membrane) is closely associated with the external surface of the cell membrane surrounding myofibers. One of the redeeming features of whole muscle grafts (and of relatively intact segments of skeletal muscle) is that the external lamina maintains its structural integrity throughout the process of necrosis and regeneration, and provides a scaffold within which the new muscle is formed.49 This feature is probably of critical importance in contributing to the excellent regeneration seen in such grafts. It not only serves as a physical barrier to orientate the newly formed myotubes and to exclude fibroblasts, but some of the molecular constituents directly influence the behavior of myoblasts, as well as stabilizing the functional mature myofiber (see below). A slow breakdown of components of the external lamina around necrotic myofibers in whole muscle grafts in rats was reported by Gulati in 1985.39 It has further been shown that laminin #2 and collagen IV in the external lamina persist in the necrotic muscle tissue at the center of whole muscle grafts in mice, in marked contrast to the situation where host muscles are damaged in situ by crush injury.50 This difference is attributed to the relatively intact vascular connections in crush injured muscles in contrast to the complete isolation of the avascular grafted tissue. It can be speculated that some serum-derived factors, such as proteases, are responsible for the rapid breakdown of these ECM components in the situation where muscle is injured.

Influence of the External Lamina The external lamina is a complex glycocalyx composed of collagen IV, laminin-entactin complexes, proteoglycans and other various minor components. In vitro studies have demonstrated a role for some of these ECM components during myogenesis. It has been shown that myoblasts attach, proliferate, migrate and differentiate into myotubes preferentially on laminin-1, as compared with interstitial ECM substrates such as collagen type 1 or fibronectin.51-54 It has also been shown that myotube formation is more frequent and extensive on Matrigel,55,56 a basement membrane-like substrate comprising mainly laminin-1, entactin and collagen type IV, than on gelatin, which is denatured collagen type 1. In the external lamina of skeletal muscle, laminin-2 (also called merosin) is the predominant form of laminin54,57,58 although the laminin #5 chain is also expressed during embryogenesis and transiently during regeneration of mature skeletal muscle.50,59 A defect

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in the laminin #2 chain of laminin-2 also leads to a severe muscular dystrophy in dy/dy mice, emphasizing the importance of the external lamina for normal muscle function.59-61 Various components of the external lamina are known to interact directly with molecules on the muscle cell surface.62 For example, laminins bind to integrins63 and specific proteoglycans, e.g., syndecans, interact with receptors for heparin sulfate-binding growth factors such as fibroblast growth factor (FGF)64-66 and m-Met, which is the receptor for hepatocyte growth factor;67, 68 both of these growth factors are potent mitogens during myogenesis. Furthermore, there are numerous modifications to the external lamina in the region of the synapse that assists in attracting ingrowing nerves to the original site of innervation.69 The combination of these interactions gives some idea of the critical importance of the external lamina in normal muscle function and during muscle regeneration. It is not known to what extent the lack of an external lamina around myoblasts which have been isolated and cultured before injection in myoblast transfer therapy might contribute to the poor survival of the donor muscle cells in this situation, in marked contrast to the excellent survival seen with sliced muscle grafts where donor myoblasts are enclosed in an essentially intact external lamina.

Identification and Behavior of Myoblasts Identification In order to study the numbers, movement and behavior of myoblasts (or satellite cells) a suitable means of reliably identifying them is required. Traditionally, satellite cells could only be accurately identified by their anatomical location between the sarcolemma and external lamina of myofibers, and this necessitated the use of electron microscopy.70,71 More recently, the use of specific antibodies to dystrophin (to mark the sarcolemma) and collagen IV (to label the external lamina) has enabled the identification of satellite cells on the basis of their position at the light microscopic level with confocal microscopy.72 However, once satellite cells are activated and move out of this classical position, they can no longer be distinguished from other mononucleated cells such as fibroblasts. These problems in identifying myoblasts are reviewed in detail elsewhere.5,73 What is required is some specific marker that would be present in/on all activated myoblasts and ideally would also be expressed by quiescent satellite cells. In the last 10 years, some promising candidate molecules have been described. In 1987, the first gene to be expressed exclusively by skeletal muscle cells, MyoD, was identified, and several related skeletal muscle specific transcription factors, myogenin, myf5 and MRF4, soon followed. In situ studies on tissue sections with mRNA probes and antibodies have demonstrated that expression of MyoD, myogenin74-77 and Myf578 increases rapidly in myoblasts in regenerating adult muscles. Initial difficulties were experienced with the development of antibodies to these proteins that were suitable for studies on sections of mature muscle, and thus relatively few studies have been reported for regenerating muscles to date.75-77 However, antibodies to the cytoskeletal protein, desmin (also found in smooth and cardiac muscle), have proved very useful as a markers for myoblasts in regenerating rodent muscles.44,76,79,80 Desmin is present in the sparse cytoplasm of satellite cells72 and is unregulated very rapidly in response to trauma. Recently, two other promising candidate molecules present on quiescent and activated satellite cells have been described: These are the cell surface proteins M-cadherin81,82 and m-Met, which is the receptor for hepatocyte growth factor.67,68,83 Hopefully, this trend will continue until a battery of suitable markers is available. Rather than using antibodies to the marker protein, another approach to identifying myoblasts is the use of transgenic mice where a reporter gene, such as lacZ, is linked to the promoter of the candidate gene, e.g., desmin.84 In this situation, expression of

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the candidate gene also results in expression of the lacZ gene, whose product (∃-galactosidase) is readily detected enzymatically by production of a blue color.

Influence of Growth Factors and Extracellular Matrix (ECM) Molecules A vast number of growth factors and some ECM molecules have been shown to affect the proliferation and fusion of myoblasts under tissue culture conditions (these are detailed in numerous reviews).5,62,67,73,85 More recently, there has been additional interest in factors such as hepatocyte growth factor, which appears to be an early mitogen for myoblasts,67,68 cytokines like interleukin 6,85-88 interferon-a89 and leukemia inhibitory factor,86,90,91 and in a factor produced by injured muscle which is a specific and potent mitogen for myoblasts92-94 and which may actually be the hepatocyte growth factor.68 All of these growth factors and ECM molecules interact in the complex in vivo environment. Many of them, such as FGF also stimulate angiogenesis (see “Revascularization” below), and many others, including proteolytic fragments of ECM molecules, stimulate the chemotaxis of inflammatory cells and myoblasts.93,95 Thus, in order to evaluate their actual importance during myogenesis, it is essential to assess the effects of the various growth factors and ECM molecules in vivo. There have been remarkably few instances of such in vivo studies in postnatal regenerating skeletal muscles. Exogenous administration of FGF-2 in a range of situations had no effect on muscle regeneration,96 indicating that FGF is probably not a limiting factor; instead, the availability of receptors and specific proteoglycans may be critical. In support of this idea, the in vivo administration of chemically substituted dextrans, which may protect heparin-binding growth factors such as FGF-2, has been shown to increase muscle repair after crush injury97 and enhance reinnervation of damaged muscles.98 The in vivo administration of LIF has also been shown to enhance muscle repair in a range of situations.80,90,91 The addition of extra macrophages improves muscle regeneration in vivo,99 as does the addition of extract from crushed muscles,4 supporting the idea that regeneration can be assisted by the exogenous administration of various factors. Earlier studies with daily intramuscular injections of the synthetic corticosteroid dexamethasone (1 mg/kg to 100 mg/kg) showed no improvement in muscle regeneration after crush injury in BALB/c mice.100 Similar administration of the cytokine interferon-# (2.25 x 103 IU/dose), resulted in impaired regeneration in SJL/J mice with persisting necrotic tissue, reduced myotube formation and increased fibrosis at 10 days after crush injury.89 Anabolic effects of exogenous IGF-I have been demonstrated in dystrophic muscles of mice, although this is probably due largely a decrease in protein degradation.101 However, exogenous administration of the thyroid hormone triodothyronine increased the severity of the dystrophy, particularly in younger mdx mice,102 probably due to metabolic effects and a modulation of myosin synthesis. The increasing number of “null” mice being engineered by silencing specific genes, combined with crossbreeding these and existing strains with inherited gene defects, provides a wealth of opportunity to dissect the in vivo importance of individual factors during the regenerative process.

Inflammatory Cell Response and Revascularization Leukocyte Infiltration Polymorphonuclear leukocytes (PML) accumulate very rapidly in damaged muscles and predominate in the first 24 hours after crush injury, but they are rapidly replaced by macrophages after this time. The rapid revascularization of PML in response to chemokines produced by tissue damage has been widely studied and is a very important event in general tissue repair. Large numbers of platelets may also be present after severe trauma, and they also produce many factors that facilitate wound repair. Tissue culture studies of chemotaxis

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in muscle95,103 and other tissues show that the PML produce soluble factors that chemoattract macrophages to the damage site. However, it should be noted that PML are not conspicuous in whole muscle grafts in the first few days due to the lack of vascular connections, since invading vessels are not evident in the graft until 72 hours after grafting.45 Macrophages are the predominant leukocyte in whole muscle grafts at all times and, as for any muscle damage, they play a critically important role during regeneration.87,104 They are essential for the effective removal of necrotic tissue and produce a vast array of growth factors and enzymes that influence many aspects of the regenerative process, including angiogenesis, the ECM environment and the chemotaxis, proliferation and differentiation of myoblasts.5,93,95,103 The question arises of exactly what attracts macrophages to the necrotic tissue in whole muscle grafts. While tissue culture studies, using Boyden chambers, show that crush injured muscle itself (in the absence of infiltrating leukocytes) does produce soluble factors that chemoattract macrophages,95 it appears that such soluble factors are not produced by whole muscle grafts, at least within the first 3 days after transplantation. This implies that an intact vasculature (or neural supply) may be required for the production of the soluble chemotactic signal. Since macrophages do eventually move out of the host vasculature into whole muscle grafts, some agent must presumably be attracting these cells to the graft. From a clinical point of view, if this signal in the muscle graft could be identified and presented to the macrophages at an earlier time, this might accelerate the inflammatory response and thereby “speed up” the process of regeneration in surgical situations. Furthermore, since leukocytes play such a critical role in regeneration, it would seem that if leukocytes were “primed” or more stimulated or “preactivated” at the time of muscle transplantation, this should also increase the efficiency of regeneration. There is evidence that the state of “activation” of host macrophages does directly influence the speed of regeneration. When whole muscle grafts were cross-transplanted between two strains of mice with strikingly different regenerative capacity—SJL/J mice have superior regeneration compared with BALB/c mice105-the pattern of regeneration reflected the strain of the host mouse, showing that it is the host environment (rather than the muscle itself) that can determine the efficiency of muscle regeneration.44 Since this is not accounted for by genetic differences between the bone marrow-derived cells from the two strains,106 it seems most likely that some factor (presumably blood-borne) in the SJL/J mice affects leukocytes so that they are in a “more activated” state. A similarly important role of the host environment was observed when muscles were transplanted between old rats (where regeneration is slower) and young rats (with superior regeneration), as it was the age of the host (and not the muscle) that determined the pattern of regeneration in long term grafts.107 Again, it seems likely that inflammatory cells might be “less active” due to some factors in the old host environment, compared with those in young animals.87,108 The questions remain: What controls the avidity of the macrophage response, and can it be enhanced?

Revascularization Autoradiographic labeling of replicating cells in whole muscle grafts showed that endothelial cell proliferation occurred first in the underlying host TA muscle (see Figs. 11.1, 11.2) and it was considered that vascular sprouts grew from the host into the graft.45 Replication of myoblasts occurs slightly ahead of the appearance of macrophages,45 and these phagocytic leukocytes move just ahead of blood vessels which sprout into the transplanted muscles.36 Macrophages and capillary sprouts are seen first at the periphery around the rim of the graft (Fig. 11.2) and, over time, they progressively move inwards toward the center of the necrotic tissue.45

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The Manipulation of Revascularization of Muscle Grafts Stimulation of angiogenesis, and thus rapid revascularization, could be particularly important for the transplantation of very large grafts where prolonged ischemia in the central necrotic area favors the proliferation of fibroblasts, and hence fibrosis.46 The use of pharmacological agents which enhance vascular growth definitely improves the revascularization of experimental muscle grafts: both clenbuterol46 and isoprenaline109 improve vascularization of grafted muscles, although isoprenaline can be subject to a dose dependent effect. Conversely, the inhibition of vascular growth into muscle grafts by the drug Propranolol definitely inhibits the revascularization of the central necrotic core of the graft.110 Exercise also influences the speed and efficiency of revascularization: both pre- and postgrafting exercise enhances revascularization.111 Apart from the effects of exercise, vascularity is also affected by aging, with a reduced blood supply,112 decreased capillary density113 and changes in vascular pathology114 being reported in older subjects. Macrophages undoubtedly play a major role in stimulating revascularization of the graft, since they are known to produce many factors which increase angiogenesis. These include factors that break down the ECM, chemotactic signals and mitogens for endothelial cells. There is a considerable research interest into factors that enhance or inhibit angiogenesis, particularly from the perspective of tumorogenesis.115,116

Denervation Effect of Denervation It is known that denervation alone does not induce regeneration, although myotubes have been reported in long-term denervated rat muscle.37 Apart from loss of function due to no electrical stimulation, the main change after denervation is atrophy of the myofibers and the movement of myonuclei to a central position. In addition, it has been shown that satellite cells are stimulated to proliferate at a low rate,117,118 although these cells do not fuse with the denervated myofibers, but instead appear to disappear from the muscle mass.119 Replication of these satellite cells was measured accumulatively from the time of denervation up to 4 weeks postoperatively.119 Such proliferation of satellite cells in denervated muscle has also been well documented by electron microscopy.37 It thus might be considered that these denervated myofibers are “primed” as proposed by Studitsky and his colleagues,120, 121 which will be discussed below. However, it has also been reported that the number of satellite cells in long-term denervated muscle decreases,122,123 which would seem to be disadvantageous to any potential regeneration.

Regeneration of Denervated Skeletal Muscle It is known that the early events of muscle regeneration, i.e., myoblast proliferation and myotube formation, are independent of innervation, as shown by muscle transplantation37,47,124 and crush injury experiments with denervated muscles.118 There is considerable interest in the effect of denervation on regeneration, particularly because of situations like Bell’s palsy where facial muscles are paralyzed. Attempts have been made to restore function to these denervated muscles by surgically reconnecting nerves and producing reinnervation. However, it appears that this is ineffective with muscles that have been denervated for a long time, as they are refractory to reinnervation. Therefore, transplantation of muscles is undertaken to replace these long-term denervated muscles. A preferable alternative to muscle transplantation would be to find a way of making the endogenous muscle regenerate and become reinnervated (discussed by Carlson).31 In 1963, Studitsky and associates in Russia carried out pioneering work on muscle regeneration120,121 and proposed that predenervation of muscle made the muscle more “plas-

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tic”, which resulted in faster and more effective regeneration after transplantation. The work of Studitsky was brought to the attention of the wider scientific community by Bruce Carlson in Ann Arbor in the mid 1970s;35 he has continued to be a leader in this field and has extensively studied denervated muscle grafts.31,48,125 Experiments to test the proposal of “plasticity” showed that there was no significant difference between the regenerative capacity of muscle which had been predenervated for 14 days compared with nondenervated control muscle grafts,48 although the denervated muscle grafts had a greater proportion (15-20%) of surviving myofibers after grafting, compared with the nondenervated grafts (2-5%). Thus, this idea has largely lost support. However, recent experiments with long-term denervated (7 months) muscles in rats would appear to potentially endorse such a concept.30 Unlike nondenervated or short-term (up to 4 months) denervated muscle grafts where the majority of the implanted myofibers undergo necrosis followed by regeneration, muscles that have been denervated for a very long time appear resistant to necrosis and most (80-95%) of the myofibers survive the ischemic conditions of transplantation.30 Thus, new muscle formation is not stimulated in these grafts. Why do these grafts not undergo necrosis? It was proposed that long-term denervation resulted in a reduction in calcium-induced necrosis due to changes in the sarcoplasmic reticulum.30 It was also proposed that these long-term denervated muscles had lost the capacity to be reinnervated, due to downregulation of a range of cell adhesion and other molecules needed for reinnervation.30 In order to force regeneration to occur in these grafts, Billington and Carlson30 soaked the muscles to be grafted in Marcaine, a local anesthetic which is known to kill muscle fibers and result in new muscle formation. The remarkable observation was made that in grafts of long-term denervated muscle which were forced to undergo necrosis, there was rapid and extensive myotube formation throughout the entire graft at 5 days, in marked contrast to the peripheral rim of myotubes seen in other grafts at this time. This suggests that these long-term denervated muscles may indeed have been “primed” as originally proposed by Studitsky. These important observations lead to the possibility that if surgical reinnervation of long-term denervated muscle was combined with the forced induction of necrosis in the endogenous muscles (perhaps by injection of the local anesthetic Marcaine), this could very successfully induce muscle regeneration and thereby facilitate reinnervation and restore function without the need for transplantation. This situation clearly requires further investigation. The nature of this fascinating myogenic response has not yet been addressed. It may well be that satellite cells are highly activated, i.e., express MyoD and myogenin or even continue to replicate (as indicated by the denervation studies of McGeachie118 which were only extended to 4 weeks), and the status of these cells should be determined. The unusual and striking formation of myotubes throughout the entire graft at 5 days strongly suggests that some potent angiogenic agent is produced by muscle in this situation, or that the myotube formation is independent of revascularization (which would be most unusual).

Ablation of Muscular Dystrophy by Denervation or Grafting In 1986, Bourke and Ontell reported that when EDL muscles of the dystrophic dy/dy mouse were transplanted, the grafted muscles appeared “healthier” with less evidence of dystrophy at 100 days than in nonoperated dy/dy muscles. A similar improvement of both the morphological126 and physiological127 properties was reported when muscles of these dystrophic mice were denervated at 2 weeks of age. Furthermore, tissue culture studies indicate that myoblasts derived from predenervated dy/dy muscles that had been allowed to reinnervate had a greater myogenic capacity in vitro than myoblasts derived from unoperated dy/dy muscles, supporting the idea that the operated muscles had been spared the progressive cycles of necrosis and regeneration that normally characterize the dy/dy muscles.128

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The dy/dy myopathy is now known to be due to a gene defect which results in a defect in laminin-2, which is a major component of the external lamina (basement membrane) in skeletal muscle.59-61 The studies described above suggest that regeneration or denervation modifies a component of the postnatal dystrophic muscle, probably at the level of the external lamina in such a way that the myofiber becomes resistant to the dystrophic process. The precise nature of this modification is yet to be elucidated. One possibility is that some basement membrane component which is normally only transiently expressed might become permanently upregulated.

Influence of the Host Environment Several lines of evidence, as outlined above, point to a crucial role for the host environment in influencing the efficiency of muscle regeneration. To recapitulate briefly, it is the age of the host (rather than the age of the grafted muscle) that determines how well the graft regenerates;107 thus, age is a factor to be considered.108 Genetics can also play a role. Some strains of mice show superior regeneration to others3 and, similarly, it is the genetic background of the host rather than that of the graft that contributes to the speed of regeneration in whole muscle grafts made between different strains of mice.44 This strain-specific difference in regenerative response is much more pronounced where the architecture of the tissue is severely disrupted in minced muscle grafts3 and crush injured muscles105 compared with the whole muscle grafts.44 This difference would seem to reflect a strain-related dependency of the myoblasts for the appropriate ECM, with BALB/c myoblasts being highly dependent on a rich ECM substrate56 and thus being severely disadvantaged in the absence of an intact external lamina, which is the situation in both minced and crushed muscles. The efficiency of the host inflammatory response (which can be influenced by age, fitness and genetics) and the capacity of the host for revascularization (which can depend on age, fitness and genetics) are aspects of the host environment that can influence the capacity for muscle regeneration.

Clinical Implications For clinical purposes, promising strategies that should be considered to improve the efficiency of new muscle formation in whole muscle grafts are: 1. Increasing the speed and avidity of the macrophage response; and 2. Stimulating revascularization; 3. Pretreatment of the graft by long-term predenervation and induced regeneration.

Acknowledgments The long term research support from the National Health and Medical Research Council of Australia to M. Grounds and fellow researchers is gratefully acknowledged.

References 1. Carlson BM. Regeneration of entire skeletal muscles. Fed Proc 1986; 45:1456-1460. 2. Carlson BM. The regeneration of minced muscles. Monographs in developmental biology. Karger, Basil 1972; 4:3-128. 3. Grounds MD. Phagocytosis of necrotic muscle in muscle isografts is influenced by the strain, age and sex of host mice. J Pathol 1987; 153:71-82. 4. Bischoff R, Heintz C. Enhancement of skeletal muscle regeneration. Dev Dyn 1994; 201:41-54. 5. Grounds MD. Towards understanding skeletal muscle regeneration. Pathol Res Pract 1991; 187:1-22. 6. Clark KM, Bindoff LA, Lightowlers RN et al. Reversal of a mitochondrial DNA defect in human skeletal muscle. Nature Genet 1997; 16:222-224.

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7. Arcila ME, Ameredes BT, DeRosimo JF et al. Mass and functional capacity of regenerating muscle is enhanced by myoblast transfer. J Neurobiol 1997; 33:185-198. 8. Dau Y, Roman M, Naviaux RK et al. Gene therapy via primary myoblasts: Long-term expression of factor IX protein following transplantation in vivo. Proc Natl Acad Sci, U S A 1992; 89:10892-10895. 9. Naffakh N, Pinset C, Montarras D et al. Long-term secretion of therapeutic proteins from genetically modified skeletal muscles. Hum Gene Ther 1996; 7:11-21. 10. Jiao S, Williams P, Safda N et al. Cotransplantation of plasmid transfected myoblasts and myotubes into rat brains enables high levels of gene expression long-term. Cell Transplant 1993; 2:185-192. 11. Day CS, Kasemkijwattana C, Menetrey J et al. Myoblast mediated gene transfer to the joint. J Orthopaedic Res 1997; 15:894-903. 12. Chiu RC-J, Zibaitis A, Kao RL. Cellular cardiomyoplasty: Myocardial regeneration with satellite cell implantation. Ann Thorac Surg 1995; 16:12-18. 13. Partridge TA. Invited review: Myoblast transfer: A possible therapy for inherited myopathies? Muscle Nerve 1991; 14:197-212. 14. Morgan JE. Cell and gene therapy in Duchenne muscular dystrophy. Hum Gene Ther 1994; 5:165-173. 15. Chamberlain JS. Dystrophin levels required for genetic correction of Duchenne muscular dystrophy. Basic Appl Myology 1997; 7:251-255. 16. Partridge T, Beauchamp J, Morgan J et al. Letter to the editor. Cell Transplant 1997; 6(2):195-196. 17. Grounds MD. Commentary on the present state of knowledge for myoblast transfer therapy. Cell Transplant 1996; 5(3):431-433. 18. Tremblay J, Guerette B. Myoblast transplantation: A brief review of the problems and of some solutions. Basic Appl Myology 1997; 7:221-230. 19. Irintchev A, Langer M, Zweyer M et al. Myoblast transplantation in the mouse: What cells do we use? Basic Appl Myology 1997; 7:161-166. 20. Fan Y, Maley M, Beilharz M et al. Rapid death of injected myoblasts in myoblast transfer therapy. Muscle Nerve 1996; 19:835-860. 21. Fan Y, Beilharz MW, Grounds MD. A potential strategy for myoblast transfer therapy: The use of sliced muscle grafts. Cell Transplant 1996; 5(3):421-433. 22. Fan Y, Grounds MD, Garlepp MJ et al. Increased survival, movement and fusion of myoblasts from sliced muscle grafts into T cell depleted and tolerised dystrophic host mice. Basic Appl Myology 1997; 7:231-240. 23. Thompson N. Autogenous free grafts of skeletal muscle. Plast Reconstr Surg 1971; 48:11-27. 24. Gierup J Hakelius L. Free autogenous muscle transplantation in five children with urinary incontinence. Z Kinderchir 1979; 104:1424-1428. 25. Harii K, Ohmori K, Torii S. Free gracilis muscle transplantation, with microneurovascular anastomoses for the treatment of facial paralysis. Plast Reconstr Surg 1976; 57(2):133-143. 26. Harrison DH. The pectoralis minor vascularized muscle graft for the treatment of unilateral facial palsy. Plast Reconstr Surg 1983; 75(2):206-213. 27. Gilbert A. Free muscle transfer. Int Surg 1981; 66:33-35. 28. Schenck RP. Rectus femoralis muscle and composite skin transplantation by micro-neurovascular anastomosis for avulsion of forearm muscles. J Hand Surg 1978; 3:60-69. 29. Freilinger G, Holle J, Carlson BM. Muscle transplantation. eds. New York: Springer Verlag, 1981:1-311. 30. Billington L, Carlson BM. The recovery of long-term denervated rat muscle after Marcaine treatment and grafting. J Neurol Sci 1996; 144:147-155. 31. Carlson BM, Billington L, Faulkner J. Studies on the regenerative recovery of long-term denervated muscle in rats. Rest Neurol Neurosci 1996; 10:77-84. 32. Ruggiero R, Niinami H, Hooper TL. Skeletal muscle ventricle for cardiac assist. Basic Appl Myology 1991; 1:129-135. 33. Sola OM, Dillard DH, Ivey TD et al. Autotransplantation of skeletal muscle into myocardium. Lab Invest 1985; 71:341-348.

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34. Carraro U, Chacques JC, Desmnos M. Eight year cardiomyoplasty: Preserved structure of myofibers and vessels of the latissimus dorsi. Basic Appl Myology 1996; 6:333-340. 35. Carlson BM, Gutmann E. Regeneration in free grafts of normal and denervated muscles in the rat: Morphology and histochemistry. Anat Rec 1975; 183:47-62. 36. Hansen-Smith FM, Carlson BM. Cellular responses to free grafting of the extensor digitorum longus muscle in rat. J Neurol Sci 1979; 41:149-173. 37. Schmalbruch H, Lewis DM. A comparison of the morphology of denervated with aneurally regenerated soleus muscle of rat. J Muscle Res Cell Motil 1994; 15:256-266. 38. Schmalbruch H. Regeneration of soleus muscles of rat autografted in toto as studied by electron microscopy. Cell Tissue Res 1977; 177:159-180. 39. Gulati AK. Basement membrane component changes in skeletal muscle transplants undergoing regeneration or rejection. J Cell Biochem 1985; 27:337-346. 40. White TP, Devor ST. Skeletal muscle regeneration and plasticity of grafts. Exerc Sport Sci Rev 1993; 21. 41. Grounds M, Partridge TA, Sloper JC. The contribution of exogenous cells to regenerating skeletal muscle: An isoenzyme study of muscle allografts in mice. J Path 1980; 132:325-341. 42. Bourke DA, Ontell M. Modification of the phenotypic expression of murine dystrophy: A morphological study. Anat Rec 1986; 214:17-24. 43. Roberts P, McGeachie JK, Grounds MD et al. Initiation and duration of myogenic precursor cell replication in transplants of intact skeletal muscles: An autoradiographic study in mice. Anat Rec 1989; 224:1-6. 44. Roberts P, McGeachie JK, Grounds MD. The host environment determines strain specific differences in the timing of skeletal muscle regeneration: Cross-transplantation studies between SJL/J and BALB/c mice. J Anat 1997; 191:585-594. 45. Roberts P, McGeachie JK. Endothelial cell activation during angiogenesis in freely transplanted skeletal muscles in mice and its relationship to the onset of myogenesis. J Anat 1990; 169:197-207. 46. Roberts P, McGeachie JK. The influence of revascularization, vasoactive drugs and exercise on the regeneration of skeletal muscle, with particular reference to muscle transplantation. Basic Appl Myology 1992; 2(1):5-16. 47. Roberts P, McGeachie JK. Skeletal muscle transplants and models of muscle regeneration. In: Green MK, Mandel TE, eds. Experimental transplantation models in small animals. Harwood Academic Publishers, Australia,1995:213-224. 48. Carlson BM. A quantitative study of muscle fiber survival and regeneration in normal, predenervated and marcaine-treated free muscle grafts in the rat. Exp Neurol 1976; 52:421-432. 49. Vracko R, Benditt EP. Basal lamina: The scaffold for orderly cell replacement. J Cell Biol 1972; 55:406-419. 50. Grounds MD, McGeachie JK, Davies MJ et al. The expression of extracellular matrix during adult skeletal muscle regeneration: How the basement membrane, interstitium, and myogenic cells collaborate. Basic Appl Myology 1998; 8:129-141. 51. Kuhl U, Ocalan M, Timpl R et al. Role of laminin and fibronectin in selecting myogenic versus fibrogenic cells from skeletal muscle cells in vitro. Dev Biol 1986; 117:628-635. 52. Goodman SL, Risse G, Von der Mark K. The E8 fragment of laminin promotes the locomotion of myoblasts over extracellular matrix. J Cell Biol 1989; 109:799-809. 53. Ocalan M, Goodman SL, Kuhl U et al. Laminin alters cell shape and stimulates motility and proliferation of murine skeletal myoblasts. Dev Biol 1988; 125:158-167. 54. Schuler F, Sorokin LM. Expression of laminin isoforms in mouse myogenic cells in vitro and in vivo. J Cell Sci 1995; 108:3795-3805. 55. Hartley RS, Yablonka-Reuveni Z. Long term maintenance of primary myogenic cultures on a reconstituted basement membrane. In Vitro Cell Dev Biol 1990; 26:955-961. 56. Maley MA, Davies MJ, Grounds MD. Extracellular matrix, growth factors, genetics: Their influence on cell proliferation and myotube formation in primary cultures of adult mouse skeletal muscle. Exp Cell Res 1995; 219:169-179.

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57. Ehrig K, Leivo I, Argraves WS et al. Merosin: A tissue-specific basement membrane protein is a laminin-like protein. Proc Natl Acad Sci U S A 1990; 87:264-3268. 58. Sewry, CA, Chevallay M, Tome FM. Expression of laminin subunits in human fetal skeletal muscle. Histochem J 1995; 27:497-504. 59. Ringelmann B, Roder C, Hallmann R et al. Expression of laminin #1, #2, #4 and #5 chains, fibronectin and tenascin-C in skeletal muscle of dystrophic 129ReJ dy/dy mice. Exp Cell Res 1998; 244: In press. 60. Xu H, Christmas P, Wu X-R et al. Defective muscle basement membrane and lack of M-laminin in the dystrophic dy/dy mouse. Proc Natl Acad Sci USA 1994; 91:5572-5576. 61. Sunada Y, Bernier SM, Kozak CA et al. Deficiency of merosin in dystrophic dy mice and genetic linkage of laminin M gene to dy locus. J Biol Chem 1994; 269:13729-13732. 62. Dodson MV, McFarland DC, Grant AL et al. Extrinsic regulation of domestic animal-derived satellite cells. Domest Anim Endocrinol 1996; 13:107-126. 63. Hodges BL, Kaufman SJ. Developmental regulation and functional significance of alternative splicing of NCAM and #7∃1 et a1 integrin in skeletal muscle. Basic Appl Myology 1997; 6:437-447. 64. Olwin BB, Hannon K, Kudla AJ. Are fibroblast growth factors regulators of myogenesis in vivo? Prog Growth Factor Res 1994; 5:145-158. 65. Gallo RL, Ono M, Povsic T et al. Syndecans, cell surface heparin sulfate proteoglycans, are induced by a proline-rich antimicrobial peptide from wounds. Proc Natl Acad Sci U S A 1994; 9:11035-11039. 66. Brickman Y, Ford MD, Small DH et al. Heparin sulfates mediate the binding of basic fibroblast growth factor to a specific receptor on neural precursor cells. J Biol Chem 1995; 270:1-8. 67. Allen RE, Sheehan SM, Taylor RG et al. Hepatocyte growth factor activates quiescent skeletal muscle satellite cells in vitro. J Cell Physiol 1995; 165:307-312. 68. Allen RE, Tatsumi R, Sheehan SM. HGF/SF is the activating factor in crushed muscle extract and is an autocrine growth factor for satellite cells. Keystone Symposium on Molecular Biology of Muscle Development 1997; 1-6:43. 69. Gatchalian CL, Schachner M, Sanes J. Fibroblasts that proliferate near denervated synaptic sites in skeletal muscle synthesize the adhesive molecules tenascin (J1), N-CAM, fibronectin and a heparin sulfate proteoglycan. J Cell Biol 1989; 108:1873-1890. 70. Mauro A. Satellite cells of skeletal muscle fibers. J Biophys Biochem Cytol 1961; 9:493-495. 71. Schultz E. A quantitative study of the satellite cell population in postnatal mouse lumbrical muscle. Anat Rec 1974; 180:589-595. 72. Zhang M, Mclennan IS. Use of antibodies to identify satellite cells with a light microscope. Muscle Nerve 1994; 17(9):987-994. 73. Grounds MD, Yablonka-Reuveni Z. Molecular and cell biology of skeletal muscle regeneration. In: Partridge TA, ed. Molecular and Cell Biology of Muscular Dystrophy. London: Chapman & Hall,1993:210-256. 74. Grounds, MD, Garrett KL, Lai MC et al. Identification of skeletal muscle precursor cells in vivo by use of MyoD1 and myogenin probes. Cell Tissue Res 1992; 267:99-104. 75. Fuchtbauer E-M, Westphal H. MyoD and myogenin are coexpressed in regenerating skeletal muscle of the mouse. Dev Dyn 1992; 193:34-39. 76. Rantanen J, Hurme T, Lukka R et al. Satellite cell proliferation and the expression of myogenin and desmin in regenerating skeletal muscle: Evidence for two different populations of satellite cells. Lab Invest 1995; 72(3):341-347. 77. Koishi K, Zhang M, Mclennan IS et al. MyoD protein accumulates in satellite cells and is neurally regulated in regenerating myotubes and skeletal muscle fibers. Dev Dyn 1995; 202(3):244-254. 78. Mouly V, Decary S, Cooper RN et al. Satellite cell proliferation: Starting point and key steps to regeneration and cell mediated therapy. Basic Appl Myology 1997; 7:167-176. 79. Helliwell TR. Lectin binding and desmin staining during bupivicaine-induced necrosis and regeneration in rat skeletal muscle. J Pathol 1988; 155:317-326.

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80. Kurek JB, Bower J, Romanella M et al. The role of leukemia inhibitory factor in skeletal muscle regeneration. Muscle Nerve 1997; 20:815-822. 81. Irintchev A, Zeschnigk M, Starzinski-Powitz A et al. Expression pattern of M-cadherin in normal, denervated and regenerating mouse muscle. Dev Dyn 1994; 199:326-337. 82. Bornemann A, Schmalbruch H. Immunocytochemistry of M-cadherin in mature and regenerating rat muscle. Anat Rec 1994; 239:1119-1125. 83. Cornelison DW, Wold BJ. C-met is expressed by quiescent, proliferating and differentiating skeletal muscle satellite cells. Mol Biol Cell 1996; 7:539A. 84. Lescaudron L, Li Z, Paulin D et al. Desmin-lacZ transgene, a marker of regenerating skeletal muscle. Neuromus Dis 1993; 3:419-422. 85. Bower JJ, White JD, Kurek JB et al. The role of growth factors in myoblast transfer therapy. Basic Appl Myology 1997; 7:177-187. 86. Austin L, Bower J, Kurek J et al. The effects of leukemia inhibitory factor and other cytokines on murine and human myoblast proliferation. J Neurol Sci 1992; 112:185-191. 87. Cannon JG. Cytokines in aging and muscle homeostasis. J Gerontol 1995; 50A:120-123. 88. Cantini M, Carraro U. Control of cell proliferation by macrophage-myoblast interactions. Basic Appl Myology 1996; 6:485-489. 89. Garrett K, Grounds MD, Maley MAL et al. Interferon inhibits myogenesis in vitro and in vivo. Basic Appl Myology 1992; 2(4):291-298. 90. Kurek JB, Bower J, Romanella M et al. Leukemia inhibitory factor (LIF) treatment stimulates muscle regeneration in the mdx mouse. Neurosci Lett 1996; 212:167-170. 91. Barnard W, Bower J, Brown MA et al. Leukemic inhibitory factor (LIF) infusion stimulates skeletal muscle regeneration after injury: injured muscle expresses LIF mRNA. J Neurol Sci 1994; 123(1-2):108-113. 92. Bischoff R. A satellite cell mitogen from crushed adult muscle. Dev Biol 1986; 115:140-147. 93. Bischoff R. Chemotaxis of skeletal muscle satellite cells. Dev Dyn 1997; 208:505-515. 94. Haugk KL, Roeder RA, Garber MJ et al. Crushed muscle extracts: A model system to investigate growth factor regulation of satellite cell activities in meat animals. Basic Appl Myology 1996; 6:163-173. 95. Grounds MD, Davies MJ. Chemotaxis in myogenesis. Basic Appl Myology 1996; 6:469-483. 96. Mitchell CA, McGeachie JM, Grounds MD. Exogenous administration of basic FGF does not enhance the regeneration of murine skeletal muscle. Growth Factors 1996; 13:1-19. 97. Gautron G, Ketzia C, Haussman I et al. Injection of a heparin sulfate-like substance in crushed muscle accelerates its regeneration. C R Acad Sci Paris Ser lll 1995; 318:671-676. 98. Aamiri A, Mobarek A, Carpentier G et al. Effets d’un dextran subtitue sur la reinnervation du muscle squelettique du rat adulte au cours de la regeneration. C R Acad Sci III 1995; 318:1037-1043. 99. Yarom R, Meyer S, Carmy O et al. Enhancement of human muscle growth in diffusion chambers by bone marrow cells. Cell Path 1982; 41:171-180. 100. Mitchell CA, Grounds MD, McGeachie JK. The effect of low dose dexamethasone on skeletal muscle regeneration in vivo. Basic Appl Myology 1991; 1:139-144. 101. Zdanowicz MD, Moyse J, Wingertzahn et al. Effect of insulin-like growth factor 1 in murine muscular dystrophy. Endocr 1995; 136:4880-4886. 102. Anderson JE, Kardami E. The effects of hyperthyroidism on muscular dystrophy in the mdx mouse: Greater dystrophy in cardiac and soleus muscle. Muscle Nerve 1994; 17:64-73. 103. Robertson TA, Maley MAL, Grounds MD et al. The role of macrophages in skeletal muscle regeneration with particular reference to chemotaxis. Exp Cell Res 1993; 207:321-331. 104. Tidball JG. Inflammatory cell response to acute muscle injury. Med Sci Sports Exerc 1995; 27:1022-1023. 105. Mitchell CA, McGeachie JK, Grounds MD. Cellular differences in the regeneration of murine skeletal muscle—a quantitative histological study in SJL/J and BALB/c mice. Cell Tissue Res 1992; 269(1):159-166. 106. Mitchell CA, Papadimitriou JM, Grounds MD. The genotype of bone-marrow derived inflammatory cells does not account for differences in skeletal muscle regeneration between SJL/J and BALB/c mice. Cell Tissue Res 1995; 208:407-413.

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107. Carlson BM, Faulkner JA. Muscle transplantation between young and old rats: Age of host determines recovery. Am Physiol Soc 1989; 25:1262-1266. 108. Grounds MD. Age-associated changes in the response of skeletal muscle cells to exercise and regeneration. Ann N Y Acad Sci 1998; 854:78-91. 109. Roberts P, McGeachie JK. The enhancement of revascularization of skeletal muscle transplants using the beta2-agonist isoprenaline. J Anat 1994; 184:309-318. 110. Roberts P, McGeachie JK. Propranolol retards revascularization and early myogenesis in regenerating skeletal muscle transplants: An autoradiographic and morphometric study in mice. J Anat 1992; 181:101-111. 111. Roberts P, McGeachie JK. The effects of pre- and posttransplantation exercise on satellite cell activation and the regeneration of skeletal muscle transplants: a morphometric and autoradiographic study in mice. J Anat 1992; 180:67-74. 112. McCully KK, Posner JE. The application of blood flow measurements to the study of aging muscle. J Gerontol 1995; 50A:130-136. 113. Coggan AR, Spina RJ, King DS et al. Histochemical and enzymatic comparison of the gastrocnemius of young and elderly men and women. J Gerontol Biol Sci 1992; 47:B71-B76. 114. Cooper LT, Cooke JP, Dzau VJ. The vascular pathology of aging. J Gerontol Biol Sci 1995; 49:B191-B195. 115. Klagsbrun M, D’Amore PA. Regulators of angiogenesis. Ann Rev Physiol 1991; 53:217-239. 116. Folkman J. New perspectives in clinical oncology from angiogenesis research. Eur J Cancer 1996; 32A:2534-2539. 117. McGeachie JK, Allbrook D. Cell proliferation in skeletal muscle following denervation or tenotomy. Cell Tissue Res 1978; 193:259-267. 118. McGeachie JK. Sustained cell proliferation in denervated skeletal muscle of mice. Cell Tissue Res 1989; 257:455-457. 119. McGeachie JK. The fate of proliferating cells in skeletal muscle after denervation or tenotomy: An autoradiographic study. Neurosci 1985; 15(2):499-506. 120. Studitsky A, Zhenevskaya R, Rumyanitseva O. The role of neurotrophic influences upon the restitution of structure and function of regenerating muscles. In: Gutmann E, Hnik P, eds. The effects of use and disuse in neuromuscular functions. Prague: Czechoslovak Academy of Science,1963:71-81. 121. Studitsky, AN. Transplantation of muscles in animals. Carlson B, ed. New Dehli: Amerind Publishing Co. Pvt. Ltd.,1988. 122. Lu D-X, Carlson BM. A quantitative study of satellite cells in long-term denervated rat extensor digitorum longus (EDL) muscle. Anat Rec 1993; 5:78. 123. Viguie CA, Carlson BM. Nuclear numbers in long-term denervated rat EDL muscle fibers. FASEB 1994; 8:A60. 124. Carlson BM. Factors influencing the repair and adaptation of muscles in aged individuals: satellite cells and innervation. J Gerontol 1995; 50A:96-100. 125. Carlson BM, Faulkner JA. The restorative potential of long-term denervated rat muscle after transplantation into an innervated site. Expl Neurol 1993; 102:50-56. 126. Moschella MC, Ontell M. Transient and chronic denervation of murine muscle: A procedure to modify the phenotypic expression of muscular dystrophy. Dev Biol 1987; 125:158-167. 127. Hermanson JW, Moschella MC, Ontell M. Effect of neonatal denervation-reinnervation on the functional capacity of the 129/ReJ dy/dy murine dystrophic muscle. Exp Neurol 1988; 102:210-216. 128. Ontell MP, Hughes D, Hauschka SD et al. Transient neonatal denervation alters the proliferative capacity of myosatellite cells in dystrophic (129REJdy/dy) muscle. J Neurobiol 1992; 23(4):407-419.

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CHAPTER 12

Myoblast Transfer as a Platform Technology of Gene Therapy and Tissue Engineering Peter K. Law

Introduction

T

he National Institute of Standards and Technology has just announced that tissue engineering will likely be the key to treating genetic diseases and degenerative disorders that accounted for 50% of the $1 trillion+ US health care cost in 1995.1-3 Among the many programs of tissue engineering, gene therapy has been hailed as the medicine of the 21st century. Despite the nearly universal belief that gene therapy will ultimately allow the treatment of currently incurable diseases and conditions, its potential remains largely unfulfilled. Only when a safe and effective gene delivery technology has been proven in humans can the full potential of gene therapy be realized. To date, more than one thousand subjects worldwide have received gene therapies from among the 200+ protocols approved. Data indicates that no single vector will serve all systems. In examining gene transfer methods mediated by particle bombardment,4,5 liposomes,6,7 calcium phosphate precipitations,7,8 and electropolation,7-9 one can conclude that transduction efficiency is extremely low and variable. The level of transgene expression depends on the promoter strength in a particular cell type. Only liposomes, together with retroviruses, adenoviruses, adeno-associated viruses and myoblasts, have been used in clinical trials.

Vectors Liposomes Cationic liposome/DNA complexes gain cellular entry via receptor-mediated endocytosis.6,10 Assuming the transgene escapes digestion by the endosome, it has no built-in mechanism to get across the nuclear membrane and is therefore nonintegrative. The minimal and transient transgene expression is the result of random targeting, integration, and regulation. Liposomes have the advantage for being nontoxic and can therefore be used in large quantities and repeatedly.11

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Viruses The viral vectors were the first to gain widespread scientific application. Notable was “the first federally approved gene therapy protocol, for correction of adenosine deaminase (ADA) deficiency, began on 14 September 1990.”12-14 Retroviral vectors can transduce dividing cells with integration into host DNA. They integrate randomly and may cause mutation and cell death. They exhibit no toxicity. Although they can house larger transgenes than adenoviruses and adeno-associated viruses, the capacity is less than 10 kb. They are unstable in primate complement and cannot be targeted to specific cell types in vivo.11,15 Adeno-associated viruses and adenoviruses have shown considerable promise and are widely used. They can accommodate a broad range of genetically modified genes; are efficiently taken up by nondividing cells in vivo; do not integrate into chromosomal DNA, thus reducing the risk of insertional mutagenesis; and are amenable to redirected tissue targeting.16 All viruses can cause harm when they revert to wild type and become replication-competent.11,17,18 Dose-dependent inflammation occurred after nasal19 or lung20 administration of the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA conjugated with adenoviral vectors. The low efficacy, if any, is what one would have expected of pioneering studies. However, the risk to benefit ratio cannot be ignored. Also, viruses produce antigens. When exposed to the host immune system, through leakage, secretion or cell death, these antigens trigger immune reactions against the transduced cells. Certain viral elements are also toxic. These three inherent problems post almost insurmountable difficulties that prohibit the safe and efficacious clinical use of viral vectors at the present except for terminal cases. To increase caution, the FDA has mandated viral vector validation of every batch to be used on humans. Single gene manipulation, often exercised ex vivo, has been used in vivo. Recombinant genes were taken up and expressed in murine skeletal myofibers21-23 and cardiac myocytes24 following intramuscular injections. Gene expression is invariably low despite different delivery conditions and methods.25 This approach lacks basis and evidence of gene integration and regulation. A more scientifically logical approach is to include viral or cellular transcriptional regulatory sequences to affect expression. In the prophylactic treatment of hemophilia A, a retroviral factor VIII cDNA conjugate was used to induce secretion of the blood-clotting factor in athymic mice from transduced implanted human skin fibroblasts.26 Both adenoviral27 and herpes simplex virus-derived28 vectors have similarly been used for in vivo transfer of factor IX cDNA to the liver. Although therapeutic levels of factor IX were obtained, the expression decayed in a few weeks, possibly due to immune response and gene inactiviation.29 Gene therapy with viral vectors has been developing rapidly, but judging from the results of cystic fibrosis and brain tumor clinical trials, it is still a young discipline.30,31 Since the main thrust of this review is on myoblast transfer therapy (MTT), additional details of nonmyoblastic single gene manipulations can be found in the books entitled GENE THERAPY—A PRIMER FOR PHYSICIANS32 SOMATIC GENE THERAPY33 and GENE THERAPY FOR NEOPLASTIC DISEASES.34

Why Myoblasts? Although genetic ailments constitute less than 2% of all human diseases, far more currently incurable diseases are the result of inadequate genetic predisposition and/or haphazard interactions between multiple genes. Symptoms precipitate when a regulatory or a structural protein is either missing or malfunctional. Without knowing these defect(s) or how they can be corrected, tissue engineering will favor genome replacement rather than single gene(s) replacement. The cell knows more than we do. Furthermore, for a gene therapy to be effective and efficient, transgene expression requires appropriate targeting into a specific cell type, integration onto a specific site on a

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Fig. 12.1 Diagram of some of the known genetic factors in Duchenne muscular dystrophy (DMD) muscle cells that differ from normal muscle cells. These include genes for membrane structural proteins that are decreased or absent in DMD—dystrophin (DIN), dystrophin-related-proteins (DRP) and dystrophin-associated glycoproteins (DAG); genes for enzymes elevated in serum levels of DMD patients—creatinine phosphokinase (CPK), aldolase (ALD) and aspartate transaminase (AST); and genes for mitochondrial (Mito) differences.

Fig. 12.2 Immunocytochemical localization of donor (stained; white arrowheads) and host (unstained; dark arrowheads) nuclei in longitudinal muscle sections. (A) and (B) are normal and dystrophic controls, respectively. (C) is from a dystrophic muscle 18 months after normal myoblast injection. A mosaic fiber (M) is demonstrated by the presence of both stained and unstained nuclei.

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specific chromosome, and regulation by factors that are the products of other genes. This chain of events involves numerous cofactors, many of which are produced transiently during embryonic development but not in adulthood. This is where the approach of single gene manipulation errs conceptually, because it cannot provide these cofactors. In complex systems, one hardly knows what they are. Again, only transfer of the whole normal genome can allow the orderly provision of these cofactors necessary for the transgene expression. Finally, secondary degenerative changes often accompany the primary protein defect. Additional structural and/or regulatory protein(s) are lost (Fig. 12.1). Even if single gene manipulation replaces the primary protein deficit, transduced cells still degenerate because of the secondary changes. These latter proteins can only be replaced by retranscribing the normal genome inserted. Myoblasts are muscle-building cells endogenous to the human body. Contained within the nucleus of each human myoblast is the normal genome with more than 100,000 normal genes that determine cell normality and cell characteristics. Less than 10% of the gene actions are known. Myoblasts are the only somatic cell type in the body capable of natural cell fusion. Through this process, they insert their nuclei, and therefore all of the normal genes, into multinucleated myofibers of the host to effect genetic repair (Fig. 12.2). The transfer of genetic material and information occurs in vivo, with the myoblasts serving as the source and the vehicle to effect gene transfer. Myoblasts are the only cells that divide extensively,35 migrate,36 fuse naturally to form syncytia,36 lose major histocompatibility complex Class I (MHC-I) antigens after fusion,37,38 and develop up to 50% of human body weight. Patients need no more than two months of immunosuppression after MTT because mature myotubes and myofibers exhibit no MHC-I antigens.37,38 These combined properties render myoblasts superior for gene transfer. Being endogenous cells, myoblasts do not produce the adverse reactions of viral vectors.

MTT Technology MTT is a platform technology of gene therapy and tissue engineering. The procedure consists of culturing large quantities of myoblasts from muscle biopsies of genetically normal human donors. Cultured myoblasts are injected into patients' muscles while the patient is under general anesthesia. An immunosuppressant is administered following the procedure to minimize donor cell rejection. The injection injury activates regeneration of host myofibers, allowing them to fuse with the injected myoblasts, thus forming genetically mosaic multinucleated myofibers (Fig. 12.2).39-41 In addition, injected myoblasts fuse among themselves, forming genetically normal myofibers.39-41 Thus, MTT delivers the normal nuclei, and therefore the whole human genome, into muscles of the genetically defective host, where the transgene is naturally and stably integrated. Other vectors could not achieve this. MTT utilizes genetic complementation, which circumvents the time consuming and expensive processes of abnormal gene identification, replacement gene synthesis, tissue targeting, gene integration, replacement, regulation, and expression, most of which are not fully understood. Cell fusion inherent in muscle regeneration is utilized to incorporate the normal nuclei into host muscle cells. Since the fusion process is a natural occurrence, there should not be any problem with specificities of integration, complementation, regulation, and expression of the normal genome inserted. It is not necessary to know which gene(s) is responsible for the defect. Furthermore, the injection of normal myoblasts directly into the host muscle eliminates any uncertainty of tissue targeting. Natural transcription of the normal genome within the donor nuclei following MTT ensures orderly replacement of any protein deficiency resulting from single gene defects or from haphazard polygenic interactions.

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Muscular Dystrophies—The Testing Ground Muscular dystrophies are genetic diseases of progressive muscle degeneration. Debilitating and fatal, these hereditary degenerative diseases deprive their sufferers of a normal quality of life and life span. A most common form, Duchenne muscular dystrophy (DMD) confines boys to wheelchairs by age 12 and claims their lives by 20. Second in prevalence only to cystic fibrosis, DMD afflicts one in every 3300 male births worldwide.42 As with any hereditary degenerative disease, DMD treatment will require repairing degenerating cells and replenishing dead cells. MTT is unique in treating the muscular dystrophies in that it transfers the normal genome to repair degenerative myofibers and it provides normal cells to replenish degenerated myofibers. As such, MTT is a combined cell/gene therapy. Potentially, not only can MTT prevent further weakening, it can also increase muscle strength. Like murine dystrophy, DMD serves as a disease model to test MTT as a cell/gene therapy in treating hereditary degenerative diseases. MTT is being developed to repair degenerating cells and to replenish degenerated cells of the muscles in all of the neuromuscular diseases affecting more than one million people worldwide. In a broad sense MTT is tested for its feasibility, safety, and efficacy to integrate the normal human genome into genetically abnormal patients. Since MTT incorporates all of the normal genes into the dystrophic myofibers to repair them, it should exert similar effects irregardless of which gene is abnormal or which protein is missing. Accordingly, MTT should be as beneficial to the murine dystrophies showing laminin #2 mutation in the dy2Jdy2J phenotypes43 as to DMD showing dystrophin deletion,44 given adjustments from mouse to human.

Animal Experiments To develop a treatment, we need to know the pathogenesis of the disease. By comparing the electric45,46 and ultrastructural properties47,48 of normal vs. dystrophic myofibers, research in the 1970s has firmly established that the genetic defects in muscular dystrophy manifest in membrane deterioration and dysfunction. Using a normal/dystrophic parabiotic mouse model with cross-reinnervation of muscles, it was demonstrated that the dystrophic nervous system would support normal muscle development.49,50 Without such knowledge, it would be imprudent to attempt strengthening dystrophic muscles with normal myogenic cell transfer. All of the developmental work of MTT was published by essentially two research teams whose approaches were disparate but complementary. While Law and associates were demonstrating the safety and efficacy of transferring normal myogenic cells into the dy2Jdy2J dystrophic mice, Partridge and associates were examining the developmental fate of donor cells in normal mice. This was at a time when neither the golden retriever muscular dystrophy (GRMD) nor the xmd canine dystrophy was known. Dystrophic dogs are available to a few laboratories that have not produced any significant results with MTT.51 It was not until 1989 that a study of MTT on mdx mice was first published.52,53 The majority of evidence in support of MTT safety and efficacy is derived from previous studies using the dy2Jdy2J mice.39-41, 54-58 Central to MTT is the correlation of genetic and phenotypic improvement at the cellular and at the whole muscle levels. Such correlation has to be derived from demonstrations that in fact there are genotypic and phenotypic improvements. These studies play an essential role in the elucidation of the mechanisms by which MTT exerts its beneficial effects.39-41,52-58 The demonstration that cultured cells survived, developed and functioned in vivo after implantation into an organ of a diseased mammal bridges the gap of knowledge between in vitro and in vivo cell biology. This was first achieved with myoblast transfer.39,40

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The original idea of MTT, and experiments testing MTT, was first published by Law in 1978.54 A deliberate attempt was made in adult dystrophic mice to produce mosaic muscles containing normal, dystrophic and mosaic myofibers from the regenerates of normal and dystrophic minced muscle mixes. This early study focused on incorporating the “missing” gene and its product(s) into genetically defective cells through cell transplantation and natural cell fusion, the result of which is strengthened mouse muscles54 having a gene defect similar to human congenital muscular dystrophy.43 The result contradicts the study of Partridge and Sloper who concluded, in transplanting normal minced muscles into normal hosts, that little or none of the regenerates was of donor origin.59 Concurrent with Law’s 1978 publication on normal/dystrophic transplant was that of Partridge, Grounds, and Sloper, which described fusion between host and donor myogenic cells of normal genotypes using skeletal muscle grafts.60 Contrary to what the latter authors previously published, they now claimed that satellite cells from donor muscle minces did survive and develop in normal host muscles. Although the study did not involve dystrophic animals, the author inferred that MTT was a distinct development with potential applicability to hereditary myopathies. In Law’s later study, muscles of newborn normal mice were grafted into recipient soleus muscles of dystrophic mice. Results obtained 6 months after the grafting indicated that the grafts survived, developed, and functioned in the dystrophic environment. The regenerates had larger cross-sectional areas and more muscle fibers than the contralateral dystrophic solei. MTT increased the mean twitch tension of adult dystrophic muscles to that of the normal.55 By 1979, the concept of replenishing lost cells and repairing degenerative cells through the production of genetic mosaicism using MTT was firmly established.55 In the same year, it was established that myoblasts cultured from muscle biopsies of adult normal rats could survive and develop in the original donor after implantation.61 MTT became the logical development, since myoblasts do not require innervation and capillary connections to survive and develop, and since myoblasts can fuse to effect genetic repair. A convenient way to obtain normal myoblasts in mice is through dissection of limb bud mesenchyme of day 12 embryos. Dissected mesenchyme was surgically implanted into the solei of dy2Jdy2J mice. Host and donors were histocompatible. Contralateral solei served as controls. Six to seven months post-operatively, the myoblast-implanted solei exhibited greater cross-sectional area, total fiber number, better cell structure, and twitch and tetanus tensions than their contralateral controls.56 Partridge’s team continued to explore factors that affected the incorporation and fusion of allogeneic muscle precursor cells in vivo.62 Their studies involved only normal mice. The implants consisted of minced muscle mixes or newborn muscles.63-65 The authors demonstrated the survival and development of donor cells in the host muscles, using electrophoretic analyses of glucose phosphate isomerases (GPI), genetic markers to identify host vs. donor cells. The year 1988 witnessed the explosive development of MTT. In the first study, primary myoblast cultures from limb bud explants of normal mouse embryos were injected into the soleus muscles of histocompatible dystrophic hosts.39 In the second study, clones of normal myoblasts were injected into the leg and intercostal muscles of histoincompatible hosts with cyclosporine A (CsA) as a host immunosuppressant.40 Using GPI as genotype markers, donor myoblasts were shown to have fused among themselves, developing into normal myofibers. They also fused with dystrophic host myogenic cells to form mosaic myofibers of normal phenotype.39,40,58 These two mechanisms of genetic complementation were shown to be responsible for improvement in muscle genetics, structure, function and animal behavior of the test dystrophic mice.39,40,54-58 Prolongation of the life spans of the myoblast-injected dystrophic mice was demonstrated.57,58 The improvement persisted despite CsA withdrawal. Partridge’s laboratory did not report implantation into abnormal muscles until 1988. Morgan et al reported the synthesis of trace amounts of phosphokinase (PhK) in about 5%

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of the myoblast-injected muscles of the PhK-deficient mice.65 Although there have been frequent claims of supplying normal muscle precursor cells to alleviate hereditary myopathies, no evidence of any structural or functional improvement after transplantation was presented. With the discovery in 1987 that the absence of the gene product dystrophin is the cause of DMD44 and mdx mouse dystrophy, a new biochemical marker became available to demonstrate MTT efficacy.41,52,53 In 1989, MTT was shown to convert mdx myofibers from dystrophin negative to positive.52,53 The study demonstrates biochemical improvement in the mdx mouse model, an additional evidence to confirm the efficacy of MTT. Given the use of inbred mice that afford histocompatible MTT, the reality is that fully matched human donors and dystrophic recipients are rarely available. MTT would thus necessitate the inclusion of host immunosuppression to facilitate myoblast survival after transfer. Cyclosporine (Cy) is the most widely documented immunosuppressant in transplantation studies.66 Availability of FK 506 in the late 80s was limited.67 Typically, host mice were primed 1 week with CsA injected subcutaneously every day at 50 mg/kg body weight before receiving myoblasts. The same CsA treatment continued for 6 months after MTT.40 Aside from donor cell survival in an immunologically hostile host, cell fusion is the key to strengthening dystrophic muscles with MTT. To improve the fusion rate between host and donor cells, various injection methods aimed at wide dissemination of donor myoblasts were tested and compared. The goal is to achieve maximum cell fusion with the least number of injections. The results indicate that delivery of myoblasts is best conducted by diagonal placement of needle into the host muscle with ejaculation of the myoblasts as the needle is withdrawn. This method of myoblast injection yields even and wide distribution of donor myoblasts with a high rate of cell fusion. Myoblasts injected perpendicular to myofiber orientation are partially distributed. Myoblasts injected longitudinally through the core of the muscles and parallel to the myofibers are poorly distributed.68 Thus, the myoblast injection method regulates cell distribution and fusion. These animal studies provide the basis for MTT clinical trials.

Clinical Trials Gene therapy encompasses interventions that involve deliberate alteration of the genetic material of living cells to prevent or treat diseases.69 According to this FDA definition, the first MTT on a DMD boy on February 15, 1990 marked the first clinical trial of human gene therapy.70 In addition to fulfilling their primary muscle-building mission, the myoblasts served as the source and the transfer vehicles of normal genes to correct the gene defects of DMD. The protocol was approved by four institutional review boards. Subjects and parents gave informed consent. The safety and efficacy of MTT was assessed by injecting the left extensor digitorum brevis (EDB) muscle of a 9 year old DMD boy with about 8 x 106 myoblasts. Donor myoblasts were cloned from satellite cells derived from a 1 g rectus femoris biopsy of the normal, adoptive father. Cy was administered for 3 months at a dose of 5-7 mg/kg body weight divided into two daily oral doses. Donor myoblasts survived, developed, and produced dystrophin in myofibers biopsied from the myoblast-injected EDB 92 days later. Dystrophin was not found in the contralateral sham-injected muscle. This first case suggests that MTT offered a safe and effective means for alleviating biochemical deficit(s) inherent in muscles of DMD.71 A misleading12 and often misquoted11, 72 “first human gene therapy” is the ADA deficiency study began on September 14, 1990,12 2 months after MTT correction of DMD gene defect was published.71 In the ADA protocol, T cells from a patient with a severe combined immunodeficiency disorder (SCID) were transduced with functional ADA genes ex vivo

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and returned to the patient after expansion through culture. In the MTT protocol, primary culture of myoblasts derived from a muscle biopsy of a normal donor was injected into a muscle of the DMD subject to produce in vivo nuclear complementation. Both gene therapies utilize cell transplantation to treat diseases. Six years after the first MTT, dystrophin was found in the myoblast-injected muscle but not in the sham-injected muscle (Fig. 12.3).73 Six years is the longest period through which any gene therapy has sustained positive results. Despite Cy withdrawal at 3 months after MTT, myofibers expressing foreign dystrophin were not rejected. Not only has the result demonstrated MTT overall safety and efficacy in this single case, it also shows stability in the integration, regulation and expression of the inserted dystrophin gene. The presence of dystrophin in the myoblast-injected but not in the sham-injected muscle provided unequivocal evidence of the survival and development of donor myoblasts in the myoblastinjected muscle. In a randomized, double-blind study involving three subjects, myoblast-injected EDBs showed increases in tensions, whereas sham-injected EDBs showed reductions.74,75 Both immunocytochemical staining and immunoblot revealed dystrophin in the myoblast-injected EDBs. Dystrophic characteristics such as fiber splitting, central nucleation, phagocytic necrosis, variation in fiber shape and size, and infiltration of fat and connective tissues were less frequently observed in these muscles. Sham-injected EDBs exhibited significant structural and functional degeneration and no dystrophin. Throughout the study, there was no sign of erythema, swelling or tenderness at the injection sites. Serial laboratory evaluations including electrolytes, creatinine, and urea did not reveal any significant changes before or after MTT. To reconcile positive results with fewer convincing ones,76-82 several issues need to be addressed. To begin with, the use of large quantities of pure live myoblasts is a prerequisite of successful MTT. Besides Law’s study,36 there is no published pictorial evidence to substantiate the purity, myogenicity and viability of the injected myoblasts as claimed. Myoblast cultures are usually contaminated with fibroblast overgrowth. MTT with such impure cultures could lead to deposition of connective tissues rather than myofiber production. Culturing 25 billion pure human myoblasts for MTT from 2 grams of muscle biopsy has been reported only by Law et al.35 Other teams work at ranges of hundreds of millions of myoblasts. In most studies 76,78-81 myoblasts were transported frozen, chilled or at room temperature for over 2 hours from the site of harvest before being injected. Since myoblasts have a high metabolic rate, they could not have survived for 2 hours without significant nutrients, oxygen and proper pH, being closely packed in saline within a vial for transport. Law’s myoblasts were injected into the subject within 30 minutes of harvest, at the same location without transport. MTT studies that reported failure 76-82 subscribed to the fallacy of making 55 to 330 injections into a muscle the size of an egg, traumatizing indiscriminately the underlying nerves, muscle, and vasculature. These injection traumas boosted macrophage access and host immune responses.83 They also induced fibrosis.84 Surviving myoblasts fused within 3 weeks in small mouse muscles.41 A nerve with multiple trauma could not regenerate soon enough through scar and connective tissues to innervate the newly formed myotubes in a large human dystrophic muscle. Stabilization of muscle contractile properties in a similar situation is achieved by 60 days in the rat, and functional return is incomplete.85 Noninnervated myotubes died within 1 week. Whatever few myotubes that developed in the unsuccessful MTT studies could not compensate for the traumatized myofibers. In Law’s study, 5-8 x 108 pure myoblasts were delivered with eight injections into the biceps brachii without nerve injury.35 Contrarily, in Mendell’s study, 55 sites, each 5 mm apart, distributed in 11 rows and 5 columns, were injected throughout the depth of each biceps of 5 to 9 year old boys.79 This was repeated monthly for 6 months. Axonal sprouts,

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Fig. 12. 3 Immunocytochemical demonstration of dystrophin in DMD muscles 6 years after MTT. Dystrophin is absent in sham-injected EDB muscle (A,C), but present in the contralateral myoblast-injected muscle (B,D). Dystrophin was immunocytochemically localized at the sarcolemma (arrows). Dystrophin is demonstrated at low (E) and high (F) magnification in normal control muscle. Cross-section; bar = 100 mm.

myotubes and neuromuscular junctions that take 6 weeks to mature86 were repeatedly traumatized by a total of 330 injections until the biceps, with or without myoblast/cyclosporine, were irreversibly damaged or destroyed. The result: no functional difference between myoblast and sham-injected muscles.79 Once injected, the myoblasts are subjected to scavenger hunt by macrophages for up to 3 weeks. This is because myoblasts exhibit MHC-I surface antigens87,88 that become absent after cell fusion.12 The latter occurs between 1-3 weeks after myoblast injection.41 An allowance in the number of injected myoblasts has to be made to satisfy the unavoidable scaven-

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ger process. As reflected in the small numbers of myoblasts injected in unsuccessful studies, it appears that either such allowance was not considered or that the teams were not able to produce larger quantities of pure myoblasts. The less successful MTT teams focused on immunosuppression to prevent T lymphocyte proliferation and antibody production without overcoming the primary hurdle of providing enough pure and live myoblasts. A basic study indicates that cyclophosphamide did not permit myoblast engraftment in the mouse.89 Without this prior knowledge, a MTT clinical trial was conducted without success using cyclophosphamide immunosuppression.78 Cy71 and potentially FK 50690 remain the immunosuppressants of choice for MTT. Results could have been more positive if either was employed in the study of Tremblay et al.77,82 All of these single muscle MTT studies had begun before the FDA established policies and regulations for cell/gene therapies. Law’s studies are the only ones that received permission for an investigational new drug application (IND) on MTT for treatment of multiple muscles. They are the only ongoing clinical trials. As a cell/gene therapy, all American MTT clinical trials must come under FDA purview. Beginning with 8 million myoblasts into a small foot muscle, Law proceeded to test 5 billion cells into 22 leg muscles, 25 billion cells into 64 body muscles, and now 50 billion cells into 82 muscles. With over 100 subjects having been treated, the complete safety of the MTT procedure has been proven. There have been no adverse reactions or side effects. MTT was studied in 32 DMD boys aged 6-14 years. Through 48 injections, 5 billion myoblasts were transferred into 22 major muscles in both lower limbs under general anesthesia. Injected muscles showed either increase in strength or no further deterioration at 9 months after MTT.36,91 Under FDA purview, MTT was completing phase II clinical trials on DMD. The whole body trial (WBT) consists of injecting 25 billion myoblasts in two MTT procedures separated by 3 to 9 months. Each procedure delivers up to 200 injections or 12.5 billion myoblasts to either 28 muscles in the upper body (UBT) or to 36 muscles in the lower body (LBT). A randomized double-blind portion of the study is conducted on the biceps brachii or quadriceps. Subjects take oral cyclosporine for 3 months after each MTT. One infantile facioscapulohumeral dystrophy and 40 DMD boys aged 6-16 have received WBT in the past 36 months with no adverse reaction. Nine months after MTT, immunocytochemical evidence of dystrophin has been demonstrated in 18 of the 20 subjects biopsied. Forced vital capacity increased by 33.3% and maximum voluntary ventilation increases by 28% at 12 months after UBT. Plantar flexion increased by 52% in force in 9 months in the ambulatory subjects. Behavioral improvements in running, balancing, climbing stairs and playing ball are noted.92-95 The latest development involves a one time injection of 50 billion myoblasts into 82 muscles with 179 skin punctures, approved for subjects with DMD, Becker MD and Limbgirdle MD. Forty subjects in this trial have experienced no adverse reaction.

Future Perspectives As a universal gene transfer vehicle with which the entire human genome can be integrated into patients’ muscles, myoblasts have shown promise in studies of the following diseases.

Cardiomyopathy Labeled cultured myoblasts engrafted and formed structures resembling desmosomes, intercalated discs, fascia adherents junctions, and gap junctions in myocardia of dogs,96 rats97 and mice98 when MTT was delivered intramuscularly96,97 or intraarterially.98 Donor muscle regenerates exhibited cardiac-like properties such as central nucleation,96 fatigue

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resistance, slow twitching, and were capable of twitch and tetanus contractions when stimulated.97 Similar results were obtained when cardiomyocytes were injected in dystrophic mice and dogs,99 rats100 and swine.101 These findings, together with established MTT safety, pave the way to MTT clinical trial in treating myocardial degeneration and dysfunction.

Insulin Resistant Diabetes Mellitus Commonly known as type II diabetes, this disease is genetically predisposed and afflicts 90% of the diabetic population. Virtually all identical siblings of these patients develop the disease, and the genetic defect can be traced to the GLUT4 gene deletion. The major sequela of insulin resistance is decreased muscle uptake of glucose, due to the moderate decrease in insulin receptors on muscle cell surface. Conceptually, MTT can add genetically normal myofibers with normal insulin receptors. It can also genetically repair the patients’ myofibers and produce normal insulin receptors on the heterokaryons. Basic research is needed to test this hypothesis on diabetic rats.

Bone/Cartilage Degeneration During embryonic development, mesenchymal progenitor cells differentiate into myoblasts, osteoblasts, chondrocytes and adipocytes under various controls of regulatory factors. Ectopic bone formation in muscle has been achieved through implantation of bone morphogenetic protein (BMP). BMP-2 was shown to convert the differentiation pathway of clonal myoblasts into the osteoblast lineage.102 This opens new ways to treat conditions of bone degeneration such as the degeneration of tooth pulp, hip, bone/joint, and long bone fractures. Given the ability to mass produce myoblasts that can be transformed into osteoblasts, and potentially chondrocytes, the difficulty of proliferating osteoblasts and chondrocytes is now overcome. Cultured autologous chondrocytes can be used to repair deep cartilage defects in the femorotibial articular surface of the human knee joint.103 The use of normal or transduced myoblasts as the source and vehicle for gene delivery has found application in the potential treatment of restenosis,104 soft tissue deformities,105 hemophilias,106,107 anemia,108 muscle trauma,109 human growth hormone deficiency110 and allograft rejection.111 MTT has produced a new frontier in medicine.

My Vision MTT implementation can benefit from development of the following programs.

Controlled Cell Fusion It will be useful to be able to control, initiate or facilitate cell fusion once myoblasts are injected. This is to minimize loss of myoblasts from macrophages whose presence is unavoidable if the patient is to have some immune protection. As the myoblasts are injected intramuscularly into the extracellular matrix (ECM), injection trauma causes the release of basic fibroblast growth factor (bFGF) and large chondroitin-6-sulfate proteoglycan (LC6SP). These latter growth factors stimulate myoblast proliferation. Unfortunately, they also stimulate the proliferation of fibroblasts that are already present in increased amounts in the dystrophic muscle. That is why it is necessary to inject fractions of myoblasts as pure as possible in MTT, without contaminating fibroblasts. Controlled cell fusion can be achieved by artificially increasing the concentration of LC6SP over the endogenous level. In addition, insulin or insulin-like growth factor 1 (IGF-1) may facilitate the developmental process, resulting in the formation of myotubes soon after myoblast injection. The use of bFGF, LC6SP and IGF-1 at optimal concentrations in the cell culture medium and in the injection medium will likely lead to greater MTT success.

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Superior Cell Lines These cell lines should be highly myogenic, nontumorigenic, nonantigenic, and will develop very strong muscles. The superior cell lines will bypass the use of immuno-suppressant, and will provide a ready access for patients who do not have a donor. A unique property of myoblasts is their loss of MHC-I antigens soon after they fuse. The immunosuppression period depends on how soon the myoblasts lose their MHC-I antigens after MTT. Even more ideal is the establishment of a myoblast cell line in which MHC-I antigens are absent. In human myoblasts cultured from normal muscle biopsies, some 91.7% of the myoblasts reacted with anti-MHC-I mAb (monoclonal antibodies). The remaining 8.3% of the myoblasts that were negative for MHC-I antigen expression were successfully separated by cytofluorometry. The lack of MHC-I antigens on these latter myoblasts may enhance survival of these myoblasts in recipients after MTT.88

Automated Cell Processors The great demand for normal and transduced myoblasts, the labor intensiveness and high cost of cell culturing, harvesting and packaging, and the fallibility of human imprecision will soon necessitate the invention and development of automated cell processors capable of producing huge quantities of viable, sterile, genetically well-defined and functionally demonstrated biologics. This invention will be one of the most important offspring of modern day computer science, mechanical engineering and cytogenetics. The intakes will be for biopsies of various human tissues. The computer will be programmed to process tissue(s) with precision controls in time, space, proportions of culture ingredients and apparatus maneuvers. Cell conditions can be monitored at any time during the process and flexibility is built in to allow changes. Different protocols can be programmed into the software for culture, controlled cell fusion, harvest and package. The outputs supply injectable cells ready for cell therapy or shipment. The cell processor will be self-contained in a sterile enclosure large enough to house the hardware in which cells are cultured and manipulated. A transport medium that can sustain the survival and myogenicity of myoblasts in package for up to 4 days will allow the cell packages to be delivered to remote points of utilization around the world. This has recently been developed.

Cell Banks The automated cell processors will constitute only a part of the cell banks. The current thought is to obtain donor muscle biopsies from young adults aged 8-22 to feed the inputs. Each donor has to undergo a battery of tests that are time consuming and expensive. From the test results and from the donor’s physical condition, one can determine if the donor cells are genetically defective or infected with viruses and/or bacteria. Human fetal tissues can potentially provide greater supplies of cells. However, aside from ethical issues surrounding abortion, it is difficult to determine the genetic normality of the cells. Muscle primordia of fetuses derived from in vitro fertilization of genetically well defined background may be an alternative. Sperm and ova can be recovered from healthy individuals that are known for their muscle strength and mass. In vitro fertilization will be followed by embryo culture to a specific developmental stage (day 26 to day 56 gestation) of the embryos. The muscle primordia that are rich in myoblasts can then be dissected out to feed the automated cell processors.

Summary Myoblasts divide profusely, and fuse during muscle regeneration, interiorizing MHC-I antigens and inserting myonuclei with the normal genome into muscles of genetically deficient recipients, where any replacement gene can be stably integrated and naturally expressed.

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Myoblasts are the natural source and vehicle for many gene therapies. Myoblast transfer therapy is now beginning US FDA phase III clinical trials for Duchenne muscular dystrophy.

Conclusion This review describes the landmark development of the first gene therapy study in humans. Through natural cell fusion, myoblast transfer transduces the human genome into dystrophic muscle cells to effect phenotype repair. The innovative cell transplantation procedure also revitalizes the degenerative organ by providing living cells of normal genotype to replenish cell loss. The result is potentially a new form of medicine. The conceptual approaches of single gene transfer and myoblast transfer toward treatment of hereditary degenerative diseases are compared. As more scientists continue to recognize myoblasts as a stable source of genes and as a safe and efficient gene transfer vehicle, MTT application will extend far beyond the treatment of neuromuscular diseases. This review provides insights to guide future development of MTT in battling against genetic and acquired diseases that presently have only diagnoses but no treatment.

Acknowledgment The author thanks Susan Kenny for typing the manuscript. Clinical trials are supported by public donations with FDA approval for cost recovery.

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43. Sunada Y, Bernier SM, Utani A et al. Identification of a novel mutant transcript of laminin a2 chain gene responsible for muscular dystrophy and dysmyelination in dy2J mice. Hum Mol Genet 1 995; 4:1055-1061. 44. Hoffman EP, Brown RH, Kunkel LM. Dystrophin: The protein product of the Duchenne muscular dystrophy locus. Cell 1987; 51:919-928. 45. Law PK, Atwood HL. Nonequivalence of surgical and natural enervation in dystrophic mouse muscle. Exp Neurol 1972; 34:200-209. 46. Law PK, Atwood HL, McComas AJ. Functional enervation in the soleus muscle of dystrophic mice. Exp Neurol 1976; 51:434-443. 47. Mokri B, Engel AG. Duchenne dystrophy: Electron microscopic findings pointing to a basic or early abnormality in the plasma membrane of the muscle fiber. Neurology 1975; 25:1111. 48. Law PK, Saito A, Fleischer S. Ultrastructural changes in muscle and motor end-plate of the dystrophic mouse. Exp Neurol 1983; 80:361-382. 49. Law PK, Cosmos E, Butler J et al. The absence of dystrophic characteristics in normal muscles successfully cross-reinnervated by nerves of dystrophic genotype: Physiological and cytochemical study of crossed solei of normal and dystrophic parabiotic mice. Exp Neurol 1976; 51:1-21. 50. Saito A, Law PK, Fleischer S. Study of neurotropism with ultrastructure of normal/ dystrophic parabiotic mice. Muscle Nerve 1983; 6:14-28. 51. Kornegay JN, Prattis SM, Bogan DJ et al. Results of myoblast transplantation in a canine model of muscle injury. In: Kakulas BA, Howell JMC, Roses AD, eds. Duchenne muscular dystrophy. Animal models and genetic manipulation. Raven Press, 1992; 203-212. 52. Partridge TA, Morgan JE, Coulton GR et al. Conversion of mdx myofibers from dystrophinnegative to positive by injection of normal myoblasts. Nature 1989; 337:176-179. 53. Karpati G, Pouliot Y, Zubrzycka-Gaarn et al. Dystrophin is expressed in mdx skeletal muscle fibers after normal myoblast implantation. Am J Pathol 1989; 135:27-32. 54. Law PK. Reduced regenerative capability of minced dystrophic mouse muscles. Exp Neurol 1978; 60:231-243. 55. Law PK, Yap JL. New muscle transplant method produces normal twitch tension in dystrophic muscle. Muscle Nerve 1979; 2:356-363. 56. Law PK. Beneficial effects of transplanting normal limb-bud mesenchyme into dystrophic mouse muscle. Muscle Nerve 1982; 5:619-627. 57. Law PK, Li HJ, Goodwin TG et al. Pathogenesis and treatment of hereditary muscular dystrophy. In: Kakulas BA, Mastaglia FL, eds. Pathogenesis and Therapy of Duchenne and Becker Muscular Dystrophy. Raven Press, 1990; 101-118. 58. Law PK, Goodwin TG, Li HJ et al. Myoblast transfer improves muscle genetics/structure; function and normalizes the behavior and life-span of dystrophic mice. In: Griggs RC, Karpati G, eds. Myoblast Transfer Therapy. Plenum Press, 1991; 75-87. 59. Partridge TA, Sloper JC. A host contribution to the regeneration of muscle grafts. J Neurol Sci 1977; 33:425-435. 60. Partridge TA, Grounds M, Sloper JC. Evidence of fusion between host and donor myoblasts in skeletal muscle grafts. Nature 1978; 273:306-308. 61. Jones PH. Implantation of cultured regenerate muscle cells into adult rat muscle. Exp Neurol 1979; 66:602-610. 62. Watt DJ. Factors which affect the fusion of allogeneic muscle precursor cells in vivo. Neuropathol Appl Neurolbiol 1982; 8:135-147. 63. Watt DJ, Lambert K, Morgan JE et al. Incorporation of donor muscle precursor cells into an area of muscle regeneration in the host mouse. J Neurol Sci 1982; 57:319-331. 64. Watt DJ, Morgan JE, Partridge TA. Long-term survival of allografted muscle precursor cells following a limited period of treatment with cyclosporine A. Clin Exp Immunol 1984; 55:419-426. 65. Morgan JE, Watt DJ, Sloper JC et al. Partial correction of an inherited biochemical defect of skeletal muscle by grafts of normal muscle precursor cells. J Neurol Sci 1988; 86:137-147. 66. Kahan BD, Bach JF. Proceedings of the second international congress on cyclosporine: Therapeutic use in transplantation. Transplant Proc 1988; 20:1-1137.

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67. Starzl TE, Thomson AW, Todo SN et al. Proceedings of the first international congress on FK506. Transplant Proc 1991; 23:2709-3380. 68. Law PK, Li H, Chen M et al. Myoblast injection methods regulates cell distribution and fusion. Transplant Proc 1994; 26:3417-3418. 69. Kessler DA, Siegel JP, Noguchi PD et al. Regulation of somatic cell therapy and gene therapy by the Food and Drug Administration. N Engl J Med 1993; 329:1169-1173. 70. Hooper C. Duchenne therapy trials starting in US and Canada. J NIH Res 1990; 2:30. 71. Law PK, Bertorini TE, Goodwin TG et al. Dystrophin production induced by myoblast transfer therapy in Duchenne muscular dystrophy. Lancet 1990; 336:114-115. 72. Karlsson S. Treatment of genetic defects in hematopoietic cell function by gene transfer. Blood 1991; 78:2481-2492. 73. Law PK. First human myoblast transfer therapy continues to show dystrophin after 6 years. Cell Transplantation 1997; 6:95-100. 74. Law PK, Goodwin TG, Fang Q et al. Long-term improvement in muscle function, structure, and biochemistry following myoblast transfer in DMD. Acta Cardiomiol 1991; 3:281-301. 75 Law PK, Goodwin TG, Fang Q et al. Myoblast transfer therapy for Duchenne muscular dystrophy. Acta Pediatr Jpn 1991; 33:206-215. 76. Gussoni E, Pavlath GK, Lanctot AM et al. Normal dystrophin transcripts detected in Duchenne muscular dystrophy patients after myoblast transplantation. Nature 1992; 356:435-438. 77. Huard J, Bouchard JP, Roy R et al. Human myoblast transplantation: Preliminary results of four cases. Muscle Nerve 1992; 15:550-560. 78. Karpati G, Ajdukovic D, Arnold D et al. Myoblast transfer in Duchenne muscular dystrophy. Ann Neurol 1993; 34:8-17. 79. Mendell JR, Kissel JT, Amato AA et al. Myoblast transfer in the treatment of Duchenne muscular dystrophy. N Engl J Med 1995; 333:832-838. 80. Miller RG, Pavlath G, Sharma K et al. Myoblast implantation in Duchenne muscular dystrophy: The San Francisco study. Neurology 1992; 42:189-190. 81. Morandi L, Bernasconi P, Gebbia M et al. Lack of mRNA and dystrophin expression in DMD patients three months after myoblast transfer. Neuromusc Disord 1995; 5:291-295. 82. Tremblay JP, Malouin F, Roy R et al. Results of a triple blind clinical study of myoblast transplantations without immunosuppressive treatment in young boys with Duchenne muscular dystrophy. Cell Transplant 1993; 2:99-112. 83. Guerette B, Asselin I, Vilquin JT et al. Lymphocyte infiltration following allo and xenomyoblast transplantation in mdx mice. Muscle Nerve 1995; 18:39-51. 84. Chen SS, Chien CH, Yu HS. Syndrome of deltoid and/or gluteal fibrotic contracture; an injection myopathy. Acta Neurol Scand 1988; 78:167-176. 85. Carlson, BM. The regeneration and transplantation of skeletal muscle. In: Seil F, ed. Nerve, organ, and tissue regeneration: Research perspectives. Academic Press, 1983; 431-454. 86. Fex S, Jirmanova I. Innervation by nerve implants of “fast” and “slow” skeletal muscles of the rat. Acta Physiol Scand 1969; 76:257-269. 87. Friedlander M, Fischman DA. Immunological studies of the embryonic muscle cell surface. Antiserum to the perfusion myoblast. J Cell Biol 1979; 81:193-214. 88. Fang Q, Chen M, Li HJ et al. MHC-1 antigens on cultured human myoblasts. Transplant Proc 1994; 26:3467. 89. Vilquin JT, Kinoshita I, Roy R et al. Cyclophosphamide immunosuppression does not permit successful myoblast allotransplantation in mouse. Neuromus Disord 1995; 5:511-517. 90. Kinoshita I, Vilquin JT, Guerette B et al. Very efficient myoblast allotransplantation in mice under FK506 immunosuppression. Muscle Nerve 1994; 17:1407-1415. 91. Law PK, Goodwin TG, Fang Q et al. Cell transplantation as an experimental treatment for Duchenne muscular dystrophy. Cell Transplant 1993; 2:485-505. 92. Law PK, Goodwin TG, Fang Q et al. Human gene therapy with myoblast transfer. Transplant Proc (In Press).

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93. Law PK, Goodwin TG, Fang Q et al. Myoblast transfer: Gene therapy for muscular dystrophy. J Cell Biochem 1995; 367. 94. Law PK, Goodwin TG, Fang Q et al. Human gene therapy with myoblast transfer. Mol Biol Cell 1996; 7:3639. 95. Law PK, Goodwin TG, Fang Q et al. Myoblast transfer therapy (MTT) phase II clinical trials. J Physiol Biochem 1997; 53:80. 96. Chiu RCJ, Zibaitis A, Kao RL. Cellular cardiomyoplasty: Myocardial regeneration with satellite cell implantation. Ann Thorac Surg 1995; 60:12-18. 97. Murry CE, Wiseman RW, Schwartz SM et al. Skeletal myoblast transplantation for repair of myocardial necrosis. J Clin Invest 1996; 98:2512-2523. 98. Robinson SW, Cho PW, Levitsky HI et al. Cell Transplant 1996; 5:77-91. 99. Koh GY, Soonpaa MH, Klug MG et al. Stable fetal cardiomyocyte grafts in the hearts of dystrophic mice and dog. J Clin Invest 1955; 96:2034-2042. 100. Li RK, Jia ZQ, Weisel RD et al. Cardiomyocyte transplantation improves heart function. Ann Thorac Surg 1996; 62:654-661. 101. Van Meter CH, Claycomb WC, Delcarpio JB et al. Myoblast transplantation in the porcine model: A potential technique for myocardial repair. J Thorac Cardiovasc Surg 1995; 110:1442-1448. 102. Katagiri T, Yamaguchi A, Komaki M et al. Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage. J Cell Biol 1994;127:1755-1766. 103. Brittberg M, Lindahl A, Nilsson A et al. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 1994; 331:889-895. 104. Morishita R, Gibbons GH, Horiuchi M et al. A gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle proliferation in vivo. Proc Natl Acad Sci 1995; 92:5855-5859. 105. Teboul L, Gaillard D, Staccini L et al. Thiazolidinediones and fatty acids convert myogenic cells into adipose-like cells. J Biol Chem 1995; 270:28183-28187. 106. Dai Y, Roman M, Naviaus RK et al. Gene therapy via primary myoblasts: Long term expression of factor IX protein following transplantation in vivo. Proc Natl Acad Sci 1994; 89:10892-10895. 107. Yao SN, Smith KJ, Kurachi K. Primary myoblast-mediated gene transfer: Persistent expression of human factor IX in mice. Gene Ther 1994; 1:99-107. 108. Hamamori Y, Samal B, Tian J et al. Persistent erythropoiesis by myoblast transfer of erythropoietin cDNA. Hum Gene Ther 1994; 5:1349-1356. 109. Alameddine HS, Louboutin JP, Dehaupas M et al. Functional recovery induced by satellite cell grafts in irreversibly injured muscles. Cell Transplant 1994; 3:3-14. 110. Barr E, Leiden JM. Systemic delivery of recombinant proteins by genetically modified myoblasts. Science 1991; 254:1507-1509. 111. Lau HT, Yu M, Fontana A et al. Prevention of islet allograft rejection with engineered myoblast expressing FasL in mice. Science 1996; 273:109-112.

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CHAPTER 13

Meniscal Allograft Transplantation Thomas R. Carter

Introduction

A

lthough the meniscus has in the past been thought to be a vestigial organ, it is presently well known to play a vital role to the optimal function of the knee.1-5 Multiple studies have shown that with excision of the meniscus, there is notable increased incidence of developing degenerative arthritis of the knee.6-11 As a result, efforts to repair an injured meniscus have gained increased attention. Unfortunately, complex and degenerative type tears are not always amenable to repair. Additionally, there have been many patients that have had previous meniscectomies prior to the development of repair or have had the meniscus excised due to the treating surgeon’s unfamiliarity with the technique. Development of meniscal allograft transplantation has been the result of efforts to delay or prevent the deleterious effects of meniscal excision in these patients.

Functions of the Meniscus The menisci serve several functions that contribute significantly to the successful performance of the knee. Numerous biomechanical studies have shown that the meniscus has appreciable load-bearing and shock absorption roles, and with its removal there is increased stress on the articular cartilage. Seedhom and Wright found the lateral meniscus to carry 70% of the lateral compartment load, and the medial meniscus 50% of the medial compartment load when the knee is in full extension.12 Ahmed and Burke directly measured the tibiofemoral pressure distribution using micro-indentation transducers and found that the menisci transmit at least 50% of the compressive load imposed on the tibiofemoral joint.13 With the meniscus removed, there would thus be a two-fold increase in the contact stresses. Baratz et al used pressure-sensitive film technique to demonstrate that peak torque contact stresses of the knee joint were increased up to 235% after meniscal excision.14 Biomechanical studies have also shown the menisci to play a role in knee stability. Wang and Walker evaluated the effects of transverse plane rotatory laxity before and after removal of the menisci in human cadaveric knees.15 In their study they concluded that the menisci serve as restraints to rotation associated with primary laxity by acting as “space filling buffers” between the tibiofemoral articular cartilage. Bargar et al found that patients with anterior cruciate ligament tears had statistically significant increased anterior laxity when the meniscus was excised.16 Isolated meniscectomy did not, however, result in appreciable change in laxity. Levy et al similarly showed in a cadaveric model that sectioning the medial meniscus and anterior cruciate ligament resulted in greater anterior tibial displacement compared to the increases from isolated ACL sectioning.17 It should be noted, though, that the combined lateral meniscectomy and ACL sectioning did not result in increased anterior Composite Tissue Transplantation, edited by Charles W. Hewitt and Kirby S. Black. ©1999 R.G. Landes Company.

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excursion compared to isolated removal of the ACL.18 It was thus Levy’s suggestion that in the ACL deficient knee the posterior horn of the meniscus acts as a wedge between the tibiofemoral articular surfaces and resists anterior excursion of the tibia related to the femur. With the medial meniscus being more secure than the more mobile lateral, this may be one of the mechanisms that produces the increased prevalence of posterior horn tears in the medial meniscus in an ACL deficient knee. By the menisci being “space fillers” in the joint they have been shown to assist in the nutrition of chondrocytes as well as increased joint lubrication by estimates of approximately 20%.19 Resistance of extreme joint flexion and extension are also thought to be a role that the menisci play.

Effects of Meniscectomy From the many important roles played by the meniscus it is easy to understand the deleterious effects that would occur to the knee by their excision. As far back as 1936, King postulated the importance of the meniscus in preventing joint arthritis.20 In his canine experiments he was able to follow the natural history of torn menisci and found the importance they play in protecting the articular hyaline cartilage and preventing its degeneration. It was, however, not until 1948 with Fairbanks’ classic article describing significant radiographic evidence of degenerative changes in knees 3-14 months after meniscectomy that a heightened awareness of meniscectomy not being a “wholly innocuous” procedure occurred.21 Multiple clinical studies have further confirmed the high incidence of unsatisfactory results following meniscectomy. Jones et al showed that many patients with medial meniscectomy had persistent medial pain and developed degenerative changes, as demonstrated by narrow medial joint space and varus alignment compared to nonoperative knees.22 Jorgenson et al reviewed the results of meniscectomy in athletes and further confirmed that arthritic changes occurred with meniscectomy.23 Fifteen years after meniscectomy, 46% had reduced their sporting activities and 89% had degenerative changes on radiographs. Tapper and Hoover noted that only 45% of men and 10% of women had symptom-free knees after meniscectomies.24 In their series, patients with partial meniscectomies had improved results compared to those with complete meniscectomies. McGinty et al and Northmore-Ball have also shown that partial meniscectomies have improved results over total meniscectomies.25,26 Due to the known sequelae of meniscus excision, efforts have increased to preserve as much of the meniscal tissue as possible. Besides attempting to perform partial rather than complete meniscectomies, there has been the development of meniscal repair techniques. Initially this involved open methods, with the subsequent development of arthroscopic measures, ranging from inside-out, outside-in, and more recently, all inside techniques.27-32 Clinical studies have shown that in stable knees in which the tear is in the periphery, i.e., the vascular region, the success rate of healing in most series ranges from 80-90%.33-36 Unfortunately, besides meniscal repair not being 100% successful, there are several types that are not amenable to repair. It is because of this type of patient, or those that have had prior meniscectomy and go on to develop pain and arthritic changes, that the need for substitution of one’s own meniscus is well demonstrated. The development of meniscal allograft transplantation was thus developed in an effort to solve this need.

Processing of Meniscal Allografts Many methods to process and preserve meniscal allografts have been used to date, with each having its own advantages and disadvantages. Graft storage, cost, cell viability, disease

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transmission, and possible antigenicity of the allograft have all been mentioned as considerations in selecting the processing technique. Due to meniscal allografts being relatively acellular, it is by and large considered “immunologically privileged”.37 There has been little evidence that immunologic matching or suppression is required for meniscal allograft transplantation. Reactive synovitis in the knee is frequently present for several months following the procedure, and although minor, does demonstrate some immune response by the host. Due to a case report of the allograft “resorbing” when placed in a rheumatoid arthritic patient, it is not recommended that the procedure be performed on those with immunologic inflammatory joint disease, due to their heightened immune response.38 The initial meniscal allografts involved fresh tissue in an effort to maintain cell viability, but this method is very impractical from a technical standpoint. The “readiness” of the recipient at any time a donor is available is one logistic problem. Screening for diseases such as the HIV virus is very difficult in such a short period of time, and while radiation can be used to assure that the virus is killed, it also would kill the graft cells and thus defeat the purpose of maintaining cell viability. The use of fresh meniscal allograft is thus not widespread. Freeze drying (lyophilization) of allografts is also a procedure that originally had interest. The method involves dehydrating the graft while freezing it in the vacuum. This method enables long term storage of the graft and is its main advantage. Once the graft is ready for use, it is thawed and rehydrated. It subsequently serves as a scaffold for the ingrowth of host cells. Unfortunately, the storage process frequently causes the collagen fibers to be damaged and alter the biomechanical properties of the graft.39 In addition, the preparation method does not destroy the HIV virus and monitoring of such entities is still required with this technique. Fresh freezing of meniscal allografts is a technique currently used by several centers for processing of their tissue. This method destroys the donor cells and denatures the histocompatibility antigens, thereby making the immune response even less likely than normal.40 The technique is simple to perform and relatively inexpensive. It readily enables storage of the graft. However, without cell survival the question is raised as to it being as efficacious as cryopreservation in maintaining graft viability and integrity. Monitoring of donor HIV virus is also necessary with this technique. Lastly, an area that has come under much interest more recently is that of cryopreservation. The method involves placing the graft in glycerol and gradually lowering the temperature to freezing so that the cells are in part maintained viable and the collagen make-up of the graft is not altered by the freezing.41,42 As with other methods, the HIV virus is not destroyed and requires monitoring. The main disadvantage of cryopreservation is the added cost and its being something that is not readily done throughout the country. Further studies will need to be performed to determine if the expense warrants the benefits of maintaining cell viability over standard fresh frozen methods. As cited previously, the concern for graft sterilization is an area of great concern. By screening the donors serologically and taking a thorough history to identify risk factors for diseases, one can help diminish disease transmission. However, with the advent of life-threatening viral diseases such as HIV and hepatitis, the need for additional sterilization methods have been noted by several orthopedic surgeons. Fortunately with the current meniscal graft preservation techniques, one is able to routinely store them for an adequate time to perform routine donor screening. Others still routinely use gamma radiation for sterilization techniques of the meniscal allografts. It has been shown that at least 3.0 MRads are required to destroy the HIV-I DNA.43 Even higher dosages are required to inactivate the free virus if it is deep within the infrastructure of the tissue.44 Unfortunately, when one gets beyond the radiation level of 2.5 MRads, significant changes occur in the meniscal allograft tissue and in particular the collagen fiber make-up.45,46 Due to the diminished success rate in grafts

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undergoing gamma radiation, this secondary sterilization method is not routinely used by most meniscal allograft transplanters at present.

Animal Studies Multiple animal studies have been performed to evaluate the feasibility and efficacy of meniscal allograft transplantation. Canham and Stanish transplanted glutaraldehyde-preserved menisci in five dog knees, and tissue culture-preserved menisci in 10 canine knees immediately after medial meniscectomy.47 In the glutaraldehyde-preserved allografts, all knees demonstrated post-operative effusions and did not demonstrate healing to the periphery at autopsy at 2 months. The tissue culture-preserved allografts had no post-operative effusions and resulted in excellent healing at the same time period post-op. Arnoczky et al have performed several studies to evaluate the cellular repopulation and healing capacity of meniscal allografts. In one canine model study they found that in deep freezing, meniscal autograft cells are killed by the preservation technique.48 They also found ready healing of the graft, and the meniscus was repopulated by host cells from the synovium. In a second canine model study, cryopreserved meniscal allografts were evaluated.49 Once again they found that healing of the graft readily occurred to the periphery by 3 months post-op. Histologically, the grafts demonstrated a decrease in the number of metabolically active cells after transplantation, but had a normal cellular distribution and activity by 3 months. Their overall assessment was that cryopreserved meniscal allografts were able to heal to the host tissues, survive within the joint environment and provide a functional replacement for the removed meniscus for up to the 6 month time period of the study. Additional studies in regard to the long term fate of the graft were needed to determine the grafts’ durability. Milachowski et al transplanted freeze-dried, gamma sterilized menisci in the knees of l5 sheep, and frozen menisci in knees of an additional 15 sheep.50 They found that while both methods of preservation had healing of the menisci to the periphery, there was a notable difference in cell activity between the two methods. The freeze-dried transplants had fully remodeled by 48 weeks, while the frozen showed little remodeling. This extensive remodeling resulted in alteration of the normal architecture of the meniscus, as well as reduction of the size. Extensive research has also been performed by Jackson et al using a goat model. In one study, autograft menisci were compared to fresh and cryopreserved allografts.51 At 6 months following surgery, they found little difference in the gross or microscopic evaluation of the implanted grafts. Once again there were excellent peripheral healing and revascularization. There was an increased concentration of water content and a decreased concentration of proteoglycan in the allograft group. The overall conclusion was that additional follow-up would be necessary to determine the long term efficacy of the grafts. In a second study they evaluated the survival of donor cell fibrochondrocytes using a DNA probe technique.52 The results demonstrated that at 4 weeks posttransplantation of the meniscus, all of the DNA was of host origin, with none of the donor remaining. The clinical significance raised by their study was whether the increased expense of maintaining cell viability was justified in regard to utilization of cryopreserved menisci. In summary, from the animal studies it is apparent that a transplanted meniscal allograft is able to readily heal to the capsule and repopulate with host cells rather than those of the donor. Remodeling does occur with variability of rate by different methods of preservation. The techniques with slower remodeling rate maintained structural integrity and functioned to a greater extent. The grafts are also able to survive in the short term and at least provide some efficacy in protecting the hyaline cartilage. However, additional research is needed to evaluate the long term durability of the grafts, as well as their ability to delay or prevent arthritis in the knee.

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Clinical Studies As can occur with any new procedure, the clinical results can vary significantly as experience is gained in regard to such factors as proper patient selection and technical considerations. Nowhere is this more evident than in meniscal allograft transplantation. While the number of reported series is still relatively small and long term follow-up is still needed, there are several definite conclusions that can be made regarding the procedure. Osteochondral allografts of the knee with the meniscus attached have been performed for a few decades, but Milachowski is given the credit for performing the first isolated meniscal allograft in May of 1984. He and his associates subsequently reported on a series involving 22 patients.50 In the series, six patients had fresh frozen grafts, while 16 received freeze-dried menisci. The follow-up was an average of only 14 months, but he did note from second look arthroscopies that the fresh frozen grafts had improved appearance compared to the freezedried. Overall failure rate, however, was comparable between the two groups, with one of the frozen and two of the freeze-dried having to be removed. Garrett reported in 1993 on his series of 43 transplants.53 Fresh menisci were used in 16 cases and cryopreserved in 27. The follow-up ranged from 2-7 years; a large percentage of the patients had complex knee difficulties, as demonstrated by only seven undergoing isolated meniscal transplants. He performed second look arthroscopies in 28 cases and found 20 of the grafts to be intact. The remaining 15 patients did not have relook arthroscopies, but he noted that they were without complaints. By analyzing those grafts that failed, he found that patients in which the femoral condyles had Outerbridge grade IV articular changes had an increased incidence of graft disruption. Van Arkel and deBoer reported on their initial experience of 23 cryopreserved meniscal transplants with a follow-up of 2-5 years.54 Satisfactory results were noted in 20 of the patients; they assessed the three failures as being due to malalignments of the knee that should have been corrected prior to the meniscal transplants. In one of the larger series by a single surgeon to date, Noyes reported a much different picture of the success of meniscal allografts.55 In his series of 96 allografts, 29 of the menisci failed prior to 2 years post-op. Using data achieved with arthroscopy and MRI scan, he found for the series that only 22% healed, 34% partially healed and 44% failed. He thus felt that caution should be used in performing the procedure, and indications were extremely narrow. On critical analysis of Noyes’ results, much information was gained by his high failure rate in efforts to prevent repeating his poor patient outcome. In all instances the meniscal allografts were irradiated with 2.5 MRads and, as noted previously, this has a detrimental effect on the collagen orientation. Secondly, a high percentage of his patients had notably arthritic knees, which similarly has been shown by others to have a negative effect on the success rate. Interestingly enough, in those patients in which he implanted the grafts in minimally arthritic knees, his success rate was approximately 70%. Finally, the grafts were not secured both anteriorly and posteriorly with bone fixation, which has also been shown to play an important role to the graft’s success. The largest cumulative series reported to date was that compiled by CryoLife, Inc. (Kennesaw, GA), a manufacturer and distributor of cryopreserved meniscal allografts, who presented their data at the meniscal transplant study group in February 1997.56 From 1989 to 1996 they accumulated information on a total of 1,023 menisci implanted in 1,015 patients by 166 surgeons. The medial meniscus was implanted in 73% of the recipients, while 27% received a lateral allograft. The mean age of the patients was 34 years, with a range of 10 to 68 years of age. Of those with a follow-up of over 2 years and implanted with bone, 98% were still intact. Only 72% of those implanted without bone and having a follow-up of over 2 years were successful.

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Indications Meniscal allograft is considered an elective procedure, and the fact that the patient has had a meniscus excised does not mean that it should be replaced in all cases. As demonstrated by the clinical studies, patient selection is of utmost importance for the success of the procedure. Knee stability and alignment, age, and degree of arthritic changes are all relevant factors in proper patient selection. The optimal patient would be a healthy, young individual having a stable knee with normal knee alignment and no chondral changes. This is the exception rather than the rule, and general guidelines can be made from the data accumulated to date in regard to patient selection. Age is one factor in which there is some controversy amongst implanting surgeons. Noyes has recommended in the past that the cut off age be 40 years old, while Rubins has implanted menisci in individuals up to the age of 73.57 It is the author’s opinion that due to the efficacy of other “time-tested” procedures, (i.e., arthroplasty) that elderly individuals are not candidates for meniscal allograft transplantation. Conversely, middle-aged individuals can physiologically be young and wish to maintain an active lifestyle. Most transplanting surgeons thus use 50-55 years as the general cut off age. The permissible degree of articular surface abnormality to be a candidate for meniscal allograft replacement was initially an area of some controversy, but is less so at the current time. Several surgeons had shown great enthusiasm for replacing the meniscus in notably arthritic knees because of symptomatic improvement in these patients. Unfortunately, though, follow-up studies demonstrated that notable joint incongruity results in increased failure of the graft. As experience has been gained, those with appreciable areas of Outerbridge grade IV chondromalacia (areas of subchondral bone exposure) have come to be considered poor candidates for the procedure and should be excluded. If there is an isolated articular defect, then possibly performing osteochondral transfer or chondral site resurfacing for grade IV chondromalacia in conjunction with meniscal allograft transplantation may be warranted. At the current time, not enough data has been accumulated to endorse this type of treatment. It should be noted that knees with less than grade IV chondromalacia are not candidates if there is notable flattening of the femoral condyles or osteophytic formation, as this can cause abrasion or extrusion of the meniscal allograft and lead to failure. An area where there is general consensus pertaining to patient selection is that of knee stability and alignment. It is well documented that there is an increased incidence of meniscal tears as well as compromising meniscal repairs occurring in knees that are anterior cruciate ligament deficient.58,59 It is therefore vitally important to perform ACL reconstruction if one is to optimize the survival of the meniscal allograft. If a mechanical malalignment of the knee is present, it is also recommended that an osteotomy to “unload” the affected compartment should be undertaken to maximize the success of the graft.60,61 For instance, if one is implanting a medial meniscus and the knee alignment is anatomically in varus, then a valgus osteotomy should be performed to transfer the forces more laterally. Whether a patient needs to be experiencing knee pain to be considered as a candidate is an area of question. The previously discussed selection factors can be objectively determined, while pain is subjective and at times difficult to determine. Although a large percent of postmeniscectomized patients developed arthritis, not all do, and thus pain is commonly used as a guide to determine which patients are developing arthritis and would benefit from the procedure. Conversely, if a patient’s pain is in excess to their physical findings, or diffuse and not localized to the involved knee compartment, they are considered poor candidates for the operation.

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Surgical Techniques Once the patient has been deemed to be a candidate for meniscal allograft transplantation, determining the size of a graft to match the recipient is necessary. Several studies have been undertaken to assess the most cost effective way of graft sizing, and it has been found that plain radiografts are the most cost effective. Garrett and Stevensen report a technique of matching the meniscal size of donors to recipients with an accuracy within 5% using standard AP radiographs.62 Carpenter et al discovered that MRI imaging consistently underestimated the meniscal allograft, whereas CT scanning and plain radiographs were more accurate.63 They found that the CT scans had no advantage over the plain radiographs in determining the size. The importance of proper meniscal sizing cannot be understated if the graft is to be a truly functional meniscus. If the graft is too small, it should not be used, due to the increased risk of tearing from excessive stresses at the meniscal capsular repair site. If it is too large, the periphery could theoretically be trimmed, but if one violates the collagen fiber make-up of the meniscus, this could alter the hoop stresses of the graft and alter the biomechanics. Although detailed explanation of the surgical procedure is beyond the scope of this text, several salient features warrant discussion for general understanding of the operation. One simple, yet vitally important component to successful healing of the graft is preparation of the meniscal capsular rim. By debriding the remaining capsular rim back to a bleeding base, healing of the allograft to the host tissue readily occurs. The technique of insertion of the allograft can be performed according to the surgeon’s preference. This may be done via arthrotomy, arthroscopic techniques, or a combination of the two with a mini-arthrotomy to insert the graft and arthroscopic preparation and fixation. Although the arthroscopic method with no bone block attachment of the graft is technically the easiest, as previously noted, the success rate of survival is diminished compared to those in which bone fixation is present. Besides the clinical results supporting this information, a study done by Chen et al has shown that bone fixation is important to diminish the contact forces across the joint.64 In their study they found a significant difference in the contact forces if the meniscus was replaced with bone plugs or bridge compared to if no bone attachment was present. Whether the graft was secured with bone plugs or a bridge did not make a difference. If the meniscal allograft was placed without bone stabilization, the subsequent contact forces of the knee were essentially the same as in a complete meniscectomized joint. In maintaining bone attachment for graft fixation, the most commonly used methods currently involve either bone plugs or a bone bridge, with the latter referred to as the “keyhole” method (Fig. 13.1).38,65 If the bone plug technique is performed, then two tunnels at the anatomic site of the previous meniscal attachment are made to stabilize the graft. In the “keyhole” method a small slot is made in the proximal tibia in line with the remnants of the anterior and posterior horns of the meniscus. The allograft is fashioned so that a bone bridge maintains the horn attachments in continuity, and subsequently inserted in this slot. In either method of fixation, once the graft is properly placed, suturing technique is then performed to repair the meniscus in the periphery. Rehabilitation following the procedure has some variability amongst treating surgeons. Due to the question of weight bearing possibly affecting the revascularization process and maintenance of cell viability in cryopreserved allografts, most authors recommend partial or nonweight bearing during the initial four weeks following the procedure. Limited range of motion is allowed during this time period to prevent excessive pull on the meniscal capsular repair, but at the same time enable movement to prevent arthrofibrosis. After 4 weeks post-op, weight bearing is routinely progressed as tolerated and no limits on range of motion are imposed. Crutch use is continued

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A

Fig. 13.1. Diagramatic illustrations of (A) done plug and (B) keyhole techniques of meniscal transplantation.

B

until the patient is ambulating with normal gait. Bicycle riding and light resistance exercises are started subsequent to this, with the author’s personal rehab protocol allowing running at three months post-op and full activities at four months. Other authors have been less aggressive in regard to their rehab, but in the author’s experience no sequelae have been found with this relatively aggressive approach.

Author’s Experience From December 1991 to June 1997 the author has performed a total of 62 meniscal allograft transplantations. All of the grafts have been prospectively studied, with the initial 34 having a follow-up of over two years. A brief review of the results of these 34 allografts is as follows. The patient group was comprised of 33 patients, who received a total of 34 cryopreserved meniscal allografts. One patient had bilateral medial meniscal transplants performed at separate operations. The average follow-up was 38.1 months with a range of 24 to 65 months. Twenty-five of the patients were male and eight were female. The average range of the patients at the time of their reconstruction was 19-50 years, with a mean of 34.4

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years. Thirty-one of the menisci were medial and three were lateral. Thirty-two of the menisci were replaced for chronic excisions, with the average time since removal being nine years (range 2-24 years). Two patients had the menisci replaced subacutely (within three months of excision). All of the patients with chronic excision had knee pain involving the involved compartment preoperatively, while the two “subacute” patients did not have pain. The degree of chondromalacia in the involved knees was variable, with the vast majority of patients having Outerbridge grade II and III chondral changes. The vast majority of the patients had complex knee problems, with only eight having the meniscus replaced alone. Twenty-four patients had an anterior cruciate ligament reconstruction performed at the same time as the meniscus replacement. One patient had a proximal tibial valgus osteotomy to unload the medial compartment, while another had a medial collateral ligament reconstruction performed at the time of the meniscus transplant. Results of the study were most encouraging. Subjective analysis found 32 of the 33 patients stated that they would undergo the procedure again if addressed with the same problem. Of those having pain preoperatively, only one did not have a noticeable lessening of pain following their surgery. The activity level of the patients was also improved in the majority of the patients. It should be noted that the patients were informed preoperatively that the purpose of meniscal transplantation was to assist in their pain and hopefully delay or prevent arthritic changes of the knee, and not to increase their activity level. Their increase in activities was taken up by themselves, due to their improvement of knee symptoms. Objective assessment in addition to obvious physical examination involved radiographic evaluation and second-look arthroscopies. Thirty-one of the allografts were evaluated via secondlook arthroscopies performed under local at 3-23 months post-op, with the vast majority being performed at six months. Six patients also had third-look arthroscopies, with two of these as part of an ongoing study in which patients have had their meniscal allografts longer than 5 year duration. One patient also had a third-look arthroscopy at 3 years post-op, after undergoing anterior cruciate ligament reconstruction on the contralateral limb. Two patients had third-look arthroscopies because of having hardware removed, with this being 14 and 22 months post-op, respectively. The sixth patient underwent reevaluation at 14 months because of departing to attend college on a basketball scholarship and wishing to know the condition of the graft. The information achieved from the arthroscopies indicated that healing of the meniscus to the capsule readily occurred. Only one patient experienced a disruption of the meniscal capsular repair site, and in retrospect this was due to an osteophyte eroding into the area of repair and causing failure of the anterior half at seven months post-op. The degree of chondromalacia was found to progress in only two patients. In both of these instances, the knees had Outerbridge grade III chondromalacia of the involved compartment at the time of the index procedure and in retrospect both had varus alignment, with the medial compartment being the involved side. Three grafts also had visible shrinkage in size. Plain radiographs were taken both preoperatively and at their most post-operative visit for all patients. Notable progression of arthritis was present in only two of the patients and correlated with those that had the varus alignment and deterioration found at the arthroscopic reevaluation. Six knees had dynamic MRI scans performed both preoperatively and at six months post-op. The MRI results demonstrated them to be of limited benefit in regard to routine assessment of the graft. Abnormal signals were found in all of the allografts, while secondlook arthroscopies showed no visible abnormalities (Fig. 13.2). Previous studies have confirmed that abnormal signals can be present following meniscal repair even in autogenous tissue and confirms the limited usefulness of evaluating meniscal allografts for possible tears after reconstruction.66,67 If wishing to determine if the meniscus maintains its position within the knee joint and “unloads” the joint, then MRI scan would be of benefit.

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Four additional patients that had only the meniscus replaced also had preoperative SPECT bone scans and the studies repeated at one year post-op. Results of the bone scans were of limited benefit at this early stage following the procedure. Increased tracer activity was found in all of the knees due to the tibial tunnels drilled for bone fixation of the grafts, and thereby made it difficult to appreciate any change in the bone activity related to the meniscus. Possibly, obtaining the SPECT bone scans several years later would demonstrate a change in bone activity. However, the cost effectiveness of performing bone scans is again raised, as the plain radiographs very likely would start to show progression in the same time frame, at a much decreased cost, if the meniscus was not providing some function. Complications of the surgical procedure were few, with only two grafts requiring excision. As previously noted, one required partial excision, while a second graft was completely removed due to the patient feeling as if it was subluxing out of the joint. At the time of its removal the graft was in fact found to be hypermobile. In retrospect it was felt to be due in part to his return to full activities against medical advice at 8 weeks post-op, which compromised healing of the graft in its proper position. Three additional patients developed mild arthrofibrosis following the procedure and required arthroscopic debridement with excellent results and no appreciable limitations of range of motion. One patient that had valgus osteotomy and another that had ACL reconstruction also required additional surgery for symptomatic hardware removal.

Summary Meniscal allograft transplantation is an area of great interest due to the limited options for those that have started to develop pain and arthritis from meniscal excision. It is a technically challenging procedure with narrow indications at present. Several questions still need to be answered pertaining to areas such as which asymptomatic patients, if any, would be appropriate candidates. Continued study is also needed to determine the long term durability of the grafts and efficacy in deferring arthritis. At the current time, the subjective results are most encouraging. It is the author’s personal feeling that the procedure can be a useful one and should be kept in the orthopedic surgeon’s armentarium for treating postmeniscectomized patients.

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References 1. Henning CE, Lynch MA. Current concepts of meniscal function and pathology. Clin Sports Med 1985; 4:259. 2. Kurosawa H, Fukuboyashi T, Nakajama H. Load-bearing mode of the knee: Physical behavior of the knee joint with and without menisci. Clin Orthop 1980; l49:283. 3. Radin EL, Lamotte F, Maquet PG. Role of the meniscus in the distribution of stress in the knee. Clin Orthop 1984; 185:291. 4. Voloshin AS, Wosk J. Shock absorption of the meniscectomized and painful knees: A comparative in vivo study. J Biomed Eng 1983; 5:157. 5. Walker PS, Erkman MJ. The role of the meniscus in force transmission across the knee. Clin Orthop 1975; 109:184. 6. Allen PR, Denham RA, Swan AV. Late degenerative changes after meniscectomy: Factors affecting the knee after operation. J Bone Joint Surg [Br] 1984; 66:666. 7. Appel H. Late results after meniscectomy in the knee joint: A clinical and radiographic follow-up investigation. Acta Orthop Scand (suppl) 1970; 133:I-III. 8. Elmer RM, Moskowitz RW, Frankel VH. Meniscal regeneration and post meniscectomy degenerative joint disease. Clin Orthop 1977; 124:304. 9. Huckell JR. Is meniscectomy a benign procedure? A long-term follow-up study. Can J Surg 1965; 8:254. 10. Johnson RJ, Kettlekamp DB, Clark W et al. Factors affecting late results after meniscectomy. J Bone Joint Surg[Am] 1974; 56:719. 11. Krause W, Pope MH, Johnson RJ et al. Mechanical changes in the knee after meniscectomy. J Bone Joint Surg [Am] 1975; 58:599. 12. Seedhom BB, Hargreaves DJ. Transmission of the load in the knee joint with special reference to the role of the menisci. II. Experimental results, discussion and conclusions. Eng Med 1979; 8:220. 13. Ahmed AM, Burke DL. In vitro measurement of static pressure distribution in synovial joints. Tibial surface of the knee. J Biomech Eng 1983; 105:226. 14. Baratz ME, Fu FH, Mengato R. Meniscal tears: The effect of meniscectomy and repair on intraarticular contact areas and stress in the human knee. A preliminary report. Am J Sports Med 1986; 14:270. 15. Wang GJ, Walker PS. Rotation laxity of the human knee. J Bone Joint Surg [Am] 1974; 56:161. 16. Bargar WL, Moreland JF, Markolf JL et al. In vivo stability testing of post meniscectomy knees. Clin Orthop 1980; 150:247. 17. Levy IM, Torzilli PA, Warren RF. The effect of medial meniscectomy on anterior-posterior motion of the knee. J Bone Joint Surg [Am] 1982; 64:883. 18. Levy IM, Torzilli PA, Gould JD et al. The effect of lateral meniscectomy on motion of the knee. J Bone Joint Surg [Am] 1989; 71:41. 19. Renstom P, Johnson RJ. Anatomy and biomechanics of the menisci. Clin Sports Med 1990; 9:523. 20. King D. The function of semilunar cartilages. J Bone Joint Surg [Am] 1936; 18:1069. 21. Fairbanks TJ. Knee joint changes after meniscectomy. J Bone Joint Surg[Br] 1948; 30:664. 22. Jones RE, Smith EC, Reisch JS. The effect of medial meniscectomy in patients older than forty years. J Bone Joint Surg [Am] 1978; 60:783. 23. Jorgenson U, Sonne-Holm S, Lauridsen F et al. Long-term follow-up of meniscectomy in athletes. J Bone Joint Surg [Br] 1980; 69:80. 24. Tapper EM, Hoover NW. Late results after meniscectomy. J Bone Joint Surg[Am] 1969; 51:517. 25. McGinty JB, Geuss LF, Marvin RA. Partial or total meniscectomy. J Bone Joint Surg [Am] 1977; 59:763. 26. Northmore-Bell MD, Dandy DJ, Jackson RW. Arthroscopic open partial and total meniscectomy. J Bone Joint Surg [Br] 1983; 65:400. 27. DeHaven KE, Black KP, Griffiths HJ. Open meniscal repair. Am J Sports Med 1989; 17:788.

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28. Graf B, Docter T, Clancy W. Arthroscopic meniscal repair. Clin Sports Med 1987; 6:525. 29. Jakob RP, Staubl HV, Zuber K et al. The arthroscopic meniscal repair. Am J Sports Med 1988; 16:137. 30. Miller DB. Arthroscopic meniscal repair. Am J Sports Med 1988; 16:315. 31. Morgan CD. The “all inside” meniscus repair. Arthroscopy 1991; 7:120. 32. Reigel CA, Mulhollan JS, Morgan CD. Arthroscopic all inside meniscus repair. Clin Sports Med 1996; 15:483. 33. Barber FA: Meniscus repair: Results of an arthroscopic technique. Arthroscopy 1987; 25. 34. Cassidy RE, Shaffer AJ. Repair of peripheral meniscus tears. Am J Sports Med 1981; 9:209. 35. Cooper D, Arnoczky S, Warren R. Arthroscopic meniscal repair. Clin Sports Med 1990; 9:589. 36. Scott GA, Jolly BL, Henning CE. Combined posterior incision and intraarticular repair of the meniscus. J Bone Joint Surg [Am] 1987; 68:847. 37. Arnoczky SP, Milachowski KA. Meniscal allografts: Where do we stand? In: Ewing JW, ed. Articular cartilage and knee joint function. Basic Science and Arthroscopy. New York: Raven. 1990:129. 38. CryoLife, Kennesaw, GA. Personal communication. 39. Jackson DW, Simon TM. Biology of meniscal allograft. In: Mow VC, Arnoczky SP, Jackson DW, eds. Knee Meniscus Basic and Clinical Foundations. New York: Raven, 1990:141. 40. Graham WC, Smith DA, McGuire MP. The use of frozen stored tendons for grafting: An experimental study. J Bone Joint Surg [Am] 1955; 37:624. 41. Arnoczky SP, McDevitt CA, Schmidt MB et al. The effect of cryopreservation in canine menisci: A morphologic and biomechanical evaluation. J Orthop Res 6; 1:1988. 42. Goble EM. Meniscal allograft reconstruction. Presented at 59th Annual Meeting of American Academy of Orthopedic Surgeons, Washington DC, February 23, 1992. 43. Fideler BM, Vangness CT, Jr., Moore T et al. Effects of gamma irradiation on the human immunodeficiency virus: A study in frozen human bone-patellar ligament-bone grafts obtained from infected cadavera. J Bone Joint Surg [Am] 1994; 76:1032. 44. Conway B, Tomford WW, Hirsch MS et al. Effect of gamma irradiation on HIV-I in a bone allograft model. Trans Ortho Res Soc 1990; 15:225. 45. Butler D, Oster D, Feder S et al. Effects of gamma irradiation on the biomechanics of patellar tendon allografts of the ACL in the goat. Trans Ortho Res Soc 16:205, 1991. 46. Yahai L, Zukor D. Irradiated meniscal allotransplants of rabbits: Study of the mechanical properties at six months post-op. Acta Orthop Belg 1994; 60:210. 47. Canham W, Stanish W. A study of the biological behavior of the meniscus as a transplant in the medial compartment of a dog’s knee. Am J Sports Med 1986; 14:376. 48. Arnoczky SP, O’Brien SJ, DiCarlo EF et al. Cellular repopulation of deep-frozen autografts: An experimental study in a dog. Orthop Trans 1988; 2:4. 49. Arnoczky SP, Warren R, McDevitt CA. Meniscal replacement using a cryopreserved allograft. Clin Orthop 1990; 252:121. 50. Milachowski KA, Weismeir K, Wirth CJ. Homologous meniscus transplantation: Experimental and clinical results. Int Orthop 1989; 13:1. 51. Jackson DW, McDevitt CA, Simon TM et al. Meniscal transplantation using fresh and cryopreserved allografts: An experimental study in goats. Am J Sports Med 1992; 20:644. 52. Jackson DW, Whelan J, Simon TM. Cell survival after transplantation of fresh meniscal allografts: DNA probe analysis in a goat model. Am J Sports Med 1993; 21:540. 53. Garrett JC. Meniscal transplantation: A review of 43 cases with two to seven year follow-up. Sports Med and Arthroscopy Rev 1993; 2:164. 54. Van Arkel ER, deBoer HH. Human meniscal transplantation: Preliminary results at two to five year follow-up. J Bone Joint Surg[Br] 1995; 77:589. 55. Noyes FR. Irradiated meniscus allografts in the human knee: A two to five year follow-up study. Presented at the Meniscal Transplant Study Group, Orlando, Florida, February 18, 1995.

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56. Mack DD, Fronk DM, McNally RT. Cryopreserved meniscal allograft reconstruction. Presented at the Meniscal Transplantation Study Group, San Francisco, CA, February 18, 1997. 57. Rubins D. Arthroscopic meniscal allograft transplantation. Presented at the Arthroscopy Association of North America 12th Annual Meeting, Palm Desert, California, April 1-4, 1993. 58. Cannon WD, Vittori JM. The incidence of healing in arthroscopic meniscal repairs in anterior cruciate ligament reconstructed knees versus stable knees. Am J Sports Med 1992; 20:176. 59. Warren RF. Meniscectomy and repair for ACL deficiency. Clin Orthop 1990; 252:55. 60. deBoer HH, Koudstaal J. Failed meniscal transplantation: A report of three cases. Clin Orthop 1994; 306:155. 61. Teitg RA. Preoperative planning for osteotomy of the proximal tibia. Sem Arthroplasty 1996; 7:133. 62. Garrett JC, Stevensen RS. Meniscal transplantation in the human knee: A preliminary report. Arthroscopy 1991; 7:57. 63. Carpenter JE, Wojtys EM, Huston LJ et al. Preoperative sizing of meniscal allografts. Arthroscopy 1993; 9:344. 64. Chen MI, Branch TP, Hutton WC. Is it important to secure the horns during lateral meniscal transplantation? A cadaveric study. Arthroscopy 1996; 12:174. 65. Shelton WR, Dukes AD. Meniscus replacement with bone anchors: A surgical technique. Arthroscopy 1994; 10:324. 66. Crues JV III, Ryu R, Morgan FW. Meniscal pathology: The expanding role of magnetic resonance imaging. Clin Orthop 1990; 252:80. 67. Rodeo SA, Potter HG, Wickiewicz TL et al. Magnetic resonance imaging of meniscal allografts: Correlation with early outcome. Presented at the 14th Annual Meeting of the Arthroscopic Association of North America, San Francisco, California, May 4-7, 1995.

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CHAPTER 14

Potential New Immunosuppressants for Composite Tissue Transplantation Daniel Jung and Barry D. Kahan

Introduction

T

he development of immunosuppressive drugs that prevent and/or control allograft rejection episodes has revolutionized the field of transplantation. Experiments performed in 1959 by Schwartz and Damashek first demonstrated the potential of the antimetabolite 6-mercaptopurine for the prolongation of canine kidney allograft survival. Due to the effects of variable absorption from the gastrointestinal tract, severe hepatic toxicity and myelosuppression, this drug could not be introduced into clinical practice. The combination of corticosteroids with azathioprine, an imidazole analog with more reproducible properties, was the most widely used clinical immunosuppressive regimen during the 1960s and 1970s, until Calne introduced cyclosporine A into the clinical arena. The fungal metabolite CsA has dramatically improved the prophylaxis of acute rejection, although beclouded by renal and hepatic toxic side effects. Tacrolimus, a macrolide immunosuppressant with in vitro potency at least ten-fold greater than CsA, was introduced in 1984, but was subsequently found to have an immunosuppressive index no broader than that of CsA. Although new compounds had been tested by the end of the 1980s— including 15-deoxyspergualin, mycophenolate mofetil, mizorbine, and brequinar—these drugs showed only additive effects with CsA. In contrast, rapamycin offers a synergistic contribution to CsA-based immunosuppression. Ongoing development of more selective agents such as antisense oligonucleotides and the introduction of new small molecule agents that specifically alter T cell function, such as FTY720, promise safer immunosuppression in the next millennium. The present review focuses on the general aspects of many new immunosuppressive agents, including the few published results in the field of composite tissue transplantation.

Cyclosporine (CsA) Analogs Two analogs of CsA have recently been evaluated in clinical trials—cyclosporine G and IMM-125—following in vitro and in vivo immunologic studies, pharmacokinetic evaluation, and safety analysis in preclinical models. Only recently has each of these drugs undergone clinical investigation as a therapeutic alternative to CsA.

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

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Cyclosporine G (CsG) Chemical Structure and Mechanism of Action OG37-325 is a natural CsA derivative which inhibits induction of lymphokine genes after T cell activation in a manner similar to that of CsA, namely binding to cyclophilin isoforms and, via this complex, binding to calcineurin with similar affinity as CsA. Thus, gene transcription of a number of the lymphokines induced early in the process of lymphocyte activation (interleukin (IL)-2, IL-3, GM-CSF and tumor necrosis factor (TNF)) is reduced. Consequently, proliferation stimulated by alloantigens (mixed lymphocyte culture), mitogens (concanavalin-A (ConA) or pokeweed mitogen), viral (MLs-1a) or bacterial (staphylococcal endotoxin B) superantigens1 is reduced to a similar extent as with CsA. Unlike CsA and its metabolite, which have been shown to alter the release of vasoactive substances such as endothelin and prostacyclin both in vitro and in vivo, the effect of CsG and its metabolite on the release of such substances has not been investigated in the primary cultures of rabbit endothelial and renal mesangial cells. The majority of the metabolites had little effect on cell growth and DNA synthesis and had no effect on the release of these analytes.2 PreClinical Animal Studies Experimental studies were performed showing CsG to be generally as effective,3,4 but occasionally less effective, than CsA5,6 for the prevention of rejection in animal kidney, heart, liver, and skin allografts. Human Pharmacokinetics CsG is rapidly absorbed in dose-dependent fashion from the gastrointestinal tract. After administration of 150 or 600 ng/ml, the maximum blood concentrations of CsG occurred at 2 to 3 hours postdose and averaged 342 ng/ml and 1179 ng/ml, respectively. Compared with the 0.4 plasma:blood ratio of CsA, the plasma:blood ratio of CsG is 0.9. CsG is primarily eliminated through biliary excretion, with only 3% of the dose excreted in the urine.7 Like CsA, CsG is metabolized through the cytochrome P-450 IIIA4 enzyme system, with the metabolites possessing less than 10% of the immunosuppressive activity of the parent molecule.8 Clinical Studies Phase II trials in kidney transplant recipients demonstrated whole blood drug levels of 200-400 ng/ml, measured using CsA-specific immunoassay. CsA was well tolerated during the first six months after transplantation.7 Multicenter clinical phase II studies on the use of CsG for prophylaxis of rejection in cadaveric renal transplantation utilized a starting dose of 10 mg/kg/d. CsG appeared to be as effective as CsA in preventing acute rejection episodes.9 Patients enrolled in the phase II study who were treated with CsG had better renal function as evidenced by a more rapid fall in serum creatinine levels within, and a lower serum creatinine at, eight weeks posttransplant.9 The serum creatinine concentrations in the CsG group were lower (despite the observation that CsG blood trough levels were 50% higher) than those in the CsA group. These indicate a more rapid recovery of renal function following transplantation in patients receiving CsG compared with those on CsA. Conclusively, CsG facilitates more rapid recovery of renal function and may induce less renal toxicity than CsA in kidney transplant patients.

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SDZ IMM-125 Chemical Structure and Mechanism of Action SDZ IMM-125 and CsA have identical molecular mechanisms and share similar levels of immunosuppressive efficiency. Human Pharmacokinetics SDZ IMM-125 is metabolized by human liver slices to primary metabolites, hydroxylated IMM-1 and IMM-9 and N-demethylated IMM-4N. However, the rate and extent of SDZ IMM-125 biotransformation is only about 25% that of CsA.10 Experimental Studies Although there are only scant clinical data about SDZ IMM-125, there are many published experimental studies. In the canine renal allograft model, SDZ-IMM-125 (20 mg/kg/d) prolongs transplant survival up to 50 days. However, at this dose there are histological changes suggestive of liver toxicity, as well as a mild anemia without evidence of nephrotoxicity.11 In a study of psoriasis patients, there were dose-dependent therapeutic effects and a significant decrease in the area of body surface afflicted by psoriasis, without any evidence of serious adverse effects.12 In the rat small bowel allograft model, host versus graft (HVG) and graft versus host (GVH) responses were alleviated, and in the rat pancreas allograft model there was a delay in the outset of allograft rejection.13

Other New Immunosuppressants Tacrolimus Chemical Structure and Mechanism of Action Tacrolimus (TRL; FK506, Fujisawa, Osaka, Japan), a polycyclic macrolide antibiotic isolated from the actinomycete Streptomyces tsukubaensis,14 has a mechanism of action similar to that of CsA. TRL inhibits calcineurin, which is responsible for dephosphorylation of the cytoplasmic component of NF-AT (nuclear factor of activated T cell), the first regulatory protein controlling the enhancer region of the IL-2 gene,15 and the subsequent steps necessary for IL-2 gene transcription and completion of the activation cascade.16 The effect during the initial hours of the G0 phase of the cell cycle demands that TRL bind the cytoplasmic immunophilin FK-binding protein (FKBP). Cell cycle analysis shows that cells are blocked during the G0/G1 transition without transcription of the immediate early genes (such as IL-2, IL-3, IL-4, IFN-g, and GM-CSF).17 Once activation has occurred, TRL does not affect T cell proliferation, and it does not inhibit the effector function of T or natural killer (NK) cells.18 TRL only inhibits T cell dependent primary humoral responses and does not affect either secondary or T cell independent B cell responses. In vivo studies showed that TRL, unlike CsA, inhibits the activation of CD4+ and CD8+ helper cell, as well as cytotoxic, T lymphocytes but does not affect antibody-dependent cell cytotoxicity or NK activities.19 PreClinical Animal Studies In experimental allograft models, TRL prolonged the survival of rat skin, heart,20 kid21 ney, liver,22 small bowel,23 and pancreatic islet transplants.24 In a small bowel transplant model, there was a greater prolongation of graft survival and prevention of GVH disease

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among animals treated with TRL than those treated with CsA.25 TRL is also effective in allocomposite tissue transplantation. When myoblast transplants in monkeys were treated with TRL, there was no significant infiltration by CD4 or CD8 lymphocytes, no production of detectable antibodies, and little mRNA expression in comparison to nonimmunosuppressed monkeys.26 In rat hindlimb allografts, there was complete prophylaxis of rejection and the expression of IL-1#, IL-2, IL-6, IFN-!, FGF, and TGF-∃ mRNAs were all depressed to levels below those seen with isografts.27 Rat limb transplants treated with TRL achieved longer times to rejection than those treated with CsA.28 Even across a strong genetic mismatch, peripheral nerve tissue transplants, which are known to be highly antigenic, enjoyed prolonged survival with TRL.29 Human Pharmacokinetics The pharmacological properties of TRL are similar to those of CsA. In human studies of pharmacokinetics, TRL showed peak blood levels at 1-3 hours after oral administration. The average oral bioavailability of TRL was 12-27%. The dose-normalized peak TRL levels were 0.1-0.5 ng/ml/kg for plasma and 40 ng/ml per 0.1 mg/kg dose for whole blood. The half life of the intravenous (i.v.) form was 9-12 hours. The oral form was rapidly absorbed independent of the presence of bile. TRL was primarily eliminated by hepatic metabolism via the P-450 IIIA cytochrome system.30,31 Coadministration of drugs that interact with the cytochrome P-450 system may affect TRL concentrations: Imidazole, verapamil, metronidazole, and erythromycin increase blood levels; while phenytoin and phenobarbital decrease blood levels.31,32 Clinical Studies Many clinical trials have compared TRL with CsA with regard to baseline immunosuppression. In a phase II study of TRL and prednisone on the prevention of cadaveric liver allograft rejection, there were higher one year patient and graft survivals, lower incidences of rejection during one month, and lower incidences of steroid-refractory rejection among patients receiving TRL compared to those receiving CsA.33 Phase III multicenter trials in Europe 34 and in the United States35 showed similar patient and graft survival rates but higher incidences of renal toxicity, altered glucose metabolism, and neurologic complications among patients in the TRL group than those in the CsA group. In renal transplantation, a phase II study showed no significant difference between the TRL and CsA groups in patient or graft survivals at one year, the rate of rejection, or the incidence of steroid rejection; however, there appeared to be less steroid-dependence and hypertension among patients receiving TRL.36 Similarly, no differences were observed among the rates of rejection-free survival in heart/lung37 transplants between patients in the TRL and CsA groups. There appeared to be a higher pancreas graft survival rate and lower rejection rate among simultaneous pancreas kidney (SPK) transplant recipients treated with TRL versus Sandimmune.38 The United States multicenter showed that there were significant correlations between TRL whole blood and plasma concentrations and the occurrence of nephrotoxicity during the first 5 weeks posttransplant. But, there was no correlation between the plasma or whole blood concentrations of TRL and the incidence of acute rejection episodes during the same period.39 The reported toxicities of TRL in liver transplant recipients include hyperkalemia (50%), acute hypertension (42%), chronic hypertension (32%), acute renal dysfunction (36%), chronic renal dysfunction (31%), acute dialysis (23%), chronic dialysis (1%), acute insulin use (36%), chronic insulin use (10%), and neurologic abnormalities (8%) including sei-

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zure, akinetic mutism, coma, aphasia, focal deficits, psychosis, and encephalopathy.40 TRL inhibits insulin mRNA transcription via its potent effects on calcineurin activity in pancreatic beta-cells, thereby causing abnormal glucose metabolism.41 Rejection Reversal TRL was also used for rescue treatment for acute refractory allograft rejection. Renal transplant patients converted to TRL (0.3 mg/kg/d) because of either uncontrolled rejection or complications attributed to CsA or steroid therapy showed a return to baseline serum creatinine levels, an improvement in histological features of the allograft upon renal biopsy, and/or a freedom from chronic dialysis. The Pittsburgh group reported that TRL rescue treatment for refractory acute rejection salvaged 74% of 77 renal allografts (mean follow-up = 14 months).42 Multicenter trials claimed that TRL treatment provided: 1. Prompt, effective reversal; 2. Good long term allograft function; 3. A low incidence of recurrent rejection; and 4. An acceptable safety profile in renal allografts.43 However, the studies were poorly controlled and there has not been FDA approval for this indication. Among patients with ongoing biopsy-proven rejection and CsA intolerance, treatment with TRL (8 mg/d) to achieve a mean 12 hour whole blood trough level of 16 ng/ml resulted in 85% actuarial patient survival and 75% pancreas graft survival over periods up to 8 months.44

Mycophenolate Mofetil Chemical Structure and Mechanism of Action Mycophenolate mofetil (MMF, previously known as RS-61443) is a semisynthetic morpholinoethyl ester of mycophenolic acid (MPA) which is produced by the fungus Penicillium glaucum. MMF noncompetitively inhibits the enzyme inosine monophosphate dehydrogenase, particularly the type II isoform, thereby blocking de novo synthesis of the purine guanosine, which is required for DNA synthesis. The proliferation of T and B cells has been claimed to be especially inhibited by MMF because these cells are dependent on a functional de novo purine synthesis pathway. T cells stimulated in the presence of MPA have been claimed to show defective synthesis of glycoprotein adhesion molecules because of dependence on guanosine intermediates for the assembly of cell surface glycoproteins. IL-2 synthesis by T cells is not impaired by MMF, which does not inhibit early events after cell activation. MPA concentrations of 1-10 nM have no effect on IL-2 synthesis by activated lymphocytes.45 Indeed, MPA is inhibitory when added to ongoing mixed lymphocyte reactions 72 hours after initiation, documenting that MPA works at a late stage in T cell activation, in contrast to CsA and TRL, which inhibit the early events. In cell cycle studies, cells treated with MPA are blocked at the G1 to S transition.46 Concentrations of 0.1 mM (100 nM) of MMF or MPA completely inhibit lymphocyte proliferation, and higher concentrations inhibit fibroblast and endothelial cell proliferation47 and decrease lymphocyte attachment. MPA also seems to decrease the pool of GTP nucleotides in human peripheral blood monocytes, but not in neutrophils. PreClinical Animal Studies MMF has been extensively studied in preclinical animal models. In pharmacokinetic studies in rats, the terminal elimination half life (t1/2 beta) was 4.74 ± 0.33 hours, and the area under the plasma concentration versus time curve (AUC) was 48.78 ± 6.01 mg/ml.

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After intraduodenal (i.d.) administration of MMF at 16.7 mg/kg, the t1/2 beta was 3.92 ± 1.05 hours, and the AUC was 38.03 ± 8.30 ug/ml. Experimental animal studies show that monotherapy with MMF (30-40 mm/kg/d) prevented the rejection of heterotopically transplanted cardiac allografts in Brown Norway (BN) to Lewis (LEW) rats. Combination therapy with MMF and CsA showed additive antirejection activity without any demonstrable increase in each agent’s toxicity.48 Similar prolongation of graft survival was seen in the canine renal49 and hepatic50 allograft models. In a rat hindlimb model of composite tissue allotransplantation, combination therapy with low dose CsA and MMF reduced the incidence of acute rejection to 11%, as compared with 64% and 100% incidences.51 MMF was claimed to prevent the onset of chronic rejection due to the inhibition of antibody formation and the reduction in the production of platelet-derived growth factor, IL-1, and IL-6 by activated macrophages. Heterotopic heart allografts in rats treated with MMF showed a lower incidence and reduced severity of proliferative arteriopathy compared to transplants in hosts treated with CsA or TRL. Combined TRL and MMF immunosuppression prolonged survival after small bowel transplantation in pigs.52 During acute rejection of rat kidney allografts, MMF resulted in better preservation of graft structure and less cellular infiltration and tubular atrophy, presumably due to a reduced expression of adhesion molecules that interact with macrophages.53 MMF inhibits primary antibody responses more efficiently than secondary responses. MPA inhibits the proliferation of human B lymphocytes transformed by Epstein-Barr virus and is not mutagenic. Clinically attainable concentrations of MPA seem to suppress the proliferation of human arterial smooth muscle cells, a property that has been alleged to decrease the risk of lymphoma development and proliferative arteriopathy in long term recipients of MMF.54 Human Pharmacokinetics MMF has a 1.5-fold greater bioavailability than the parent compound, MPA.55 After its rapid oral absorption, MMF is hydrolyzed in the liver to the active metabolite MPA, which is metabolized principally by hepatic glucuronyl transferase to form an inactive phenolic glucuronide (MPAG), which is secreted into the bile. Intestinal glucuronide converts the inactive compound back into active MPA—enterohepatic recycling—which may explain the gastrointestinal toxicity of the drug.56 Clinical Studies Three large multicenter studies, each including 500 kidney transplant patients, showed a statistically significant reduction in the incidence of allograft rejection episodes in MMF groups, with concomitant reduction in the use of antilymphocyte antibody therapy.57 The phase I/II escalating dose (100 to 3500 mg/d) clinical trial in kidney transplant patients failed to document organ toxicity or bone marrow depression in patients treated with MMF;58 rather, MMF produced gastrointestinal side effects of mild ileus, gastritis, nausea, vomiting, and primarily diarrhea. Clinical trials in psoriatic patients suggested that MPA was safe at doses of 20-60 mg/kg/d, with frequent gastrointestinal symptoms, particularly during the first year of treatment, and the occurrence of herpes zoster or simplex viral infections in 10-25% of patients.59 A randomized, double-blind, multicenter, placebo-controlled study of the addition of MMF to a regimen of CsA and oral corticosteroids for the prevention of acute renal allograft rejection enrolled 491 patients. After six months, there was a 46.4% rate of biopsyproven rejection for placebo, 17.0% for 2 g MMF, and 13.8% for 3 g MMF. The spectrum of adverse events included the gastrointestinal tract, the hemic system, and opportunistic infections.60 In heart transplantation, MMF is safe and appears to be at least as effective as

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azathioprine (AZA) for the prevention of rejection episodes.61 The United States randomized, double-blind, placebo-controlled trial showed that the 2 g daily MMF dose was better tolerated and demonstrated a safety profile similar to that of AZA, in contrast to the slightly more effective 3 g dose.62 Rejection Reversal A multicenter trial of conversion to MMF (2-3.5 g/d) at a mean of 20 weeks after transplantation for refractory liver allograft rejection showed responses in 21/23 patients with steroid- and OKT3-resistant acute rejection. The report claimed resolution of rejection in 14 and improvement in 7 patients.63 A randomized, open-label, multicenter study for the treatment of refractory acute cellular renal allograft rejection compared the efficacy and safety of MMF to AZA. Although treatment with MMF seemed to reduce graft loss and death by 45% at 6 months and reduced the risk of experiencing a subsequent biopsy-proven rejection episode or treatment failure by almost 50%,64 the results did not merit approval of MMF for this indication.

Antisense Oligonucleotides Mechanism of Action Therapeutic antisense oligonucleotides may inhibit the expression of critical proteins in many ways: binding to DNA to form a triplex; translational arrest; inhibition of RNA processing; promotion of the degradation of the targeted mRNA by RNAse H-dependent mechanisms, due to binding of target mRNA or pre-mRNA through Watson-Crick base pairing; or by nonspecific binding to proteins. Antisense oligonucleotides have been used to help define the role of oncogenes, such as c-myc or c-myb, in cell proliferation and maturation and to demonstrate the dependence of Th1 helper T cell proliferation on IL-2, and Th2 helper T cells on IL-4, synthesis. The mechanism of action to inhibit product expression may depend on the cell type targeted, the particular mRNA, and/or the chemical nature of the oligonucleotide. Phosphorothioate oligodeoxynucleotides (PS-oligos), in which sulfur atoms are substituted for nonbridging oxygen atoms in the phosphate backbone, demonstrate greatly increased stability toward serum and cellular nucleases and, therefore, have been used for in vivo applications. Experimental Studies The specificity and application of antisense oligonucleotides have been shown in animal models for their ability to selectively block disease-causing genes and thereby inhibit production of disease-associated proteins. A 20-mer phosphodiester oligonucleotide that inhibits the production of a gene product that is essential to the growth of human papilloma virus65 was shown to inhibit viral replication in, and gene expression by, human hepatoma cell lines. In vivo antisense oligonucleotides directed against the 5’-region of the preS gene of the duck hepatitis B virus inhibited viral replication and gene expression.66 Mice with severe combined immunodeficiency that had been infected with Philadelphia leukemia virus displayed a 50% cure rate upon treatment with a combination of cyclophosphamide and bcr/abl antisense oligonucleotides.67 Antisense oligonucleotide molecules have been shown to enter neoplastic CNS cells in tissue culture and inhibit cell proliferation without showing detectable toxicity at concentrations exceeding the expected therapeutic concentrations.68,69 Treatment of human melanoma cells and solid tumors with antisense oligonucleotides targeted to c-myc inhibits their growth and is associated with the induction of apoptosis.70

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In transplantation, a series of PS-oligos, specific for intercellular adhesion molecule-1 (ICAM-1) mRNA, have been shown to inhibit mRNA induction and protein expression of ICAM-1 molecules by mouse endothelioma cells in vitro. In vivo administration via a 7 day osmotic pump prolonged the survival of heart allografts in dose-dependent fashion. Furthermore, combination therapy with antilymphocyte serum rapmycin (SRL) and brequinar (BQR) also produced longer survival rates than PS-oligo monotherapy.71 In combination with an anti-LFA-1 mAb, the ICAM-1 antisense PS-oligos induced donor-specific transplantation tolerance. Thus, ICAM-1 antisense PS-oligos proffer a new method of nontoxic, gene-targeted immunosuppressive treatment for organ transplantation.72 Ongoing phase I/ II studies are seeking to determine the safety and efficacy of this drug for the prevention of acute rejection of cadaveric renal transplantation. Human Pharmacokinetics Pharmacokinetic studies of several PS-oligos demonstrate that they are well absorbed from parenteral sites, distribute broadly to all peripheral tissues, do not cross the bloodbrain barrier, and are eliminated primarily by slow metabolism.

Rapamycin Chemical Structure and Mechanism of Action Rapamycin (SRL, rapamycin; Rapamune, Wyeth-Ayerst, Princeton, NJ), a natural fermentation product (macrolide antibiotic) produced by Streptomyces hygroscopicus, is structurally related to FK506. Although originally evaluated as an anti-fungal agent, SRL has powerful immunosuppressive activities. Its mechanism of action is distinct from that of other immunosuppressants. In contrast to CsA and TRL, which inhibit T cell stimulation by reducing cytokine production, SRL inhibits cytokine-induced signal transduction pathways in the G1 phase of the cell cycle, resulting in marked suppression of IL-2-driven T cell proliferation. SRL inhibits lymphocyte proliferation without affecting nucleotide or cytokine synthesis.73,74 Although SRL and TRL bind to a family of cytoplasmic and membrane bound proteins called FKBPs, they seem to act via distinct molecular mechanisms. SRL blocks signals transduced from IL-2 receptor (IL-2r) to the nucleus75 via phosphorylation of p70S6 kinase, an enzyme that is critical to signal translation for cytokines.76,77 SRL-treated isolated human peripheral blood mononuclear cells display a significantly decreased activity of p70S6 kinase.78 In addition, SRL dramatically diminishes the kinase activity of the cdk4/cyclin D and cdk2/cyclin E complexes that normally peak in the mid-to-late G1 phase of the cell cycle, possibly by preventing the elimination of p27 kip, a negative regulatory protein for cyclin-dependent kinases, inhibiting the phosphorylation of retinoblastoma protein and suppressing the activity of cdk2 and cyclin A.79 The overall effect of these actions is interference with the progression of T cells from the G1 to the S phase of the cycle.80,81 TRL seems to interfere with the action of SRL, probably by displacing it from its FKBPs. In vitro exposure to SRL inhibits T cell and thymocyte proliferation induced by ConA, phytohemagglutinin, anti-CD3 antibody, and alloantigen-driven MLR, as well as anti-IgM antibodypokeweed-mitogen driven cell activation, far more potently than CsA.82 SRL also blocks IgG, IgM, and IgE synthesis by unpurified B cells much more effectively than CsA. In addition, at 10- to 100-fold higher concentrations than those needed to inhibit T cell proliferation,83 SRL prevents antibody-dependent cellular cytotoxicity (ADCC) and the cytolytic effects of natural killer (NK) cells and IL-2 activated killer (LAK) cells.

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PreClinical Animal Studies The immunosuppressive effects of SRL have been investigated in several animal models. Although SRL dispersed in cremophor EL/ethanol for oral administration or suspended in carboxymethyl cellulose displays immunosuppressive potency,84 a greater efficiency is documented upon i.v. administration with a polysorbate polyethanol glycol vehicle both for heart and kidney allografts from Wistar-Furth (WF) to Buffalo (BUF) rats,85 as well as for mouse heart allografts. The efficacy of SRL for prevention of acute rejection exceeds that of TRL and CsA. In addition, SRL blocks skin allo- and xenograft rejection, as well as the destruction of heart, kidney, liver, small bowel, or pancreaticoduodenal grafts,86-90 islet cell rejection in mice, and total limb transplant rejection in rats, although in the last setting it has been claimed to be less effective than TRL.91,92 SRL displays a highly synergistic effect with CsA in vivo to prolong graft survival in many animal models.93 In vitro the two drugs synergize to reduce mitogen-induced lymphocyte proliferation, IL-2, and IL-6-induced proliferation of responsive cell lines, and generation of cytotoxic T cells. A pharmacokinetic-pharmacodynamic study using a reversephase HPLC method to determine SRL concentrations among rabbit recipients of heterotopic heart allografts suggested that the optimal range of SRL blood levels was 10-60 mg/l.94 Using higher doses delivered i.v., SRL displayed nonlinear pharmacokinetics. The drug appears to distribute widely outside the blood compartment. Its long terminal half life (130 hours) indicates slow clearance.95 Human Pharmacokinetics In renal transplant patients the pharmacokinetic parameters show rapid absorption, with 70-75% of the patients reaching peak concentrations within 1 hour, and a relatively long terminal half life, averaging between 57 and 62 hours. The oral clearance seems low, with large intersubject variability, but, due to the bioavailability of the drug, the actual clearance was only below 15% of the oral-dose clearance.96 SRL is at least in part metabolized by the cytochrome P450 IIIA system by both rat and human liver and rat intestinal microsomes. Clinical Studies Phase I clinical studies in renal transplant patients revealed the safety of administration of single doses of SRL orally, to at least 34 mg/m2 and 15 mg/m2, respectively. Multiple doses of SRL (escalating from 0.5-13 mg/m2/d) given orally for 14 days showed the drug to be safe and well tolerated. In phase II trials for the prophylaxis of acute renal allograft rejection, SRL administered in combination with CsA was well tolerated and did not potentiate the nephrotoxic properties or hypertensive properties of CsA, but did show the major toxic effects of reducing platelet counts and increasing hyperlipidemia.97 The multicenter phase II trial showed that SRL (1 or 3 mg/m2/d) in combination with CsA and prednisone reduced the incidence of acute rejection episodes from 40% to 7-10% among renal transplant recipients. Rejection Reversal As rescue therapy, SRL has been more effective for mouse heart grafts or rat heart allograft rejection than higher doses of CsA and TRL,98 even beginning on posttransplant day 5, at which time the rat heart allograft had become heavily infiltrated with mononuclear cells expressing high levels of mRNA for interleukins and cytokines. SRL also seems to have an effect on chronic allograft rejection because of its ability to antagonize both cytokines activating immune cells and growth factors that may be responsible for the proliferation of

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vascular smooth cells. SRL also prevented GVHD when administered at the time of transplantation of allogenic bone marrow and spleen cells, thereby prolonging survival in mouse recipients; in addition, SRL prolonged survival even when administered after the mice had experienced the onset of GVHD.99 Successful myoblast allotransplantation in MDX mice was obtained with SRL.100

Leflunomide Chemical Structure and Mechanism of Action Leflunomide has been used in patients with rheumatoid arthritis and has only recently been considered for use in transplantation. The immunosuppressive activities of leflunomide seem to be as potent as those of CsA.101,102 Leflunomide is derived from a series of compounds synthesized as agricultural herbicides by Hoechst AG. Although leflunomide inhibits the proliferation of both lymphocytic and nonlymphocytic cells, not all cells are equally sensitive.103 B cells and B cell lines appear to be most susceptible (IC50s = 1-10 mM), while T cells and T cell lines are somewhat more resistant (IC50s = 1-50 mM).104 The anti-proliferative activity of leflunomide has been attributed to two distinct effects: inhibition of de novo pyrimidine nucleotide synthesis via inhibition of dihydroorotate dehydrogenase(DHO-Dhase) and a secondary inhibition of tyrosine kinases,105 particularly the Lck and Fyn families which are associated with the transduction of many growth factor receptor signals, including IL-2, IL-3, and TGF-#, but not IL-1. Thus, leflunomide resembles SRL in its suppression of T cell responses to IL-2. These findings suggest that the agent principally inhibits the G1 to S phase of the cell cycle where proliferative responses to IL-2 were blocked.106,107 Leflunomide appeared to depress the enhanced immunological activity usually observed in rheumatoid patients and to normalize CD4/CD3 ratios, as well as to increase the numbers of resting B cells.103 Although leflunomide partially inhibits IL-2 production, it does not affect IL-2 receptor expression, and the administration of exogenous IL-2 fails to restore T cell proliferative responses. In addition to interference with pyrimidine biosynthesis and inhibition of IL-2 receptor-associated tyrosine kinase activity, leflunomide may suppress these immune reactions in vitro, partially by promoting the production of TGF-∃1 production and inhibition of IL-2 production, since TGF-∃1 is immunosuppressive for both T and B lymphocyte proliferation and antibody formation.108 Leflunomide seems to inhibit the in vitro generation of murine B cell plaque-forming colonies (PFCs) in response to the T-dependent antigen SRBC, even when added as late as day four of a five day assay. Leflunomide (IC50 = 5-10 mM) also inhibited murine B cell proliferation upon stimulation with anti-IgM mAb, LPS, or PMA and calcium inophore when added after 24 hours of a four day assay. Thus, it appears that the activity of leflunomide on the humoral response may be derived in part from its effect on B cell proliferation. After hamster to rat heart transplantation, recipient rats rapidly developed anti-hamster IgM xenoantibodies. Leflunomide at a dose of 20 mg/kg/d (days 0 to 21) completely inhibits xenoantibody formation, suggesting that this drug acts directly on B lymphocytes.109 PreClinical Animal Studies The ability of leflunomide to control acute rejection was first reported by Kuchle et al110 in nonvascularized skin and vascularized kidney transplant models. They found equal efficacy of comparable doses of leflunomide and CsA, but only leflunomide was able to prolong skin graft survival when given as late as 5-10 days posttransplant.111 Leflunomide was able to prevent acute rejection of cardiac and small intestine transplants and the development of allospecific antibodies. Rat corneal allograft survival was significantly prolonged

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with leflunomide.112 Leflunomide seemed to potentiate the actions of other immunosuppressants. Despite the strong immunological challenge of a rat neovascularized myocutaneous model, the combination of leflunomide with CsA controlled allorejection.113 SRL and leflunomide seemed to “synergize” with CsA to induce transplantation tolerance in a strong strain combination in rats; the early mechanisms of tolerance seemed to depend on anergy, and the later mechanism on graft accommodation.114 The side effects of leflunomide treatment included interstitial nephritis, ulcerations of the stomach and jejunal mucosa, as well as anemia and anorexia, which have been attributed to the generation of an aniline metabolite following hepatic oxidation of the parent compound. Therefore, the interesting biologic properties of leflunomide seem to be overridden by its propensity for gastric toxicity, emaciation, and anemia, combined with its embryo toxicity and its metabolic conversion to the toxic compound trifluoroaniline. All of these factors have beclouded clinical evaluation until an active nontoxic analog can be discovered. Pharmacokinetics After oral absorption, it is converted in the blood into a stable active opening form, ATT1726, which represents more than 40% of the total metabolites found in animal and human serum. In rodents the half life of ATT1726 is 10-30 hours, which is 10-fold longer than that in humans. The pharmacokinetic characteristics of leflunomide show significant individual variations.

Mizorbine Chemical Structure and Mechanism of Action Mizorbine or bredinine (MZB, 4-carbamoyl-1-B-D-ribofuranoyl-imidazolium-5-olate) is a hydrophilic imidazole nucleoside antibiotic first isolated from the soil fungus Eupenicillium brefeldianum. It was found to affect de novo purine synthesis by inhibiting inosine monophosphate dehydrogenase (IMPDH), which converts IMP to xanthine monophosphate, an intermediate in the synthesis of GMP, and thereby depletes intracellular guanine nucleotide pools (GTP) in lymphocytes, with subsequent inhibition of B and T cell proliferation. MZB was first used clinically more than a decade ago in Japan and was found to be nonmyelotoxic and nonhepatotoxic. There have been no blinded, randomized trials to compare the effects of MZB versus AZA for rejection prophylaxis or steroid sparing. In vitro MZB (1-50 mg/ml) inhibits human peripheral blood T cell responses to stimulation with alloantigen, anti-CD3 mAb, or pharmacologic mitogens by 10-100% in dosedependent fashion. Measurement of purine ribonucleotide pools by HPLC showed that exposure to MZB decreased the intracellular GTP levels. Indeed, addition of GTP reversed the antiproliferative effects of MZB. MZB failed to alter early events after T cell activation, as assessed by steady-state mRNA levels of c-Myc, IL-2, c-Myb, histamine, and cdc2 kinase, as well as surface IL-2 receptor expression. However, cell cycle analysis revealed decreased numbers of cells in the S, G2, and M phases, suggesting a G1/S blockade. MZB has been most widely used in combination immunosuppressive regimens with CsA or corticosteroids.115 PreClinical Animal Studies Studies on in vitro lymphocyte responses demonstrated that MZB (2.5-10 mg/ml) produces dose-dependent inhibition of thymidine incorporation into DNA in canine peripheral blood lymphocytes stimulated with PHA, ConA, or pokeweed mitogen. In addition, MZB significantly reduced the total white blood cell and mononuclear cell count during

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days 5 through 9 of treatment. MZB given at the time of PPD injection significantly suppressed the development of erythema and induration. Furthermore, it reduced hemagglutinin production in response to sheep RBC injections. These experiments suggest effects of MZB on both cell-mediated and humoral immune responses.116 Studies with a short course of MZB show long term viability of tracheal allografts in dogs, as demonstrated by graft patency and epithelialization.117 More commonly, MZB has been studied in combination with CsA in canine renal, heart, and pancreas allografts. In a study of canine renal allografts, the addition of CsA to a regimen produced significantly longer survival than in allografts treated with CsA alone.118 Preliminary results comparing MZB with AZA times in combination with prednisolone in canine kidney transplantation showed that MZB produced less of an elevation of hepatic enzymes, relatively fewer side effects, and longer survival times.119 Pharmacokinetic studies in animal models demonstrated that MZB is readily absorbed orally and is excreted by the kidneys, with a serum half-life of approximately four hours.120 Renal insufficiency, such as seen during renal allograft rejection, was associated with elevated serum levels of the drug and with hemorrhagic gastroenteritis, but not with myelosuppression or hepatotoxicity.121,122 Human Pharmacokinetics In renal transplant patients with normal transplant function (serum creatinine <100 mmol/l), the Cmax was 0.5-1.3 mg/l, tmax 2-4 h, t1/2 3.2-6.2 h, and Cmin <0.05 mg/l.123 Over the therapeutic range of MZB, plasma concentrations ranged from 0.1 to 3 mg/mL.124 There was no correlation between the oral dose and the trough level, Cmax, or the AUC, and there was a poor correlation between the Ccr and the serum MZB concentration. Furthermore, individual patients display a wide range for absorption of MZB.125 Clinical Studies When used as a single agent in a randomized clinical trial in renal transplantation, MZB showed an 80.7% graft survival rate at one year in 61 patients, compared with 63.9% graft survival among 84 patients treated with AZA as a single agent.126 When MZB is used in triple immunosuppressive therapy in cadaveric renal transplantation, the incidence of rejection episodes seems to be reduced and the incidence of leukopenia markedly diminished. Further, in contrast to AZA, there is no contraindication to the concomitant use of MZB and allopurinol.127,128 Although the 61 patients receiving haploidentical renal transplants under treatment with MZB, CsA, and prednisone had similar graft survival rates and serum creatinine concentrations to the group receiving azathioprine, CsA, and prednisone, the MZB regimen was associated with significantly less bone marrow suppression (p<0.005) and systemic infection (p<0.02).129

Brequinar Sodium Chemical Structure and Mechanism of Action Brequinar sodium (BQR, 6-fluoro-2-(2'-fluoro-1,1'-biphenyl-4-yl)-3-methyl-4-quinoline carboxylic acid sodium), a synthetic difluoroquinolone carboxylic acid derivative originally developed as an antineoplastic drug,130 is a noncompetitive inhibitor of dihydroorotate acid dehydrogenase(DHO-DH) in the de novo biosynthetic pathway of pyrimidine nucleotide. The enzyme DHO-DH catalyzes the oxidation of dihydroorotic acid to orotate. Because lymphocytes depend on the de novo pyrimidine pathway for synthesis of RNA and DNA, the action of BQR during the proliferative S phase of immune response is more potent

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than effects on purine synthesis.131 Although this differential sensitivity to BQR allows low doses of the drug to be used for immunosuppression, it has been associated with a high incidence of myelodepression. BQR inhibits both primary T cell dependent and independent B lymphocyte-mediated immune reactions in vivo and anamnestic contact sensitivity responses to DNFB, while not affecting the ability of host to respond to a secondary challenge with an unrelated antigen.132 PreClinical Animal Studies In experiments with 6, 12, and 24 mg/kg BQR treatment for rat heart, kidney, or liver allografts administered three times weekly for a period of 4 weeks after surgery, the compound prevented the occurrence of allograft rejection. BQR (12 mg/kg) reversed ongoing rat liver allograft rejection when administered at days 6, 7, or 8 following transplantation. Indeed, not only did the treatment disrupt the rejection process, but it also induced permanent acceptance of the graft. In rat small bowel allograft recipients, BQR was more effective in blocking HVG than GVHD responses. In combination with perfusion of the allograft with an anti-#∃ T cell receptor monoclonal antibody, BQR provided potent immuno-suppression.133 BQR has displayed synergistic interactions with other immunosuppressive drugs—such as CsA, TRL, SRL, and MMF—that resulted in prolonged graft survival at reduced drug doses.134-137 A study of the effect of BQR on accelerated allograft rejection in presensitized recipients showed that the titer of antidonor antibodies was reduced at 7 days postgrafting, especially the quantities of IgG1 and IgG2b.138 Human Pharmacokinetics BQR is a water-soluble drug with high oral bioavailability (greater than 90%).139 The peak in plasma is achieved at 2 to 4 hours after oral administration. Approximately 20-30% of the drug is eliminated in the urine and 60% in the feces, presumably due to biliary excretion. The predominant role of nonrenal routes of excretion is an advantage for kidney transplantation. The primary difficulties associated with the administration of BQR to man is its narrow therapeutic window for prevention of graft rejection versus the appearance of side effects, possibly due to the long plasma half life of the drug.58 Clinical Studies Phase I clinical trials of BQR in cancer, psoriasis, or rheumatoid arthritis patients demonstrated that the agent was safe when administered intravenously in large single doses at infrequent intervals. The adverse effects included myelosuppression, nausea, vomiting, diarrhea, mucositis, and skin rash.140,141 Phase I studies in allograft recipients showed that single doses of BQR over the dose range of 0.5-4 mg/kg were not associated with any serious toxicities except minor complaints of headache, diarrhea, and pain. Treatment with multiple 0.5-2 mg/kg doses of BQR produced only headaches.139 BQR significantly reduced the incidence of steroid-resistant rejection and resource utilization in primary renal transplant patients compared with AZA.142 But a multicenter study failed to document the benefit of addition of BQR to a CsA-Pred regimen. Experimentally, BQR has effective immunosuppressive activity, synergistic interaction in combination with CsA, ability to prevent antibody formation, good bioavailability, and quantitative methods of monitoring drug blood levels. Phase I trials of BQR have been completed, and the extension of these studies for a more careful examination of the efficacy of the drug may depend upon biochemical modification of the parent drug.143 BQR will potentially be used as a component of future regimens to prevent graft rejection and particularly to dampen humoral responses.

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Deoxyspergualin Chemical Structure and Mechanism of Action 15-Deoxyspergualin (DSG, 1-amino-19-guanidine-11-hydroxy-4, 9, 12-triazanonadecane-10, 13-dione) is a polyamine derivative of spergualin, a natural anticancer drug produced by Bacillus laterosporus, that contains spermine- and guanidine-like structures. Although initially evaluated as an antitumor agent, subsequent studies have documented immunosuppressive properties. Because DSG only poorly inhibits lymphocyte proliferation, it seems to interfere at a later stage in the activation process. In vitro analysis of DSG action suggested blockade of antigen presentation by reducing IL-1 production and by downregulating the expression of MHC Class I antigen and induction of a suppressor cell population.144 In monocytes, DSG has been reported to inhibit the generation of superoxide radicals and hydrolytic enzymes, expression of MHC Class II antigens, and secretion of IL-1.145 When DSG is added to in vitro cultures undergoing cytotoxic T lymphocyte (CTL) differentiation, it almost completely suppresses the development of alloantigen-specific CTLs with variable effect on LAK cells and no effect on NK cell killing.146 DSG has potent effects on humoral immunity. In rodents, DSG inhibits antibody responses to T cell-dependent antigens, including sheep RBC, keyhole limpet hemocyanin, immunotoxins, or alloantigens.147 In humans, DSG inhibits antibody responses to monoclonal antibodies as well as T cell-independent antigens such as DNP-lipo-polysaccharide (LPS) or DNP-Ficoll.148 Thus inhibition of humoral immunity by DSG does not depend upon an effect on T cells but rather a direct inhibition of either B cell development or accessory cell function. The biochemical mechanism of action of DSG seems to depend upon binding to a member of the Hsp70 family of heat shock proteins, heat shock cognate 70 (Hsc70),149 which plays a role in the binding and intracellular transport of antigenic peptides within APCs. Binding of DSG to Hsp70 may interfere with antigen processing and/or presentation.150 Furthermore, the action of hsp facilitates translocation of proteins from the cytoplasm to the nucleus and may explain the effect of DSG to block ( light chain expression, surface immunoglobulin expression,151 and subsequently, B cell differentiation and antibody production. PreClinical Animal Studies Experimental animal studies of DSG in mice showed prolonged survival of heart and thyroid152 allografts compared to CsA. Using peripheral nerve allotransplantation in the rat, DSG therapy appeared to provide better preservation of nerve conduction than CsA.153 DSG prolonged rat skin allograft survival.154 Following transplantation of allogenic bone marrow into lethally mediated mice, DSG blocked the development of GVHD, resulting in prolonged host survival. DSG produces a dose-dependent increase in the median survival time of heterotopically transplanted rat cardiac, liver, and pancreas allografts,155 both as monotherapy and in combination with CsA. Pharmacokinetics DSG has a very short plasma half life with a range of 4-30 minutes due to renal elimination. The drug is poorly absorbed via the gastrointestinal tract, with an oral bioavailability of only 3-6%, demanding that it be administered parenterally. DSG has major acute toxicities, including myelosuppression and, in some animal models, gastrointestinal toxicity.156 Clinical Studies Clinical trials with DSG have been confined largely to pilot investigations. A phase I study in kidney transplantation showed that the maximum tolerated dose was 500 mg/ml

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administered by a three hour intravenous infusion. Bone marrow suppression was the doselimiting toxicity, with leukopenia, thrombocytopenia, and nausea as major side effects occurring with greater frequency and severity at DSG doses )6 mg/kg/d.157 In a phase II open label, randomized trial, DSG successfully reversed 59% of rejection episodes compared with 62% for OKT3.158 Rejection Reversal A pilot study of DSG rescue therapy in refractory acute renal allograft rejection showed that the serum creatinine levels of 3/6 patients improved, and 5/6 kidneys continued to function.159 Groth reported that pancreatic islet cell transplantations under DSG treatment showed several weeks to several months of functional graft survival.160 Upon simultaneous transplantation of pancreatic islets and a kidney into a type I insulin-dependent diabetic(IDDM) endstage renal disease patient, induction therapy with DSG and anti-lymphocyte globulin, followed by maintenance therapy with CsA and azathioprine beginning on day 11, also showed long term graft function.161 These favorable effects of DSG may be explained because macrophages are the effector cells responsible for primary nonfunction of neovascularized cellular transplants such as pancreatic islets, and DSG inhibits several macrophage functions. DSG also reduces preformed antibodies against xenografts, proffering a significant advantage in ABO-incompatible grafts in transplant recipients or in allografting patients with high levels of panel-reactive antibodies.162 Further DSG showed inhibitory effects on rebound antibody formation after plasmapheresis in a renal transplant recipient on primary responses to immunotoxins in cancer patients. DSG humoral immunosuppression may prove useful for blocking the human anti-mouse antibody (HAMA) response observed after OKT3 treatment to transplant patients. In conclusion, DSG has a novel mechanism of action. Although its toxicities do not appear to overlap those of CsA, its toxicity profile and the requirement for parenteral administration have limited its widespread clinical introduction.

FTY720 Chemical Structure and Mechanism of Action A new compound with immunosuppressive properties was chemically modified from the moiety ISP-1 purified from the culture filtrates of Isaria sinclairii. This 2-amino2-2-(4-octylphenyl) ethyl-1,3-propanediol hydrochloride, FTY720, has less toxicity and greater activity than the original substance ISP-1. Its mechanism of action is mediated by apoptotic cell death. After a single oral administration of FTY (10 mg/kg) to rats, the total number of peripheral lymphocytes decreased significantly by 3 hours, while the number of polymorphonuclear cells increased and bone marrow cells and thymocytes were unaffected.163 FTY720 seems to specifically affect the trafficking of T lymphocytes, particularly CD4 positive cells. The reduction of circulating lymphocytes after a single dose lasted at least one week, while the bone marrow was not affected.164 In vitro culture of lymphocytes with large doses of FTY720 showed an increased number of dead cells, as well as features characteristic of apoptosis, such as absence of microvilli, chromatin condensation, and formation of apoptotic bodies by electron microscopy, and chromosomal DNA by agarose gel electrophoresis.163 In vitro the drug inhibits lymphocyte proliferation only at high doses, whether stimulated by allogenic cells or by ConA, and does so much less effectively than CsA. In vitro treatment of human mononuclear cells with FTY720 resulted in a dose-dependent reduction of cell viability. Rapid acceleration of cell death in human mononuclear cells was seen as early as 2 hours after incubation with FTY720.

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PreClinical Animal Studies FTY720 administered for 15 days to rats induced significantly prolonged survival of liver or heart allografts. A pretransplant or immediate posttransplant 2-day course of FTY720 also prolonged allograft survival. In mongrel to beagle kidney transplantation, long term graft survival was obtained by daily administration of FTY720, although at a higher dose than required for rat recipients. FTY720 induced long-lasting unresponsiveness by low dose (0.1-0.3 mg/kg) treatment in the lethal GVH response system. It has been claimed that FTY720 provides more effective immunosuppression than CsA or other immunosuppressive drugs for prevention of GVH response, because FTY720 selectively inhibits immunocompetent T cells but not hematopoietic progenitor cells, including immature lymphocytes.165 Doses of more than 0.1 mg/kg significantly prolong survival of heterotopic cardiac allografts in rats, and doses of 10 mg/ kg induced survival for more than 100 days in 3/5 recipients.166 In a study on the rat skin allografts, administration of FTY720 prevented acute rejection in both MHC-compatible and MHC-incompatible transplants at similar doses to those that were effective upon intraperitoneal administration, indicating good bioavailability of this compound. In canine kidney and rat liver allograft models, pretransplant treatment with FTY720 (5 mg/kg) induced a remarkable prolongation of recipient survival.163 FTY720 significantly potentiated the effects of CsA on graft survival.167 Subtherapeutic doses of each drug showed a remarkable advantage in increasing skin graft survival times,168 which may reflect distinct mechanisms of immunosuppressive action. FTY720 seems to be a promising drug for clinical application for prevention of acute rejection and induction of long term acceptance in transplantation.

Prospectus Compared to the situation 30 years ago, there is a large number and variety of new immunosuppressants. However, each individual agent displays a narrow therapeutic window between adequate immunosuppression and toxicity. Thus there is a need for either rational combinations, or for entirely new immunosuppressive drugs that are not only more potent but also act more specifically upon the immune system with fewer side effects. This chapter has identified synergistic immunosuppressive regimens to achieve maximum pharmacologic immunosuppression utilizing drugs with distinct mechanisms of action and nonoverlapping toxicities. During the next millennium, the primary goal of research in immunosuppression must be the development of a method to induce specific immunological tolerance of clonotypic lymphoid cells directed against foreign donor epitopes without affecting the rest of the immune repertoire. Though composite tissue transplantation has not been performed clinically, some of the new immunosuppressants that have effectively prolonged graft survival may be feasible for this purpose. Controlled clinical trials will be needed to prove that these new drugs offer a clear advantage over conventional immunosuppression for composite tissue transplantation.

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123. Zaoui P, Serre-Deveaubais F, Bayle F et al. Clinical use of mizorbine and pharmacologic monitoring assessment in renal transplantation. Transplant Proc 1995; 27(1):1064-1065. 124. Sonda K, Takahashi K, Tanabe K et al. Clinical pharmacokinetic study of mizorbine in renal transplantation patients. Transplant Proc 1996; 28(6):3643-3648. 125. Kokado Y, Takahara S, Ishibashi M et al. Pharmacokinetics of mizorbine in renal transplant patients. Transplant Proc 1994; 26(4):2111-2113. 126. Ichikawa Y, Ihara H, Takahara S et al. The immunosuppressive mode of action of mizorbine. Transplantation 1984; 38:262-267. 127. Lee HA, Slapak M, Raman GV et al. Mizorbine as an alternative to azathioprine in triple therapy immunosuppressant regimens in cadaveric renal transplantation: Two successive studies. Transplant Proc 1995; 27(1):1050-1051. 128. Dayton JS, Turka LA, Thompson CB et al. Comparison of the effects of mizorbine with those of azathioprine, 6-mercaptopurine, and mycophenolic acid on T lymphocyte proliferation and purine ribonucleotide pools. Mol Pharmacol 1992; 41:671-676. 129. Mita K, Akiyama N, Nagao T et al. Advantages of mizorbine over azathioprine in combination therapy with cyclosporine for renal transplantation. Transplant Proc 1990; 22:1679-1681. 130. Murase N, Starzl TE, Demetris AJ et al. Hamster-to-rat heart and liver xenotransplantation with FK506 plus antiproliferative drugs. Transplantation 1993; 55:701-706. 131. Eiras-Hreha G, Cramer DV, Cosenza C et al. Brequinar sodium: Monitoring immunosuppressive activity. Transplant Proc 1993; 25(3;suppl2):32-36. 132. Jaffee BD, Jones EA, Loveless SE et al. The unique immunosuppressive activity of brequinar sodium. Transplant Proc 1993; 25(suppl2):19-22. 133. Wang M, Stepkowski SM, Kahan BD. Donor pretreatment with anti-T cell receptor monoclonal antibodies prevents graft-versus-host disease in brequinar-treated small bowel allograft recipients. Transplant Proc 1995; 27(1):383. 134. Cramer DV, Chapman FA, Jaffee BD et al. The prolongation of concordant hamster-to-rat cardiac xenografts by brequinar sodium. Transplantation 1992; 54:403-408. 135. Kahan BD, Chou T, Tejpal N et al. Synergistic effects of cyclosporine analogs—A, D, G, IMM-125—with rapamycin and/or brequinar. Transplant Proc 1994; 26(5):3021-3024. 136. Stepkowski SM, Tu Y, Chou TC et al. Synergistic interactions of cyclosporine, rapamycin, and brequinar on heart allograft survival in mice. Transplant Proc 1994; 26(5):3025-3027. 137. Stepkowski SM, Kahan BD. The synergistic activity of the triple combination: Cyclosporine, rapamycin, and brequinar. Transplant Proc 1993; 25(suppl2):29-31. 138. Mozaki S, Ito T, Kamiike W et al. Effective suppression of brequinar sodium on accelerated allograft rejection in presensitized recipients. Transplant Proc 1995; 27(1):451-452. 139. Sher LS, Eiras-Hreha G, Kornhauser DM et al. Safety and pharmacokinetics of brequinar sodium in liver allograft recipients on cyclosporine and steroids. Hepatology 1993; 18:746. 140. Schwartsmann G, Dodion P, Vermorken JB et al. Phase I study with brequinar sodium in patients with solid malignancies. Cancer Chemother Pharmacol 1990; 25:345-351. 141. Arteaga CL, Brown TD, Kuhn JG et al. Phase I clinical and pharmacokinetic trial of brequinar sodium. Cancer Res 1989; 49:4648-4653. 142. Dunn JF, Hatch J, Precht A et al. Brequinar sodium significantly reduces the incidence of steroid-resistant rejection and resource utilization in primary renal transplant patients compared with azathioprine. Transplant Proc 1996; 28(2):955-957. 143. Makowka L, Cramer DV. Brequinar sodium, a new immunosuppressive drug for transplantation. Transplant Sci 1992; 2:50-54. 144. Waaga AM, Krzymanski M, Ulrichs K et al. In vitro analysis of the mode of action of the immunosuppressive drug 15-deoxyspergualin. Archivum Immunologiae et Therapiae Experimentalis 1996; 44(2-3):155-163. 145. Waaga AM, Ulrichs K, Krzysmanski M et al. The immunosuppressive agent 15-deoxyspergualin induces tolerance and modulates MHC-antigen expression and IL-1 production in the early phase of rat allograft responses. Transplant Proc 1990; 22:1613-1614. 146. Kerr PG, Atkins RC. The effects of deoxyspergualin on human cytoxic T lymphocytes: CTL, NK cells and LAK cells. Kidney Int 1990; 38:557-558.

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147. Fujii H, Takada T, Nemoto K et al. Deoxyspergualin directly suppresses antibody formation in vivo and in vitro. J Antibiot(Tokyo) 1990; 43:213-219. 148. Makino M, Fujiwara M, Watanabe H et al. Immunosuppressive activities of deoxyspergualin. II. The effect on the antibody responses. Immunol Pharmacol 1987; 14:115-122. 149. Ramos EL, Nadler SG, Grasela DM et al. Deoxyspergualin: Mechanism of action and pharmacokinetics. Transplant Proc 1996; 28(2):873-875. 150. Hansen LK, Houchins JP, O’Leary JJ. Differential regulation of HSC70, HSP70, HSP90a and HSP90b mRNA expression by mitogen activation and heat shock in human lymphocytes. Exp Cell Res 1991; 192:587-596. 151. Gething MJ, Sambrook J. Protein folding in the cell. Nature 1992; 355:33-45. 152. Kamata K, Okubo M, Masaki Y et al. Effect of the 15-deoxyspergualin on the survival of thyroid allografts in mice. Transplant Proc 1989; 21:1099-1103. 153. Muramatsu K, Doi K, Kawai S. Immunosuppressive effect of 15-deoxyspergualin applied to peripheral nerve allotransplantation in the rat. Exp Neurol 1995; 132(1):82-90. 154. Waaga AM, Krzymanski M, Ulrichs K et al. Induction of specific tolerance by 15-deoxyspergualin treatment after rat skin and kidney transplantation in rats. Analysis of effectivity of various protocols of DOS application. Archivum Immunologiae et Therapiae Experimentalis 1996; 44(2-3):143-153. 155. Schubert G, Stoffregen C, Timmermann W et al. Comparison of the new immuno-suppressive agent 15-deoxyspergualin and cyclosporine after highly allogenic pancreas transplantation. Transplant Proc 1987; 19:3978-3979. 156. Amemiya H, Suzuki S, Niiya S et al. A new immunosuppressive agent, 15-deoxyspergualin, in dog renal allografting. Transplant Proc 1989; 21:3468-3470. 157. Amemiya H, Suzuki S, Ota K et al. Multicentre clinical trial of antirejection pulse therapy with deoxyspergualin in kidney transplant patients. Int J Clin Pharmacol Res 1991; 11:175-182. 158. Okubo M, Tamura K. 15-Deoxyspergualin “rescue therapy” for methylprednisolone-resistant rejection renal transplants as compared with anti-T cell monoclonal antibody (OKT3). Transplantation 1993; 55:505. 159. Matas AJ, Gores PF, Kelley SL et al. Pilot evaluation of 15-deoxyspergualin for refractory acute renal transplant rejection. Clin Transplant 1994; 8:116. 160. Groth CF. Deoxyspergualin in allogenic kidney and xenogenic islet transplantation: Early clinical trials. Ann N Y Acad Sci 1993; 685:193-195. 161. Gores PF, Najarian JS, Stephanian E et al. Insulin dependence in type I diabetes after transplantation of unpurified islets from single donor with 15-deoxyspergualin. Lancet 1993; 341;19-21. 162. Jindal RM, Tepper MA, Soltys K et al. Deoxyspergualin C: A novel immunosuppressant. Mt Sinai J Med 1994; 61(1):51-56. 163. Suzuki S, Enosawa S, Kafefuda T et al. A novel immunosuppressant, FTY720, with a unique mechanism of action, induces long-term graft acceptance in rat and dog allotransplantation. Transplantation 1996; 61(2):200-205. 164. Enosawa S, Suzuki S, Kafefuda T et al. Induction of selective cell death targeting on mature T-lymphocytes in rats by a novel immunosuppressant, FTY720. Immunopharmacology 1996; 34(2-3):171-179. 165. Masubuchi Y, Kawaguchi T, Ohtsuki M et al. FTY720, a novel immunosuppressant, possessing unique mechanisms. IV. Prevention of graft versus host reactions in rats. Transplant Proc 1996; 28(2):1064-1065. 166. Hoshino Y, Suzuki C, Ohysuki M et al. FTY720, a novel immunosuppressant possessing unique mechanisms. II. Long-term graft survival induction in rat heterotopic cardiac allografts and synergistic effect in combination with cyclosporins A. Transplant Proc 1996; 28(2):1060-1061. 167. Kawaguchi T, Hoshino Y, Rahman F et al. FTY720, a novel immunosuppressant possessing unique mechanisms. III. Synergistic prolongation of canine renal allograft survival in combination with cyclosporine A. Transplant Proc 1996; 28(2):1062-1063.

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168. Chiba K, Hoshino Y, Suzuki C et al. FTY720, a novel immunosuppressant possessing unique mechanisms. I. Prolongation of skin allograft survival and synergistic effect in combination with cyclosporine in rats. Transplant Proc 1996; 28(2):1056-1059.

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CHAPTER 15

Rationale for Local Immunosuppression in Composite Tissue Allografting Scott A. Gruber, Mansour V. Shirbacheh and Jon W. Jones

Introduction

I

n contrast to visceral organ transplants, composite tissue allografts (CTAs) are modules composed of various tissues of predominantly ectodermal and mesodermal derivation, and may consist of all or part of the following: skin, subcutaneous tissue, connective tissue, muscle, bone, cartilage, bone marrow, and neurovascular tissue. Although CTAs can theoretically include complex modules derived from anywhere on the body, the vast majority of experimental work has focused on the limb and its individual components. CTAs have tremendous potential clinical application for functional and structural reconstruction of major peripheral tissue defects following birth, major burns, traumatic injuries, or oncological diseases.1 In many of these acquired and congenital tissue defects, the availability of autogenous donor tissue may be severely restricted by unacceptable donor site deformities in terms of function and/or appearance,2 and autografting or use of prosthetic methods would provide a less precise and functional reconstruction than would be possible with the successful use of CTAs.3 Unfortunately, despite advances made during the past decade with regard to: 1. Refinements of microsurgical technique and replantation surgery; 2. Introduction of more specific and potent immunosuppressive agents; 3. Development of better techniques of organ preservation; 4. Expansion of extrarenal visceral organ transplantation; and 5. Improved understanding of the cellular mechanisms of allograft rejection, CTAs continue to remain one of the last frontiers in clinical organ transplantation. In September 1991, the Rehabilitation Research and Development Service of the Department of Veterans Affairs held a 2 day workshop on CTAs, entitled “Can Limbs be Transplanted in Humans in Five Years,” to provide future directions, options, and recommendations regarding limb transplantation research.4 It was concluded that the possible toxicities associated with immunosuppression of CTAs would have to be minimal, while the effectiveness in preventing graft rejection and allowing biomechanical and physiological functional return maximal, prior to clinical consideration. With regard to functional restoration, electrophysiologic studies in rat5 and primate6 peripheral nerve allograft models have demonstrated that host axons may regenerate across major histocompatibility barriers in a Composite Tissue Transplantation, edited by Charles W. Hewitt and Kirby S. Black. ©1999 R.G. Landes Company.

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manner indistinguishable from that of isografts if adequate immunosuppression is provided to prevent rejection. Along these lines, the neuromuscular and physical performance of cyclosporine A (CsA)-treated rat CTAs was not significantly different from that of syngeneic controls,7 and several investigators have documented sensory and motor reinnervation of CsA-treated primate upper extremity CTAs on both neurophysiologic testing and histologic examination.8-10 Finally, immunosuppression of canine vascularized bone11 and knee joint12 allografts with high dose CsA has permitted functional recovery, as assessed by weightbearing ability, gait, and range of active and passive movement, equivalent to that of autografts. Therefore, it would appear that the single most important obstacle currently preventing limb transplantation from becoming a clinical reality is not the inability to restore function, but rather the lack of specific, safe, and effective immunosuppressive therapy. While chronic systemic administration of relatively high doses of nonspecific immunosuppressive agents is readily accepted in the visceral organ transplant recipient faced with poor quality of life or death, this would at present be unacceptable in the patient requiring musculoskeletal reconstruction.13 With this as a background, transplantation of composite tissues in man has been performed in a limited fashion over the past few years. Guimberteau et al14 presented a one year follow-up of two cases of vascularized digital flexion system (living tendon) transplantation with CsA immunosuppression. CsA was withdrawn after 6 months without rejection. In 1995, Hofmann and colleagues15 successfully transplanted a vascularized femoral diaphysis under CsA immunosuppression, apparently the first report of vascularized allogenic grafting of fresh, perfused long bone segments in man. Two more patients were subsequently transplanted, and one of the three has demonstrated primary bone healing and continued vascularization of the graft.16 Moreover, sciatic nerve allografting under CsA immunosuppression has yielded successful results for up to 3 years posttransplant (personal communication, Dr. Susan Mackinnon, Washington University School of Medicine, St. Louis, MO). Finally, a renal transplant patient successfully received a vascularized skeletal muscle allograft and autologous split-thickness skin grafting for reconstruction of a large scalp defect after multiple resections, irradiation, and chemotherapy for refractory squamous cell carcinoma.17

Rat CTA Models Much information concerning the immunological aspects of CTAs has been derived from the vascularized rat hindlimb allograft model. Prior to the introduction of CsA, recipients treated with various combinations of immunosuppressive doses of azathioprine, 6-mercaptopurine (6-MP), and prednisone all died of drug side effects before the onset of macroscopic signs of rejection.18 Although the use of CsA as primary immunosuppressant for rat limb transplants has produced long term, rejection-free survival in some cases,19-21other investigators have noted early22 or delayed23,24 skin rejection, and at the very least, moderate to high dose, continuous therapy would appear to be necessary to prevent rejection across major histocompatibility barriers.21,25 This notion is supported by the fact that discontinuation of CsA administration has also resulted in rapid rejection of rat vascularized2 and nonvascularized26 muscle allografts, peripheral nerve allografts,5 and vascularized bone allografts.13 More recent studies of vascularized allogeneic limb transplantation in the rat utilizing newer immunosuppressants have not significantly improved this overall picture. In one report,27 rapamycin did not prevent rejection when given alone or in combination with CsA, and produced significant toxicity at high doses. Intramuscular administration of a single huge dose (10 mg/kg) of tacrolimus (FK506) on the day of surgery followed by once weekly maintenance dosing (3 mg/kg) induced indefinite allograft acceptance in one study,28

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but the majority of recipients developed Pneumocystis carinii pneumonia. In another report,29 FK506 was given daily for 2 weeks posttransplantation at clinically relevant doses (0.32-0.64 mg/kg/d) by intramuscular injection, and produced an immunosuppressive effect similar to that of CsA, with early rejection of the grafted skin occurring in the majority of animals. Using ten-fold higher doses administered orally, Fealy et al27,30 found that FK506 significantly prolonged allograft survival, prevented rejection without toxicity, and suppressed intragraft cytokine mRNA expression of IL-1, IL-2, IL-6, !-interferon, basic fibroblast growth factor, and transforming growth factor ∃ to below isograft levels. Finally, mycophenolate mofetil was able to both prevent24 and completely reverse3 established acute rejection, although in the former case animals suffered early weight loss (ameliorated with saline injections and dietary supplementation) and moderate bone marrow toxicity with long term therapy. In follow-up studies, the San Francisco group obtained long term graft survival in 89% of animals using a combination regimen of low dose mycophenolate mofetil and low dose CsA, with a significantly reduced incidence of rejection and severity of anemia when compared with high dose mycophenolate mofetil monotherapy.31

Canine and Primate CTA Models In two early reports of canine limb transplantation in which 6-MP/azathioprine was used alone32 or in various combinations with anti-lymphocyte serum and hydrocortisone,33 rejection was manifested initially by skin ulcerations and could not be prevented or reversed without significant systemic drug toxicity. As a result, deaths from generalized, pulmonary, or uncontrollable wound sepsis accompanied by pancytopenia occurred in the majority of animals. Unfortunately, more recent studies of hand, partial hand, and neurovascular free flap transplantation in nonhuman primates have stressed the need for using continuous, high dose, CsA-based immunosuppression to ensure long term allograft survival to an even greater extent than in the above mentioned rodent studies.9,34,35 Skanes et al34 found that, in addition to administering a tapering dose of methylprednisolone beginning at 10-15 mg/kg/d, it was necessary to give massive parenteral (25 to 48 mg/kg/d) or oral (91-115 mg/kg/d) doses of CsA to maintain serum levels in the 820-1500 ng/ml range by radioimmunoassay (RIA) (2- to 3-fold greater than clinically recommended levels) in order to prevent rejection. Side effects included gingival hyperplasia, abnormal hair growth, and multiple subcutaneous abscesses. Similarly, Stevens and colleagues35 noted that 10 of 12 monkeys developed rejection episodes despite maintaining median whole blood trough CsA levels in the 420-1253 ng/ml range by specific RIA and required anti-rejection treatment with high dose steroids or a cocktail of monoclonal antibodies. In this series, five animals developed renal dysfunction, seven were noted to have elevated liver enzymes, seven died of sepsis and/or lymphoid tumor development, and anorexia and weight loss occurred universally.

Rejection of CTAs In both large and small animal models, it has become clear that vascularized CTAs elicit nonsynchronized immune responses of varying intensity among their tissue components, with skin and muscle being the most antigenic, bone of intermediate immunogenicity, and cartilage and tendon the least antigenic.2,8,10,12,20,22-24,32,33 As stated above, a negative association between the degree of cytokine expression/activity in the skin of vascularized rat hindlimb allografts and ultimate graft survival was noted by the Stanford group.30 Although the relative antigenicity of skin has been well studied in nonvascularized models,36 our understanding of skin immunogenicity in primarily vascularized grafts is not as advanced. Interestingly, Lee et al37 demonstrated that when transplanted as a whole, the vascularized rat limb allograft was rejected more slowly and generated lower immune re-

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sponses than did its individual components transplanted separately, suggesting that the immune system may be overwhelmed to a certain degree by a large antigen load. Although Sobrado et al38 concluded that cell-mediated immunity is the major mechanism of CTA rejection, and that the humoral arm of the immune response appears to play a less important role, Lee and coworkers37 found that the relative magnitude of the cell-mediated versus humoral response elicited varied with the individual tissue type and whether or not the graft was vascularized. In more recent work, the Boston group demonstrated that total body irradiation of the donor followed by a “waiting period” of two or more days before transplantation significantly delayed rejection in a strongly immunogenic vascularized heterotopic rat knee allograft model without the use of concomitant systemic immunosuppressive therapy.39 This regimen completely abolished host cellular, but not humoral, immune responses at 2 weeks posttransplant when rejection became manifest. Indeed, local immunosuppression in the form of graft pretreatment may be a helpful adjunct in prolonging survival of CTAs, perhaps by decreasing the antigenicity of donor marrow and/or other tissue components and by preventing graft versus host disease. Clearly, a better understanding of the immunogenic mechanisms of CTAs and of related issues such as the potential for donor leukocyte migration, graft versus host disease, and microchimerism needs to be obtained and combined with a less toxic means of host treatment in order to move transplantation of these nonvital tissues into the clinical realm.

Local Immunosuppression One approach toward reducing the drug-specific and general adverse consequences of systemic immunosuppression in CTA recipients, and thereby improving the clinical feasibility of the procedure, is the utilization of local drug administration systems to establish a more selective presence of currently available nonspecific immunosuppressive agents in the transplanted limb or limb component, with a concomitant reduction in systemic drug exposure.40 Conflicting reports regarding the effectiveness of local treatment of canine and human renal allografts with a variety of antimetabolite and corticosteroids appeared in the late 1960s.41-43 With the exception of experimental and clinical studies demonstrating the efficacy of local graft irradiation,44,45 further examination of local immunosuppression was abandoned for 15 years, awaiting technological advances in drug delivery systems and a better understanding of both the cellular events within the rejecting allograft and the pharmacokinetics of targeted drug delivery.46 In 1986, Ruers et al47 demonstrated that continuous intraarterial (i.a.) infusion of prednisolone in rat renal allograft recipients produced a significant increase in graft survival when compared with same dose systemic administration. This work was rapidly followed by multiple reports of favorable experiences with local immunosuppressive therapy in rat heterotopic cardiac, renal, and pancreatic islet cell allograft models.40,48 In these studies, budesonide, CsA, rapamycin, 16,16-dimethyl PGE2, FK506, and anti-T cell monoclonal antibody were infused directly into the transplanted organ for up to 2 weeks via an Alzet osmotic minipump implanted in the abdominal cavity. In each case, allograft survival was significantly prolonged over that achievable with same dose by intravenous (i.v.) administration. Coincident with this work, several investigators demonstrated long term survival of rodent skin allografts treated topically with CsA,49,50 and more recently, with combination corticosteroid and CsA therapy.51 Along these lines, Inceoglu et al52 showed a significant improvement in rat hindlimb allograft survival and a decreased incidence of skin rejection when using a combination of topical fluocinolone acetonide and low dose systemic CsA therapy compared with either treatment given alone. Other targeting systems, such as aerosol inhalation, drug-impregnated polymer rods, controlled release drug matri-

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ces, liposomes, gene transfer, and cellular cotransplantation, have all been effectively used in small animal models to direct immunosuppressants to the transplanted organ of interest.53 Although the rat models mentioned above elegantly demonstrate the efficacy of local immunosuppression, they have several limitations when one considers the potential for eventual clinical application. The osmotic pumps utilized for drug delivery are inaccessible within the abdomen, cannot be emptied of and refilled with drug, and can only infuse drug for a two or four week interval. As a result, long term considerations such as pump compatibility, drug stability, and catheter-induced thrombosis and infection cannot be evaluated. Furthermore, repeated blood sampling to assess regional and systemic pharmacokinetics is not practical. In view of these limitations, Gruber et al54 developed a canine renal allograft model of local immunosuppression utilizing programmable, implantable pumps and biocompatible catheters to deliver drugs directly to the transplanted kidney via i.a. infusion. This model more closely resembled the situation in man anatomically and physiologically, and could be applied directly to man with little modification. Long term arterial catheter placement with background heparin infusion was technically feasible and did not compromise autotransplant renal function or histologic integrity.55 6-MP was infused intrarenally with both a four-fold increase in local drug concentration and an 80% decrease in systemic drug delivery, to produce an overall pharmacokinetic advantage of 30-fold.56 Turning to allotransplants, Gruber and colleagues57 find that intrarenal dosing of heparin to the point of producing systemic anticoagulation was limited by failure of the transplanted kidney to eliminate drug and did not prolong canine renal allograft survival. In contrast, i.a. 6-MP therapy more effectively prolonged survival than same dose i.v. treatment with reduced systemic drug concentrations and toxicity (hepatotoxicity and myelosuppression), confirming the previous pharmacokinetic studies in autografts.58 Local administration of increasing doses of prednisolone against a background of oral azathioprine merely produced a shift in the cause of death from rejection to systemic drug toxicity without significantly affecting overall survival.59 Finally, infusion of mizoribine directly into the kidney allograft produced lower systemic drug levels than same dose i.v. administration, in agreement with pharmacokinetic studies in autografts. At local doses required to achieve immunosuppression, the transplanted kidney was not able to extract enough drug to prevent death from gastrointestinal tract toxicity, so that an overall survival benefit was not realized. However, combination of a subtherapeutic dose of oral CsA with i.a., but not i.v., mizoribine infusion conferred a significant survival advantage with lower systemic mizoribine concentrations, suggesting mediation via a local immunosuppressive effect.60 More recently, we have developed a rabbit CTA model of local immunosuppression utilizing pump/catheter-based i.a. infusion of a forelimb transplant. Preliminary studies indicate that, when compared with the same dose i.v. infusion, i.a. CsA delivery produces steady-state drug levels in skin and muscle that are significantly higher in the locally-treated limb, but equivalent in the contralateral limb. The relatively low blood flow in the extremity no doubt contributes to produce a considerable regional pharmacokinetic advantage of i.a. CsA infusion that is not achievable with other high flow target organs such as the liver and kidney.61 While further study is needed, this data provides a direction for future efforts in composite tissue transplantation.

Conclusion

The studies by Gruber et al56,58 with 6-MP represented both the first attempt by anyone to validate the principles governing the pharmacokinetics of target-aimed drug delivery in a large-animal model and the first clear demonstration of the efficacy of local immunosuppressive therapy in a large animal model. Subsequently, three other groups of investigators have demonstrated the beneficial effects of locoregional drug delivery in canine unilateral

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lung and liver transplant models, utilizing aerosolized CsA, portal venous administration of CsA, and systemic administration of liposomal FK506, respectively.62-64 In light of these results and those in the rodent models described above, as well as the recent demonstration that intragraft mechanisms appear to be important in regulating virtually all phases of allograft rejection, including initial sensitization, lymphocyte recruitment, and transmigration of host cells into the graft,65 it is clear that the concept of local immunosuppression deserves reexploration and application to CTAs in a large animal, clinically relevant model.

References 1. Llull R, Beko KR II, Black KS et al. Composite tissue allotransplantation: Perspectives concerning eventual clinical exploitation. Transplant Rev 1992; 6:175. 2. Tan CM, Yaremchuk MJ, Randolph MA et al. Vascularized muscle allografts and the role of cyclosporine. Plast Reconstr Surg 1991; 87:412. 3. van den Helder TB, Benhaim P, Anthony JP et al. Efficacy of RS-61443 in reversing acute rejection in a rat model of hindlimb allotransplantation. Transplantation 1994; 57:427. 4. Gruber SA, Black KS, Hewitt CW. Rehabilitation Research and Development Service, Department of Veteran Affairs. Composite Tissue Transplantation Workshop, State of the Art. Washington, D.C. 1991. 5. 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. 6. Bain JR, Mackinnon SE, Hudson AR et al. Preliminary report of peripheral nerve allografting in primates immunosuppressed with cyclosporin A. Transplant Proc 1989; 21:3176. 7. Black KS, Hewitt CW, Aniel M et al. Neuromuscular capabilities in long-term composite tissue allografts. Transplant Proc 1988; 20 (Suppl. 2):269. 8. 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. 9. Stark GB, Swartz WM, Narayanan K et al. Hand transplantation in baboons. Transplant Proc 1987; 19:3968. 10. Stevens HPJD, Hovius SER, Vuzevski VD et al. Immunological aspects of allogeneic partial hand transplantation in the rhesus monkey. Transplant Proc 1990; 22:2006. 11. Aebi M, Regazzoni P, Perren SM et al. Microsurgically revascularized bone allografts with immunosuppression with cyclosporine: Preliminary report of the effect in an animal model CSC. Transplantation 1986; 42:564. 12. Doi K, DeSantis G, Singer DI et al. The effect of immunosuppression on vascularized allografts. J Bone Joint Surg 1989; 71B:576. 13. Paskert JP, Yaremchuk MJ, Randolph MA et al. The role of cyclosporin in prolonging survival in vascularized bone allografts. Plast Reconstr Surg 1987; 80:240. 14. Guimberteau JC, Baudet J, Panconi B et al. Human allotransplant of a digital flexion system vascularized on the ulnar pedicle: A preliminary report and 1-year follow-up of two cases. Plast Reconstr Surg 1992; 89:1135. 15. Hofmann GO, Kirschner MH, Buhren V et al. Allogenic vascularized transplantation of a human femoral diaphysis under cyclosporin A immunosuppression. Transpl Int 1995; 8:418. 16. Kirschner MH, Hofmann GO. Preliminary results in the transplantation of allogeneic vascularized femoral diaphyses under immunosuppression. Transplantation 1996; 8:48. 17. Brennan DC, Jones TR. Cadaveric allogenic skeletal muscle transplantation in a renal transplant patient: Results and implications. Abstract Annual Meeting American Society of Transplant Physicians 1997; 16:108. 18. Doi K. Homotransplantation of limbs in rats. Plast Reconstr Surg 1979; 64:613. 19. 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.

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20. Black KS, Hewitt CW, Hwang JS et al. Dose response of cyclosporine-treated composite tissue allografts in a strong histoincompatible rat model. Transplant Proc 1988; 20 (Suppl. 2):266. 21. Fritz WD, Swartz WM, Rose S et al. Limb allografts in rats immunosuppressed with cyclosporin A. Ann Surg 1984; 99:211. 22. Press BH, 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; 6:313. 23. Hotokebuchi T, Arai K, Takagishi K et al. Limb allografts in rats immunosuppressed with cyclosporine: As a whole-joint allograft. Plast Reconstr Surg 1989; 83:1027. 24. Benhaim P, Anthony JP, Lin LY-T et al. A long-term study of allogeneic rat hindlimb transplants immunosuppressed with RS-61443. Transplantation 1993; 56:911. 25. 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. 26. Gulati AK, Zalewski AA. Muscle allograft survival after cyclosporin A immunosuppression. Exp Neurol 1982; 77:378. 27. Fealy MJ, Umansky WS, Bickel KD et al. Efficacy of rapamycin and FK 506 in prolonging rat hindlimb allograft survival. Ann Surg 1994; 219:88. 28. 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. 29. Kuroki H, Ikuta Y, Akiyama M. Experimental studies of vascularized allogeneic limb transplantation in the rat using a new immunosuppressive agent, FK506. Transplant Proc 1989; 21:3187. 30. Fealy MJ, Most D, Huie P et al. Association of down-regulation of cytokine activity with rat hindlimb allograft survival. Transplantation 1995; 59:1475. 31. Benhaim P, Anthony JP, Ferriera L et al. Use of combination of low dose cyclosporine and RS-61443 in a rat hindlimb model of composite tissue allotransplantation. Transplantation 1996; 61:527. 32. Goldwyn RM, Beach PM, Feldman D et al. Canine limb homotransplantation. Plast Reconstr Surg 1966; 37:184. 33. Lance EM, Inglis AE, Figarola F et al. Transplantation of the canine hindlimb. J Bone Joint Surg 1971; 53A:1137. 34. Skanes SE, Samulak DD, Daniel RK. Tissue transplant for reconstructive surg. Transplant Proc 1986; 18:898. 35. Stevens HP, Hovius SE, Heeney JL et al. Immunologic aspects and complications of composite tissue allografting for upper extremity reconstruction: A study in the rhesus monkey. Transplant Proc 1991; 23:623. 36. Rosenberg AS, Singer A. Cellular basis of skin allograft rejection: An in vivo model of immune-mediated tissue destruction. Annu Rev Immunol 1992; 10:333. 37. Lee WP, Yaremchuk MJ, Pan YC et al. Relative antigenicity of components of a vascularized limb allograft. Plast Reconstr Surg 1991; 87:401. 38. Sobrado L, Pollak R, Robichaux WH et al. The survival and nature of the immune response to soft-tissue and composite-tissue allografts in rats treated with low dose cyclosporine. Transplantation 1990; 50:381. 39. Lee WPA, Randolph MA, Weiland AJ et al. Prolonged survival of vascularized limb tissue allografts by donor irradiation. J Surg Res 1995; 59:578. 40. Gruber SA. Locoregional immunosuppression of organ transplants. Immunol Rev 1992; 129:5. 41. Retik AB, Dubernard JM, Hester WJ et al. A study of the effects of intraarterial immunosuppressive drug therapy on canine renal allografts. Surgery 1966; 60:1242. 42. Terz JJ, Crampton R, Miller D et al. Regional infusion chemotherapy for prolongation of kidney allografts. J Surg Res 1969; 9:13. 43. Kountz SL, Cohn RB. Initial treatment of renal allografts with large intrarenal doses of immunosuppressive drugs. Lancet 1969; 1:338.

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44. Gergely NF, Coles JC. Prolongation of heterotopic cardiac allografts in dogs by topical radiation. Transplantation 1970; 9:193. 45. Halperin EC, Delmonico FL, Nelson PW et al. The use of local allograft irradiation following renal transplantation. J Radiat Oncol Biol Phys 1984; 10:987. 46. Eckman WW, Patlak CS, Fenstermacher JD. A critical evaluation of the principles governing the advantages of intraarterial infusions. J Pharmacokinet Biopharm 1974; 2:257. 47. Ruers TJM, Buurman WA, Smits JFM et al. Local treatment of renal allografts, a promising way to reduce the dosage of immunosuppressive drugs. Transplantation 1986; 41:156. 48. Gruber SA. Local immunosuppressive therapy in organ transplantation. Transplant Proc 1994; 26:3214. 49. Lai C-S, Wesseler TA, Jr, Alexander JW et al. Long-term survival of skin allografts in rats treated with topical cyclosporine. Transplantation 1987; 44:83. 50. Black KS, Nguyen DK, Proctor CM et al. Site-specific suppression of cell-mediated immunity by cyclosporine. J Invest Dermatol 1990; 94:644. 51. Llull R, Lee TP, Vu AN et al. Site-specific immune suppression with topical cyclosporine. Synergism with combined topical corticosteroid added during the maintenance phase. Transplantation 1995; 59:1483. 52. Inceoglu S, Siemionow M, Chick L et al. The effect of combined immunosuppression with systemic low dose cyclosporin and topical fluocinolone acetonide on the survival of rat hindlimb allografts. Ann Plast Surg 1994;33:57. 53. Gruber SA. Local Immunosuppression of Organ Transplants. Gruber SA, ed. Austin: RG Landes Co., 1996. 54. Gruber SA, Cipolle RJ, Canafax DM et al. An implantable pump for intrarenal infusion of immunosuppressants in a canine autotransplant model. Pharm Res 1988; 12:781. 55. Gruber SA, Burke BA, Canafax DM et al. Feasibility of vascular catheter placement for intrarenal infusion in a canine autotransplant model. Transplant Proc 1989; 21:1125. 56. Gruber SA, Canafax DM, Erdmann GR et al. The pharmacokinetic advantage of local 6-mercaptopurine infusion in a canine renal transplant model. Transplantation 1989; 48:928. 57. Gruber SA, Cipolle RJ, Tzardis P et al. Pharmacodynamics of local heparin infusion in a canine renal allograft model. J Pharmacol Exp Ther 1990; 252:733. 58. Gruber SA, Hrushesky WJM, Cipolle RJ et al. Local immunosuppression with reduced systemic toxicity in a canine renal allograft model. Transplantation 1989; 48:936. 59. Gruber SA, Hrushesky WJM, Canafax DM et al. Local prednisolone infusion of canine renal allografts. Transplantation 1989; 48:1072. 60. Gruber SA, Erdmann GR, Burke BA et al. Mizoribine pharmacokinetics and pharmacodynamics in a canine renal allograft model of local immunosuppression. Transplantation 1992; 53:12. 61. Collins JM. Pharmacologic rationale for regional drug delivery. J Clin Oncol 1984; 2:498. 62. Dowling RD, Zenati M, Burckart GJ et al. Aerosolized cyclosporine as single agent immunotherapy in canine lung allografts. Surgery 1990; 108:198. 63. Ko S, Nakajima Y, Kanehiro H et al. The significance of local immunosuppression in canine liver transplantation. Transplantation 1994; 57:1818. 64. Ko S, Nakajima Y, Kanehiro H et al. The enhanced immunosuppressive efficacy of newly developed liposomal FK506 in canine liver transplantation. Transplantation 1995; 59:1384. 65. Gruber SA. The case for local immunosuppression. Transplantation 1992; 54:1.

CHAPTER 16

Long Term Limb and Nerve Allograft Survival with FK506 Immunosuppression Neil F. Jones and Esther Voegelin

Introduction

E

xperimental limb transplantation has used different animal models across different immunologic barriers and a variety of immunosuppressive agents.1-24 Following the first limb transplantations with parabiosis,1 blood transfusion,2,3 anti-lymphocyte serum,4 6-mercaptopurine,5,6 azathioprine3,5 and steroids;3,6,7 cyclosporine (CsA) produced significant prolongation of survival of composite limb allografts in rats depending on the dosage and duration regimen.8-22 Although the surgical transplantation of composite tissue allografts can be performed using various immunosuppressive agents, preventing chronic rejection in such transplants remains problematic. Furthermore, significant toxic side effects (hepatotoxicity, nephrotoxicity), particularly for nonlife-threatening conditions, make elective composite tissue transplantation still unacceptable. In addition, composite tissue allografts containing skin and muscle are substantially more antigenic than solid organ transplants and thus require even higher immunosuppressive doses.6,23,27,28 Several newer drugs, including FK506, RS-61443 and rapamycin, as well as combination immunosuppressive therapy, have subsequently been evaluated for their effectiveness in experimental composite tissue allotransplantation.29-34

Mechanism of FK506 FK506 is a macrolide antibiotic isolated from Streptomyces tsukubaensis in Osaka, Japan in 1984 with potent immunosuppressive properties.35 Although FK506 has an entirely different molecular structure than that of cyclosporine, its immunosuppressive properties are similar. However, FK506 is reported to be up to 100 times more potent than cyclosporine.35 FK506 suppresses cell-mediated and humoral immune responses, including alloantibody production to blood transfusion in animal models.36 It inhibits the mixed lymphocyte reaction assay, interleukin 2 formation by T lymphocytes, and inhibits formation of other soluble mediators including interleukin 3 and interferon !.37 FK506 strikingly prolongs survival of various organs and skin grafts in rodents, rabbits, dogs, primates and man.38 Moreover, FK506 can reverse cardiac or renal allograft rejection in rats39 and dogs,40 and has been shown to reverse acute or early chronic liver rejection in man.41 When used in combination Composite Tissue Transplantation, edited by Charles W. Hewitt and Kirby S. Black. ©1999 R.G. Landes Company.

Composite Tissue Transplantation

206

Table 16.1. Limb allograft transplantation: Experimental groups Group

Treatment

Dosage mg/kg

Duration in days

Number of animals

0 1

Isografts controls Allografts, no immunosuppression Cyclosporine FK506 FK506 FK506 FK506 FK506 Cyclosporine RS-61443

-

-

15

25 1 2 20 1 2 25 30

1-14 1-14 1-14 1 1-14, then twice weekly 1-14, then twice weekly 1-14, then twice weekly 1-14, then twice weekly

6 8 30 10 9 9 10 9 10

2 3 4 5 6 7 8 9

with either whole body irradiation or with the novel immunosuppressive drug deoxyspergualin (a polyamine derivative), FK506 very effectively prolongs xenograft survival in rodents.42,43 Beneficial effects of FK506 for the control of autoimmune diseases have been observed in rodent models of insulin-dependent diabetes, rheumatoid arthritis, posterior uveitis, allergic encephalomyelitis, and glomerulonephritis.38 Although FK506 is well tolerated by rodents, minor renal impairment and hyperglycemia have been reported in addition to thymic medullary atrophy in rats.44 However, in dogs, FK506 caused anorexia and vascular lesions in various organs, especially the heart and gastrointestinal tract.40,45 In renal allografted baboons, hyperglycemia and lethal emaciation have been reported in one study46 but no major side effects have been observed in other centers.47,48 The most frequent adverse side effects of FK506 in clinical trials have been renal impairment, abnormalities in glucose metabolism, and neurotoxicity (tremor, paresthesia and sleep disorders).41 Recent data have unveiled molecular mechanisms demonstrating interactions between FK506 and its primary binding protein and calcineurin.49 Calcineurin is understood to be responsible for the toxicity of FK506 and cyclosporine. With a more precise understanding of the mechanism of FK506 at the molecular level, it is more likely that the drug can be further modified to increase its efficacy without increased toxicity.

Limb Transplantation in Rats Immunosuppressed with FK506 We have performed one of the largest series of experimental limb transplantations ever reported to compare the efficacy of short term and long term immunosuppression to prevent the rejection of a limb transplant across the strongest histocompatibility barrier in rats (ACI ∀ Lewis) using the conventional immunosuppressive agent cyclosporine 53 and the newer immunosuppressive agents FK506 and RS-61443. One hundred thirty-three hindlimbs from ACI rats (RT1a) have been transplanted orthotopically to Lewis rats (RT11) using the model first described by Lipson et al.50 This combination represents a very strong histocompatibility, differing in Class I and Class II antigens of the rat MHC system (RT1 system).51 The donor limb was transected at midfemur level and the skin stripped away except over the foot and ankle, and the recipient thigh skin was used as an envelope to cover the transplanted composite allograft. After osteosynthesis, the femoral artery, vein, and nerve and the sciatic nerve were repaired by standard microsurgical techniques.

Long-Term Composite Tissue Allograft Survival with FK506 Immunosuppression

207

In addition to a control group of isografts (Group 0) and a control group consisting of allografts without immunosuppression (Group 1), animals were divided into 8 other groups based on the type and dosage of immunosuppression (Table 16.1). The time of rejection was defined as the first change in the skin after erythema and edema but before progressing toward epidermolysis, desquamation, or even eschar formation.52 Because the composite allograft was transplanted into the recipient-derived skin envelope, the underlying musculoskeletal tissues were protected and could be observed long after skin rejection had occurred. Biopsies were obtained for histology at the time of rejection of the donor foot skin. In selected animals, the foot was then amputated through the ankle joint, and biopsies of the muscle, bone and cartilage were obtained 3 months after skin rejection. After euthanasia, a total of 354 biopsies from 79 animals including the donor skin, gastrocnemius muscle, the femoral bone at the site of osteosynthesis and the knee joint were harvested. Three hundred thirty biopsies from 64 animals could be included in the analysis.53 By modifying the grading system for cardiac muscle rejection by Billingham et al54 and descriptions of tissue rejection in composite limb allografts by other authors,50,55-58 a new classification system for rejection in composite limb allotransplantation was developed.53 This system consists of four grades: Grade 0 = normal; Grade I = mild rejection; Grade II = moderate rejection; and Grade III = severe rejection. Specific histologic criteria of rejection in skin, muscle, articular cartilage and bone are highlighted (Table 16.2). After grading the rejection of each biopsy, a mean rejection grading (MRG) was developed for skin, muscle, cartilage and bone for the different treatment groups and analyzed statistically. Of 133 successful limb transplants, 108 animals could be included for analysis. A 14 day course of FK506 prolonged the survival of a limb allograft for 54-95 days across the ACI ∀ Lewis histocompatibility barrier. Allograft survival was dose dependent: Mean rejection times were significantly prolonged in animals receiving 2 mg/kg FK506 when compared with animals receiving 1 mg/kg FK506.52 Comparison of those animals receiving a 14 day course of FK506 (Groups 3 and 4, Table 16.1) with those animals receiving a 14 day course of cyclosporine (CsA) (Group 2, Table 16.1) demonstrated that FK506 is significantly more effective in prolonging rejection than CsA. Even a single 20 mg/kg dose of FK506 given on the first postoperative day (Group 5, Table 16.1) was as effective in preventing the rejection of the skin component of a limb allograft as a 14 day course of 1 mg/kg FK506, a finding similar to that reported by Arai et al.59 When rejection did occur, however, the process was much more rapid, and histology revealed chronic ongoing rejection in the musculoskeletal tissues. Administration of FK506 twice weekly, especially at the dose of 2 mg/kg, was very effective in allowing long term survival of limb allografts. However, all the animals receiving 1mg/kg FK506 twice weekly (Group 6, Table 16.1) eventually developed rejection, and all the animals receiving 2 mg/kg FK506 twice a week (Group 7, Table 16.1) lost weight and died without signs of skin rejection after approximately 300 days. This phenomenon was similar to that reported by Arai et al59 in that 6 of 8 of their long term survivors died at 214-240 days posttransplantation from Pneumocystis carinii pneumonia, possibly related to chronic graft versus host disease. Histologic and bacterial examination of animals in Group 7 of our study did reveal bacterial pneumonia, but apart from weight loss, there were no other signs of graft versus host disease (GVHD).

Normal

Mononuclear focal infiltration, basal cell vacuolation.

Suprabasal bulla formation, mixed infiltrate.

Edema, vasculitis, necrosis.

Grade 0

Grade 1

Grade 2

Grade 3

Skin

Scar formation >90% connective tissue. Very small fibers and poor myelination. Severe endoneurial infiltration of mononuclear cells.

Increased endoneurial connective tissue or fibrosis. Decreased caliber and poor myelination. Infiltration of mononuclear cells into perineurium

Perineurial fibrosis, nearly normal fiber caliber and myelinated fibers. Perineurial infiltrate of mononuclear cells.

Normal

Nerve

Diffuse aggressive polymorphous infiltrate, hemorrhage, vasculitis, necrosis or fibrosis.

Multifocal aggressive infiltration with myocyte necrosis.

Focal perivascular or diffuse but sparse interstitial infiltrate, or focal aggressive infiltration.

Normal

Muscle

Necrosis of the articular cartilage, with or without mononuclear infiltrate.

Multifocal mononuclear infiltration with rough surface of the joint.

Focal erosion of cartilage, granulation tissue.

Normal

Articular Cartilage

Table 16.2. Histological grading system for skin, nerve, muscle, cartilage and bone rejection

Edema, vasculitis, necrosis.

Nonvascularized intertrabecular space, irregular cortical bone, locally nonviable woven bone.

Periosteal infiltrate and reaction.

Normal

Bone

208 Composite Tissue Transplantation

Long-Term Composite Tissue Allograft Survival with FK506 Immunosuppression

209

Animals receiving CsA for 14 days and then twice weekly (Group 8, Table 16.1) showed signs of rejection of the skin component of the limb transplant with a mean rejection time of 61.6 days. All recipient animals immunosuppressed with RS-61443 (Group 9, Table 16.1) also showed signs of rejection while still receiving immunosuppression with a mean rejection time of 43.6 days. The ACI ∀ Lewis mismatch in rats is a very strong histocompatibility barrier49 and differs from previously described weaker transfers such as Fisher ∀ Lewis and Brown Norway ∀ Lewis.58 Lack of standardization in the immunogenic disparity of the rat strains used and lack of standardization in the grading system of rejection have made it difficult to compare the relative potency of different immunosuppressive agents. From this large series of limb transplantations, a histologic grading system was defined to assess the severity of rejection in the different component tissues of a transplanted limb allograft (Table 16.2).53 In contrast to the soft tissues, bone only shows rejection across a major histocompatibility barrier,60 although Class I and II antigens are presented on bone cells.61 The histologic changes seen in bone and cartilage in this study were similar to those described in previous papers on vascularized joint transplantation.23,57,61 All three immunosuppressive agents were able to prolong the rejection of the skin component of a limb transplant compared with nonimmunosuppressed controls. Based on the mean rejection time of the skin component of a limb allograft and also on the histologic grading system, FK506 was more effective than CsA in preventing the rejection of a limb transplant. All of the component tissues showed severe rejection in those animals immunosuppressed by a short term 14 day course of CsA at an average follow-up of 44 days (Table 16.3). This indicates that long term survival of a limb allograft across a major histocompatibility barrier cannot be achieved by using a short term course nor even a long term intermittent course of CsA. A 14 day course of FK506, 1mg/kg, was able to protect bone and cartilage but not muscle, whereas a 14 day course of FK506, 2 mg/kg, prevented rejection of all three tissues, muscle, cartilage and bone. There was no statistically significant difference between long term intermittent immunosuppression with CsA and RS-61443; both were significantly inferior to FK506. Using intermittent immunosuppression with 2 mg/kg FK506 twice weekly, there were minimal signs of rejection on long term follow-up at 10 months, but all the animals died of pneumonia after weight loss. The histologic study also demonstrated that, irrespective of whether CsA or FK506 was used for immunosuppression, the hierarchy of rejection seems to be skin > muscle > bone > cartilage. This is different from the mixed lymphocyte results of Lee et al,23 who believed that muscle was more antigenic than skin.

Other Studies of Limb Transplantation with FK506 Immunosuppression

Arai et al62 examined the effect of FK506 on rat limb allografts across the Brown Norway ∀ F344 histocompatibility barrier and demonstrated that a 14 day course of 1mg/kg FK506 intramuscularly prolonged allograft survival to 150 days. A single dose of 50 mg/kg FK506 on the day of operation prolonged allograft survival to 104 days. Three of 10 animals treated with 1 and 5 mg/kg i.m. for 14 days postoperatively developed GVHD between 100 and 150 days (Table 16.4). In another study, Arai et al63 demonstrated that a single injection of FK506 between 2-50 mg/kg on the day of transplantation significantly prolonged allograft survival in a dose dependent manner. In addition, delayed treatment with a single 10 mg/kg injection of FK506 7 or 10 days posttransplantation reversed the early signs of skin rejection and prolonged limb allograft survival for 50 days. This delayed single dose treatment with 10 mg/kg FK was nearly as effective as the prophylactic treatment at day 0 with regard to the length of allograft survival. Although a single dose of 50 mg/kg on day 0

No immunosuppression Cyclosporine 25 mg/kg, 14 days FK506 1 mg/kg, 14 days FK506 2 mg/kg, 14 days FK506 20 mg/kg, 1 day FK506 1 mg/kg, 14 days, then twice weekly FK506 2 mg/kg, 14 days, then twice weekly Cyclosporine 25 mg/kg, 14 days, then twice weekly RS-61443 30 mg/kg, 14 days, then twice weekly

1 2 3 4 5 6 7 8 9 6.8 30 54.2 95 64 149 296 61.6 43.6

MRT in days

Drug

FK506 i.m.

Rat-strain

ACI (RT1a) Lew (RT11)

Authors

Jones et al 199552 1996 53

0

2

1 2 20 1

Dosage mg/kg/day 0-14 1-14 0 0-14, then 2x weekly 0-14, then 2x weekly 6

10

30 10 9 9

Duration days # Rats

7 ± 0.7

296 ± 30

45 ± 9.5 122 ± 159 64 ± 33.2 149 ± 83.7

Limb survival days

19.8 44.4 156 130 127 190 300

Biopsy days post-op

Table 16.4. Experimental limb transplantation and FK506 immunosuppression

Dosage and drug

Groups

3 2.68 1.3 2.3 1.16 0.67

muscle

around 300 days: weight loss and death

Complications/Others

3 2.8 2 3 2 0.89

skin

2.8 1.96 0 1.4 0.16 0

bone

MRG (Range from 0-3; 0 = no rejection, 3 = severe rejection)

2.57 0.76 0 0 0.83 0.11

cartilage

Table 16.3. Mean rejection times (MRT) and mean histological grading (MRG) for different experimental groups of limb allografts.

210 Composite Tissue Transplantation

FK506 i.m.

FK506 i.m.

FK506 i.m.

BN (RT1n) F344 (RT11)

BN (RT1n) F344 (RT11)

Lew (RT1l) PVG (RT1c)

Arai 198962

Arai 198963

Kuroki 198964

CsA

Drug

Rat-strain

Authors

0.32 0.64 0 15

10 then 3

0 10

2 10 50

0.2 1 5 0 2 10 50

Dosage mg/kg/day

0-14 0-14 0-14 0-14

day 0 once a week

day 7 day 10

day 0 day 0 day 0

day 0 day 0 day 0

0-14 0-14 0-14

8 8 8 8

8

8 6 6

8 8 8

6 6 6 6 6 6 6

Duration days # Rats

33.9 ± 49.8 ± 11.8 ± 31.0 ±

4.5 5.0 1.7 3.2

11.4 ± 0.7 56.7 ± 20.4 46.0 ± 4.2

16.4 ± 2.8 51.3 ± 6.2 104.4 ± 17.2

14.7 ± 2.5 149.5 ± 63.9 101.7 ± 8.5 10.9 ± 1.1 16.0 ± 2.8 51.3 ± 6.2 104.4 ± 17.2

Limb survival days

* 1,2,3,4,6,8,11,14 and 20 weeks

no problems <200 days, 6/8 GVHD, 4/8 died * or ** : 1** day 214 , 3** day 228, 233, 242 (P. carinii) 2/8 signs of rejection 3 weeks after discontinuation of FK days >280, >290, 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)

Lew (RT1l) PVG (RT1c)

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

Composite Tissue Transplantation

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

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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.

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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

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Long-Term Composite Tissue Allograft Survival with FK506 Immunosuppression

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

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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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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

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

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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 (p<0.05) (Table 17.2). The only detectable MMF toxicity in this study was a moderate bone marrow suppression, which appears to be species specific and limited to the rat.15 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

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cells/ml x 1000, p<0.05) but no difference was present between autograft, CsA-treated, and MMF-treated animals (4.9 vs. 5.4 vs. 5.1, respectively). Analysis of leukocyte differential counts revealed a significant decrease in the mean percentage of lymphocytes in MMF-treated rats (39.4%) relative to both CsA-treated rats (66.7%) and preoperative baseline values (77.7%) (p<0.005). Bone marrow suppression due to MMF does not occur in other animal species, and has yet to be a significant toxicity in human patients enrolled in clinical trials and receiving MMF doses as high as 30-50 mg/kg/d.15,18,20,28

Reversing Acute Rejection Both early and delayed rejection continue to be a major obstacle in clinical organ transplantation, despite successful primary immunosuppression. Therefore, effective rescue therapy to reverse rejection is essential to the long term success of any allotransplant. MMF has been used successfully for this purpose both experimentally and clinically. In 1990, the first study using MMF (30 mg/kg/d) to reverse established rejection was reported in a rat model of cardiac allotransplantation.29 The following year, Platz et al30 confirmed these findings in canine kidney transplants, demonstrating an 87.5% rate of reversed acute rejection. Dogs in that study received high initial doses of MMF (160 mg/kg/d for 3 days) on the day that acute rejection was diagnosed, in addition to baseline immunosuppression with CsA and prednisone. More recently, a multicenter human trial evaluating the effect of MMF on refractory acute rejection of kidney allografts revealed a 69% rescue rate in patients who had not responded to antirejection therapy using either steroids or monoclonal OKT3 antibody.18,31 In addition to these renal transplant studies, refractory rejection of human liver allografts has also been treated successfully using MMF with minimal morbidity.32 The phenomena of both early and delayed rejection can also be expected to occur with limb or other composite tissue transplants. In the limb allograft model, acute rejection occurs in a graded, progressive, and reproducible fashion that results in severe, complete rejection by postoperative day 12-13 if left untreated (Fig. 17.1; compare with MMF-treated allograft, Fig. 17.2). In such a case, by postoperative day (POD) 7 signs of acute rejection are both clinically and histologically evident (Fig. 17.3). Grossly, edema and exudate formation is present, with moist desquamation of the skin. Histologically, there is evidence of dyskeratosis, vacuolization of the epidermis with cleft formation between the epidermis and dermis, and a mononuclear cell infiltrate throughout the dermis. If these same rats (POD 7) are then treated with a daily dose of MMF (30 mg/kg/d), a marked decrease in limb edema is observed by POD 9 with complete resolution grossly by POD 11-13. Histologic examination on POD 42 demonstrates resolving rejection in all animals and complete resolution of rejection by POD 76-105 (p=0.001) (Fig. 17.4). If allograft rejection is instead allowed to progress to POD 9 before treatment with MMF (30 mg/kg/d), considerable reduction of the limb edema is again observed after 2 days of treatment (POD 11) with complete resolution grossly in 5-6 days (POD 14-15). As with grafted animals left untreated until POD 7, histologic analysis after 5 weeks of MMF therapy shows diminishing signs of rejection and complete recovery by POD 60-79 (p<0.01) (Fig. 17.5).

Combination Mycopenolate Mofetil and Low-Dose Cyclosporine to Prevent Rejection As mentioned above, composite tissue allografts are significantly more antigenic than organ transplants, requiring toxic doses of conventional immunosuppressants (e.g., CsA) to prevent rejection. In fact, in recent studies in primates, long term survival of either hand or composite mandibular allografts could not be achieved, despite the use of very high doses of CsA and serum levels 2-3 times higher than considered therapeutic and safe in humans.33,34

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A

B

Fig. 17.1. (A) Untreated rat hindlimb allograft (BN to F344) 13 days posttransplant, demonstrating severe acute rejection. Findings include exudation, moist desquamation, edema, hair loss, and epidermal necrosis. (B) The histologic skin analysis from the same animal on posttransplantation day 13 (hematoxylin and eosin stain). Massive edema and frank epidermal necrosis is evident (Grade 4+ rejection).

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A

B

Fig. 17.2. (A) Hindlimb allograft (BN to F344) treated with MMF (30 mg/kg/d) 231 days posttransplantation. There is no evidence of rejection. The skin appears normal and good hair growth is present. (B) the histologic findings on skin biopsy from the same animal posttransplant day 231 (hematoxylin and eosin stain). The epidermis is normal without evidence of rejection (Grade 0 rejection).

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A

B

Fig. 17.3. (A) Hindlimb allograft (BN to F344) 7 days posttransplantation prior to any immunosuppressive therapy. Acute rejection is characterized by edema throughout the allograft. (B) The histologic findings (hematoxylin and eosin stain) include significant epidermal-dermal clefting, vacuolization, dyskeratosis, and keratinocyte necrosis (Grade 3 rejection).

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A

B

Fig. 17.4. (A) Hindlimb allograft (BN to F344) showing complete resolution of edema posttransplant day 90. (B) Skin biopsy of the limb allograft on the same day (hematoxylin and eosin stain) demonstrates normal epidermis, with complete resolution of edema, dyskeratosis, and vacuolization (Grade 0 rejection).

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Fig.5. (Top) Histopathologic grading of limb allograft skin biopsies 7 (Group 2) or 9 (Group 3) days after untreated rejection, corresponding to the day MMF (RS-61443) therapy was started. (Middle) Histopathologic grading of limb allograft skin biopsies after 5 weeks of treatment with MMF. (Bottom) Histopathologic grading of limb allograft skin biopsies at time animals were sacrificed.

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Unlike organ transplantation, the clinical application of composite tissue allografts would involve conditions that are not life threatening. Therefore, if immunosuppressive drugs are to be used in such a case to prevent rejection, significant toxic side effects would be particularly unacceptable. One way to address this problem is to use a combination of two or more drugs, both at subtherapeutic doses, to prevent rejection. Since CsA and MMF have differing mechanisms of action and toxicity profiles, it was reasoned that subtherapeutic doses of each drug, given together, might provide effective immunosuppression with less toxicity. Based upon well established protocols for preventing rejection using CsA or MMF separately, a study was performed35 comparing the efficacy of subtherapeutic CsA and MMF doses given alone with that of combination therapy. Rats received either CsA (1.5 mg/kg/d) of MMF (15 mg/kg/d) following limb allograft transplantation. The third group of allografts, treated daily with a combination of CsA (1.5 mg/kg/d) and MMF (15 mg/kg/d) therapy, showed significantly less rejection than those groups receiving only CsA or MMF alone at 12 and 31 weeks postoperatively. Furthermore, at 12 and 31 weeks posttransplant, allografts that received combination therapy had a 94% and 89% rejection-free survival, respectively (Fig. 17.6). In comparison, most animals that received subtherapeutic doses of either CsA or MMF alone developed acute immunologic rejection (64% and 100%, respectively) (Fig. 17.7). Hematocrits measured at the time of sacrifice in the animals treated with MMF + CsA demonstrated only minor anemia compared to the previous studies using MMF alone at higher doses (34.7 vs. 27.0, p=0.0001). The synergistic effects between MMF and CsA in preventing rejection while minimizing toxicity may provide a significant advance in our ability to produce effective, relatively nontoxic immunosuppression for these highly antigenic composite tissue allotransplants.

Functional Outcomes: Nerve Regeneration The two critical requirements forming obstacles to the clinical application of composite tissue transplantation as a viable reconstructive option are: 1. The ability to prevent rejection long term; and 2. The degree of functional recovery achievable. Neural regeneration is required if such composite tissue allografts are to develop useful function. Primary repair of the sciatic nerve during hindlimb transplantation in the rat serves as a useful model for studying such return of function. In 1994, the first neural regeneration studies using MMF as primary hindlimb allograft immunosuppression were reported.37 Hindlimb allotransplantation was performed between rat strains with a strong histocompatibility locus antigenic mismatch. The sciatic nerve was repaired with 10-0 nylon perineural interrupted stitches and the rats were treated with either CsA (10 mg/kg/d x 20 days, then 2X/week) or MMF (30 mg/kg/d) for 13 months. Untreated autografts served as controls. Sciatic nerve specimens were harvested at sacrifice for quantitative morphometric analysis of the regenerating nerves. The nerves were fixed immediately in 2.5% glutaraldehyde with 100 mm cacodylate buffer (pH 7.3) and processed for both toluidine blue histology and intermediate voltage (x4,000-18,000) electron microscopy (EM). Specimens were obtained from contralateral (unoperated control) sciatic nerves both proximal and distal to the donor-recipient sciatic to sciatic coaptation site. Axon diameter, myelin thickness, and ratio of axon diameter to myelin thickness were measured. The degree of skin rejection in the limb allografts was also quantified (grade 0 = no rejection, grade 4 = severe rejection). By 13 months postoperatively, autograft controls showed indefinite survival without any sign of clinical or histologic rejection. CsA-treated rats initially were rejection-free, but developed a progressive mild to moderate rejection in 83% of the animals at approximately 5

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Fig. 17.6. Comparison of the rejection-free survival among low dose CsA therapy, low dose MMF (RS) therapy, and combination low dose CsA + MMF therapy. Both the duration of, and the percentage of animals showing, rejection-free survival are greater for those receiving combination CsA + MMF therapy.

Fig. 17.7. Percentage of animals unable to sustain long term (>200 days posttransplantation) rejection-free survival. As expected, autograft controls (Group 1) showed no rejection while untreated allograft controls (Group 2) all had rapid, severe rejection. Animals receiving combination CsA + MMF low-dose therapy (Group 5) demonstrated a significantly lower rate of rejection than animals receiving either CsA (Group 3) or MMF (Group 4) at the same subtherapeutic doses.

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months postoperation (mean rejection grade = 2.0). In contrast, MMF-treated rats had >8 months rejection-free survival in 83% of the animals (mean rejection grade = 0.17). Protective sensation was regained within 50-60 days posttransplantation, with partial functional return (e.g., weight bearing, task-oriented motion, toe spread) in all rats. Functional recovery in autograft, CsA-treated, and MMF-treated rats was identical clinically, although normal function was never fully attained secondary to flexion contractures and muscle disuse atrophy. Both light microscopy (toluidine blue) and EM studies of the limb sciatic nerves demonstrated advanced regeneration based on axon, myelin, extracellular matrix, and nonmyelinated nerve morphologic criteria. Relative to unoperated controls, however, transplanted sciatic nerve segments in all three groups displayed smaller myelinated axons with greater size variability, reduced myelin sheath thickness, disorganized nonmyelinated axons, expanded extracellular matrix, and ongoing axonal resorption and remodeling. Sciatic nerve biopsies proximal to the nerve-nerve coaptation site (i.e., recipient animal sciatic nerve) had intermediate ultrastructural features that resembled both the control segments and the transplanted donor graft segments. Quantitatively, axon diameter and myelin sheath thickness were measured in autograft and immunosuppressed allograft nerve specimens in >300 individual axons. When comparing control (unoperated) versus distal donor versus proximal recipient nerve segments across all treatment groups, nerve specimens from the control group had a greater axonal diameter (9.80 ± 2.73 mm) than either recipient (proximal) nerves (7.22 ± 2.92 mm) or donor (distal) nerves (5.48 ± 2.43 mm). Similarly, the control group specimens had greater myelin thickness (2.10 ± 0.63 mm) than either the recipient (1.55 ± 0.77 mm) or the donor nerves (0.91 ± 0.35 mm). When expressed as ratios of axonal diameter to myelin thickness for each axon, control segments had the lowest ratio, indicating a thicker myelin sheath, on average, for a given axonal diameter. The largest ratio was in donor (distal) nerves. Each of these differences was statistically significant by analysis of variance (Scheffe’s S test, p<0.01). When donor nerve segments were analyzed according to treatment group (i.e., autograft vs. CsA vs. MMF), axonal diameters in donor nerves from CsA-treated rats were smaller than in either autografts (p=0.0001) or MMF-treated rats (ANOVA, p=0.001). This same finding was also evident in CsA-treated donor nerve segments for myelin thickness (p=0.0056 vs. autograft and p=0.0001 vs. MMF). There were no significant differences in these parameters between the autograft and MMF-treated groups in the donor segments. No significant differences existed among the three groups in the recipient or control nerve segments. Therefore, quantitative assessment of the degree and quality of nerve regeneration of sciatic nerves within rat hindlimb allografts reveals advanced regeneration that is consistent with clinical evaluations of functional return. However, even mild subacute rejection can significantly impair neural regeneration. The delayed acute rejection observed in CsA-treated animals may have adversely affected the degree of neural regeneration in comparison to animals immunosuppressed with MMF (all rejection-free). In other studies, the degree of neural regeneration seen in the rat is far more complete than that seen in humans. Consequently, the results of neural regeneration studies must be interpreted carefully.

Conclusion Composite tissue allotransplantation has tremendous potential application for the reconstruction of complex acquired or congenital defects. As with any allograft, the ultimate goal of treatment involves both long term rejection-free survival and significant functional recovery of the graft. Mycophenolate mofetil, an immunosuppressive drug with significant clinical application in organ transplantation, has provided an experimental foundation for the eventual clinical use of composite tissue allografts. Before such clinical use is realized,

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future studies will need to address such issues as graft versus host disease, maximization of functional recovery, other combination immunosuppressive therapies, prevention of delayed rejection, and effective immunosuppressive therapy in nonhuman primate models.

References 1. Buncke HJ, Schulz WR. Experimental digital amputation and reimplantation. Plast Reconstr Surg 1965; 36:62-70. 2. Buncke HJ, Buncke CM, Schulz WR. Immediate Nicoladoni procedure in the rhesus monkey, or hallux-to-hand transplantation, utilizing microminiature vascular anastomoses. Br J Plast Surg 1966; 19:332-337. 3. Komatsu S, Tamai S. Successful replantation of a completely cut-off thumb. Case report. Plast Reconstr Surg 1968; 42:374-377. 4. Harii K, Ohmori K, Ohmori S. Successful clinical transfer of ten free flaps by microvascular anastomoses. Plast Reconstr Surg 1974; 53:259-270. 5. Murray JE. Organ transplantation (skin, kidney, heart) and the plastic surgeon. Plast Reconstr Surg 1971; 47:425-431. 6. Sakai A, Yakushiji K, Mashimo S. Lymphocyte stimulation by allogeneic tissue cells in rats: With special reference to differential survival of skin and kidney allografts. Transplant Proc 1980; 12:74-81. 7. Skanes SE, Samulack DD, Daniel RK. Tissue transplantation for reconstructive surgery. Transplant Proc 1986; 18:898-900. 8. Birinyi LK, Baldwin WM, Tilney NL. Differential effects of heterologous antisera on the survival of cardiac and skin allografts in rats. Transplantation 1981; 32:336-338. 9. Fritz WD, Swartz WM, Rose S et al. Limb allografts in rats immunosuppressed with cyclosporine A. Ann Surg 1984; 199:211-215. 10. 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. 11. Lee WA, Gu L, Miksztal AR et al. Bioavailability improvement of mycophenolic acid through amino ester derivatization. Pharmacol Res 1990; 7:161-166. 12. Nelson PH, Eugui E, Wang CC et al. Synthesis and immunosuppressive activity of some side-chain variants of mycophenolic acid. J Med Chem 1990; 33:833-838. 13. Eugui EM, Mirkovich A, Allison AC. Lymphocyte-selective antiproliferative and immunosuppressive effects of mycophenolic acid in mice. Scand J Immunol 1991; 33:175-183. 14. Allison AC, Almquist SJ, Muller CD, et al. In vitro immunosuppressive effects of mycophenolic acid and an ester pro-drug, RS-61443. Transplant Proc 1991; 23(2 Suppl 2):10-14. 15. Eugui EM, Mirkovich A, Allison AC. Lymphocyte-selective antiproliferative and immunosuppressive activity of mycophenolic acid and its morpholinoethyl ester (RS-61443) in rodents. Transplant Proc 1991; 23(2 Suppl 2);15-18. 16. European Mysophenolate Mofetil Cooperative Study Group. Placebo-controlled study of mycophenolate mofetil combined with cyclosporine and corticosteroids for prevention of acute rejection. Lancet 1995; 345:1321-1325. 17. Halloran P, Mathew T, Tomlanovich S et al. Mycophenolate mofetil in renal allograft recipients. A pooled efficacy analysis of three randomized, double-blind, clinical studies in prevention of rejection. Transplantation 1997; 63:39-47. 18. Sollinger HW, Deierhoi MH, Belzer FO et al. RS-61443-A phase I clinical trial and pilot rescue study. Transplantation 1992; 53(2):428-432. 19. Deierhoi MH, Kauffman RS, Hudson SL et al. Experience with mycophenolate mofetil (RS61443) in renal transplantation at a single center. Ann Surg 1993; 217:476-484. 20. Kirklin JK, Bourge RC, Naftel DC et al. Treatment of recurrent heart rejection with mycophenolate mofetil (RS-61443): Initial clinical experience. J Heart Lung Transplant 1994; 13:444-450. 21. Barry JM. Immunosuppressive drugs in renal transplantation. A review of regimens. Drugs 1992; 44:554-566.

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22. Goldblum R. Therapy of rheumatoid arthritis with mycophenolate mofetil. Clin Exp Rheumatol 1993; 11:S117-119. 23. Yocum DE. Cyclosporine, FK506, rapamycin, and other immunomodulators. Rheum Dis Clin North Am 1996; 22:133-134. 24. Tressler RJ, Garvin LJ, Slate DL. Anti-tumor activity of mycophenolate mofetil against human and mouse tumors in vivo. Int J Cancer 1994; 57:568-573. 25. Lee WPA, Yaremchuck MJ, Pan YC et al. Relative antigenicity of components of a vascularized limb allograft. Plast Reconstr Surg 1991; 87:401-411. 26. Doi K. Homotransplantation of limbs in rats. A preliminary report on an experimental study with nonspecific immunosuppressive drugs. Plast Reconstr Surg 1979; 64:613-621. 27. Benhaim P, Anthony JP, Lin L et al. A long-term study of allogeneic rat hindlimb transplants immunosuppressed with RS-61443. Transplantation 1993; 56:911-917. 28. Platz KP, Sollinger HW, Hullett DA et al. RS-61443—A new, potent immunosuppressive agent. Transplantation 1991; 51:27-31. 29. Morris RE, Hoyt EG, Murphy MP et al. Mycophenolic acid morpholinoethylester (RS-61443) is a new immunosuppressant that prevents and halts heart allograft rejection by selective inhibition of T and B cell purine synthesis. Transplant Proc 1990; 22:1659-1662. 30. Platz KP, Bechstein WO, Eckhoff DE et al. RS-61443 reverses acute allograft rejection in dogs. Surgery 1991; 110(4):736-740, discussion 740-741. 31. Sollinger HW, Belzer FO, Deierhoi MH et al. RS-61443 (mycophenolate mofetil). A multicenter study for refractory kidney transplant rejection. Ann Surg 1992; 216:513-518. 32. Klintmalm GB, Ascher NL, Busuttil RW et al. RS-61443 for treatment-resistant human liver rejection. Transplant Proc 1993; 25:697. 33. van der Helder TBM, Benhaim P, Anthony JP et al. Efficacy of RS-61443 in reversing acute rejection in a rat model of hindlimb allotransplantation. Transplantation 1994; 57:427-433. 34. Gold ME, Randzio J, Kniha H et al. Transplantation of vascularized composite mandibular allografts in young cynomolgus monkeys. Ann Plast Surg 1991; 26:125-132. 35. Hovius SER, Stevens HPJD, van Nierop PWM et al. Allogeneic transplantation of the radial side of the hand in the rhesus monkey: I. Technical aspects. Plast Reconstr Surg 1992; 89:700-709. 36. Benhaim P, Anthony JP, Ferreira L et al. Use of combination of low-dose cyclosporine and RS-61443 in a rat hindlimb model of composite tissue allotransplantation. Transplantation 1996; 61:527-532. 37. Benhaim P, Anthony JP, Lewis JC et al. Improved sciatic nerve regeneration in rat hindlimb allografts immunosuppressed with RS-61443. Surg Forum 1994; 45:717-719. 38. Taylor DO, Ensley RD, Olsen SL et al. Mycophenolate mofetil (RS-61443): Preclinical, clinical, and three-year experience in heart transplantation. J Heart Lung Transplant 1994; 13:571-582.

CHAPTER 18

Long Term Prevention of Rejection and Combination Drug Therapy James P. Anthony, Robert D. Foster and Stephen J. Mathes

Introduction

F

or composite tissue allotransplantation to ever be applied clinically, the method of preventing rejection of such allografts must be both reliable and safe to the patient. Currently, the immunosuppressive drugs proven clinically to prevent rejection of organ transplants are also the most effective means of preventing rejection in models of composite tissue allografts (CTAs).1,2 However, the experimental results are not uniformly favorable. Despite the efficacy of immunosuppressive drugs (e.g., cyclosporine, FK506) in the acute phase of transplantation, their effectiveness in long term CTA survival is less well defined, particularly in preventing rejection of the skin component of the transplant3,4 (Table 18.1). Furthermore, CTAs are significantly more antigenic than organ transplants 58 (especially the skin and muscle components), requiring increased immunosuppression (often 2-3 times greater in animal models) to maintain transplant viability. Since all immunosuppressive drugs have potentially severe side effects at high doses, a method for minimizing the drug requirements is essential before composite tissue allotransplantation can be useful in humans.

Long Term Prevention of Rejection Any discussion on the long term survival of CTAs must first define “long term survival.” Ultimately, the long term goal would be an allograft that demonstrates no sign of rejection to any component of the graft, and any anti-rejection regimen used should be nontoxic to the organism. Unlike organ transplants, CTAs consist of a wide variety of tissue types. The antigenicity of the component tissues has been shown to differ significantly relative to each other.9 Any CTA containing skin and muscle is substantially more antigenic than any solid organ transplant, and thus requires higher immunosuppressant doses. Therefore, any analysis of the long term survival of a limb allograft should specifically address the skin/muscle viability of that graft. Although experimental limb allotransplantation has been attempted using numerous immunosuppressive regimens including parabiosis,10 blood transfusion,11 anti-lymphocyte serum,12 6-mercaptopurine,13-14 azathioprine,13 and steroids,14 the only study describing prolonged CTA survival prior to 1976 was by Lance et al,15 who transplanted hindlimbs between unrelated beagles. It was not until the introduction of cyclosporine A (CsA) that long term CTA survival became widely achievable. In 1976, Borel demonstrated the immunosuppressive activity of CsA, a lipophilic cyclic polypeptide derived Composite Tissue Transplantation, edited by Charles W. Hewitt and Kirby S. Black. ©1999 R.G. Landes Company.

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Table 18.1. Progress in long term CTA survival PreCsA Era Goldwyn et al. Doi

Average Limb Allograft Survival* 18 days 15 days

CsA Black et al. Benhaim et al.

152 days 155 days

Other immunosuppressive drugs (FK506, MMF) Buttemeyer et al. Benhaim et al.

149 days 241 days

* including rejection-free skin component

from the fungus Tolypocladium inflatums.16 CsA functions by inhibiting the production of lymphokines such as interleukin 2 (IL-2), as well as the response of precursor cytotoxic effector lymphocytes to IL-2. In 1982, Black et al17 were the first to report prolonged rat limb allograft survival (101 ± 13 days) in four rats who received high dose CsA (25 mg/kg/d) for only the first 20 days posttransplant. A follow-up report in 1983 supported these findings,18 with allograft survival ranging from 50-70 days in four of five recipients. The fifth rat survived greater than 225 days and apparently had become tolerant to the donor limb. Further studies by the same group delineated a dose-dependent increase in allograft survival in order to establish a regimen for maximum graft survival.19 However, throughout these studies and other studies using CsA as the primary CTA immunosuppression, our definition of “long term survival” failed to be met, since reliable, long term rejection-free transplant survival was not received. Only sporadic reports of indefinite, rejection-free limb allograft survival using CsA immunosuppression have been made, regardless of the CsA dosing schedule. Furnas et al20 used CsA in doses of 8 mg/kg twice a week, with only one of five rats maintaining limb survival (>400 days) without rejection. Three of the five rats in that study developed skin rejection by post-operative day 66 to 238. Black et al1 reported comparable results for CsA administered subcutaneously at the same dose, but orally administered CsA at 8 mg/kg/d resulted in skin rejection in all rats at a mean of 174 days posttransplant. Similarly, CsA administered at 10 mg/kg/d s.c. for 20 days, followed by twice a week dosing thereafter, was unable to induce indefinite rejection-free survival, with five of six rats developing mild to moderate rejection 32-35 weeks after transplantation. Finally, Fritz et al,21 using CsA at 10 mg/kg/d s.c. for 113 days posttransplant, prevented rejection in five of seven rats, with the remaining two animals rejecting the skin component of their grafts. Neither increasing the daily CsA dose nor extending the total number of days of treatment has been the solution to preventing long term limb graft rejection. Using CsA as high as 15-25 mg/kg/d s.c., Hotokebuchi et al22 and Kuroki et al23 observed early rejection of limb allografts at an average of 31-59 days posttransplantation. Only two published reports have shown long term survival with high dose CsA therapy. In the first, Black et al24 produced

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rejection-free survival of limb allografts in six rats receiving CsA for 189 days. However, unspecified animal morbidity was noted at these doses. In the second study, Hewitt et al25 described eight long term limb allograft survivors (206-701 days) without the need for continued immunosuppression. These animals were exposed to various CsA regimens and represented the long term survivors from a combination of previous studies. Therefore, the interpretation of these findings is difficult. Of note, the strain combinations for grafting (donors: Lewis x Brown Norway, recipients: Lewis) were unique compared to all other studies in the literature on limb allografting. The chimeric donors may provide insight into these isolated incidents of tolerance induction. In rats treated with CsA for more than the acute phase of transplantation (10 mg/kg/d s.c. x 20 days, then twice per week), allograft survival without clinical evidence of rejection was evident for the first 16 weeks posttransplantation. However, by post-operative day 116-140, four of six animals developed mild rejection of the grafted skin, which progressed to moderate rejection in five of six animals by the day of sacrifice (post-op day 225).26 Results of recent studies in primates reflect the truly limited role that CsA would most likely play in human composite tissue transplantation. Hovius et al27 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 post-operative day 21-144 (mean: 66 days). Gold et al28 performed 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 post-operative day 13 without allograft rejection. Skanes et al,29 in a study of allogeneic neurovascular skin flaps and hand transplants in baboons, observed that the mean serum CsA concentration necessary to prevent rejection was more than twice the level recommended clinically in humans. To achieve this CsA concentration, the required CsA doses were 25-29 mg/kg/d s.c. and 35-48 mg/kg/d i.v., producing significant CsA-related toxicity to the animals. The fact that rather high doses of CsA were inadequate to ensure long term CTA survival in these primate studies is troubling, since much lower doses of CsA in humans have been shown to be toxic to both the kidney and liver. With the relative success of CsA established, a growing list of newer immunosuppressive drugs, proven useful in the clinical setting of organ transplantation, have been evaluated for their usefulness as first-line drugs to prevent CTA rejection. FK506 (Fujisawa), a potent macrolide antibiotic isolated from the soil fungus Streptomyces tsukubaensis, has a similar mechanism of action to that of CsA, with greater than 10 times the potency.30 Analogous to early studies using brief courses of CsA, several authors have been able to demonstrate prolonged limb allograft survival after only a 14 day course of FK506. Arai et al31 produced mean limb allograft survival times of 102-150 days in rats receiving FK506 at 1-5 mg/kg/d for 14 days. Kuroki et al23 produced somewhat shorter mean survival times (34-50 days) using lower FK506 doses (0.32 to 0.64 mg/kg/d for 14 days), but, in a later study32 reported a 19% (5 of 27 rats) long term limb survival rate in rats receiving the same dose of FK506. However, the term “survival” in that study referred to grafted tissues other than the skin; the skin was rejected in ∗50 days. An interesting phenomenon highlighted by Arai’s studies33 is the ability of a single large dose of FK506 to prolong limb allograft survival. Doses of 2, 10, and 50 mg/kg induced mean limb survivals of 16, 51, and 104 days, respectively. Moreover, long term therapy with a single initial FK506 dose of 10 mg/kg followed by 3 mg/kg once a week was able to produce limb survival rates of >200 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, p<0.05) but no difference was present between autograft, CsA-treated, and MMF-treated animals (4.9 vs. 5.4 vs. 5.1, respectively). Analysis of leukocyte differential counts revealed a significant decrease in the mean percentage of lymphocytes in MMF-treated rats (39.4%) relative to both CsA-treated rats (66.7%) and preoperative baseline values (77.7%) (p<0.005).26 Bone marrow suppression due to MMF is not known to occur in other animal species and has yet to be a significant toxicity in human patients enrolled in clinical trials receiving MMF doses as high as 30-50 mg/kg/d.39-41

Combination Drug Therapy Based on the CTA survival rates using drug monotherapy outlined above, the use of two or more drugs, each at subtherapeutic doses, to prevent CTA rejection appears essential to the future of composite tissue allotransplantation. Currently, little experience has been documented in the literature. Fealy et al,37 in a study comparing the efficacy of CsA, rapamycin (SRL), and FK506 in preventing rat hindlimb rejection, found that combining CsA with SRL resulted in no significant prolongation of survival from that seen with CsA administered alone. It is noteworthy that, in that same study, SRL as monotherapy (using several

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different doses) was found to be ineffective in prolonging limb survival compared to administering no treatment at all. Additional such studies have used low dose CsA combined with various immuno-modulating agents. Inceoglu et al43 had observed from prior studies that combining systemic CsA with glucocorticoids failed to provide significantly prolonged limb allograft survival without the incidence of serious infection. They reasoned that fluocinolone acetonide (FA), a topical glucocorticoid, when applied over the allograft limb skin, would enhance the immunosuppressive effect of low dose CsA synergistically at the level of the skin while avoiding the systemic glucocorticoid-related side effects. In fact, using CsA (4 mg/kg/d s.c.) plus FA (6 mg/cm2/d), the incidence of skin rejection was delayed (first sign of rejection: 32-51 days) compared to animals treated with low dose CsA alone (first sign of rejection: 12-25 days). Unfortunately, no comparison to full strength CsA doses was made. Yeh et al44 recently published a study combining low dose CsA with leflunomide (LEF), a unique antiproliferative agent. LEF inhibits the proliferation of both T and B cell lines, the proliferation of T cells to mitogenic cytokines, and the response of B cells to mitogenic stimuli.45 It is thought that these effects are produced primarily by inhibiting pyrimidine biosynthesis, blocking production of uridine monophosphate. It has been shown to be effective in preventing or reversing heart, kidney, intestine, lung, and skin allograft rejection in animal models.46,47 In addition, CsA and LEF appear to work synergistically in preventing allograft rejection in rats and dogs and in hamster to rat xenograft rejection.48-50 Using myocutaneous grafts across a major histocompatibility mismatch, LEF combined with CsA (10 mg/kg/d and 5 mg/kg/d, respectively) prevented rejection in five of six allografts for the duration of the study (60 days). By comparison, allografts treated with either LEF (10 mg/kg/d) or CsA (5 mg/kg/d) alone survived only 28 and 24 days, respectively. Since CsA and MMF function through different pathways with unrelated toxic manifestations, it was reasoned that subtherapeutic doses of each drug, given together, might provide effective immunosuppression with less overall toxicity. A number of other investigators had confirmed the efficacy of combining MMF and CsA to improve the rejectionfree survival of highly antigenic transplants. Canine intestinal allograft survival has been prolonged using therapeutic doses of MMF and CsA with prednisone.51 Interestingly, these authors found that when subtherapeutic doses of MMF and CsA were used, intestinal allograft survival was not prolonged beyond that of either full dose MMF or CsA alone, indicating that while MMF and CsA may have a synergistic effect when given together, this effect is best manifested when therapeutic doses of both MMF and CsA are given. Bechstein et al,52 comparing canine liver allograft survivals, demonstrated similar results. The first studies using combination drug therapy to prevent rejection in rat hindlimb allografts were performed by Benhaim et al.53 The efficacy of subtherapeutic CsA and MMF doses given alone with that of combination therapy was evaluated. Rats received either CsA (1.5 mg/kg/d) of MMF (15 mg/kg/d) following limb allograft transplantation. A third group of allografts, treated daily with a combination of CsA (1.5 mg/kg/d) and MMF (15 mg/kg/ d), showed significantly less rejection than those groups receiving only CsA or MMF alone at 12 and 31 weeks post-operatively. Furthermore, at 12 and 31 weeks posttransplant, allografts that received combination therapy had a 94% and 89% rejection-free survival, respectively. In comparison, most animals that received subtherapeutic doses of either CsA or MMF alone developed acute immunologic rejection (64% and 100%, respectively). The toxic side effects of MMF in this same study revealed only a minor anemia compared to the previous studies using MMF alone at higher doses (34.7 vs. 27.0, p=0.0001). Experimental combination drug therapy regimens will most likely be employed more often in the future, as the incidence of toxic side effects of the individual drugs are more readily documented in animal studies and combination drug dosages are better defined.

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Conclusion The introduction of immunosuppressive drugs represents the most significant step towards realizing the clinical application of composite tissue allotransplantation since the birth of microsurgery. Prolonged survival of CTAs is more readily achievable as a result, allowing us to better understand the nature of rejection and providing valuable information about the quality and degree of functional recovery. As our knowledge about the immunology behind CTA survival/rejection increases, combination drug regimens directed towards optimizing survival while minimizing the deleterious effects of the therapy will be further refined. Although tolerance of the grafts without requiring immunosuppression is the ultimate goal, immunosuppressive drugs will most likely play a significant role towards achieving that objective.

References 1. Black KS, Hewitt, Fraser LA et al. Composite tissue (limb) allografts in rats. II. Indefinite survival using low-dose cyclosporine. Transplantation 1985; 39:365-368. 2. Buttemeyer R, Jones NF, Min Z et al. Rejection of the component tissue of limb allografts in rats immunosuppressed with FK506 and cyclosporine. Plast Reconstr Surg 1996; 97:139-148. 3. Murray JE. Organ transplantation (skin, kidney, heart) and the plastic surgeon. Plast Reconstr Surg 1971; 47:425-431. 4. Sakai A, Yakushiji K, Mashimo S. Lymphocyte stimulation by allogeneic tissue in rats: With special reference to differential survival of skin and kidney allografts. Transplant Proc 1980; 12:74-81. 5. Skanes SE, Samulack DD, Daniel RK. Tissue transplantation for reconstructive surgery. Transplant Proc 1986; 18:898-900. 6. Sakai A, Yakushiji K, Mashimo S. Lymphocyte stimulation by allogeneic tissue cells in rats: With special reference to differential survival of skin and kidney allografts. Transplant Proc 1980; 12:74-81. 7. Skanes SE, Samulack DD, Daniel RK. Tissue transplantation for reconstructive surgery. Transplant Proc 1986; 18:898-900. 8. Birinyi LK, Baldwin WM, Tilney NL. Differential effects of heterologous antisera on the survival of cardiac and skin allografts in rats. Transplantation 1981; 32:336-338. 9. Lee WPA, Yaremchuck MJ, Pan YC et al. Relative antigenicity of components of a vascularized limb allograft. Plast Reconstr Surg 1991; 87:401-411. 10. Schwind JV. Homotransplantation of extremities of rats. Radiology 1962; 78:806-809. 11. Lapchinsky AG, Eingorn AG, Uratkov EF. Homotransplantation of extremities in tolerant dogs observed up to seven years. Transplant Proc 1973; 5:773-779. 12. Poole, Bowen JE, Batchelor JR. Prolonged survival of rat allografts due to immunological enhancement. Transplantation 1976; 22:108-111. 13. Goldwyn RM, Beach PM, Feldman D et al. Canine limb homotransplantation. Plast Reconstr Surg 1966; 37:184-185. 14. Doi K. Homotransplantation of limbs in rats. A preliminary report on an experimental study with nonspecific immunosuppressive drugs. Plast Reconstr Surg 1979; 64:613-621. 15. Lance Em, Inglis AE, Figarola et al. Transplantation of the canine hindlimb. Surgical techniques and methods of immunosuppression for allotransplantation—A preliminary report. J Bone Joint Surg (AM) 1971; 53:1137-1149. 16. Borel JF, Feurer C, Gibler HU et al. Biological effects of cyclosporine A: A new antilymphocytic agent. Agents Actions 1976; 6:468-475. 17. Black KS, Hewitt CW, Fraser LA et al. Cosmas and Damian in the laboratory. N Engl J Med 1982; 306:368-369. 18. Hewitt CW, Black KS, Fraser LA et al. Cyclosporine-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.

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19. Hewitt CW, Black KS, Frase LA et al. Composite tissue (limb) allografts in rats. I. Dosedependent increase in survival with cyclosporine. Transplantation 1985; 39:360-364. 20. Furnas DW, Black KS, Hewitt CW et al. Cyclosporine and long-term survival of composite tissue allografts (limb transplants) in rats with historical notes on the role of plastic surgeons in allotransplantation. Transplant Proc 1983; 15(4 Suppl 1):3063-3068. 21. Fritz WD, Swartz WM, Rose S et al. Limb allografts in rats immunosuppressed with cyclosporine A. Ann Surg 1984; 199:211-215. 22. Hotokebuchi T, Arai K, Takagishi K et al. Limb allografts in rats immunosuppressed with cyclosporine: As a whole-joint allograft. Plast Reconstr Surg 1989; 83:1027-1036. 23. Kuroki H, Ikuta Y, Akiyama M. Experimental studies of vascularized allogeneic limb transplantation in the rat using a new immunosuppressive agent, FK 506: Morphological and immunological analysis. Transplant Proc 1989; 21:3187-3190. 24. Black KS, Hewitt CW, Hwang JS et al. Dose response of cyclosporine-treated composite allografts in a strong histocompatible rat model. Transplant Proc 1988; 20 (2 Suppl 2):266-268. 25. Hewitt CW, Black KS, Dowdy et al. Composite tissue (limb) allografts in rats. III. Development of donor-host lymphoid chimeras in long-term survivors. Transplantation 1986; 41:39-43. 26. Benhaim P, Anthony JP, Lin L et al. A long term study of allogeneic rat hindlimb transplants immunosuppressed with RS-61443. Transplantation 193; 56:911-917. 27. Hovius SER, Atevens HPJD, van Nierop PWM et al. Allogeneic transplantation of the radial side of the hand in the rhesus monkey: I. Technical aspects. Plast Reconstr Surg 1992; 89:700-709. 28. Gold ME, Randzio J, Kniha H et al. Transplantation of vascularized composite mandibular allografts in young cynomolgus monkeys. Ann Plast Surg 1991; 26:125-132. 29. Skanes SE, Samulack DD, Daniel RK. Tissue transplantation for reconstructive surgery. Transplant Proc 1986; 18:88-900. 30. Sawada S, Suzuki G, Kawase Y et al. Novel immunosuppressive agent, FK506: In vitro effects on the cloned T cell activation. J Immunol 1987; 139:1797-1801. 31. Arai K, Hotokebuchi T, Miyhara H et al. Prolonged limb allograft survival with short-term treatment with FK506 in rats. Transplant Proc 1989; 21:3191-3193. 32. 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. 33. Arai K, Hotoebuchi T, Miyahara H et al. Limb allografts in rats immunosuppressed with FK506. I. Reversal of rejection and definite survival. Transplantation 1989; 48:782-786. 34. Nelson PH, Eugui E, Wang CC et al. Synthesis and immunosuppressive activity of some side-chain variants of mycophenolic acid. J Med Chem 1990; 33:833-838. 35. Eugui EM, Mirkovich A, Allison AC. Lymphocyte-selective antiproliferation and immunosuppressive effects of mycophenolic acid in mice. Scand J Immunol 1991; 33:175-183. 36. Allison Ac, Almquist SJ, Muller CD et al. In vitro immunosuppressive effects of mycophenolic acid and an ester pro-drug, RS-61443. Transplant Proc 1991; 23(2 Suppl 2):10-14. 37. 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. 38. Muramatsu K, Doi K, Akino T et al. Nerve-regenerating effect of 15-deoxyspergualin, peripheral nerve allotransplants in rat. Acta Orthop Scand 1996; 67:399-402. 39. Eugui EM, Mirkovich A, Allison AC. Lymphocyte-selective antiproliferative and immunosuppressive activity of mycophenolic acid and its morpholinoethyl ester (RS-61443) in rodents. Transplant Proc 1991; 23:(2 Suppl 2):15-18. 40. Sollinger HW, Deierhoi MH, Belzer FO et al. RS-61443—A phase I clinical trial and pilot rescue study. Transplantation 1992; 53(2):428-432. 41. Kirklin JK, Bourge RC, Naftel DC et al. Treatment of recurrent rejection with mycophenolate mofetil (RS-61443): Initial clinical experience. J Heart Lung Transplant 1994; 13:444-450.

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42. Platz KP, Sollinger HW, Hullett DA et al. RS-61443, a new potent immunosuppressive agent. Transplantation 1991; 51:27-31. 43. Incegoglu S, Siemionow M, Chick L et al. The effect of combined immunosuppression with systemic low-dose cyclosporine and topical fluocinolone acetonide on the survival of rat hindlimb allografts. Ann Plast Surg 33:57-65. 44. Yeh LS, Gregory CR, Griffey SM et al. Effects of leflunomide and cyclosporine on myocutaneous allograft survival in the rat. Transplantation 1996; 62:861-877. 45. Cao WW, Kao PN, Chao AC et al. Mechanism of the antiproliferative action of leflunomide. J Heart Lung Transplant (In Press). 46. Morris RE, Huang X, Cao WW et al. Leflunomide (HWA 486) and its analogy suppress Tand B- cell proliferation in vitro, acute rejection, ongoing rejection, and antidonor antibody synthesis in mouse, rat, and cynomolgus monkey transplant recipients, as well as arterial intimal thickening after catheter injury. Transplant Proc 1995; 27:445-447. 47. Kuchle CCA, Thoenes GH, Langer HK et al. Prevention of kidney and skin rejection in rats by leflunomide, a new immunomodulating agent. Transplant Proc 1991; 23:1083-1086. 48. Yuh D, Morris RE, Hoyt G et al. Leflunomide prolongs pulmonary allograft and xenograft survival. J Heart Lung Transplant (In Press). 49. Morris RE, Huang X, Shorthouse R et al. Use of cyclosporine (CsA), mycophenolic acid (MPA), rapamycin (SRL), leflunomide (LFM) or deoxyspergualin (DSG) for prevention and treatment of obliterative airway disease (OAD) in new arrival models (abstract). J Heart Lung Transplant 1995; 14:S65. 50. Lirtzman RL, Gregory CR, Griffey SM et al. Combination leflunomide and cyclosporine immunosuppression prevents MLR mismatched allograft rejection in mongrel dogs. Transplant Proc (In Press). 51. D’Alessandro AM, Rankin M, McVey J et al. Prolongation of canine intestinal allograft survival with RS-61443, cyclosporine and prednisone. Transplantation 1993; 55:695-702. 52. Bechstain WO, Schilling M, Steele DM et al. RS-61443/cyclosporine combination therapy prolongs canine liver allograft survival. Transplant Proc 1993; 25:702-703. 53. Benhaim P, Anthony JP, Ferreia L et al. Use of combination of low-dose cyclosporine and RS-61443 in a rat hindlimb model of composite tissue allotransplantation. Transplantation 1996; 61:527-532.

CHAPTER 19

Efficacy of Rapamycin and FK506 in Prolonging Rat Hindlimb Allograft Survival James Chang, Yvonne L. Karanas and Barry H.J. Press

Introduction

A

dvances in microsurgical reconstruction have made limb transplantation technically possible. Parallel advances have occurred in the long term survival of transplanted vital organs. Therefore, the replacement of limbs and composite tissue in humans may become a reality once immunosuppressive regimens become fully safe and therapeutic. Recently, newer immunosuppressive agents have been tested in heart, liver, and kidney transplantation and are now available for use in limb transplantation. The purpose of this chapter is to review the mechanisms, toxicities, and current research pertaining to two related drugs, FK506 and rapamycin, in animal models of hindlimb transplantation. The current era of hindlimb transplantation began with Goldwyn et al, who were able to document prolonged survival of limb allografts in a canine model using azathioprine and 6 mercaptopurine.1 Two separate groups, Hewitt et al2 and Kim et al,3 reported prolonged rat limb allograft survival with cyclosporine A. Other researchers have tested cyclosporine in combination with other agents, such as prednisone.4 Until recently, cyclosporine remained the standard for comparison with other immunosuppressive agents in hindlimb transplantation models. Two other drugs have been isolated and have offered promise for improved limb allograft immunosuppression, FK506 and rapamycin. Both of these agents have been used clinically in the past decade in human vital organ transplantation with significant success. While there has been great experimental and clinical enthusiasm for both agents, research data concerning their efficacy in hindlimb transplantation has been limited. In order to update the progress thus far, all published experimental work on the efficacy of FK506 and rapamycin in hindlimb allotransplantation is reviewed here.

History FK506 (tacrolimus) was discovered at Fujisawa Corporation in 1984 as part of a mixed lymphocyte reaction screening process to develop an alternative to cyclosporine A.1 FK506 is a natural actinomycete (Streptomyces tsukubaensis) product with a hydrophobic macrolactam structure unrelated to cyclosporine. Like cyclosporine, FK506 is hepatically metabolized. Based on pioneering work in Japan and at the University of Pittsburgh in the Composite Tissue Transplantation, edited by Charles W. Hewitt and Kirby S. Black. ©1999 R.G. Landes Company.

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past 10 years, FK506 has become a standard agent of immunosuppression in human vital organ transplantation. Optimized immunosuppression may be achieved at doses much lower than cyclosporine.

Mechanism

The mechanism of FK506 immunosuppression is similar to that of cyclosporine.5 In short, cytokine synthesis is inhibited via FK506-FKBP binding with calcineurin/calmodulin complexes, which blocks calcium-dependent signal transduction. This resulting decrease in serine/threonine phosphatase activity prevents cytokine gene activation. FK506 suppresses transcription of interleukin (IL)-2, -3, -4, -5, interferon (IFN)-!, tumor necrosis factor (TNF)-a and granulocyte-macrophage colony stimulating factor (GM-CSF). In addition, gene expression of IL-2 and IL-7 is also inhibited. FK506 also blocks T cell cycle progression during the G0-G1 stage. Its overall effect is inhibition of lymphokine cytokine synthesis and T cell division early in the cell cycle.

Toxicity FK506 has toxic effects on the kidney, resembling the limitations of cyclosporine. Increased thromboxane A2 and endothelin-related vasoconstriction of the renal vasculature may lead to reduced blood flow to glomeruli and to the renal cortex.5 Fung et al reviewed the incidence of toxic effects in their transplant patients at the University of Pittsburgh.1 35.5% of liver transplant patients using FK506 required insulin to treat hyperglycemia. Only 12% required insulin long term. Neurotoxic effects have ranged from insomnia and tremors to seizures and coma. However, the direct neurotoxic effect of FK506 is difficult to assess in these patients with marginal hepatic function and multiple possible causes of encephalopathy. To date, it remains unknown if FK506 will increase the risk for secondary malignancies. A 1.4% incidence of posttransplantation lymphoproliferative disorder (PTLD) was reported by Fung et al. In addition, immunosuppression with FK506 led to an increased incidence of infection with cytomegalovirus (CMV)—20%.

Vital Organ Transplantation Although FK506 is used widely in many transplant centers, including our program at Stanford, the greatest experience with FK506 in solid organ transplantation remains at the University of Pittsburgh Medical Center. While a full discussion of the experimental and clinical data available on FK506 is beyond the scope of this chapter, several points may be summarized here: 1 1. Both intravenous and oral forms are available; 2. Frequent monitoring of plasma levels is important to minimize toxicities; 3. Optimal therapeutic serum trough levels of FK506 may be 100 times lower than cyclosporine; 4. Outcomes with FK506 in liver transplantation at Pittsburgh were significantly better when compared to cyclosporine controls;8 5. FK506 was able to rescue rejection in grafts failing under cyclosporine therapy; 6. Clinical trials using FK506 continue in human heart, lung, liver, kidney, and pancreas transplant cohorts at multiple institutions.

Hindlimb Research Several groups have investigated the efficacy of FK506 in rat hindlimb allo-transplantation models. Kuroki et al performed hindlimb transplantation between Lewis (RT1l) and PVG (RT1c) rats.9 Normal rejection without immunosuppressive support occurred uniformly in 4 to 6 days. Intramuscular injection of FK506 at a dosage of 0.32 mg/kg/d for 14

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days was equally effective as cyclosporine A at a dosage of 15 mg/kg/d (mean survival time: 34 days vs. 31 days). Therefore, FK506 was 40-50 times more potent than cyclosporine. FK506 administered in a similar fashion at a higher dosage of 0.64 mg/kg/d allowed even longer prevention of rejection, with a mean survival time of 50 days. Their work demonstrated that continued daily dosing of FK506 could prevent rejection across a major histoincompatibility barrier. Further work by Arai et al documented that single dosing of intramuscular FK506 perioperatively could increase the period of graft survival in a dose dependent manner in Brown Norway (RT1n) limbs transplanted into Fisher (RT1l) recipients.10 Two mg/kg, 10 mg/kg, and 50 mg/kg single doses increased hindlimb survival to 16 days, 51 days, and 104 days respectively. Interestingly, long term hindlimb survival with continued administration of FK506 resulted in possible chronic graft versus host disease (GVHD), with 6 of 8 rats dying around 270 days of Pneumocystis carinii pneumonia. Early weight loss, common in the postoperative rats, resolved over time in most animals. Min and Jones at the University of Pittsburgh and later at the University of California, Los Angeles, applied their considerable experience with FK506 to a similar hindlimb transplantation model.11 114 ACI (RT1a) donor to Lewis (RT1l) recipient hindlimb transplantations were performed with varying regimens of FK506 administered intramuscularly. In this large series, 14 days of FK506 immunosuppression also prolonged survival in a dose dependent fashion. The longest period of graft survival (mean survival time: 296 days) was achieved with 2 mg/kg/d for 2 weeks, followed by 2 mg/kg two times a week. All nine rats in this group eventually died around 300 days with weight loss and pneumonia, again suggesting possible graft versus host disease. In a related paper by the same group, the authors devised a classification system to describe the rejection process based on histologic grading of hematoxylin and eosin stained tissues.12 Each component of the transplanted hindlimb, skin, muscle, cartilage, and bone, was examined separately. The histologic grading system, based on similar systems used for cardiac transplantation, was divided into four grades. While each tissue component had a separate, specific grading system, the histologic findings can be generalized as follows: Grade 0: Normal; Grade I: Early inflammatory cell infiltration; Grade II: Continued inflammatory cell infiltration with early tissue destruction; and Grade III: Edema with vasculitis and tissue necrosis. Using this grading system, the relative antigenicity and time course to rejection of each tissue component was described. Skin was the earliest to reject, followed in order by muscle, bone, and cartilage. With these studies, FK506 has replaced cyclosporine as the standard of immuno-suppression in rat hindlimb allotransplantation models. While long term graft survival now seems possible with continued FK506 therapy, the problems of infection and graft versus host disease remain limiting factors. Therefore, attention has been directed toward rapamycin as an alternative immunosuppressive agent.

Rapamycin History Rapamycin (Sirolimus) follows FK506 as another natural product derived from an actinomycete, Streptomyces hygroscopicus.5 It was originally discovered as an antifungal at Ayerst Research in the 1970s and was found to be a better immunosuppressant than antibiotic because of its involuting effect on lymphoid tissue. Rapamycin is also structurally unrelated to cyclosporine, but as a lipophilic macrolide shares some molecular similarities to FK506.

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Mechanism Rapamycin is best characterized as an inhibitor of cytokine or growth factor action rather than an inhibitor of cytokine or growth factor synthesis.5 It has been shown to inhibit stimulation of T cells by IL-2, -4, and -6. Rapamycin also blocks protein synthesis in T cells by inhibiting a specific 70 kDa kinase. T cell cycle progression is blocked later in the cell cycle than by FK506, at the G1 to S phase. Unlike FK506, rapamycin blocks calciumindependent signaling pathways in both T and B cells. In addition, rapamycin seems to have some inhibitory effect on cytokine function in nonimmune cells, including fibroblasts, endothelial cells, and smooth muscle cells.

Toxicity Unlike the wealth of human clinical data compiled on FK506, the experience with rapamycin thus far remains limited. In vivo human data regarding toxicities attributed to this newer agent is unavailable. Most information has been derived from experimental studies in animal models. Rapamycin is thought to be less nephrotoxic than cyclosporine, but has been shown to cause diabetes in rats. While fulminant gastrointestinal vasculitis has been found in dogs, nonhuman primates have exhibited few complications in rapamycin transplantation trials.13

Vital Organ Transplantation There is an enlarging body of evidence that although FK506 and rapamycin are structurally similar, their in vitro and in vivo profiles differ significantly.14 FK506 and rapamycin bind to the same immunophilin receptors, leading to original theories that the two agents were antagonistic. More recent experimental evidence suggests that FK506 and rapamycin, as well as cyclosporine and rapamycin, may act synergistically to promote immunosuppression at lower dosages.14,15 Rapamycin has been shown to be effective in preventing acute and chronic rejection in skin, heart, kidney, pancreas, and small bowel transplantation in rodent to nonhuman primate models.14 Furthermore, rapamycin effectively reversed allograft rejection. Calne et al achieved long term immunosuppression (mean survival time >100 days compared to 7.4 days in control) in rat heart allografts using 50 mg/kg/d of rapamycin intramuscularly for 14 days.13 Morris et al reported that intraperitoneal rapamycin was more potent than both FK506 and cyclosporine in preventing rat and mouse heart allograft rejection.15 In their study, a brief course of intraperitoneal rapamycin led to long term graft survival. Rapamycin has a profound effect on the immune system, making it an attractive agent for long term immunosuppression. Increasing data suggests that its mechanisms and in vivo effects differ considerably from FK506. Rapamycin seems to prevent the posttransplant graft vasculopathy associated with both chronic rejection and FK506 administration.14 For these reasons, rapamycin represented a novel candidate for testing in hindlimb allotransplantation models.

Hindlimb The earliest documented work on rapamycin in hindlimb allotransplantation was a short abstract published in 1991. In this report, Aboujaode et al described their work transplanting Buffalo (RT1b) limbs onto Wistar Furth (RT1u) recipient rats.16 Either cyclosporine, rapamycin, or a combination of the two agents was infused intravenously via an osmotic mini-pump each day for 14 days. Rapamycin and cyclosporine were equally effective in preventing allograft rejection in their model. Interestingly, subtherapeutic doses of each agent combined acted synergistically to improve allograft survival. No mention was made of toxicities or complications in this series.

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Fealy et al in our laboratory have recently tested the efficacy of rapamycin in a rat hindlimb transplantation model: Brown Norway (RT1n) donor to Lewis (RT1l) recipient rats.17 Rapamycin was compared to cyclosporine and to the newer standard, FK506. Midfemur amputation was performed in the Brown Norway donor rats after isolation and transection of the femoral neurovascular structures and the sciatic nerve. Bone fixation was performed to the recipient Lewis rats using 22 gauge stainless steel needles as intramedullary rods. The femoral artery, vein, nerve, and sciatic nerve were anastomosed using 10-0 nylon sutures with microsurgical technique. Ischemic times ranged from 58-98 minutes. Postoperatively, the rats were protected with wide neck collars to prevent automutilation of the transplanted limbs. Rapamycin was administered at 3 mg/kg/d intraperitoneally and was increased to prevent allograft rejection. Our laboratory compared seven experimental groups: I-III, increasing doses of intraperitoneal rapamycin; IV, intraperitoneal cyclosporine + rapamycin; V, oral FK506; VI, oral FK506 for 14 days followed by high dose FK506 salvage therapy; and VII, continuous oral FK506 for greater than 90 days. Controls included intraperitoneal carriers alone and intraperitoneal cyclosporine alone. Treatment with rapamycin did not significantly prevent limb allograft rejection. Animals tested with 3.0, 4.5, and 6.0 mg/kg/d of intraperitoneal rapamycin for 14 days underwent rejection at an average of 9-11 days, compared to control rats without immunosuppression whose hindlimb transplants survived an average of 4 days. Alternatively, cyclosporine alone and in combination with rapamycin increased survival to up to an average of 19 days, though no significant synergism was found. The best results were found with FK506. Oral FK506 at 6 mg/kg/d for 14 days resulted in prolonged survival up to an average of 28 days. Salvage therapy with 10 mg/kg//d of oral FK506, if started within 3 days of rejection, was successful in reversing rejection. Furthermore, continuous FK506 prevented limb allograft rejection entirely for the greater than 90 days tested. Complications were limited to weight loss in all groups, which eventually resolved. In the rapamycin cohort, three deaths resulted from wound sepsis; organisms cultured included E. coli, Proteus, Staphylococcus aureus, Pseudomonas, and Group D Streptococcus. FK506 rats all survived except for one death of unknown cause. In summary, rapamycin did not prevent rejection in our rat hindlimb transplant model but had significant side effects, with three deaths from wound infection. FK506 prevented rejection, both with continuous infusion and with salvage therapy. In addition, there was no significant toxicity with FK506. Fealy et al in our laboratory examined the role of cytokine activity in relation to FK506 and rapamycin immunosuppression.18 The same major immunohistoincompatibility rat hindlimb allograft model (Brown Norway (RT1n) to Lewis (RT1l)) was used. Study groups included control untreated allografts, allografts treated with oral FK506 (6 mg/kg/d for 14 days), and allografts treated with intraperitoneal rapamycin (4.5 mg/kg/d for 14 days). Further controls were Lewis to Lewis isograft and untreated Brown Norway hindlimb tissue. Grafted limbs were biopsied with a 3 mm skin punch on postoperative days 2, 5, 7, 10 and 13. Biopsies were full thickness including skin, subcutaneous tissue, and muscle. In situ hybridization using our previously described colorimetric nonisotopic technique was performed on all specimens. Briefly, 6 micron tissue slices were hybridized with cytokinespecific probe sequences and, through a series of antibody and enzymatic reactions, development of blue color indicated a positive intracellular cytokine mRNA signal. Cytokines tested were transforming growth factor beta (TGF-∃1), platelet derive growth factor alpha (PDGF-#), basic fibroblast growth factor (∃FGF), interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 6 (IL-6), and gamma interferon (IFN-!). Positive cell staining within one square millimeter of tissue at x100 magnification was quantified.

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Our laboratory found that untreated allografts were rejected at a mean survival time of 4.75 days. FK506-treated allografts were not rejected in the 2 week treatment period. As in our previous study, rapamycin did not prevent rejection, though it did delay onset of rejection to a mean survival time of 8.7 days. Most important, upregulation of cytokine expression occurred in relation to clinical signs of rejection. In untreated allografts, all 7 cytokines tested exhibited mRNA upregulation on day 5, paralleling clinical rejection. Rapamycintreated specimens had high cytokine mRNA peaks between days 7 and 13, also approximately at the time of rejection. Cells expressing these cytokines ranged from mononuclear cells in the early phase to mononuclear cells, epithelial cells, and fibroblasts in the later phase. FK506-treated allografts, which did not reject, had extremely low levels of cytokine mRNA expression throughout the two weeks tested. This correlated with the understanding that FK506 causes immunosuppression by downregulating cytokines involved in the rejection process.

Conclusions FK506 and rapamycin are actinomycete-derived agents that have shown great promise for immunosuppression. Both drugs have been used in human vital organ transplantation, and data continues to be compiled regarding their clinical profiles. Great differences in efficacy and toxicity exist between these two structurally related agents. Our data using these two immunosuppressive drugs in a rat hindlimb transplantation model correlates with data derived from other experimental models. First, FK506 is able to prolong rat limb allograft survival indefinitely. Short dosing regimens have also prolonged limb allograft survival. Secondly, in our studies, rapamycin has lead to disappointing results in the same rat hindlimb transplantation model. Limb allograft survival times were minimally prolonged compared to control rats receiving no immunosuppression. Finally, our in situ hybridization data has shown that upregulation of cytokine mRNA parallels limb allograft rejection for controls, FK506-treated specimens, and rapamycin-treated specimens. FK506 is known to inhibit cytokine synthesis. The lack of cytokine mRNA in our FK506 rat hindlimb specimens (and the high levels found in rejecting control and rapamycin-treated specimens) suggests an important temporal role these cell mediators may play in the rejection process. Therefore, at the present time, FK506 remains the standard for immunosuppression in experimental hindlimb transplantation. What is the future of human limb allotransplantation? It will depend on the development of a truly safe immunosuppressive regimen. FK506 has led to promising results in the rat models described here, but significant toxicities still exist. Rapamycin has been extremely disappointing in our limited studies, but it deserves further study with varying dosages and routes of administration before it is completely condemned. Even newer agents such as leflunomide and 15-deoxyspergualin are currently being tested in cardiac transplantation animal models and will warrant experimentation.19 Perhaps a combination of several of these newer agents, including FK506 and rapamycin, will eventually allow surgeons to one day transplant functional limbs with minimal risk to their patients.

References 1. Goldwyn RM, Beach PM, Feldman et al. Canine limb transplantation. Plast Reconstr Surg 1996; 37:184-185. 2. Hewitt CW, Black KS, Fraser LA et al. Composite tissue (limb) allografts in rats: I. Dose dependent increase in survival with cyclosporine. Transplantation, 1985; 39:360-364. 3. Kim SK, Aziz S, Oyer P et al. Use of cylosporin A in allotransplantation of rat limbs. Ann Plast Surg 1984; 12:249-255.

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4. Press BHJ, Sibley RK, Shons AR. Modification of experimental limb allograft rejection with cyclosporine and prednisone: A preliminary report. Transplant Proc 1983; 15:3057-3062. 5. Morris RE. Mechanisms of action of new immunosuppressive drugs. Ther Drug Mon 1995; 17:564-569. 6. Fung JJ, Alessiani M, Abu-Elmagd K et al. Adverse effects associated with the use of FK506. Transplant Proc 1991; 23:3105-3108. 7. Nossa GJV. Summary of the first international FK506 congress: Perspectives and prospects. Transplant Proc 1991; 23:3371-3375. 8. Fung JJ, Starzl TE. FK506 in solid organ transplantation. Ther Drug Mon 1995; 17:592-595. 9. Kuroki H, Ikuta Y, Akigama M. Experimental studies of vascularized allogeneic limb transplantation in the rat using a new immunosuppressive agent FK506: Morphogeneic and immunologic analysis. Transplant Proc 1989; 21:3187-3190. 10. Arai K, Hotokebuchi T, Miyahari H et al. Limb allografts in rats immunosuppressed with FK506: Reversal of rejection an indefinite survival. Transplant Proc 1989; 48:782-786. 11. Min Z, Jones NF. Limb transplantation in rats: Immunosuppression with FK506. J Hand Surg 1995; 20A:77-87. 12. Buttemeyer 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. 13. Calne RY, Lim S, Samaan A et al. Rapamycin for immunosuppression in organ allografting. Lancet 1989; 2: 227. 14. Morris RE. Rapamycin: FK506s fraternal twin or distant cousin? Immunol Today 1991; 12:137-140. 15 Morris RE, Wu J, Shorthouse R. A study of the contrasting effects of cyclosporine, FK506 and rapamycin on the suppression of allograft rejection. Transplant Proc 1990; 22:1638-1641. 16. Aboujaqude M, Chen H, Wu J et al. Efficacy of rapamycin in limb transplantation in the rat. Clin Invest Med 1991; 14A:146. 17. 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. 18. Fealy MK, Most D, Huie P et al. Association of down regulation of cytokine activity with rat hindlimb allograft survival. Transplantation 1995; 59:1475-1480. 19. Nair RV, Morris RE. Immunosuppression in cardiac transplantation: A new era in immunopharmacology. Curr Opin Cardiol 1995; 10:207-217.

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Allogeneic Vascularized Transplantation of Human Knee Joints

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CHAPTER 20

Allogeneic Vascularized Transplantation of Human Knee Joints Gunther O. Hofmann

Introduction

T

he allogeneic vascularized grafting of total joints has been a matter of discussion for decades. Until 1996, no clinical attempts have been made to perform such a transplantation. Since we started our clinical transplantation program in April of last year, four human knee joints have been transplanted.

History

The first transplantation of human knee joints was reported by Erich Lexer1,2 in 1908. These graftings of human bone and joint tissue were performed without organ preservation techniques, without vascular pedicles, and graft reperfusion. Transplantation immunology and the phenomenon of acute and chronic graft rejection were unknown, as well as immunosuppressive drugs and antibiotics. Therefore, Lexer’s attempts were doomed to fail. Meanwhile, various groups had performed experimental knee joint transplantation in different animal systems employing orthotopic and heterotopic techniques, using various immunosuppressive protocols.3-17 The good clinical results of our first vascularized transplantation of human femoral diaphyses,18-20 in combination with the experimental experiences of our group and others,21-27 gave the initial impetus for the clinical transplantation program.28,29

Indications The indication for the transplantation of a human knee joint might be considered in severe traumatic destruction of bone and soft tissue around the knee (Figs. 20.1 and 20.2). A complete loss of the extensor apparatus makes the implantation of a total knee joint arthroplasty (TKA) impossible. The only alternatives in these situations are an above knee amputation or arthrodesis with shortening of the leg. The last procedure may at least result in a stable, weight-supporting leg. Mobility at the level of the former knee joint is definitively lost. In these situations, a joint transplantation may be at least the only alternative for young patients (age below 45 years).

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Fig. 20.1. Severe destruction of a knee joint due to a direct trauma in a young patient. Complete loss of the extensor appartus.

Trauma Management The strategy of treatment of such injuries is subdivided into four steps: Step 1: Eradication of infection; Step 2: Restoration of soft tissue coverage; Step 3: Preparation for transplantation; Step 4: Transplantation.

Eradication of Infection As all the patients with these disorders suffer from an acute contamination or infection of the injured knee joint, a radical debridement has to first be performed. All necrotic parts of bone and soft tissues have to be removed. Open reduction and stabilization of high and lower leg is performed using external fixators. For soft tissue management, wound conditioning is performed using temporary colloid wound closure techniques until the microbiological swabs are clean.

Restoration of Soft Tissue Coverage This is achieved using local (gastrocnemius) and free pedicle flaps (latissimus dorsi). The procedure of definitive closure of the soft tissue coverage may be combined with a switch in the osteosynthesis technique.

Preparation for Transplantation Subsequently, a change in the surgical procedure is performed. The external fixators are removed. Intramedullary nails are implanted in the femur and tibia (Fig. 20.3). A simple arthroplasty made of polyethylene is attached at the top of the nails (Fig. 20.4). A soft tissue expander is additionally placed inside the former knee joint cavity. This arrangement en-

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Fig. 20.2. Roentgenogram demonstrating the extent of osseous destruction at the site of distal femur, proximal tibia and patella.

ables assisted passive motion using a CPM (continuous passive motion) device during the time on the waiting list, to avoid contractures of the soft tissues.

Transplantation Usually, multi-organ donors are used for harvesting knee joint allografts. Therefore, the fundamental criteria for postmortal multi-organ donation are to be respected: 1. Cerebral death; 2. Unrestricted agreement to explanation; 3. No exclusions due to risk factors; 4. Negative serological tests (HIV-I and -II, HbsAg, HCV).

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Fig. 20.3. Roentgenogram demonstrating the temporary substitution of the lost knee joint using intramedullary nails and a hinge arthroplasty made of PE (polyethylene) attached at the top of the nails.

Since bone and joint transplantation has no vital indication, additional safety criteria for the recipients are defined. No blood or blood derivatives are used for the donor prior to explanation, and donor age must be below 45 years.

Transplantation Transplantation is performed fresh and with a cold ischemia time of 24 hours. In situ perfusion is performed with 4 liters of UW (University of Wisconsin) solution, using a separate catheter inserted in the common iliac artery of the donor’s leg in an anterograde direction. The graft is procured with a long vascular pedicle of the superficial femoral artery

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Fig. 20.4. Intraoperative view of the temporary hinge arthroplasty. Same situation as in Fig. 20.3.

and vein. Special interest is focused on the joint capsule remaining intact. Within the allogeneic transplantation protocol, the AB0 compatibility between donor and recipient is respected. The HLA compatibility is ignored, due to logistic restrictions in donor acquisition. A negative serological cross-match should have excluded preformed cytotoxic antibodies to avoid the danger of hyperacute rejection. An additional criterion is the geometrical compatibility between donor’s and recipient’s knee joint. To avoid soft tissue problems, the donor’s knee joint should be a little bit smaller (about 90% in both planes) than the recipient’s contralateral site. The surgical procedure of the transplantation consists principally of five steps: 1. Superficial femoral artery and vein are prepared in the adductorial canal and wound with vessel loops; 2. The transplantation site is prepared: All temporary arrangements (spacer, hingearthroplasty) are removed. The intramedullary nails are withdrawn and another debridement and lavage of the joint cavity is performed; 3. The osteosyntheses are performed using the already inserted nails in a dynamically interlocking technique using a special compression device inside the nails (ICN®, Osteo-Stryker); 4. The anastomoses between the graft’s vascular pedicle and the recipient’s superficial femoral vessels are performed in end to side technique employing 6.0 nonabsorbable sutures (Seralene®, Serag-Wiessner); 5. Sutures of the following muscles and tendons re-establish the mobility of the grafted joint—quadriceps tendon into quadriceps muscle, tractus ileotibialis, gastrocnemic muscle, biceps femoris muscle, pes anserinus.

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Immunosuppression Due to the fact that all the grafted tissues, such as bone, bone marrow, cartilage, synovial membrane, ligaments, and menisci can be rather immunogeneic structures, immunosuppression is started immediately following reperfusion of the graft. During the first three days, i.v. quadruple-induction therapy with cyclosporine A (1 mg/kg b.w.), azathioprine (1.5 mg/kg b.w.), ATG (4 mg/kg b.w.), and cortisone (120 mg) is administered. An oral double-drug maintenance therapy is following with cyclosporine A (6 mg/kg b.w.) and azathioprine (1.0 mg/kg b.w.).

Follow-Up Different technical methods are employed for the follow-up of the transplanted patients: 1. Clinical chemistry—cyclosporine trough levels, inflammation parameters including leukocytes, CRP and procalcitonine; 2. Angiography by DSA technique is performed during the first week following transplantation to demonstrate macroscopic perfusion of the graft’s arterial pedicle; 3. The following are performed by noninvasive Duplex sonography—scintigraphy by SPECT-technique to evaluate the microscopic perfusion of the graft and intact cellular metabolism of the grafted cells by tracer uptake; arthroscopies of the transplanted knee joints performed half a year after transplantation and in cases of suspected rejection, to allow biopsies of the synovial membrane, the cartilage, and the grafted bone; roentgenograms by standard technique to show signs of bone healing and osseous consolidation of the osteotomies between the graft and host femur and tibia.

Results To date, four allogeneic transplantations of vascular human knee joints have been performed. Three of them were successful. The patients are mobilized, pain free, and using their stable legs under full weight-bearing conditions (Fig. 20.5). The range of motion varies from complete extension up to 120 degrees of flexion. Two of them returned to work. One of them is highly active in sports. The grafts are perfused. All grafted tissues are vital in biopsies. The osteotomies are completely healed (Fig. 20.6). Immunosuppression with a double-drug maintenance, without discontinuation, seems to be mandatory. In one case, the reduction of immunosuppression to cyclosporine monotherapy resulted in an acute rejection crisis which had to be managed by steroids (250 mg cortisone for three days). One graft was lost due to recurrence of a previous infection from destroyed knee in the transplanted one. The immunosuppression had to be stopped. Within one week, the perfusion of the graft vanished. The patient is on the waiting list for retransplantation.

Discussion and Overview According to our first experiences, the allogeneic vascularized transplantation of human knee joints is rather encouraging.30,31 Never the less indication should be limited to the following situations: 1. Patients younger than 45 years; 2. Completely destroyed joint; 3. Large bone defect; 4. Deficient extensor mechanism. Patients fulfilling these characteristics cannot be treated with TKA in order to re-establish a mobile weight-bearing extremity. Furthermore, all other indications such as osteoarthritis, rheumatoid arthritis and malignant bone tumors should be treated by TKA. Joint

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263 Fig. 20.5. 31 year old male patient, one year following allogeneic vascularized transplantation of a right knee joint. Unrestricted mobility and weight bearing.

Fig. 20.6. Roentgenograms of the same patient. Solid osseous consolidation of the femoral and tibial osteotomies.

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transplantation is not a competitive method to TKA. The cost of transplantation is the risk of immunosuppression and, therefore, this procedure is limited to specific unique cases. A number of questions concerning total knee joint transplantation remain:30,31 1. Graft procurement— What is the adequate perfusion solution? What is the maximum tolerable cold ischemia time? 2. The problem of the immune monitoring— What are the signs of an acute or chronic rejection in a joint? How do we manage the rejection crisis? What is the best specific immunosuppressive? How long should they be applied? 3. The grafted joint has no proprioception— Will this lead to an early osteoarthritis? In contrast to all of these open questions, the surgical procedure for the restoration phase, as well as for the transplantation, seems to be solved. With the designed management protocol, we were able to reduce the transplantation time of 14 hours (first case) to 6 hours (last case). Intramedullary nailing seems to be the osteosynthesis of first choice. Periosteal graft perfusion via the graft pedicle remains completely intact. Allogeneic vascularized transplantation of complete human joints is a challenging new procedure. For the immediate future, it should be limited to the clinical study designs. An open and serious discussion and informed consent with the patient is mandatory because of the necessary immunosuppression.

References 1. Lexer E. Substitution of whole or half joints from freshly amputated extremities by free plastic operation. Surg Gynecol Obstet 1908; 6:601-607. 2. Lexer E. Joint transplantation and arthroplasty. Surg Gynecol Obstet 1925; 40:782-809. 3. Judet H, Padovani JP. Transplantation d’articulation complète avec rétablissement circulatoire immédiat par anastomose artérielle et veineuse chez le chien. Rev Chir Orthop 1973; 59:125-138. 4. Reeves B. Studies on vascularized homotransplants of the knee joint. J Bone Joint Surg 1968; 50B:226-227. 5. Reeves B. Orthotopic transplantation of vascularized whole knee-joints in dogs. Lancet 1969; 8:500-502. 6. Goldberg VM, Porter BB, Lance EM. Transplantation of the canine knee joint on vascular pedicle. J Bone Joint Surg 1973; 55A:1314. 7. Goldberg VM, Porter BB, Lance EM. Transplantation of the canine knee joint on a vascular pedicle. J Bone Joint Surg 1980; 62A:414-424. 8. Walker N. Das vaskularisierte Knochentransplantat zur Überbrückung großer Knochendefekte. Handchir 1981; 13:100-102. 9. Yaremchuk MJ; Sedacca T, Schiller AL et al. Vascular knee allograft transplantation in a rabbit model. Plast Reconstr Surg 1983; 71:461-472. 10. Siliski JM, Simpkin S, Green CJ. Vascularized whole knee joint allografts in rabbits immunosuppressed with cyclosporine A. Arch Orthop Trauma Surg 1984; 103:26-35. 11. Yaremchuk MJ, Weiland AJ, Randolph MA et al. Experimental vascularized bone allograft transplantation. In: Aebi, M, Regazzoni P, eds. Bone Transplantation. Berlin: Heidelberg, NewYork: Springer-Verlag, 1989:88-89. 12. Doi K, De Santis G, Singer DJ et al. The effects of immunosuppression on vascularized allografts. J Bone Joint Surg 1989; 71B:576-582. 13. Innis PC, Randolph MA, Paskert JP et al. Vascularized bone allografts: In vitro assessment of cell-mediated and humeral responses. Plast Reconstr Surg 1991; 87:315-325.

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14. Schäfer D, Rosso R, Fricker R et al. Functional and morphological results of canine vascularized total knee transplantation under cyclosporine A as compared to autologous vascularized knee replantation. Surg Forum 1993; 44:597-607. 15. Lee WPA, Pan YC, Kesmarky S et al. Experimental orthotopic transplantation of vascularized skeletal allografts: Functional assessment and long-term survival. Plast Reconstr Surg 1995; 95:336-353. 16. Boyer MI, Danska JS, Nolan L et al. Microvascular transplantation of physeal allografts J Bone Joint Surg 1995; 77B:806-814. 17. Rosso R, Schäfer D, Fricker R et al. Functional and morphological outcome of knee joint transplantation in dogs depends on control of rejection. Transplantation 1997; 63:1723-1733. 18. Chiron P, Colombier IA, Tricoir Jl et al. Une allogreffe massive vascularisée de diaphyse femoral chez l’homme. Int Orthop 1990; 14:269-272. 19. Hofmann GO, Kirschner MH, Bühren V et al. Allogeneic vascularized transplantation of a human femoral diaphysis under cyclosporine A immunosuppression. Transpl Int 1995; 8:418-419. 20. Kirschner MH, Hofmann GO. Vorläufige Ergebnisse der Transplantation allogener gefäßgestielter Femurdiaphysen unter Immunsuppression. Transplantations medizin 1996; 8:48-53. 21. Burwell RG, Gowland G. Studies in the transplantation of bone II. The changes occurring in the lymphoid tissue after homografts and antigraft of fresh cancellous bone. J Bone Joint Surg 1961; 43B:820-843. 22. Burwell RG. Studies in the transplantation of bone. V. The capacity of fresh and treated homografts of bone to evoke transplantation immunity. J Bone Joint Surg 1963; 45B:386-401. 23. Friedlaender GE, Strong DM et al. Studies on the antigenicity of bone. J Bone Joint Surg 1976; 58A:854-858. 24. Urovitz EP, Langer F, Gross AE et al. Cell-mediated immunity in patients following joint allografting. American Orthopedic Research Society, Transaction 22 1976; 1:132. 25. Friedlaender GE, Strong DM, Sell KW. Studies on the antigenicity of bone. II Donor-specific anti-HLA-antibodies in human recipients of freeze-dried allografts. J Bone Joint Surg 1984; 66A:107-112. 26 Kirschner MH, Menck J, Hofmann GO. Anatomical bases of a vascularized allogeneic knee joint transplantation: Arterial blood supply of the human knee joint. Surg Radiol Anat 1996; 18:263-269. 27. Hofmann GO, Falk C, Wangemann T. Immunological transformations in the recipient of grafted allogeneic human bone. Arch Orthop Trauma Surg 1997; 16:143-150. 28. Hofmann GO, Kirschner MH, Wagner FD et al. First vascularized knee joint transplantation in man. Transplantations medizin 1996; 8:46-47. 29. Hofmann GO, Kirschner MH, Wagner FD et al. Allogeneic vascularized grafting of a human knee joint with postoperative immunosuppression. Arch Orthop Trauma Surg 1997; 116:125-128. 30. Mankin HJ, Doppelt S, Tomford W. Clinical experience with allograft implantation. Clin Orthop 1983; 174:69-86. 31. Brauns L, Hofmann GO, Kirschner MH et al. Abstossungs monitoring und immuno-suppressive Therapie nach gefäßgestielter, allogener Knie-gelenkstransplantation. Transplantation medizin 1997; 9:148-152.

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Clinical Transplantation of Skin Using Immunosuppression Bruce M. Achauer and Victoria Vander Kam “In 1817, Sir Astley Cooper performed the first successful human transplantation.” Freshwater, 19781

Introduction

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he history of transplantation, plastic surgery and skin grafting is virtually identical. The accessibility of skin grafts and the need to treat wounds has stimulated humans since as early as 2500 BC. The Indian tilemakers, who developed the Indian forehead flap, reportedly did so with skin grafts from the buttock to the nose. They appear to be less well documented than their flap procedures. There is some dispute about their clinical success. In any case, this technique was lost. The next high point of the skin grafting story took place in 1804 when Baronio successfully performed skin transplants in sheep (reviewed in ref. 2). In the early 1800s, the Indian method was rediscovered, leading Cooper to take a healthy piece of skin from an amputated thumb and place it over the stump. This was the first successful human skin graft.3 Buenger reported free skin grafts for nasal reconstruction. Somewhere lost in the crowd were successful cases done by Americans, Johnathan Mason Warren of Boston in 1840 and Joseph Pancoast of Philadelphia in 1844. In 1869, skin grafting became more relevant when a young intern named Reverdin, under the mentorship of Felix Guyon, took pinch grafts of epidermal islands to speed wound healing.4 This technique was popularized in London by Pollock, who is noted for treating burn wounds, and by Hamilton in the United States.2 In 1871, Ollier harvested grafts that had some dermal component. Placing them adjacent to each other resulted in healing of a higher quality graft with less scarring. He called this “dermoepidermic” grafting.5 In 1874, Thiersch studied skin grafting and concluded that thin strips of graft were preferable to Reverdin’s techniques. Full thickness skin grafting was performed and popularized by Wolfe in 1875. An ophthalmologist from Scotland, he used a full thickness graft on a burn scar contracture of the eyelid. In 1870, LeFort used a full thickness autograft unsuccessfully and later performed a full thickness skin homograft. Krause popularized the use of full thickness grafts in 1893.2 The process of graft take was outlined well by these early investigators. Thiersch used the term “inosculation” to describe the direct connection of host and graft vessels. This was earlier (1865) described by Bert as “abouchement,” which proved that wound circulation established itself as early as eighteen hours after application of the graft. Plasmatic circulation, which is the diffusion of oxygen into the graft by the host plasma, probably helps the graft survive and results in weight gain of up to 40%. Circulation in grafts can be demonstrated on the second day. Composite Tissue Transplantation, edited by Charles W. Hewitt and Kirby S. Black. ©1999 R.G. Landes Company.

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Zoografting became popular in the late 1900s and early 20th century. Grafts were taken from a wide variety of animals including rabbits 6 and pigs with leg ulcers.7 Transplantation of large pieces of skin from one site to another in sheep with burns was described by Baronio.8 In his classic textbook on plastic surgery, Davis discussed zoografts which were commonly used at the time.9 He noted that when the wound was totally healed, suddenly and with no apparent cause, the graft began to melt away and soon disappeared. In 1932, Padgett used skin allografts from family and unrelated donors for patients with severe burns.10 He realized that they would not survive permanently, but they remained long enough for the donor sites to re-epithelialize and be reharvested. The grafts from family members seemed to survive longer than those from unrelated donors, but it was hard to quantitate. In 1937, James Barret Brown of St. Louis performed skin grafts between monozygotic twins with permanent survival.11 It was in the 1940s that Gibson and Medawar conducted their classic experiment on burn patients.12 These studies demonstrated that a second set of allografts supplied to an individual resulted in rejection at an accelerated rate compared with the first set. This proved the existence of memory in the immune response. They ushered in a period of experimentation in which the immune response was studied. Skin grafting was the focus of virtually all of these experiments. Eventually, the Transplantation Proceedings developed as a supplement to the Journal of Plastic and Reconstructive Surgery. This also led some frustrated plastic surgeons, unable to obtain a permanent take with skin grafts, to experiment with organs which were putatively less antigenic, i.e., kidneys. After a series of experimental renal transplants in animals, plastic surgeon Joe Murray performed the first successful human renal transplant. This work resulted in his being awarded the Nobel prize. Major burns stimulated demand for the ability to cover large burn surface areas with grafts. This led to the use of immunosuppression following allografting in an effort to prolong survival or eliminate rejection. In 1974, Burke reported on extensively burned children.13 These children sustained burns of 91%, 72% and 91% total body surface area (TBSA). After allografts were placed, they were treated with azathioprine, 5.6 mg/kg for 3 days, then 1.5 mg/kg daily thereafter. Immunosuppression was continued until the allograft covered less than 20% of the burn wound. The drug was withheld when white counts fell below 3500. Patients were treated in a bacteriologically controlled nursing unit. Donors were living relatives who were tissue typed to determine the best match. All three patients survived. In 1975, eleven cases were reported, seven of whom survived.14 This was a major contribution to burn care, but proved to be very demanding and was eventually discontinued as a routine treatment for large burns.

Skin as an Immune Organ In 1868, a medical student named Paul Langerhans discovered dendritic cells of the epidermis. These cells, named after him, are one of the large family of cells expressing Class II major histocompatibility complex (MHC) antigens critical to antigen processing and presentation to specific T lymphocytes. They contain “tennis racket” shaped organelles called Birbeck granules that appear to result from the internalization of Class II MHC cell surface molecules. Langerhans’ cells are major antigen presenting cells of the skin. They play a key role in the peripheral immune system. They are involved in the induction of cell mediated immunity and are required for the induction of cutaneous delayed type hypersensitivity responses. They are critical to epidermal allosensitivity responses and to epidermal allosensitization such as graft versus host reaction. Langerhans’ cells originate from the bone marrow and can be depleted by UV light, radiation and steroids.15 Keratinocytes, as well as melanoctyes, are immunologically active. Keratinocytes account for more than 90% of epidermal cells. In addition to serving as a physical barrier, they may

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help in initiating cell-mediated immune responses. They can synthesize interleukin 1. They function as an early warning system. Melanocytes represent 2-5% of epidermal cells and may contribute to the immune function by secreting biologic response modifiers, especially related to inflammation.15

Clinical Application When cyclosporine (CsA) became available for research purposes our laboratories began work on a rat burn model to study allografts with CsA immunosuppression. This work was inspired by Burke’s work in the seventies with azathioprine and anti-thymocyte globulin (ATG). Our early work demonstrated that there was a dose response curve for CsA in a rat model. A minimum dose leading to prevention of graft rejection (8 mg/kg/d orally or 2.6 mg/kg/d intravenously) was determined. Preparatory research was done first on a burn model, followed by a burn plus bacteria model and finally a very large burn injury model in rats. The animal studies were very encouraging and led to long term allograft survival.16-18 The first clinical application of CsA as an immunosuppressant for skin transplantation was reported in 1986.19 An eleven year old male presented with an 85% TBSA burn, most of which was full thickness. The day before primary excision, CsA was initiated using the doses described above. Frozen skin allografts were meshed 1.5: 1 and applied over very widely meshed autografts. No attempt was made to match cadaver donor grafts with the recipient. In fact, the donors represented not only a variety of blood and Rh type differences, but a variety of races as well. CsA was continued for 120 days. The patient sustained two episodes of well documented sepsis, which responded promptly to antibiotics with appropriate leukocytosis and immediate recovery. After discharge, CsA was discontinued. Some grafted areas became quite firm and hyperkeratotic. These eventually became soft and pliable. No subsequent grafts were required. Others have subsequently tried similar regimens with success. Mindikoglu20 reported a patient with prolonged allograft survival using CsA. Drug therapy was used for three months. There was no evidence of graft rejection nor any side effects related to the CsA. Twelve days after discontinuation of CsA, rejection was noted. The burns were subsequently covered with autografts during two operations. In 1990, Sakabu et al21 reported a series of three patients with massive burns who were treated with allografts and CsA. They reported prolongation of allograft survival in these extensively burned patients. Ultimately, the patients rejected the allografts and they were replaced by sequential autografts accomplished by recropping the limited donor sites. The performance of the allografts responded closely to therapeutic blood levels of CsA. The burn patients had unpredictable metabolism of the CsA and needed to be monitored closely. These patients had massive injuries: 85, 95 and 97% TBSA. Grafting and follow-up were very well documented. There were no unusual sepsis problems. Frame et al reported three cases in 1989.22 These patients had extensive burns of 35%, 60% and 25% TBSA. Cyclosporine was used for three weeks, three months and three weeks respectively. Allografts survived during treatment with CsA, but rejected after discontinuation of the drug, at 12 days in case one, 5-7 days in case 2. Case 3 demonstrated no visible evidence of rejection. In this case, a meshed auto/allograft technique was used. Eldad reported two cases in which CsA treatment failed to extend skin allograft survival in the burn patient.23 Two children with extensive burns (85 and 95% TBSA) were treated with fresh, family-related skin allografts. These grafts were rejected during CsA treatment after 14-18 days. One child survived, while the other succumbed with Candida sepsis. Interestingly, a case report from the same institution reported prolonged skin allograft survival on the hand of a child with aplastic anemia who was being treated with low dose CsA for 50 days. No rejection occurred.24

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Results obtained clinically with CsA have been variable. This may be due to variations in dosing or metabolism of CsA. It may be due to differences in combinations of grafts or graft techniques. Another possibility may be alterations in the individual patients’ immune systems due to multiple blood transfusions. Further study may have clarified the reasons. However, due to the potential for side effects from systemic use of CsA, some investigators have shifted their attention to the study of allografting accompanied by topical immunosuppression.

Topical Immunosuppression CsA has been studied extensively for its use as a topical immunosuppressant. When applied topically, it is possible to induce site-specific immune suppression of local T cell mediated immune responses involved in skin allograft rejection. The use of CsA topically reduces toxicity as compared to its use systematically. Allograft survival may be extended with topical CsA alone or in combination with steroidal anti-inflammatory agents which can produce synergistic results.25 The combined use of topical CsA and silver sulfadiazine (SSD) on Pseudomonas-infected allografts was studied in a rat model.26 This drug combination significantly prolonged allograft survival and controlled infection. Fujita has examined the effect of FK506 ointment on rat skin allograft survival.27 The histologic grade of rejection was greatly reduced when FK506 was used in comparison to animals without FK506. Blood concentration of FK506 was low (0.5 mg/ml) thereby minimizing the adverse, systemic effects of the drug. This work suggests that the topical administration of FK506 to skin allografts may be useful and effective in the suppression of skin allograft rejection.

Skin Modification Richters et al demonstrated that skin preserved with 85% glycerol does not produce a reaction to human purified blood T cells in culture reactions. Human, purified T cells did not proliferate when cultured with allogeneic-treated skin cells, whereas untreated cells induced a distinct response. A moderate response was obtained after adding T cells and viable antigen presenting cells to the allogeneic-treated skin cells.28 Wu et al29 treated skin allografts with anti-∃2 microglobulin monoclonal antibody (∃2 mAb) and irradiation with ultraviolet C (UV-C) light. Five burn patients were grafted with allografts that were treated with either ∃2 mAb or ∃2 mAb and UV-C irradiation. They were compared with untreated allografts from the same source. The survival times of the treated specimens were increased by 35%. If the UV-C irradiation was added, the mean rejection time was then lengthened by 100%. No immunosuppression was used.

Conclusions Skin transplantation has a long and interesting history. Currently, the use of allografts with systemic immunosuppression is reserved for life threatening burn injuries only. Topical immunosuppression offers great promise for the future to advance the practice of skin transplantation. The transplantation of skin is a fascinating field with tremendous potential for further research.

References 1. Freshwater MF, Krizek TJ. George David Pollock and the development of skin grafting. Ann Plast Surg 1978; 1:96-102. 2. Hauben DJ, Baruchin A, Mahler D. On the history of the free skin graft. Ann Plast Surg 1982; 9:242-245.

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3. Zimmerman LM, Veith I. Great ideas in the history of surgery. 2nd ed. Baltimore: Williams and Wilkins, 1961; 400. 4. Davis JS. The story of plastic surgery. Ann Plast Surg 1941; 113:641. 5. Chick LR. Brief history and biology of skin grafting. Ann Plast Surg 1988; 21:358-363. 6. Coze PC. De l’emploi des greffes epidermiques pratiquees avec des lambeaux de peau de lapin, pour la guerison des plaies rebellen. Compt Rend Acad Sci 1872; 74:642. 7. Raven TF. Skin grafting from the pig. Br Med J 1887; 2:623. 8. Baronio G. Delgli Innesti Animali. Milan, Stamperia e Fonderia del Genio, 1804. 9. Davis JS. Plastic Surgery: Its principles and practices. Philadelphia: Blackiston, 1919. 10. Padgett EC. Is iso-grafting practicable? South Med J 1932; 25:895-900. 11. Brown JB. Homografting of skin with report of success in identical twins. Surgery 1937; 1:558-563. 12. Gibson T, Medawar PB. Fate of skin homografts in man. J Anat 1943; 77:299-310. 13. Burke JF, May JW, Albright N. Temporary skin transplantation and immunosuppression for extensive burns. N Engl J Med 1974; 290:267-269. 14. Burke JF, Quinby WC, Bondoc CC et al. Immunosuppression and temporary skin transplantation in the treatment of massive third degree burns. Ann Surg 1975; 182:183-197. 15. Salmon JK, Armstrong CA, Ansel JC. The skin as an immune organ. West J Med 1994; 160:146-152. 16. Achauer BM, Hewitt CW, Black K et al. CsA prolongs skin allografts in a rat burn model. Transplant Proc 1983; 15(1):3073-3076. 17. Hewitt CW, Black KS, Achauer BM et al. Cyclosporine and skin allografts for the treatment of thermal injury: I. Extensive graft survival with low-level long-term administration and prolongation in a rat burn model. Transplantation 1988; 45:8-12. 18. Black KS, Hewitt CW, Achauer BM et al. Cyclosporine and skin allografts for the treatment of thermal injury: II. Development of an experimental massive third degree burn model demonstrating extensive graft survival. Transplantation 1988; 45:13-16. 19. Achauer BM, Hewitt CW, Black KS et al. Long-term skin allograft survival after shortterm cyclosporine treatment in a patient with massive burns. Lancet 1986; 1(4):13-15. 20. Mindikoglu AN Cetinkale O. Prolonged allograft survival in a patient with extensive burns using cyclosporine. Burns 1993; 19:70-72. 21. Sakabu SA, Hansbrough JS, Cooper ML et al. Cyclosporine A for prolonging allograft survival in patients with massive burns. J Burn Care Rehab 1990; 11:410-418. 22. Frame JD, Sanders R, Goodacher TE et al. The fate of meshed allograft skin in burned patients using cyclosporine immunosuppression. Br J Plast Surg 1989; 42:27-34. 23. Eldad A, Benmeir P, Weinberg A et al. Cyclosporine A treatment failed to extend skin allograft survival in two burn patients. Burns 1994; 20:262-264. 24. Lusthaus S, Kaufman T, Livoff A et al. A case of prolonged allograft survival after a short term treatment with CsA in a child with aplastic anemia. Eur J Plast Surg 1991; 14:200-202. 25. Llull R, Lee TP, Vu AN et al. Site-specific immune suppression with topical cyclosporine. Synergism with combined topical corticosteroid added during the maintenance phase. Transplantation 1995; 59(10):1483-1485. 26. Lai CS, Miskell PH, Gonce SJ et al. Combined use of topical cyclosporine A and silver sulphadiazine on allografts infected with Pseudomonas in burned rats. Burns 1987; 13:181-184. 27. Fujita T, Takahashi S, Yagihashi A et al. Prolonged survival of rat skin allograft by treatment with FK506 ointment. Transplantation 1997; 64:922-925. 28. Richters CD, Hoekstra MJ, Van Baane J. Immunogenicity of glycerol-preserved human cadaver skin in vitro. J Burn Care Rehab 1997; 18:228-233. 29. Wu J, Barsoni D, Armato U. Prolongation of survival of alloskin grafts with no concurrent general suppression of the burned patients immune system: A preliminary clinical investigation. Burns 1996; 22:35-38.

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The Clinical Future of Composite Tissue Transplantation Robert D. Foster and James P. Anthony

Introduction

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espite the considerable advances in reconstructive plastic surgery, current methods of microvascular reconstruction fall short of recreating many native tissues in terms of function, sensation, and/or aesthetic ideal. Even the most impressive composite flap reconstructions currently performed do not meet the definition of optimal reconstruction. For example, in mandibular reconstruction, immediate restoration of composite mucosa, mandible, and soft tissue defects is now possible using a variety of osteocutaneous free flaps, including iliac crest,1 radial forearm,2 scapula,3 and the fibula.4 Although each provides reliable bone stock with overlying skin for mucosal lining, all require the placement of prosthetic implants to restore dentition, all fail to replace missing muscles of mastication, and most important, none is capable of restoring lost sensation. Particularly for areas of the body requiring very specialized functions (i.e., hand, tongue, pharynx), current reconstructive methods remain largely inadequate. These areas of highly specialized functions may be best reconstructed with composite tissue allotransplants (CTAs). One of the first CTAs to be performed in humans may be the larynx. Although more than 6,000 people in the United States suffer total larynx loss secondary to tumor removal, trauma, or congenital defects each year, there is currently no clinical or experimental method of laryngeal reconstruction which can fully replace the larynx. The optimal method of larynx reconstruction would provide the necessary sensory-motor recovery to prevent pulmonary aspiration, the structural rigidity needed to preclude airway collapse, and the motor control of the vocal folds necessary for the production of speech, in a package the size of a fist.5-8 Prior attempts at laryngeal reconstruction have focused on re-establishing the airway connection between the oral cavity and the trachea using a variety of materials, including freeze-dried cadaver cartilage, jejunal free flaps, and alloplastic implants.9-12 These methods are all ultimately unsatisfactory because they are either nonvascularized, and therefore subject to infection (preserved cartilage and alloplastic implants), and/or insensate, rendering the patient prone to pulmonary aspiration (all of the above methods). Moreover, none of these reconstructions restores vocal fold function. While there are several methods of postlaryngectomy voice restoration, including esophageal speech, electronic devices, and various types of indwelling valves,13-15 none of these provide normal voice quality and all require the patient to live with a permanent, unaesthetic, and socially debilitating tracheostomy. Composite Tissue Transplantation, edited by Charles W. Hewitt and Kirby S. Black. ©1999 R.G. Landes Company.

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The complexity of the anatomy of the tongue and the sophistication of its movements serve to define the greatest challenge in oral cavity reconstruction: functional return following near-complete and total glossectomy. The complex set of muscles is enveloped by an even more complex mucosal lining containing mucous and serous glands, general sensory end organs, and special taste sensory end organs. Despite the introduction of multiple reconstructive techniques over the past 20 years, the primary reconstructive goals following total glossectomy remain inadequate in restoring speech, taste, and intraoral sensation. Over the past 30 years, tremendous progress has occurred in the fields of immunology, drug immunosuppression and human organ allotransplantation. Today more than 20,000 organ transplants are performed in the United States each year, including kidney, liver, pancreas, heart, lung, and small bowel. Within this context, there has been renewed interest in the development of human cadaver allogeneic composite tissue transplants for complex reconstructions such as the larynx and the tongue. Composite tissue allografts could be the answer to the reconstructive challenges outlined above, providing customized tissue with the appropriate skin, muscle, nerves, bone, and fascia necessary to replace functional tissue that has been removed or destroyed.

Composite Tissue Transplantation: Past and Present Historical Perspective With the notable exception of Cosmas and Damian, two physicians said to have achieved permanent acceptance of a cadaveric lower limb allograft in the third century A.D., much of the initial clinical data on tissue transplantation dates back to the 19th and early 20th centuries. Incipient attempts at allogeneic skin grafting were described in the 1870s,16 with several subsequent reports concluding that the success of skin allografts was comparable to autografting.17 The importance of donor-recipient compatibility was first suggested in 1918,18 although this remained an area of considerable controversy until the 1930s when it became generally accepted that allografts had a significantly higher rejection rate. The landmark experiments of Peter Medawar describing allograft rejection in 1944 using a skin model in rabbits19 were a direct extension of the clinical skin graft studies he performed with the noted plastic surgeon Thomas Gibson on burned RAF pilots during the Battle of Britain in World War II.20 Since the first successful transplantation of a kidney allograft between identical twins was reported by another plastic surgeon, Joseph Murray, in 1955,21 subsequent clinical and basic science research in the field of transplantation has focused largely on solid organs. Nevertheless, a number of studies have been performed to better understand the mechanisms by which CTAs are rejected, and a variety of pharmacologic and immunologic approaches have been used to study tissue allograft acceptance. The composite tissue allograft most often used experimentally has been the limb allograft. Because limb allografts contain nearly every type of nonorgan tissue (i.e., skin, subcutaneous tissue, muscle, bone, bone marrow, cartilage, lymph nodes, nerve, and vessels), they are an excellent model for studying CTAs. Furthermore, limb allografts are clearly more antigenic than organ transplants, since higher immunosuppressant doses are required to prevent rejection.22-25 In particular, the skin and muscle appear to be highly antigenic.26 It is generally believed that if successful immunosuppression can be achieved with limb allografts, nearly any CTA should be feasible in immunologic terms. The primary obstacles to clinical application of limb and other CTAs relate both to the efficacy and toxicity of long term immunosuppression and to the functional recovery that will be obtained in these patients; the technical obstacles have been largely solved by the same advances in microsurgical techniques that now allow relatively routine replantation of amputated limbs.

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In 1976, Poole, Bowen, and Batchelor27 first described orthotopic vascularized limb allografts in rats. Long term limb allograft survival was achieved through immunologic enhancement consisting of preoperative anti-donor alloantiserum and donor renal allografting. Limb allografts from the donor strain were transplanted 42-156 days following kidney transplant, with five of seven animals displaying minimal or no rejection 23-207 days following limb transplant. In 1979, Doi28 described results of limb allografting across two inbred strains of rats that provided a strong antigenic mismatch at the major histocompatibility complex (MHC). Untreated allografts were rejected completely at an average of 12.5 days posttransplant, and, while he found azathioprine and prednisolone effective in prolonging survival, all of the limbs were ultimately rejected. Larger animal studies have generated similar results. Goldwyn et al29 demonstrated only modest prolongation of hindlimb allograft survival in dogs receiving 6-mercaptopurine and azathioprine. The mean limb survival was only 18 days (range 11-28 days), with most animals succumbing to infection and/or drug toxicity. Lance et al30 improved on these results using a combination of anti-lymphocyte serum, azathioprine, and hydrocortisone. Limb allografts survived 53-112 days in four beagles; however, all animals died due to either sepsis or pancytopenia secondary to bone marrow suppression. Interestingly, though, three additional animals in this study received a tolerance-inducing regimen that variably included high dose, short term immunosuppression (∗ 2 weeks), thymectomy, splenectomy, and injection of “tolerance antigen” (donor leukocytes or splenocytes). Two of the three animals displayed no signs of rejection at 7 and 13 months posttransplant, whereas the third animal developed skin rejection at day 105.31 Whole-joint knee allografts in dogs immunosuppressed with anti-lymphocyte serum, azathioprine, and steroids have also produced similar results.32,33 As a group, the results of these studies (prior to the use of cyclosporine A) demonstrated long term limb allograft survival too limited to justify clinical application. It was clear that more effective and less toxic immunosuppression was required before further progress could be made.

The First Significant Step Forward: Cyclosporine and Newer Immunosuppressive Agents The introduction of cyclosporine A (CsA) (Sandoz Pharmaceuticals, East Hanover, NJ) in the 1970s marked a significant event not only in the development of organ allotransplantation but composite tissue allotransplantation as well. CsA functions by inhibiting interleukin 2, a potent stimulator of T lymphocyte proliferation, suppressing cytotoxic T cells and activating suppressor T cells. Black et al,34 in 1982, were the first to publish a series of reports on the efficacy of CsA in rat hindlimb allotransplants across a strong MHC antigenic mismatch. Their first study demonstrated an average limb allograft survival of 101 ± 13 days in four rats that received short term (20 days), high dose CsA (25 mg/kg/d) following transplantation. A second report in 1983 supported these findings, with four out of five recipients on the same CsA regimen displaying limb allograft survival rates ranging from 50-70 days and the fifth rat apparently developing tolerance (limb survival >225 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

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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.

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Fig. 22.1. Canine anatomy relevant to laryngeal transplantation.

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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.

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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

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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.

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29. Goldwyn RM, Beach PM, Feldman D et al. Canine limb homotransplantation. Plast Reconstr Surg 1966; 37:184-195. 30. Lance EM, Inglis AE, Figarola F et al. Transplantation of the canine hind limb. Surgical technique and methods of immunosuppression for allotransplantation—A preliminary report. J Bone Joint Surg [Am] 1971; 53:1137-1149. 31. Porter BB, Lance EM. Limb and joint transplantation. A review of research and clinical experience. Clin Orthop 1974; 104:249-274. 32. Reeves B. Orthotopic transplantation of vascularised whole knee-joints in dogs. Lancet 1969; 1:500-502. 33. Goldberg V, Porter BB, Lance EM. Transplantation of the canine knee joint on vascular pedicles. J Bone Joint Surg [Am] 1973; 55:1314. 34. Black KS, Hewitt CW, Fraser LA et al. Cosmas and Damian in the laboratory. N Engl J Med 1982; 306:368-369. 35. Hewitt CW, Black KS, Fraser LA et al. Cyclosporine-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. 36. 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. 37. Furnas DW, Black KS, Hewitt CW et al. Cyclosporine and long-term survival of composite tissue allografts (limb transplants) in rats with historical notes on the role of plastic surgeons in allotransplantation. Transplant Proc 1983; 15(4 Suppl 1):3063-3068. 38. 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. 39. Benhaim P, Anthony JP, Lin L et al. A long-term study of allogeneic rat hindlimb transplants immunosuppressed with RS-61443. Transplantation 1993; 56:911-917. 40. Hotokebuchi T, Arai K, Takagishi K et al. Limb allografts in rats immunosuppressed with cyclosporine: As a whole-joint allograft. Plast Reconstr Surg 1989; 83:1027-1036. 41. 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. 42. Black KS, Hewitt CW, Hwang JS et al. Dose response of cyclosporine-treated composite allografts in a strong histoincompatible rat model. Transplant Proc 1988; 20(2 Suppl 2):266-268. 43. Hewitt CW, Black KS, Dowdy SF et al. Composite tissue (limb) allografts in rats. III. Development of donor-host lymphoid chimeras in long-term survivors. Transplantation 1986; 41:39-43. 44. Hovius SER, Stevens HPJD, van Nierop PWM et al. Allogeneic transplantation of the radial side of the hand in the rhesus monkey: I. Technical aspects. Plast Reconstr Surg 1992; 89:700-709. 45. Gold ME, Randzio J, Kniha H et al. Transplantation of vascularized composite mandibular allografts in young cynomolgus monkeys. Ann Plast Surg 1991; 26:125-132. 46. van den Helder TBM, Benhaim P, Anthony JP et al. Efficacy of RS-61443 in reversing acute rejection in a rat model of hindlimb allotransplantation. Transplantation 1994; 57:427-433. 47. Benhaim P, Anthony JP, Ferreira L et al. Use of combination of low-dose cyclosporine and RS-61443 in a rat hindlimb model of composite tissue allotransplantation. Transplantation 1996; 61:527-532. 48. Buttemeyer 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. 49. Parkman R. Graft-versus-host-disease. Annu Rev Med 1991; 42:189-197. 50. Sullivan KM. Graft-versus-host-disease. In: Blume KG, Petz LD, eds. Clinical Bone Marrow Transplantation. New York: Churchill Livingstone, 1983;88791-129.

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51. Tanaka K, Sullivan KM, Shulman HM et al. A clinical review: Cutaneous manifestations of acute and chronic graft-versus-host disease following bone marrow transplantation. J Dermatol 1991; 18:11-17. 52. Arai K, Hotokebuchi T, Miyahara H et al. Prolonged limb allograft survival with shortterm treatment with FK506 in rats. Transplant Proc 1989; 21:3191-3193. 53. 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. 54. Ferreira LM, Anthony JP, Mathes S et al. Complications of allogeneic microsurgical transplantation of a limb (composite tissue) in rats. Rev Assoc Med Bras 1995; 41:213-218. 55. Mackinnon SE, Hudson AR, Bain JR et al. The peripheral nerve allograft: an assessment of regeneration in the immunosuppressed host. Plast Reconstr Surg 1987; 79:436-446. 56. Bain JR, Mackinnon SE, Hudson AR et al. The peripheral nerve allograft in the primate immunosuppressed with cyclosporine A: I. histologic and electrophysiologic assessment. Plast Reconstr Surg 1992; 90:1036-1046. 57. Mackinnon SE, Hudson AR. Clinical application of peripheral nerve transplantation. Plast Reconstr Surg 1992; 90:695-699. 58. Hare GM, Evans PJ, Mackinnon SE et al. Effect of cold preservation on lymphocyte migration into peripheral nerve allografts in sheep. Transplantation 1993; 56:154-162. 59. Evans PJ, Awerbuck DC, Mackinnon SE et al. Isometric contractile function following nerve grafting: A study of graft storage. Muscle Nerve 1994; 17:1190-1200. 60. Midha R, Mackinnon SE, Evans PJ et al. Rejection and regeneration through peripheral nerve allografts: Immunoperoxidase studies with laminin, S100, and neurofilament antisera. Restor Neurol Neurosci 1994; 7:45-57. 61. Strasberg SR, Mackinnon SE, Hare GMT et al. Reduction in peripheral nerve allograft antigenicity with warm and cold temperature preservation. Plast Reconstr Surg 1996; 97:152-160. 62. Mackinnon SE. Nerve allotransplantation following severe tibial nerve injury. J Neurosurg 1996; 84:671-676. 63. Nakao Y, Mackinnon SE, Hertl MC et al. Monoclonal antibodies against ICAM-1 and LFA-1 prolong nerve allograft survival. Muscle Nerve 1995; 18:93-102. 64. Nakao Y, Mackinnon SE, Strasberg SR et al. The immunosuppressive effect of monoclonal antibodies to ICAM-1 and LFA-1 on peripheral nerve allograft in mice. Microsurgery 1995; 16:612-620. 65. Anthony JP, Allen DB, Trabulsy PP et al. Canine laryngeal transplantation: Preliminary studies and a new heterotopic allotransplantation model. Eur Arch Otorhinolaryngol 1995; 252:197-205. 66. Cober S, Randolph MA, Lee WPA. Induction of tolerance to skin allografts via intrathymic injection of donor alloantigen. Presented at the 42nd Annual Meeting of the Plastic Surgery Research Council, 1997; 42:29. 67. Rubin JP, Cober S, Butler PEM et al. In utero induction of transplantation tolerance without immunosuppression in a large animal model. Presented at the 42nd Annual Meeting of the Plastic Surgery Research Council, 1997; 42:42. 68. Pearson TC, Darby CR, Bushell AR et al. The assessment of transplantation tolerance induced by anti-CD4 monoclonal antibody in the murine model. Transplantation 1993; 55:361-367. 69. Dono K, Maki T, Wood ML et al. Induction of tolerance to skin allografts by intrathymic injection of donor splenocytes. Transplantation 1995; 60:1268-1273. 70. Saitovich D, Bushell A, Mabbs DW et al. Kinetics of induction of transplantation tolerance with a nondepleting anti-CD4 monoclonal antibody and donor-specific transfusion before transplantation. Transplantation 1996; 61:1642-1647. 71. Foster RD, Anthony JP. A model for reliable hindlimb transplantation in the mouse. (Submitted). 72. Ildstad ST, Wren SM, Bluestone JA et al. Characterization of mixed allogeniec chimeras. Immunocompetence, in vitro reactivity, and genetic specificity of tolerance. J Exp Med 1985; 162:231-244.

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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.

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

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Index

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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