Islet Transplantation and Beta Cell Replacement Therapy

Islet Transplantation and

Beta Cell Replacement Therapy

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Islet Transplantation and

Beta Cell Replacement Therapy

Edited by

A. M. James Shapiro

University of Alberta Edmonton, Alberta, Canada

James A. M. Shaw

University of Newcastle Newcastle upon Tyne, UK

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Informa Healthcare USA, Inc. 52 Vanderbilt Avenue New York, NY 10017  2007 by Informa Healthcare USA, Inc. Informa Healthcare is an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8247-2862-9 (Hardcover) International Standard Book Number-13: 978-0-8247-2862-5 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequence of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Islet transplantation and beta cell replacement therapy / edited by A. M. James Shapiro, James A. M. Shaw. p. ; cm. Includes bibliographical references. ISBN-13: 978-0-8247-2862-5 (hardcover : alk. paper) ISBN-10: 0-8247-2862-9 (hardcover : alk. paper) 1. Islands of Langerhans–Transplantation. 2. Pancreatic beta cells–Transplantation. 3. Diabetes–Treatment. I. Shapiro, A. M. James. II. Shaw, James A. M. [DNLM: 1. Diabetes Mellitus, Type 1–surgery. 2. Islets of Langerhans Transplantation. 3. Insulin-Secreting Cells–transplantation. WK 815 I82 2007] RD599.5.I84I82 2007 2007022615 617.50 570592–dc22 Visit the Informa web site at www.informa.com and the Informa Healthcare Web site at www.informahealthcare.com

Foreword

As James Shapiro points out in the first chapter of this superbly edited book on islet transplantation and beta-cell replacement therapy for diabetes, the concept is old but the realization is recent. Within five years of the late 19th century experimental observations of von Mering and Minkowski, definitively showing that diabetes was inevitable after extirpation, and that the pancreas had to have an “internal secretion” regulating blood glucose levels, attempts began to supply the unidentified and obviously missing substance to diabetic patients by injecting pancreatic extracts or by transplanting pancreatic fragments. Clinical success with a pancreatic extract (the insulin of Banting and Best) was achieved decades before complete reversal of diabetes by islet transplants in animal models. In the interim, immediately vascularized pancreas transplants were clearly shown to reverse diabetes, and the clinical application that began in 1966 continues to this day. However, pancreas transplants are associated with a relatively high incidence of surgical complications, and the entire gland is transplanted solely to supply one cell—the only one missing in type 1 diabetes: the beta cell. Thus, if there ever was a solid-organ transplant that could be replaced by a cellular transplant, pancreas for islet transplantation was it. If there was ever an indication for a wholesale transfer from major to minimally invasive surgery, this is it. The rationale for and progress towards this goal is laid out in this book. As noted by Shapiro, the morbidity associated with pancreas transplants in the early days was an incentive to develop islet transplantation. Indeed, many thought the transition would be rapid. However, as the results of pancreas transplants improved, the immediate need for clinical application of islet transplantation diminished, probably delaying its development. Likewise, the promise of islet transplantation was probably partially responsible for the underuse of pancreas transplants, even when the results did improve. Progress in clinical islet transplants has reached a point where the results approach those of pancreas transplants, with surgical complications truly minimized. This book is not only a practical guide for the current application of islet transplantation, but also a blueprint for the future.

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The patients currently selected for beta-cell replacement therapy are those for whom being immunosuppressed is judged less onerous than remaining diabetic, or those already in need of immunosuppression—that is, diabetic recipients of a kidney transplant. Expansion of islet transplantation to the general diabetic population will require reduction of the side effects of immunosuppression or a practical method of inducing immunological tolerance. Expansion to all diabetics will require a source of beta cells other than the limited number of deceased human donors, either from human stem cells or by use of xenografts. Leaders in the field, including Shapiro, have contributed chapters on each of these topics as well as on the current technical aspects and challenges of islet transplantation. A bonus is the inclusion in the first chapter of the final reminiscence of the father of islet transplantation, Paul Lacy. This book is a monument to the hope of every diabetic—to be free of the need for exogenous insulin with the least risk possible. David E. R. Sutherland, MD, PhD Professor and Head, Division of Transplantation Director, Diabetes Institute for Immunology and Transplantation Department of Surgery University of Minnesota Minneapolis, Minnesota, U.S.A.

Preface

Transplantation of pancreatic segments to replace the “internal secretion” missing in diabetes was first attempted in 1893, several years before the discovery of insulin. Despite ongoing refinement of insulin formulations and delivery devices over the last 85 years, including recent progress towards the closed loop, bioartificial pancreas, insulin replacement by conventional injection therapy remains inextricably linked to hypoglycemia. Transplantation of whole pancreas, together with its blood supply, can entirely prevent significant hypoglycemia while normalizing overall glycemic control. This major invasive procedure is necessarily associated with morbidity and mortality and will remain suitable for only a minority. The turn of the millennium heralded a new dawn in the management of Type 1 diabetes, with realization of reproducible insulin independence, and liberation from disabling hypoglycemia following transplantation of isolated islet cells. This book is dedicated to further refinement of all aspects of this procedure, with the aim of achieving the best possible outcomes for future recipients and providing these outcomes to suitable candidates worldwide. A successful clinical program is dependent on a truly multidisciplinary approach. Our goal in editing this first edition of Islet Transplantation and Beta Cell Replacement Therapy is to bring together leading proponents from each discipline critical to current and future success in the field. The contributing authors have excelled in providing a uniformly accessible text for all involved in clinical transplantation and the underpinning basic research. The approach is practical and focused on improving clinical outcomes for those with Type 1 diabetes. A wealth of experience is provided for those setting up new programs. We are confident that the level of detail and insight presented within a unified, structured format will ensure that this volume is equally well-thumbed in established centers of excellence. In the early chapters, a historical perspective is followed by an excellent exposition of the true impact and far-reaching consequences of hypoglycemia for those living with all forms of exogenous insulin replacement therapy. The subsequent chapters outline the key role of the endocrinologist in holistic assessment and selection of islet transplant recipients, in addition to facilitating a truly informed decision agreed to by both the patient and the multidisciplinary team that takes into account risks as well as potential benefits. v

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Clinical success cannot be achieved without the highest quality pancreas resection and preservation in tandem with expert islet isolation, validation, and, ultimately, transplantation. Each of these challenges is addressed in turn in Chapters 4 through 7. This section is completed by a practical guide to safe and effective management of islet immunosuppressant regimens. True understanding of the factors underlying attrition of islet function over time will only be gained by enhanced graft monitoring posttransplantation. The importance and future promise of in vivo islet imaging, in addition to metabolic monitoring of the recipient, are addressed next. This is followed by hard-earned advice on setting up a new clinical islet transplant program, outlining potential models and pitfalls for a costeffective and sustainable integrated approach. The parallel requirement for optimally designed and implemented clinical trials is explored in Chapter 12. Chapter 13, “Clinical Outcomes and Future Directions in Islet Transplantation,” highlights the most recent clinical milestone—reproducible insulin independence following transplantation of islets purified from a single donor pancreas. The importance of maintaining islets in culture enabling pre-operative stabilization and induction, in addition to successful transplantation of islets purified at a distant isolation facility, is discussed next as a prelude to Chapter 15, which provides a vision into the future of islet immunosuppressant protocols. Potential approaches for those already immunosuppressed with a functional renal graft are specifically considered. The last four chapters address the need for new sources of beta cells to meet future clinical needs through xenotransplantation, generation of new insulin-producing cells from adult tissue, and, ultimately, stem cell banks. Finally, the multitude of ways in which gene therapy may impact on clinical practice in islet transplantation and beta cell replacement within the foreseeable future are reviewed. We are extremely grateful to all of the contributing authors, without whom our aspirations to realize a truly state-of-the-art work in these pioneering days would have foundered. We also wish to acknowledge the invaluable support and enthusiasm of our associate editors Camillo Ricordi and Jonathan Lakey, in addition to Dana Bigelow and her tireless team at Informa Healthcare. This book is dedicated to the memory of Paul Lacy, whose prescient words, recorded from his final public lecture and printed in our opening chapter, continue to resonate for all of us lucky enough to be involved in trying to shape future chapters for those with diabetes. A. M. James Shapiro James A. M. Shaw

Contents

Foreword David E. R. Sutherland Preface v Contributors ix

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1. A Historical Perspective on Experimental and Clinical Islet Transplantation 1 A. M. James Shapiro 2. Hypoglycemia in Type 1 Diabetes: The Need for a New Approach Stephanie A. Amiel

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3. Patient Selection and Assessment: An Endocrinologist’s Perspective 57 Peter A. Senior 4. The Surgical Aspects of Pancreas Procurement for Pancreatic Islet Transplantation 81 Neal R. Barshes, Timothy C. Lee, Ian W. Udell, Christine A. O’Mahony, F. Charles Brunicardi, John A. Goss, and A. M. James Shapiro 5. Pancreas Preservation for Islet Isolation 99 Mohammadreza Mirbolooki and Jonathan R. T. Lakey 6. Aspects and Challenges of Islet Isolation 115 Mohammadreza Mirbolooki, Jonathan R. T. Lakey, Tatsuya Kin, Travis Murdoch, and A. M. James Shapiro 7. Percutaneous Portal Vein Access: Radiological Aspects Richard J. Owen

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8. Care of the Islet Transplant Recipient: Immunosuppressive Management and Complications 147 Raquel N. Faradji, Pablo Cure, Camillo Ricordi, and Rodolfo Alejandro 9. Islet Graft Monitoring and Imaging Christian Toso and Thierry Berney

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10. Metabolic Measures of Islet Function and Mass After Islet Transplantation 193 R. Paul Robertson vii

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11. Challenges in Setting Up a New Islet Transplant Program Paul R. V. Johnson

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12. Key Factors to Consider in Setting Up Clinical Trials in Islet Cell Transplantation: A Nursing Coordinator’s Perspective 215 Barbara S. DiMercurio 13. Clinical Outcomes and Future Directions in Islet Transplantation Faisal Al-Saif and A. M. James Shapiro 14. Culture and Transportation of Human Islets Between Centers Hirohito Ichii, Antonello Pileggi, Aisha Khan, Chris Fraker, and Camillo Ricordi

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15. Development and Application of Contemporary Immunosuppression in Human Islet Transplantation 269 Dixon B. Kaufman 16. Pig Islet Xenotransplantation—Update and Context Daniel R. Salomon 17. Approaches to b-cell Regeneration and Neogenesis Susan C. Campbell and Wendy Macfarlane 18. Stem Cell Approaches for b-cell Replacement Enrique Roche and Bernat Soria 19. Diabetes Gene Therapy 327 Peter S. Chapman and James A. M. Shaw Index

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283 293

Contributors

Rodolfo Alejandro Department of Medicine, University of Miami Leonard M. Miller School of Medicine, and Clinical Islet Transplant Program, Cell Transplant Center, Diabetes Research Institute, Miami, Florida, U.S.A. Faisal Al-Saif Department of Surgery, Walter C. Mackenzie Health Center, University of Alberta, Edmonton, Alberta, Canada Stephanie A. Amiel London, U.K.

King’s College School of Medicine, King’s College,

Neal R. Barshes Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, U.S.A. Thierry Berney Department of Surgery, University of Geneva Hospital, Geneva, Switzerland F. Charles Brunicardi Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, U.S.A. Susan C. Campbell Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle, U.K. Peter S. Chapman Matthew J. Ryan Veterinary Hospital, University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A. Pablo Cure University of Miami Leonard M. Miller School of Medicine, and Clinical Islet Transplant Program, Cell Transplant Center, Diabetes Research Institute, Miami, Florida, U.S.A. Barbara S. DiMercurio Adult General Clinical Research Center, University of Colorado Health and Science Center, Denver, Colorado, U.S.A. Raquel N. Faradji Department of Medicine, University of Miami Leonard M. Miller School of Medicine, and Clinical Islet Transplant Program, Cell Transplant Center, Diabetes Research Institute, Miami, Florida, U.S.A.

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Chris Fraker DeWitt Daughtry Family Department of Surgery, University of Miami Leonard M. Miller School of Medicine, and Clinical Islet Transplant Program, Cell Transplant Center, Diabetes Research Institute, Miami, Florida, U.S.A. John A. Goss Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, U.S.A. Hirohito Ichii DeWitt Daughtry Family Department of Surgery, University of Miami Leonard M. Miller School of Medicine, and Clinical Islet Transplant Program, Cell Transplant Center, Diabetes Research Institute, Miami, Florida, U.S.A. Paul R. V. Johnson Oxford Islet Transplant Program, Nuffield Department of Surgery, University of Oxford, and Department of Pediatric Surgery, John Radcliffe Hospital, Oxford, U.K. Dixon B. Kaufman Department of Surgery, Division of Transplantation, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, U.S.A. Aisha Khan DeWitt Daughtry Family Department of Surgery, University of Miami Leonard M. Miller School of Medicine, and Clinical Islet Transplant Program, Cell Transplant Center, Diabetes Research Institute, Miami, Florida, U.S.A. Tatsuya Kin Department of Surgery, Walter C. Mackenzie Health Center, University of Alberta, Edmonton, Alberta, Canada Jonathan R. T. Lakey Department of Surgery, Walter C. Mackenzie Health Center, University of Alberta, Edmonton, Alberta, Canada Timothy C. Lee Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, U.S.A. Wendy Macfarlane School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, U.K. Mohammadreza Mirbolooki Department of Surgery, Walter C. Mackenzie Health Center, University of Alberta, Edmonton, Alberta, Canada Travis Murdoch Department of Surgery, Walter C. Mackenzie Health Center, University of Alberta, Edmonton, Alberta, Canada Christine A. O’Mahony Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, U.S.A. Richard J. Owen Department of Radiology, University of Alberta, Edmonton, Alberta, Canada

Contributors

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Antonello Pileggi DeWitt Daughtry Family Department of Surgery, University of Miami Leonard M. Miller School of Medicine, and Clinical Islet Transplant Program, Cell Transplant Center, Diabetes Research Institute, Miami, Florida, U.S.A. Camillo Ricordi DeWitt Daughtry Family Department of Surgery, University of Miami Leonard M. Miller School of Medicine, and Clinical Islet Transplant Program, Cell Transplant Center, Diabetes Research Institute, Miami, Florida, U.S.A. R. Paul Robertson Pacific Northwest Research Institute and the Departments of Medicine and Pharmacology, University of Washington, Seattle, Washington, U.S.A. Enrique Roche Instituto de Bioingenieria, Universidad Miguel Hernandez, Elche, Alicante, Spain Daniel R. Salomon Department of Molecular and Experimental Medicine, The Scripps Research Institute, Center for Organ and Cell Transplantation, Scripps Health, La Jolla, California, U.S.A. Peter A. Senior Division of Endocrinology, University of Alberta, Edmonton, Alberta, Canada A. M. James Shapiro Department of Surgery, Walter C. Mackenzie Health Center, University of Alberta, Edmonton, Alberta, Canada James A. M. Shaw Diabetes Research Group, Institute of Cellular Medicine, University of Newcastle, Newcastle upon Tyne, U.K. Bernat Soria CABIMER, Andalusian Center for Molecular Biology and Regenerative Medicine, Seville, Andalucia, Spain Christian Toso Department of Surgery, University of Alberta Hospital, Edmonton, Alberta, Canada, and Department of Surgery, University of Geneva Hospital, Geneva, Switzerland Ian W. Udell Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, U.S.A.

1 A Historical Perspective on Experimental and Clinical Islet Transplantation A. M. James Shapiro Department of Surgery, Walter C. Mackenzie Health Center, University of Alberta, Edmonton, Alberta, Canada

INTRODUCTION It is always sobering to reflect back on history and appreciate how much was accomplished with seemingly primitive tools, and especially to realize that very few ‘original’ ideas have not been considered, attempted, tried, and perhaps dismissed many years before their time. Such is definitely the case with islet transplantation for diabetes. The first clinical attempt at islet transplantation in the treatment of diabetes occurred on December 20th, 1893, 28 years before the discovery of insulin, and was published in the following year in the British Medical Journal (1) (Fig. 1). Dr WatsonWilliams and his surgical colleague Mr. Harsant, working at the Bristol Royal Infirmary in England, transplanted three pieces of freshly slaughtered sheep’s pancreas, “each the size of a Brazil nut,” into the subcutaneous tissues of a 15-year-old boy dying from uncontrolled ketoacidosis. Their

Figure 1 Original article header in the British Medical Journal, December 1894. Source: From Ref. 1. 1

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operation, performed under chloroform anesthesia, was completed “within twenty minutes of the death of the sheep.” This seemingly simple experiment is remarkable from many aspects— not least the fact that Watson-Williams and Harsant’s first instincts were to use xenograft pancreas tissue and not human tissue as a potentially unlimited source. Secondly, their transplant took place without any immunosuppression, in the hope that tolerance to xenogeneic tissue would naturally occur. It was not until almost 50 years later that the mechanisms underlying allograft rejection, the concepts underlying immunological tolerance, and how the immune system could be in check with powerful non-specific immunosuppression began to emerge. Nonetheless, a major hope and focus of research remains the induction of immunological tolerance to xenogeneic tissues, and this still seems like an almost insurmountable barrier. In Watson-Williams and Harsant’s case, there was temporary improvement in the patient’s clinical condition before the tissue was rapidly rejected and death occurred three days later. Even Watson-Williams and Harsant’s idea was not new, for Oscar Minkowski had already carried out a similar procedure in a pancreatectomized dog in the previous year (1892), and had described a temporary reduction in glycosuria (2). These experiments were published just three years after Joseph von Mering and Oscar Minkowski had discovered that the pancreas was linked to diabetes by surgical removal of a dog’s pancreas with onset of polyuria and glycosuria (3) (Fig. 2). The first clinical attempts to transplant fragmented pancreatic tissue as allografts in patients with diabetes is attributed to the pioneering surgeon Frederick Charles Pybus from Newcastle-upon-Tyne, England, where in July 1916 Pybus procured cadaveric human pancreas fragments and transplanted these subcutaneously in two patients (4). One of the two patients showed temporary reduction in glycosuria before rejection ensued (Fig. 3). Four years later, on October 31st, 1920, the idea occurred to Frederick Banting that ligation of the pancreatic duct in dogs might lead to acinar degeneration and enhanced recovery of the “internal secretions” for treatment of diabetes (5). The effect was dramatic, and ongoing studies by Banting, Best, Collip, and Macleod rapidly led to the introduction of exogenous insulin into clinical practice in 1922. By the following year, Eli Lilly and Company was producing insulin in virtually unlimited quantities for the widespread treatment of diabetics (6). In 1923, it appeared that diabetes had been ‘cured’ by insulin. It only slowly became apparent that while insulin could prevent the devastating and rapidly fatal death sentence from ketoacidosis, it really only provided a very protracted stay of execution, with diabetes becoming an incurable illness with most patients developing one or more end-stage debilitating or life-shortening secondary complication.

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Figure 2 Oscar Minkowski discovered the link between the pancreas and diabetes (1892).

Thus the idea of endocrine replacement therapy lay fallow, and there was little incentive to continue with futile attempts to transplant fragments of pancreatic tissue. It was not until the late 1960’s that Paul E. Lacy

Figure 3 Original article header in The Lancet, 1924, published eight years after the experiments took place. Source: From Ref. 4.

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initiated islet transplantation studies in mice using islets isolated from the pancreas rather than the large pieces of devascularized tissue used previously. Extracting islets from the pancreas was not an easy task. The human pancreas contains 1–14.8 million islets of mean diameter 157 µm constituting only 0.8–3.8% of the total mass of the gland (7,8). In 1911, Bensley stained islets within the guinea pig pancreas using a number of dyes, and was able to pick free the occasional islet for morphological study (9). Armed with watchmaker’s forceps, hypodermic needles, and a binocular microscope, Claus Hellerstro¨m developed methods in 1964 for free-hand micro-dissection of small numbers of islets for biochemical and physiological study (10). These techniques were effective in an obese, hyperglycemic strain of mouse with uniquely large islets, but were impractical in most other species. Prompted by a need for large-scale isolation to further in vitro studies, Moskalewski introduced a mechanical and enzymatic method of dispersion of pancreatic tissue in 1965 using bacterial collagenase derived from Clostridium histolyticum (11). Although the collagenase destroyed many islets, it did permit complete separation of islets from surrounding acinar tissue, with demonstrable viability in culture and appropriate b-cell degranulation in hyperglycemic challenge. Paul E. Lacy and colleague Dr. Kostianovsky introduced two further modifications in 1967 that considerably improved islet yield and recovery (12).a Mechanical disruption of the pancreas by ductal injection of a balanced salt solution greatly increased the subsequent penetration of collagenase, with consequential enhanced islet release. They further discovered that islets could be separated from digested acinar tissue by differential density elutriation on discontinuous sucrose gradients, but these islets failed to release insulin in response to a glucose challenge, which was presumed to be the result of hyperosmolar sucrose injury from cellular dehydration and islet exhaustion. Lindall et al. found that replacement of sucrose gradients with Ficoll (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), a high molecular weight polymer of sucrose, led to more efficient islet separation, and David Scharp and colleagues further showed that dialyzed Ficoll provided islets that responded appropriately in vitro (13,14). These preliminary studies paved the way for transplantation studies in diabetic rodents. Younoszai et al. in 1970 were the first to demonstrate amelioration of the diabetic state in rats by intraperitoneal implantation of allografted islets, with improvement in glycuria but only temporary improvement in glycemia (15). Two years later, Ballinger and Lacy showed

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Paul E. Lacy gave his final address, three weeks before he passed away, at a historic meeting in Philadelphia. A complete transcript of his account of the progress and challenges of islet isolation and transplantation concludes this chapter.

A Historical Perspective on Experimental and Clinical Islet Transplantation

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sustained improvement (but not complete correction) of chemical diabetes in rats receiving 400–600 islets delivered intraperitoneally or intramuscularly, with graft excision inducing return of diabetes (16). It was not until Rechard and Barker transplanted larger numbers of islets (800–1200) into the peritoneal cavity in 1973 that chemically induced diabetes was effectively cured for the first time (17). Searching for optimal sites for islet implantation, Kemp et al. found that intraportal embolization of only 400–600 rodent islets to an intrahepatic site resulted in complete reversal of diabetes within 24 hours, whereas a similar islet load placed intraperitoneally or subcutaneously was inadequate (18). Portal embolization was thus recognized to be the most efficient site for islet implantation in the rodent, with the benefit of high vascularity, proximity to islet-specific nutrient factors, and physiological first-pass insulin delivery to the liver. It has subsequently become apparent that once embolized to the liver, islets undergo a process of angiogenesis and neovascularization to form a microvascular network and to re-establish nutritional blood supply. In the mouse, capillary sprouts and arterioles arise within 2 to 4 days, interconnect by day 6, and the process is completed by day 10 to 14 (19). These vessels are of host origin, pierce the islet, and branch into capillaries within the centre of the graft (20). Furthermore, it was shown that a physiological “core-to-mantle” perfusion is reinstated for optimal intercellular b-to-alpha/delta sensing and signaling for optimal insulin and glucagon control (21). Similar techniques of islet isolation and purification were not successful when applied to the more dense and fibrous pancreas of larger animals, including the human gland. Mirkovitch et al. were the first to reverse pancreatectomy-induced diabetes by intrasplenic autotransplantation of partially digested pancreatic tissue in dogs (22); intravenous glucose tolerance tests were indistinguishable from normal controls, even if less than half the pancreas was used for tissue digestion. Warnock and others subsequently showed that islet autografts prepared by enzymatic digestion and mechanical dispersion could reliably reverse the diabetic state in dogs (23). Simon Griffin et al. further showed that up to three recipients could be normalized by one donor graft when non-purified pancreatic tissue was infused intrasplenically (24). Unfortunately the human spleen is not distensible as in the dog, where the spleen serves as an important role in autotransfusion in the face of life-threatening hemorrhage. Attempts to transplant impure or partially purified tissue intrasplenically in humans have met with considerable morbidity, including splenic rupture (25), wedge splenic infarction, and portal vein thrombosis in a high proportion of recipients, although insulin independence has been achieved in the autograft setting (26). Investigators resorted to intraportal transplantation of impure pancreatic homogenates in dogs and ultimately in humans, leading to

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disastrous outcomes, including disseminated intravascular coagulation, portal vein thrombosis, and the sequelae of portal hypertension, hepatic infarction, and, in some cases, liver failure (27–29). Mehigan et al. found that the addition of heparin and aprotinin to the tissue preparation at the time of transplantation could ameliorate the risk of disseminated intravascular coagulation (30). Recent progress has occurred in the science of islet isolation, based on evolution of an enzymatic pancreatic dissociation process that provides more consistent high yields of viable human islets for transplantation. The techniques used currently evolved in a strong international collaborative effort with a select number of islet isolation laboratories. The early history and development of the current state-of-the-art isolation methods are reviewed below. Recent methods have increased the efficiency of the process, and have had major impact in enhancing the consistency and quality of highly purified islet preparations for safe transplantation into patients. The evolution towards current techniques is outlined below. Improvement in the isolation and purification of islets from the large animal pancreas became a major focus of intensive study in several laboratories, using the canine pancreas as the pre-clinical model. Intraductal injection of collagenase directly into the pancreatic duct was shown by Horaguchi and Merrell, and subsequently by Noel et al., to be the most effective way to dissociate the pancreas for high yield islet isolation, with up to 57% recovery of the total islet mass (31,32). Trans-ductal collagenase delivery, whether by direct injection (33) or continuous perfusion (34,35), was able to cleave the islet–acinar interface more readily than any method described previously, but still led to significant islet destruction through inadvertent islet enzyme penetration (36). However, the process did permit successful isolation of islets from the pig (37), monkey (38), and human pancreas (33). The approach was further refined to allow precise control of the temperature and perfusion pressure (39). Lakey et al. demonstrated that retrograde intraductal Liberase-HI (Roche Pharmaceuticals, Indianapolis, Indiana, U.S.A.) delivery using a recirculating controlled perfusion system provided superior human islet recovery and survival when compared to syringe loading (40). By providing control over perfusion pressures and Liberase temperature during loading of the enzyme into the pancreas, the recirculating controlled perfusion system more effectively delivers the Liberase to the interface of the islet–acinar interface resulting in a greater separation of islets from the surrounding exocrine tissue (40) A major advance came with the introduction of a semi-automated dissociation chamber and process originally developed by Ricordi et al. in 1988, modifications of which have now become the universal standard for successful high yield large animal and human islet isolation (41). The

A Historical Perspective on Experimental and Clinical Islet Transplantation

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Ricordi continuous digestion chamber and automated shaker.

collagenase-distended pancreas is placed inside a stainless steel chamber containing glass marbles and a 500 µm mesh screen and mechanically dissociated by gentle agitation, with tissue samples evaluated sequentially to determine the end-point before liberated islets become fragmented by overdigestion (Fig. 4). This novel approach minimized trauma to the islets in a continuous digestion process with the collection of free islets as they are liberated from the digestion chamber. A comparison of manual and automated methods of islet isolation clearly demonstrated superiority of the automated method (35,42–44). Since the introduction of this technique, many laboratories around the world have utilized this system for the isolation of islets from canine, pig, and human islets. Large-scale purification of human islets of suitable quality for safe transplantation into the human portal vein was enhanced considerably by the introduction of an automated refrigerated centrifuge system (COBE 2991) by Lake et al., which permitted rapid large volume Ficoll gradient processing in a closed system 600 ml bag (45) (Fig. 5A). When Ficoll is made up in Euro-Collins solution (Euro-FicollÔ), hypertonic exposure of the exocrine component reduces osmotic swelling and enhances differential isletexocrine density improving purification, but results in significant b-cell stress with degranulation and loss of insulin content (46,47). Until recently, a major limitation to successful pancreatic digestion has been the source, quality, and variability in collagenase activity, and contaminants in various enzyme blends. A new class of highly purified enzyme blend (Liberase), containing collagenase I and II, thermolysine, clostripain, and clostridial neutral protease, with low endotoxin activity, has

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Figure 5 (A) COBE 2991Ô cell apheresis system. (B) Final packed cell volume is less than 4 cc Processor (Gambro BCT, Inc., Lakewood, Colorado, U.S.A.) after purification.

consistently provided enhanced islet yield, viability, and function from the human pancreas, and has been an important advance to the field (48–51). Refinements in manufacture have largely eliminated the lot-to-lot variability in crude enzyme effectiveness for islet isolation (50,51). Liberase has proven to be superior to crude collagenase preparations by consistently yielding large numbers of islets without compromising the functional viability (51) (Fig. 6). Despite the key advances in collagenase quality, intraductal enzyme delivery, automated dissociation, and purification outlined above, inconsistency remains in the overall success of the islet isolation procedure, which may reflect variability in donor-related factors (donor inotropic need,

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Figure 6 Low-endotoxin Liberase (Roche Pharmaceuticals, Indianapolis, Indiana, U.S.A.) collagenase—significant improvement in consistency and yield (blend of type I and type II collagenase with thermolysine).

duration of cardiac arrest, hyperglycemia, age, and obesity in the donor, in addition to the skills of the local procurement team) (52). EARLY CLINICAL TRIALS OF ISLET TRANSPLANTATION Prior to the introduction of the Edmonton Protocol in the year 2000, a total of 447 human islet allografts, and 3,185 fetal or neonatal islet allografts and xenografts were carried out in 79 institutions, as reported to the Islet Transplant Registry (53–55). These results were interesting as the excellent success of islet transplantation in small and large animals in the laboratory and of human islet autotransplantation after pancreatectomy stood in contrast to the striking lack of success of islet allotransplantation in the treatment of patients with autoimmune Type 1 diabetes. The first series of clinical islet allograft transplants in Type 1 diabetic patients immunosuppressed with azathioprine and corticosteroids were reported by Najarian et al. in 1977, and followed shortly after reports of successful cure of diabetes by islet transplantation in rats (56). It was anticipated that human islet transplantation would supercede whole pancreas transplantation, which was associated with appalling morbidity and mortality rates in that particular time. While the initial attempts at islet transplantation appeared to be safe, these efforts were largely ineffective. Of seven patients transplanted with dispersed pancreatic tissue into the peritoneal cavity or via the portal vein, no patient achieved insulin independence, although some were able to reduce insulin requirements for limited periods (56). The first C-peptide negative Type 1 diabetic to achieve sustained insulin independence by one year after islet transplantation occurred in 1978 in Zurich, Switzerland

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after single donor-to-recipient transplantation of non-purified islet tissue embolized to the spleen, simultaneous with kidney transplantation (57). Despite a number of anecdotal reports since 1979, only 35 Type 1 diabetic patients have attained insulin independence after islet allograft transplantation, according to data registered with the International Islet Transplant Registry, as of December 1997 (58). Activity in clinical islet transplantation could be subdivided into five categories: (i) islet autografts in patients undergoing total pancreatectomy; (ii) islet allografts after total pancreatectomy; (iii) islet allografts in Type 1 diabetic patients; (iv) fetal islet allografts or xenografts in Type 1 diabetics; and (v) islet allografts in Type 2 diabetics. Success may be judged in terms of patient survival, graft survival (C-peptide production), attainment of insulin independence, effect upon glycemic control (glycosylated HbA1c), overall quality of life, and impact upon secondary diabetic complications.

ISLET AUTOGRAFTS The remarkable success of islet autotransplantation had a major impact on overall progress and attitudes towards islet transplantation established beyond doubt the concept of insulin independence after islet transplantation in the clinical setting. Indeed, the literature suggested that, after total pancreatectomy for chronic pancreatitis and intraportal infusion of purified or unpurified pancreatic digest, approximately 50% of patients will be rendered independent of insulin. The first islet autotransplant following pancreatectomy for chronic pancreatitis was carried out in Minnesota in 1977 (59), and over the subsequent 20 years a world experience has been accrued in 189 patients in 22 centers (7,58). Most patients underwent total or near-total pancreatectomy for intractable pain in chronic pancreatitis without pancreatic duct dilatation. Pyzdrowski et al. reported a limited series of intraportal islet autografts in whom all recipients became insulin independent after transplantation, with documentation of functional intrahepatic islets on liver biopsy staining positive for insulin, glucagons, and somatostatin, and with evidence of intrahepatic insulin secretion on hepatic vein catheterization (60). Reviewing the experience of 69 islet autografts reported to the Islet Transplant Registry, 80% of patients became insulin independent for longer than one week, and 61% maintained insulin independence beyond one year (58). The longest follow-up of insulin independence in islet autografts was more than 13 years (61,62). The best predictor of insulin independence in islet autografts is the number of islets transplanted, with a transplant mass exceeding 300,000 islets associated with an insulin independence rate of 74% at two years post-transplant (63). Farney et al. further showed in a series of 29 islet autografts that 21% of patients lost graft function between 3 and 24 months after intraportal islet embolization where a median of 148,000 islets were

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transplanted, but if a median of 384,500 islets were given there were no late graft failures beyond 2 years, with a maximal follow-up of over 12 years (64). Most centers have used non-purified pancreatic digest for islet autotransplantation because the fibrotic and atrophic nature of grafts scarred by chronic pancreatitis typically yields low tissue volume (usually 5 mls or less). There is also concern that further purification of an already marginal islet transplant mass may render the exercise futile. While complications of portal vein thrombosis, disseminated intravascular coagulopathy, and fatality have been described after islet autotransplantation previously, the risks have been minimized in recent years by systemic heparinization and better characterization of the dispersed grafts (7,30). An accepted approach was to Ficoll-purify pancreatic digests exceeding 15 ml in volume to further lower the risk of portal vein thrombosis (7). The introduction of low-endotoxin collagenase (Liberase) may also be critical in minimizing the acute risk of physiological perturbations associated with infusion of non-purified islet preparations. While many different sites have been tried for islet autotransplantation, the optimal site appears to be through portal venous embolization. Attempts to embolize to the spleen led to significant life-threatening complications of splenic infarction, rupture, and even gastric perforation (26).

ISLET ALLOGRAFTS AFTER PANCREATECTOMY A unique series of nine islet allografts were completed at the University of Pittsburgh in 1989 in patients undergoing abdominal exenteration with multi-visceral resection for malignancy, followed by cluster transplantation of liver, kidney, and bowel (65). Islets were isolated from a single multivisceral donor pancreas in the majority of cases, and infused intraportally after liver reperfusion. Over 50% of recipients achieved and maintained insulin independence until their demise from recurrent malignancy. The series represented an unusual opportunity to complete islet allografts in the absence of an autoimmune diabetes background, which may have contributed to the preservation of the functional reserve of these grafts. Other major factors contributing to the success of the cluster–islet transplantation experience included: (i) embolization of non-purified islet preparations; and (ii) the use of steroid-free immunosuppression (high dose tacrolimus monotherapy), which represented the first experience with less diabetogenic immunosuppression (66).

ISLET ALLOGRAFTS IN TYPE 1 DIABETES A total of over 447 attempts to treat Type 1 diabetes with islet allografts were reported to the Islet Transplant Registry between 1974 and 2000, 394

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of which occurred within the most recent decade (53). Mainstay immunosuppression was largely based on the combination of glucocorticoids, cyclosporine, and azathioprine, with anti-lymphocyte serum induction (67). The majority of these grafts were combined islet–kidney transplants, since it was felt inappropriate to initiate new immunosuppression in islet-alone recipients who would not have otherwise required therapy to sustain another solid organ kidney or liver graft. Under these protocols, fewer than 10% of patients were able to discontinue insulin therapy for longer than one year, although 28% had sustained C-peptide secretion at one year post transplant (54). These disappointing results contrasted with the success of islet autografts and partial success of islet allografts in non-diabetic pancreatectomized recipients where glucocorticoid-free immunosuppression was combined with unpurified islet preparations (66). A key question remained unanswered: were the previous poor results of islet allografts in Type 1 diabetic recipients a result of poor control of alloimmune pathways, or did they reflect recurrence of autoimmune diabetes? Insulin independence was only rarely achievable under glucocorticoid and cyclosporine-based immunosuppression. C-peptide secretion diminished to zero over time in most cases, suggesting islet graft loss from acute rejection or possible recurrence of autoimmune diabetes. Results of whole pancreas transplantation indicate that stable graft function is achievable over time, even with lower dose maintenance immunosuppression, suggesting that prevention of autoimmune destruction might be more readily achieved than prevention of alloimmune rejection. Autoimmune recurrence after whole pancreas transplantation only appeared to be a challenge when no immunosuppression was given, as occurred in a livingdonor hemi-pancreas transplant between identical twins, where autoimmune recurrence led to graft loss within two months. Detailed analysis identified four “common characteristics” associated with improved success (cold ischemia < 8 hours; transplant mass > 6,000 IE/ Kg; intraportal delivery; and ALG/ATG induction, but not OKT3) (55,68); 29% of this subgroup were independent of insulin, and 46% had HbA1c levels of less than 7%, which in the context of the DCCT trial suggests that tight glycemic control afforded by islet transplantation might slow progression of secondary diabetic complications (69). In the late 1990s, results of clinical islet transplantation in patients with T1DM improved under cyclosporine, glucocorticoid, and azathioprine immunosuppression, together with anti-IL2 receptor induction and antioxidants. Combined data from the Giessen and Geneva (GRAGIL consortium) groups reported a 50% rate of C-peptide secretion and 20% insulin independence rate at one year (70,71). Islets were cultured for a mean of two days, and mean islet implant mass was 9,000 IE/ kg, derived from single donors in half of cases. Two of ten patients achieved insulin

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independence after single-donor islet infusions, but it took 6–8 months to achieve independence, and both were recipients of shipped islets from a central islet isolation site (70). The University of Milan further reported experience with two immunosuppressant protocols in Type 1 diabetic islet after kidney recipients [anti-lymphocyte serum (ALS) þ cyclosporine þ azathioprine þ prednisone in the first Era (1989–1996) vs. anti-thymocyte globulin (ATG) þ cyclosporine þ mycophenolate þ metformin together with antioxidants in the second Era (1998–2001)] (72). Rejection rates were low in both eras (3/21 vs. 3/20 in era one vs. era two, respectively). Rates of insulin independence were enhanced from 33% to 59% with the elimination of prednisone and addition of mycophenolate and metformin. Over 50% of patients maintained insulin independence beyond one year with the newer protocol, possibly as a result of more effective immunosuppression coupled with anti-inflammatory, less diabetogenic, and improved insulin action with the newer protocol. The recent era of the Edmonton Protocol and subsequent protocol modifications is outlined in subsequent chapters of this book. LIVING DONOR ISLET TRANSPLANTATION The early era of clinical islet allotransplantation was fraught with difficulties as outlined above. In an attempt to overcome the poor islet viability and yields obtained from these early cadaveric islet isolations, Dr. David Sutherland and colleagues in Minnesota attempted two living donor islet transplants, the first of which took place on September 27th, 1977 (73). Islets were isolated from an HLA-identical sister and transplanted intraportally without purification into her diabetic brother, a recipient of a previous kidney transplant from a different sibling. The recipient became C-peptide positive for six weeks, but never achieved insulin independence. A further attempt at living donor islet transplantation was carried out by the same group on July 12th, 1978. This recipient achieved only temporary insulin independence during the third week post-transplant, then promptly rejected the islets during an episode of renal rejection. Both of these early attempts, while heroic, were largely considered as technical failures. The Minnesota Group then turned their attention to living donor segmental vascularized pancreas grafts, and have reported a high rate of success now in approximately 150 cases, including recent experience with laparoscopic, minimally invasive retrieval surgery in the living donor (74,75). The first successful living donor islet transplant, with sustained attainment and maintenance of insulin independence, was carried out in Kyoto, Japan by Matsumoto and colleagues in January, 2005 (76,77). This was a motherto-daughter transplant in which the recipient had chronic pancreatitis in the absence of autoimmunity. Insulin independence was maintained for at least 7 months, and the early results are encouraging.

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FETAL ISLET ALLOGRAFTS OR XENOGRAFTS IN TYPE 1 DIABETES A surprisingly large number of attempts to transplant fetal or neonatal islet allografts and xenografts has occurred mainly in the Far East in human Type 1 diabetic recipients. In fact, the number of fetal and neonatal islet transplants actually exceeds the number of adult islet allografts by a factor of ten. A total of 3,185 cases have been published or registered since 1977, but the true cumulative total is now estimated to exceed 5,000 (55,58). Access to human fetal tissue is clearly more readily available in China and Eastern Europe, where over 96% of these transplants have been carried out. Turchin et al. reported on their experience in 1,500 human fetal and neonatal porcine islet transplants carried out in Kiev, and found a reduction in hypoglycemic episodes (78). Insulin independence after human fetal or neonatal dispersed pancreas tissue transplantation was reported by Hu et al. in 48 recipients from 54 hospitals in China, with delayed progression of microvascular secondary complications in patients with good graft function after 29 months of followup (79,80). Insulin independence after human fetal islet transplantation has been reported in 9 further recipients in other centers (81–83). Unfortunately, despite this extensive experience, these apparently successful outcomes must be interpreted with caution, as the majority of grafts have been poorly characterized in terms of transplant mass and pre-transplant C-peptide negativity. Tuch et al. recovered human fetal islet grafts with persistent b-cells in three patients 9–14 months after transplantation, but could not demonstrate immunoreactive C-peptide in peripheral blood and found histological changes of islet rejection (84). Groth et al. detected porcine C-peptide in the urine from 200 to 400 days after transplantation in four of ten patients transplanted with fetal porcine islet clusters, but could not document C-peptide in serum (85). Some investigators used non-human xenogeneic islet tissue derived from bovine, porcine, and rabbit sources, with implantation to a variety of sites, including muscle, spleen, bone marrow, and even direct intracerebral implantation (55,86). Most of the transplants were performed without adjuvant immunosuppression. Based on current evidence, human fetal islet transplants are not protected from autoimmune attack (87). The issues of rejection (88), immaturity of the human fetal pancreas, and ethical issues surrounding recovery of human fetal tissue remain significant challenges for this approach in the cure of diabetic patients.

PANCREAS TRANSPLANTATION While this book is largely about islet transplantation, it is important that the progress achieved in islet transplantation is placed in context with the now excellent results attainable in whole pancreas transplantation. Dramatic improvement in outcome has occurred in clinical vascularized pancreas transplantation since the first procedure was carried out by Kelly and Lillehei

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at the University of Minnesota in 1966 (89). The earliest attempts met with dismal mortality rates in excess of 60% and graft survival of only 3% at one year, related to uncontrolled sepsis from failure of duodenal anastomotic healing in the face of high dose steroids (90,91). In 1983, two crucial developments immediately enhanced the success of the procedure. First, the introduction of cyclosporine provided enhanced immunologic potency and reduced sepsis with better tissue healing through its steroid sparing potential; secondly, both Cory and Sollinger described techniques for bladder drainage of pancreatic exocrine secretions, which provided better immune monitoring through urinary amylase assessment, a lower anastomotic leak rate with reduced gram negative sepsis (92–94). Subsequent improvement in outcome led to endorsement of simultaneous pancreas-kidney transplantation as recommended treatment of the Type 1 diabetic presenting in non-reversible renal failure (95). Up till 2000, pancreas transplantation was currently the only treatment of Type 1 diabetes that consistently restores sustained endogenous secretion of insulin responsive to near-normal levels, leading to correction of HbA1c, far surpassing the impact of intensive insulin management achievable in the DCCT trial (96,97). Currently, there have been more than 25,000 whole pancreas transplants performed worldwide, the majority of which have been carried out as simultaneous pancreas-kidney (SPK) grafts, although numbers of solitary pancreas transplants are increasing. At the time the Edmonton Protocol moved forward in 2000, the actuarial survival of patients and of functional pancreas grafts (with complete insulin independence) was 94% and 89% at one year and 81% and 67% at five years, respectively, according to registry data (98). The results of pancreas-alone grafts were inferior to simultaneous pancreas-kidney grafts. However, pancreas-alone transplantation led to excellent outcomes in carefully selected individuals under tacrolimus-based immunosuppression, with one-year graft survival at 80% to 90%, with corresponding patient survival as high as 95% (99–101). Compared with kidney-alone transplantation in Type-1 diabetics, patient survival improved by at least 10% by five years and by up to 59% at ten years following combined transplantation (102,103). Freedom from insulin, blood glucose monitoring, and dietary restriction improves the overall quality of life for the diabetic undergoing successful pancreas-kidney transplantation, but scores generally fail to match those of the general non-diabetic healthy population by one year posttransplant (104–106). Quality of life improvement is particularly evident in patients with hypoglycemic unawareness, brittle diabetes, or gastroparesis (107). Recent advances in surgical technique, immunosuppression, and posttransplant monitoring have had major impact in reducing the morbidity of patients undergoing simultaneous pancreas-kidney and solitary pancreas transplantation. A recent return to enteric exocrine drainage by graft duodenojejunal anastomosis has dramatically reduced complications of urinary tract infection, urethritis, urethral stricture, and metabolic acidosis, and therefore the need to perform enteric conversion in up to 33% of cases (108–111).

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THE HISTORY OF ISLET TRANSPLANTATION BY PAUL E. LACY PHILADELPHIA, PENNSYLVANIA, U.S.A. DECEMBER 2ND, 2004 Paul E. Lacy passed away on February 15th, 2005. Approximately three months before he passed away, he gave his final, remarkable public lecture where most of the leaders in the islet transplant field were in attendance. The lecture was recorded, and a verbatim extract is provided below.

Paul E. Lacy giving his final lecture at the NDRI Meeting in Philadelphia, on December 2nd 2004, three weeks before his death.

I was asked whether I would be using PowerPoint for this lecture but they forgot that when I started we were still using the magic lantern and I haven’t progressed much beyond that. What I have planned is an overview of islet transplantation. I will not be going in to details, and I will not be mentioning some people not because their work wasn’t great because it was great, but I’m just trying to give a feeling of how it all came about. You can’t transplant islets unless you know how to isolate them. There was a man named Moskalewski in the mid-1960s who reported you could take the guinea pig pancreas, incubate it with collagenase, chop it and let it settle in a cylinder and in saline, and the islets would accumulate at the bottom. Well I tried it. Yes you could. You could get some islets from the guinea pig pancreas. So we tried it on the rat, which is the one we used for research on diabetes. With that we got a few but not very many. So we worked on the process for quite a while, and then came upon the idea that the islets are not connected to the pancreatic ducts. So we simply injected a salt solution into the pancreatic ducts, which blew

A Historical Perspective on Experimental and Clinical Islet Transplantation

up the acinar tissue, separated it from the islets, then we incubated with collagenase, chopped it, and there were hundreds of islets. You could see them under the dissecting microscope, you could hand-pick individual islets so they were absolutely pure. Now this made it possible to do fundamental studies on insulin formation, storage, and release in many laboratories, and it was wonderful. It did raise the curiosity question, and it was curiosity, what would happen if you transplanted these islets into diabetic rats in an inbred strain where you wouldn’t have to worry about rejection? We tried it. It worked. It reversed the diabetes. Young David Scharp came along at that time—he was a postdoctoral fellow training in surgery—and he looked at the different sites one used to transplant the islets. He found that the liver was the most ideal in terms of the smallest number of islets needed to reverse diabetes, and you put them in by injecting into the portal vein that drained into the liver. They were large enough to be trapped in the liver, small enough that they didn’t damage the liver. So that then opened up other questions as to what would happen with the complications of diabetes as they occur in the rodent. Konrad Federlin in Germany led the way in this area, and many others as well, and demonstrated that it would prevent these complications if you transplanted the islets ahead of time, and would reverse the early complications almost entirely in rats. So now it was a question of what you do now? Well, I thought it would be a perfect opportunity to see could you transplant islets in rats without having to give continuous immunosuppression. And at time there was a theory, a theory proposed by Snell, in which he suggested that maybe passenger leukocytes, antigen-presenting cells, carried along with the transplant, they were responsible for initiating rejection. So if you wanted to prevent rejection, you got rid of the passenger leukocytes. Pure theory, very little to support it. But it was one, as we had pure islets, we thought we could test. So, with Joe Davey, who was Head of Immunology at our institution at the time, we started. And we thought that with pure islets, that had no lymph nodes, no ducts, no nothing, they were absolutely pure, maybe we could just use those and that that would work. No it didn’t. When you transplanted between strains they were rejected. So if the theory was correct that meant there were still passenger leukocytes in the islets that we had to get rid of. So we tried different approaches, one of them was to inject silica, sand, into the donor animals, remove the islets hoping it would kill off macrophages. It helped a little, not very much. Then we read of Kevin Lafferty’s original publication, in which he stated he could take the mouse thyroid, incubate it in 95% oxygen for three weeks, then transplant it from one strain of mouse to another, no immunosuppression. He suggested he was killing the passenger leukocytes within the thyroid. We tried it immediately. Unfortunately, within three days the islets were dead. They could not tolerate the high oxygen concentration.

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I happened to see an article by Terasaki in California. He had been shipping lymphoid spleen cells across the country at room temperature. He found that when they went from California to New York that they would not stimulate an immune reaction. Well that was exactly what we wanted. So I said OK, let’s try it. So we took islets in a Petri dish in tissue culture, rat islets, and put them on the top of the desk and left them at room temperature. Looking at them each day, they didn’t disintegrate, they were there, they were intact, so we said, “what the heck, let’s transplant them, and give an injection of an antibody to lymphocytes at the time we transplanted.” We did. It worked. It completely prevented rejection without the need for any further immunosuppression. Now the immunologists were a little suspicious of that, thinking that maybe you mixed up the white rats! So we knew you couldn’t mix up rats and mice, so we tried it, rat islets into mice across the species barrier. Same procedure, it worked. No question. Now the question was what to do next, because that raised the possibility that if you ever transplanted islets into diabetic patients, that maybe you could use this method or a modification of the method to produce tolerance in the recipient, and that tolerance was specific for the tissue you put in. You could put in other tissue and it would be rejected. That would be ideal for the child. So David Scharp and I talked about what to do. We decided to go on and now try to isolate human islets. Many others had gone on with this, and used syringes and needles to disrupt the collagenase-digested pancreas and obtained some islets. Others used perfusion to disrupt the pancreas and obtained some islets, but it needed to be improved. So we used the old cow pancreas as the model for the human, because it is big and it has lots of collagen within it. But one of the problems with the collagenase digestion is that the collagen breaks down and you have “goo” and that traps the islets. So, my wife said, “Why don’t you use Velcro?” Well that was way long time ago. I didn’t know what Velcro was! So as you know it has little hooks, and we put the pancreas on the hooks and sure enough it held it in place and held back the “goo”, but it was a little traumatic and it held back the islets as well. So David and I talked and we thought about it. And then we thought you need a way of chopping the digested pancreas as rapidly as possible. Well, if you think about it, you chop normally with a pair of scissors as we did in the rat, but we can’t do that with the human pancreas or with the old cow pancreas. So it brought to mind my mother’s hand-operated meat grinder that was in the attic. We brought it in, changed the force plate on it, put the pancreas through, and sure enough there were islets. Now to separate those initially we used a flour sifter. You see we moved from the sewing room to the kitchen for these elegant tools! The wire that goes around on the flour sifter was a little hard on the islets, so David then developed a way to separate them on Ficoll gradients by centrifugation.

A Historical Perspective on Experimental and Clinical Islet Transplantation

Now that brought us to the point where we had to have human pancreases. Lee Ducat saved the day, as we could only get maybe one or two human pancreases locally. No way could we develop a method with just those few pancreases. She established a network called the National Diabetes Research Interchange (NDRI), obtained the pancreases, and we got underway. About this time also the elegant work of Ray Rajotte was done, in which he cryopreserved the islets in animals and in man. So that made it possible, even though we were getting few islets, that you could bring together different isolations, cryopreserve them, and give them all then to a patient, which we did. The patients had no C-peptide, no indication of insulin secretion prior to transplantation even with stimulation. After transplantation, they did not come off insulin, but they had C-peptide. There was no question there was function there, but it was not good enough. About that time a young man came to my laboratory to learn about islet transplantation, and that was Camillo Ricordi. The day after he arrived, we took him around to look at apartments, and on the way there he saw a jeep, “Is that a jeep?” “Yes, that’s a jeep.” “We don’t sell those in Italy.” He kept yapping about the jeep, and just couldn’t get it out of his system. I don’t think he heard a thing about the apartments. For the next three days he went from dealership to dealership, and finally he bought a jeep, and was delighted with it. It took a few more days till he got it all out of his system. Then he began working in the lab and it was elegant. He developed a way to gently digest a pancreas and get islets. We had an extra human pancreas that David and I knew we couldn’t get islets out with the meat grinder method we were using, so we gave it to him and he got islets. We dropped everything and turned to this method, which is now used in nearly all laboratories in the world. Now having many many islets, hundreds of thousands, it was possible to transplant into patients, and they would come off insulin. This was done in many laboratories. In coming off insulin, you followed them and a year later only about 15% were still off insulin. No one had any idea what was going on. But there was a young man in Edmonton, Canada, James Shapiro and his associates, had an idea what to do. They tried it. It worked. I am very very proud of him for what he’s done because he rejuvenated the area and the field of islet transplantation. Now, at this time, as he will tell you, 85% of the patients are still off insulin one year later, not 15%, which is marvelous. From my standpoint there are two problems that still remain. One of them is to be able to transplant the islets without the need for continuous immunosuppression. Approaches for this are to try to induce tolerance in the recipient as I told you about in the rat, another is encapsulation of the islets to protect them from the immune system, and remarkable advances have been made in both those areas.

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The second major problem from my standpoint is the source of islets, the continued supply of islets. How are you going to do this? You will hear about stem cells to make islets, making islets in vitro from duct cells and other cells. This is an approach that I think very well will work, and it will need more effort and more time to do it. Another approach is to use xenografts of islets, pig islets. Carl Groth was the first to transplant pig islets into diabetic patients. There is now lots of work being done in that area. Now, finally I have no doubt it will be possible to do all these things, and it will be possible to transplant islets into a diabetic child without continuous immunosuppression. For me this journey has been a delightful one. Of course there have been peaks, and of course there have been terrible valleys that have been deep and wide, but it has been a wonderful, wonderful journey. I feel so very privileged to have been a part of a therapeutic approach from the bench to the bedside of the patient. I also feel privileged in having worked with so many, many, wonderful people, and for having established friendships that I shall always cherish. Thank you.

Paul E. Lacy, M.D. (1924–2005)

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Williams P. Notes on diabetes treated with extract and by grafts of sheep’s pancreas. British Medical Journal 1894; 2:1303. Minkowski O. Weitere Mitteilungen u¨ber den diabetes mellitus nach extirpation des pankreas. Berl Klin Wochenschr 1892; 29:90. von Mering J, Minkowski O. Arch Exp Pathol Pharmakol 1889; 26:371. Pybus F. Notes on suprarenal and pancreatic grafting. Lancet 1924:550. Banting F. Extract from Banting’s Notebook 2am October 31st. Academy of Medicine notebook, Archives of Toronto University, Canada 1920 . Bliss M. The discovery of insulin. Toronto:McClelland and Stewart Limited, 1982. Robertson GS, Dennison AR, Johnson PR, London NJ. A review of pancreatic islet autotransplantation. Hepatogastroenterology 1998; 45(19):226. Bretzel RG, Hering BJ, Federlin KF. Islet cell transplantation in diabetes mellitus—from bench to bedside. Exp Clin Endocrinol Diabetes 1995; 103 (Suppl 2):143. Bensley RR. Studies on the pancreas of the guinea pig. Am J Anat 1911; 12:297. Hellerstro¨m C. A method for the microdissection of intact pancreatic islets of mammals. Acta Endocrinol 1964; 45:122. Moskalewski S. Isolation and culture of the islets of langerhans of the guinea pig. Gen Comp Endocrinol 1965; 5:342. Lacy P, Kostianovsky M. Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes 1967; 16:35. Lindall A, Steffes M, Sorenson R. Immunoassayable insulin content of subcellular fractions of rat islets. Endocrinology 1969; 85(2):218. Scharp DW, Kemp CB, Knight MJ, Ballinger WF, Lacy PE. The use of ficoll in the preparation of viable islets of langerhans from the rat pancreas. Transplantation 1973; 16(6):686. Younoszai R, Sorensen R, Lindall A. Homotransplantation of isolated pancreatic islets. Diabetes 1970; 19(suppl 1):406. Ballinger WF, Lacy PE. Transplantation of intact pancreatic islets in rats. Surgery 1972; 72(2):175. Reckard CR, Ziegler MM, Barker CF. Physiological and immunological consequences of transplanting isolated pancreatic islets. Surgery 1973; 74(1):91. Kemp C, Knight M, Scharp D, Ballinger W, Lacy P. Effect of transplantation site on the result of pancreatic islet isografts in diabetic rats. Diabetologia 1973; 9:486. Menger MD, Wolf B, Hobel R, Schorlemmer HU, Messmer K. Microvascular phenomena during pancreatic islet graft rejection. Langenbecks Arch Chir 1991; 376(4):214. Vajkoczy P, Menger MD, Simpson E, Messmer K. Angiogenesis and vascularization of murine pancreatic islet isografts. Transplantation 1995; 60 (2):123. Menger MD, Vajkoczy P, Beger C, Messmer K. Orientation of microvascular blood flow in pancreatic islet isografts. J Clin Invest 1994; 93(5):2280.

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22. Mirkovitch V, Campiche M. Successful intrasplenic autotransplantation of pancreatic tissue in totally pancreatectomized dogs. Transplantation 1976; 21: 265. 23. Warnock GL, Rajotte RV, Procyshyn AW. Normoglycemia after reflux of islet-containing pancreatic fragments into the splenic vascular bed in dogs. Diabetes 1983; 32(5):452. 24. Griffin SM, Alderson D, Farndon JR. Comparison of harvesting methods for islet transplantation. Br J Surg 1986; 73(9):712. 25. Gores PF, Najarian JS, Stephanian E, Lloveras JJ, Kelley SL, Sutherland DE. Transplantation of unpurified islets from single donors with 15- deoxyspergualin. Transplant Proc 1994; 26(2):574. 26. White SA, London NJ, Johnson PR, et al. The risks of total pancreatectomy and splenic islet autotransplantation. Cell Transplant 2000; 9(1):19. 27. Shapiro AM, Lakey JR, Rajotte RV, et al. Portal vein thrombosis after transplantation of partially purified pancreatic islets in a combined human liver/islet allograft. Transplantation 1995; 59(7):1060. 28. Walsh TJ, Eggleston JC, Cameron JL. Portal hypertension, hepatic infarction, and liver failure complicating pancreatic islet autotransplantation. Surgery 1982; 91(4):485. 29. Froberg MK, Leone JP, Jessurun J, Sutherland DE. Fatal disseminated intravascular coagulation after autologous islet transplantation. Hum Pathol 1997; 28(11):1295. 30. Mehigan DG, Bell WR, Zuidema GD, Eggleston JC, Cameron JL. Disseminated intravascular coagulation and portal hypertension following pancreatic islet autotransplantation. Ann Surg 1980; 191(3):287. 31. Horaguchi A, Merrell RC. Preparation of viable islet cells from dogs by a new method. Diabetes 1981; 30(5):455. 32. Noel J, Rabinovitch A, Olson L, Kyriakides G, Miller J, Mintz DH. A method for large-scale, high-yield isolation of canine pancreatic islets of Langerhans. Metabolism 1982; 31(2):184. 33. Gray DW, McShane P, Grant A, Morris PJ. A method for isolation of islets of Langerhans from the human pancreas. Diabetes 1984; 33(11):1055. 34. Rajotte RV, Warnock GL, Evans MG, Ellis D, Dawidson I. Isolation of viable islets of Langerhans from collagenase-perfused canine and human pancreata. Transplant Proc 1987; 19(1 Pt 2):918. 35. Warnock GL, Kneteman NM, Evans MG, Dabbs KD, Rajotte RV. Comparison of automated and manual methods for islet isolation. Can J Surg 1990; 33(5):368. 36. van Suylichem PT, Wolters GH, van Schilfgaarde R. Peri-insular presence of collagenase during islet isolation procedures. J Surg Res 1992; 53(5):502. 37. Ricordi C, Finke EH, Lacy PE. A method for the mass isolation of islets from the adult pig pancreas. Diabetes 1986; 35(6):649. 38. Gray DW, Warnock GL, Sutton R, Peters M, McShane P, Morris PJ. Successful autotransplantation of isolated islets of Langerhans in the cynomolgus monkey. Br J Surg 1986; 73(10):850. 39. Warnock GL, Cattral MS, Rajotte RV. Normoglycemia after implantation of purified islet cells in dogs. Can J Surg 1988; 31(6):421.

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40. Lakey JR, Warnock GL, Shapiro AM, et al. Intraductal collagenase delivery into the human pancreas using syringe loading or controlled perfusion. Cell Transplant 1999; 8(3):285. 41. Ricordi C, Lacy PE, Scharp DW. Automated islet isolation from human pancreas. Diabetes 1989; 38Suppl 1:140. 42. Ao Z, Lakey JR, Rajotte RV, Warnock GL. Collagenase digestion of canine pancreas by gentle automated dissociation in combination with ductal perfusion optimizes mass recovery of islets. Transplant Proc 1992; 24(6):2787. 43. Toomey P, Chadwick DR, Contractor H, Bell PR, James RF, London NJ. Porcine islet isolation: prospective comparison of automated and manual methods of pancreatic collagenase digestion. Br J Surg 1993; 80(2):240. 44. Ricordi C, Rastellini C. Automated method for pancreatic islet separation. In:Ricordi C, ed. Methods in Cell Transplantation. Austin, Tx:RG Landes, 1995:433. 45. Lake SP, Bassett PD, Larkins A, et al. Large-scale purification of human islets utilizing discontinuous albumin gradient on IBM 2991 cell separator. Diabetes 1989; 38 Suppl 1:143. 46. Lakey JR, Cavanagh TJ, Zieger MA. A prospective comparison of discontinuous EuroFicoll and EuroDextran gradients for islet purification. Cell Transplant 1998; 7(5):479. 47. Brandhorst H, Brandhorst D, Brendel MD, Hering BJ, Bretzel RG. Assessment of intracellular insulin content during all steps of human islet isolation procedure. Cell Transplant 1998; 7(5):489. 48. Gill JF, Chambers LL, Baurley JL, et al. Safety testing of Liberase, a purified enzyme blend for human islet isolation. Transplant Proc 1995; 27(6):3276. 49. Linetsky E, Selvaggi G, Bottino R, et al. Comparison of collagenase type P and Liberase during human islet isolation using the automated method. Transplant Proc 1995; 27(6):3264. 50. Linetsky E, Bottino R, Lehmann R, Alejandro R, Inverardi L, Ricordi C. Improved human islet isolation using a new enzyme blend, liberase. Diabetes 1997; 46(7):1120. 51. Lakey JR, Cavanagh TJ, Zieger MA, Wright M. Evaluation of a purified enzyme blend for the recovery and function of canine pancreatic islets. Cell Transplant 1998; 7(4):365. 52. Lakey JR, Warnock GL, Rajotte RV, et al. Variables in organ donors that affect the recovery of human islets of Langerhans. Transplantation 1996; 61 (7):1047. 53. Brendel M, Hering B, Schulz A, Bretzel R. International Islet Transplant Registry Report. University of Giessen, Germany , 1999:1. 54. Bretzel RG, Brandhorst D, Brandhorst H, et al. Improved survival of intraportal pancreatic islet cell allografts in patients with type-1 diabetes mellitus by refined peritransplant management. J Mol Med 1999; 77(1):140. 55. Hering B, Ricordi C. Islet transplantation for patients with Type 1 diabetes: results, research priorities, and reasons for optimism. Graft 1999; 2(1):12. 56. Najarian JS, Sutherland DE, Matas AJ, Steffes MW, Simmons RL, Goetz FC. Human islet transplantation: a preliminary report. Transplant Proc 1977; 9(1): 233.

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57. Largiader F, Kolb E, Binswanger U, Illig R. [Successful allotransplantation of an island of Langerhans]. Schweiz Med Wochenschr 1979; 109(45):1733. 58. Brendel MD, Hering BJ, Schultz AO, Bretzel RG. Islet Transplant Registry Newsletter No. 8, 1998. 59. Najarian JS, Sutherland DE, Matas AJ, Goetz FC. Human islet autotransplantation following pancreatectomy. Transplant Proc 1979; 11(1):336. 60. Pyzdrowski KL, Kendall DM, Halter JB, Nakhleh RE, Sutherland DE, Robertson RP. Preserved insulin secretion and insulin independence in recipients of islet autografts. N Engl J Med 1992; 327(4):220. 61. The international islet transplant registry report. Newsletter No. 7 1996; 6(1):1. 62. Robertson RP, Lanz KJ, Sutherland DE, Kendall DM. Prevention of diabetes for up to 13 years by autoislet transplantation after pancreatectomy for chronic pancreatitis. Diabetes 2001; 50(1):47. 63. Sutherland DE, Gores PF, Hering BJ, Wahoff D, McKeehen DA, Gruessner RW. Islet transplantation: an update. Diabetes Metab Rev 1996; 12(2):137. 64. Farney AC, Hering BJ, Nelson L, et al. No late failures of intraportal human islet autografts beyond 2 years. Transplant Proc 1998; 30(2):420. 65. Tzakis AG, Ricordi C, Alejandro R, et al. Pancreatic islet transplantation after upper abdominal exenteration and liver replacement. Lancet 1990; 336(8712):402. 66. Ricordi C, Tzakis AG, Carroll PB, et al. Human islet isolation and allotransplantation in 22 consecutive cases. Transplantation 1992; 53(2):407. 67. Boker A, Rothenberg L, Hernandez C, Kenyon NS, Ricordi C, Alejandro R. Human islet transplantation: update. World J Surg 2001; 25(4):481. 68. Hering B, Brendel M, Schultz A, Schultz B, Bretzel R. International Islet Transplant Registry. Newsletter 1996; 6(7):1. 69. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of longterm complications in insulin dependent diabetes mellitus. N Engl J Med 1993; 329:977. 70. Oberholtzer J, Benhamou P, Toso C, et al. Human islet transplantation network for the treatment of type 1 diabetes: first (1999-2000) data from the Swiss-French GRAGIL Consortium. Americal Journal of Transplantation 2001; 1(1):182. 71. Oberholzer J, Triponez F, Mage R, et al. Human islet transplantation: lessons from 13 autologous and 13 allogeneic transplantations. Transplantation 2000; 69(6):1115. 72. Maffi P, Bertuzzi F, Guiducci D, et al. Per and peri-operative management influences the clinical outcome of islet transplantation. Americal Journal of Transplantation 2001; 1(1 Suppl 1):181. 73. Sutherland DE, Goetz FC, Najarian JS. Living-related donor segmental pancreatectomy for transplantation. Transplant Proc 1980; 12(4 Suppl 2):19. 74. Gruessner RW, Sutherland DE, Drangstveit MB, Bland BJ, Gruessner AC. Pancreas transplants from living donors: short- and long-term outcome. Transplant Proc 2001; 33(1-2):819. 75. Tan M, Kandaswamy R, Sutherland DE, Gruessner RW. Laparoscopic donor distal pancreatectomy for living donor pancreas and pancreas-kidney transplantation. Am J Transplant 2005; 5(8):1966.

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76. Matsumoto S, Okitsu T, Iwanaga Y, et al. Follow-up study of the first successful living donor islet transplantation. Transplantation 2006; 82(12):1629. 77. Matsumoto S, Okitsu T, Iwanaga Y, et al. Insulin independence after livingdonor distal pancreatectomy and islet allotransplantation. Lancet 2005; 365 (9471):1642. 78. Turchin I, Tronko M, Komissarenko V. Experience of 1.5 thousand transplantations of b-cell cultures to patients with diabetes mellitus. Fifth International Congress on Pancreas and Islet Transplantation. Miami, Florida, 1995. 79. Hu YF, Cheng RL, Shao AH, et al. The influences of islet transplantation on metabolic abnormalities and diabetic complications. Horm Metab Res 1989; 21(4):198. 80. Hu YF, Gu ZF, Zhang HD, Ye RS. Fetal islet transplantation in China. Transplant Proc 1992; 24(5):1998. 81. Chastan P, Berjon JJ, Gomez H, Meunier JM. Treatment of an insulindependent diabetic by homograft of fetal pancreas removed before the tenth week of pregnancy: one-year follow-up. Transplant Proc 1980; 12(4 Suppl 2): 218. 82. Bojko N, Chooklin S, Perejaslov A. Results of clinical islet transplantation after allotransplantation of pancreatic islet cell cultures to diabetic patients. Acta Diabetologica 1997; 34:148(abstract). 83. Valente U, Ferro M, Barocci S, et al. Report of clinical cases of human fetal pancreas transplantation. Transplant Proc 1980; 12(4 Suppl 2):213. 84. Tuch BE, Sheil AG, Ng AB, Trent RJ, Turtle JR. Recovery of human fetal pancreas after one year of implantation in the diabetic patient. Transplantation 1988; 46(6):865. 85. Groth CG, Korsgren O, Tibell A, et al. Transplantation of porcine fetal pancreas to diabetic patients. Lancet 1994; 344(8934):1402. 86. Komissarenko VP, Turchin IS, Komissarenko IV, Efimov AS, Benikova EA. Transplantation of an islet cell culture of human and animal fetal pancreases as a treatment method in diabetes mellitus. Vrach Delo 1983(4):52. 87. Sundkvist G, Bergqvist A, Weibull H, et al. Islet cell antibody reactivity with human fetal pancreatic islets. Diabetes Res Clin Pract 1991; 14(1):1. 88. Djordjevic PB, Brkic S, Lalic NM, et al. Human fetal pancreatic islet transplantation in insulin-dependent diabetics:possibilities of early detection of transplant destruction. Glas Srp Akad Nauka [Med] 1994; 44:83. 89. Kelly WD, Lillehei RC, Merkel FK, Idezuki Y, Goetz FC. Allotransplantation of the pancreas and duodenum along with the kidney in diabetic nephropathy. Surgery 1967; 61(6):827. 90. Bartlett S. Pancreatic transplantation after thirty years: still room for improvement. J Am Coll Surg 1996; 183:408. 91. Sutherland D, Kendall D, Goetz F, Najarian J. Pancreas transplantation in humans. In: Flye MW, ed. Principles of organ transplantation. Philadelphia: W.B. Saunders Company, 1989. 92. Nghiem DD, Gonwa TA, Corry RJ. Metabolic effects of urinary diversion of exocrine secretions in pancreatic transplantation. Transplantation 1987; 43(1):70.

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93. Cook K, Sollinger HW, Warner T, Kamps D, Belzer FO. Pancreaticocystostomy: an alternative method for exocrine drainage of segmental pancreatic allografts. Transplantation 1983; 35(6):634. 94. Sollinger HW, Knechtle SJ, Reed A, et al. Experience with 100 consecutive simultaneous kidney-pancreas transplants with bladder drainage. Ann Surg 1991; 214(6):703. 95. American Diabetes Association. Position statement: pancreas transplantation for patients with diabetes mellitus. Diabetes Care 1992; 15:1668. 96. Ryan EA. Pancreas transplants: for whom? Lancet 1998; 351(9109):1072. 97. Stratta RJ. Vascularised pancreas transplantation. BMJ 1996; 313(7059):703. 98. Sutherland DE. Pancreas transplantation. J Diabetes Complications 2001; 15 (1):10. 99. Bartlett ST, Schweitzer EJ, Johnson LB, et al. Equivalent success of simultaneous pancreas kidney and solitary pancreas transplantation. A prospective trial of tacrolimus immunosuppression with percutaneous biopsy. Ann Surg 1996; 224(4):440. 100. Gruessner RW, Sutherland DE, Najarian JS, Dunn DL, Gruessner AC. Solitary pancreas transplantation for nonuremic patients with labile insulindependent diabetes mellitus. Transplantation 1997; 64(11):1572. 101. Sutherland DE, Gruessner RW, Dunn DL, et al. Lessons learned from more than 1,000 pancreas transplants at a single institution. Ann Surg 2001; 233(4):463. 102. Tyden G, Bolinder J, Solders G, Brattstrom C, Tibell A, Groth CG. Improved survival in patients with insulin-dependent diabetes mellitus and end-stage diabetic nephropathy 10 years after combined pancreas and kidney transplantation. Transplantation 1999; 67(5):645. 103. Kumar A, Newstead CG, Lodge JP, Davison AM. Combined kidney and pancreatic transplantation. Ideal for patients with uncomplicated type 1 diabetes and chronic renal failure. BMJ 1999; 318(7188):886. 104. Gross CR, Limwattananon C, Matthees BJ. Quality of life after pancreas transplantation: a review. Clin Transplant 1998; 12(4):351. 105. Matas AJ, McHugh L, Payne WD, et al. Long-term quality of life after kidney and simultaneous pancreas-kidney transplantation. Clin Transplant 1998; 12 (3):233. 106. Adang EM, Kootstra G, Engel GL, van Hooff JP, Merckelbach HL. Do retrospective and prospective quality of life assessments differ for pancreaskidney transplant recipients? Transpl Int 1998; 11(1):11. 107. Kendall DM, Rooney DP, Smets YF, Salazar Bolding L, Robertson RP. Pancreas transplantation restores epinephrine response and symptom recognition during hypoglycemia in patients with long-standing type I diabetes and autonomic neuropathy. Diabetes 1997; 46(2):249. 108. Kuo PC, Johnson LB, Schweitzer EJ, Bartlett ST. Simultaneous pancreas/ kidney transplantation—a comparison of enteric and bladder drainage of exocrine pancreatic secretions. Transplantation 1997; 63(2):238. 109. Newell KA, Bruce DS, Cronin DC, et al. Comparison of pancreas transplantation with portal venous and enteric exocrine drainage to the standard technique utilizing bladder drainage of exocrine secretions. Transplantation 1996; 62(9):1353.

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110. Sollinger HW, Sasaki TM, D’Alessandro AM, et al. Indications for enteric conversion after pancreas transplantation with bladder drainage. Surgery 1992; 112(4):842. 111. Sollinger HW, Ploeg RJ, Eckhoff DE, et al. Two hundred consecutive simultaneous pancreas-kidney transplants with bladder drainage. Surgery 1993; 114(4):736.

2 Hypoglycemia in Type 1 Diabetes: The Need for a New Approach Stephanie A. Amiel King’s College School of Medicine, King’s College, London, U.K.

INTRODUCTION When blood glucose concentrations fall too low to support normal brain function, confusion, abnormal behaviors, coma, and seizures may ensue. It is not surprising that hypoglycemia, low blood glucose, ranks with blindness and renal failure as a fear for patients with Type 1 diabetes (1) and is widely believed to be a major limitation to the ability to achieve targets for glycemic control (2). The major trial that demonstrated unequivocally the link between lower mean blood glucose concentrations and reduced risk of longterm vascular complications (The Diabetes Control and Complications Trial, or DCCT) showed a three-fold increase in hypoglycemic episodes severe enough to render the patient incapable of self-management in those randomly assigned to receive intensive diabetes therapy (3). The negative associations shown between risk of hypoglycemia and mean glycated hemoglobin (HbA1c, a measure of mean blood glucose concentrations over about 2 months) are compatible with the logical assumption that running blood glucose concentrations lower increases the risk of overshooting into frank hypoglycemia (4). In the DCCT, there was also a direct association between the use of intensive insulin therapy and the risk of severe hypoglycemia, irrespective of HbA1c achieved (Fig. 1) (5). Although more modern methods of diabetes management can achieve improved mean blood glucose concentrations without increasing the risk of severe hypoglycemia (6–8), expectations have also increased. Patients and professionals now expect to achieve the more rigorous glucose targets needed in the fight against long-term complications but, at the same time, are far less accepting 29

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Figure 1 The rate of severe hypoglycemia from the Diabetes Control and Complications Trial, plotted against HbA1c. Note that as HbA1c rises, the risk of severe hypoglycemia increases, but the risk is higher during intensified therapy for any given HbA1c. Source: From Ref. 3.

of the lifestyle restrictions imposed on people using insulin therapy than before. Despite major improvements in treatment regimens (6–9), the introduction of designer insulins with (slightly) more physiological action profiles (e.g., 10,11) and advances in home blood glucose monitoring (12,13), severe hypoglycemia remains a major problem in the appropriate management of insulin-deficient diabetes mellitus. Furthermore, avoiding intensified insulin therapy and not maintaining near-normoglycemia does not provide protection against severe hypoglycemia; all insulin deficient diabetic patients are at risk. DEFINITIONS OF HYPOGLYCEMIA Definitions of hypoglycemia remain controversial. Professional fear of hypoglycemia has led the American Diabetes Association recently to define hypoglycemia in diabetes treatment as any blood glucose less than 4 mmol/L, which means that healthy people often fall into this category (14)! It is true that the healthy body’s own mechanisms for maintaining blood glucose concentrations are activated as soon as blood glucose concentrations start to fall, with a reduction in endogenous insulin secretion and, at an arterialized plasma glucose of around 3.6 mmol/L, an increase in pancreatic glucagon secretion, together mediating an increase in endogenous glucose production primarily

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from the liver as a defense against a more profound glucose fall (15). A slightly greater hypoglycemia is required to trigger adrenergic and autonomic activation, leading to subjective awareness of a low blood glucose concentration. More profound hypoglycemia still triggers growth hormone and cortisol responses (15,16). The effects of the neuro-humoral responses are to increase endogenous glucose production, both directly and indirectly (by provision of additional substrate for gluconeogenesis), and to reduce glucose uptake by “inessential” peripheral tissues, such as muscle and fat, diverting available glucose to the brain and heart. In healthy individuals, these mechanisms are so efficient that severe hypoglycemia, with clinically significant cognitive dysfunction, does not occur, at least outside extremes of behavior, such as marathon running. In experimental studies, where the circulating glucose concentration is forced down, usually by insulin infusion, cognitive dysfunction is first detectable at an arterialized plasma glucose concentration of around 3 mmol/L (17). The initial cortical dysfunction will be manifest as slowed or less accurate performance of specific cortical function tasks, but has been interpreted as relevant to the sort of impairment that increases the risk of driving and other accidents. Dangerously impaired performance can be demonstrated in driving simulations at these sorts of glucose concentrations (18). An increasing range of cortical functions becomes impaired as plasma glucose declines further (19–21). Furthermore, although personality traits persist at the level of hypoglycemia used in ethical research studies (22), mood changes, including an increase in feelings of anger, have been demonstrated (23). Because of the probable relevance of slowed reaction times on formal testing to clinically important events, such as impaired intellectual performance and slowed reactions while undertaking potentially dangerous activities, a blood glucose of less than 3 mmol/L is often used to define clinically significant hypoglycemia in research studies of new therapies. For therapeutic purposes, it is probably useful to differentiate between the lower end of the target glucose range, which should be over 4 mmol/L (24), and that which constitutes hypoglycemia, requiring immediate intervention. A clinical definition for the latter may be set at <3.5 mmol/L, depending upon the circumstances at the time, the activity the person is performing, and the patient’s recent experience of confusional episodes. If, for research purposes, one needs a definition that includes hypoglycemia with potentially serious adverse outcomes, a concentration of <3 mmol/L is appropriate.

INSULIN SECRETION AND THE ABSENCE OF HYPOGLYCEMIA Insulin as a Controller of Normoglycemia Insulin is the cause of hypoglycemia. Insulin is normally secreted by b-cells within the islets of Langerhans scattered throughout the pancreas. In healthy individuals, the secretion of insulin is rigorously linked to glucose

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metabolism within the b-cell and therefore to the glucose supply to the b-cells; in other words, insulin secretion is controlled essentially by blood glucose concentrations (Fig. 2). This system only fails in exceptional circumstances. An abnormality in the molecular mechanisms for glucose metabolism in the b-cell can result in abnormal blood glucose control. For example, in the monogenic diabetes condition MODY-2, the glucokinase enzyme is abnormal (25,26). Glucose phosphorylation in the b-cell is slowed, so the release of insulin occurs at higher glucose concentrations than usual, resulting in mild, chronic hyperglycemia. A genetic mutation of the glucokinase gene that increases its activity has the reverse effect, causing hypoglycemia (27). Genetic mutations in the genes controlling the adenosine triphosphate (ATP)-sensitive potassium channel, normally closed by ATP generated from intracellular glucose metabolism, can result in either persistent under- or over- secretion of insulin, causing diabetes or hypoglycemia, according to the effect of the mutation on the function of the channel (28,29). Much more commonly, diabetes results from reduction in b-cell mass, either by autoimmune destruction, as in Type 1a diabetes, or by other mechanisms, as occurs in the evolution of Type 2 disease (30). Treatment comprises administration of exogenous insulin and/or drugs that increase insulin secretion by non-physiological means. At any time, if blood insulin

Glucose

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Figure 2 Schematic of a pancreatic b-cell showing, in simplified form, the link between blood glucose concentration and insulin secretion. Glucose is taken into the cell via a specialized glucose transporter. In the cell, the glucose is initially phosphorylated by glucokinase, and the phosphorylated glucose passes along the metabolic pathway, generating adenosine triphosphate (ATP) molecules in direct proportion to the amount of glucose molecules metabolized. ATP closes specific ATPsensitive inwardly rectifying potassium channels in the cell membrane, causing depolarization and opening of voltage-gated calcium channels. The rise in intracellular calcium is integral to the release of insulin molecules, which is in direct proportion to the rate of glucose metabolism. Abbreviation: SUR, sulfonylurea receptor.

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concentrations are sustained independent of the physiological b-cell regulatory mechanism—either by the use of insulin secretagogues, such as sulfonylureas and metiglinides, or by injection of exogenous insulin— hypoglycemia can ensue because of the loss of the responsive link between blood glucose concentration and circulating insulin concentrations. The importance of the normal controls of circulating insulin levels in the defense against hypoglycemia is great. Even in established diabetes, residual endogenous insulin secretion seems to protect against hypoglycemia. Absence of detectable C-peptide, a marker of complete loss of endogenous insulin, is a major association of increased frequency of severe hypoglycemia (4). Furthermore, failure of insulin clearance, e.g., because of insulin antibodies, hypothyroidism, or renal or liver disease, is accompanied by greater risk of hypoglycemia (31–33). The Anti-Insulins Glucagon is the primary anti-hypoglycemic hormone. The link between glucagon secretion and blood glucose is less clear than with insulin, as glucagon secretion can be stimulated by non-glucose metabolites. However, for the glucagon-secreting pancreatic α-cell to respond to hypoglycemia, it needs to detect an acute fall in insulin secretion from adjacent functional b-cells (34,35). Thus the insulin-deficient diabetic patient has lost the first two defenses against hypoglycemia, by virtue of artificial elevation of plasma insulin concentrations and an impaired ability to mount a glucagon response to a falling blood glucose concentration, secondary to the loss of cross-talk between the b and α-cells in the islets. In Type 1 diabetes, glucagon responses to acute hypoglycemia are commonly lost within the first five years (31). The remaining components of the counter-regulatory neuro-endocrine response to hypoglycemia are common responses to other stressors that threaten the survival of the individual. The molecular mechanisms by which they are engaged are not fully understood but central control is likely. Neurons that respond to changes in glucose supply by altering their firing rate have been demonstrated, and appear to use mechanisms for glucose sensing akin to those of the pancreatic b-cell (36,37). Networks of neurons specifically responding to hypoglycemia or biochemical glucoprivation have been documented in animal studies and their activation associated with hormonal responses and/or behavioral (feeding) responses (38). Hypothalamic neuronal metabolism plays a major role (39,40). In humans, neuro-imaging studies have shown specific areas of the brain to be activated in association with the different components of the stress response, including the hypothalamus (41), brain areas known to be involved in monitoring the body’s state, e.g., the anterior cingulate cortex, and those involved in motivational behaviors, including feeding, such as the prefrontal dopaminergic pathways through the ventral striatum (42–44).

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WHY DOES SEVERE HYPOGLYCEMIA OCCUR? In healthy individuals, the prevention of severe hypoglycemia is endogenous and automatic. In those with diabetes, loss of the endogenous ability to regulate insulin is universal. Failure of the glucagon response to hypoglycemia is detected within the first five years of Type 1 diabetes and has also been described in late Type 2 diabetes (31,45). Deficiencies in the rest of the neuro-endocrine response have been described in those with Type 1 diabetes of longer disease duration (31) and in patients with a history of severe hypoglycemia (46). Moreover, impaired neuro-endocrine response has been shown to predict severe hypoglycemia (47). Unable to mount the normal counter-regulatory response to hypoglycemia, the diabetic patient is dependent upon subjective awareness of the hypoglycemia at a time when sufficient cortical function is maintained for a logical and integrated response. The symptoms of hypoglycemia are thus critically important to the prevention of severe hypoglycemia in a diabetic individual. The symptoms of hypoglycemia are traditionally described as either “adrenergic/autonomic” (shaking, sweating, palpitations, feelings of anxiety, nausea), “neuroglycopenic” (difficulty in concentration or speaking, confusion, tiredness, or dizziness), or “non-specific” (all-important hunger, blurred vision, weakness). These logical classifications have been substantiated by statistical analyses of reported symptoms and, in factorial analysis, cluster with others in the same grouping (48). Symptoms of hypoglycemia can be very variable, although most individuals with diabetes describe individually specific, and often quite subtle, changes in their self-awareness that alert them to hypoglycemia. Studies of reported symptoms and signs in children show different clusterings from those of adults, whether based upon child or parental report, in which behavioral changes are more prominent (49). This is despite the increased vigor of the counter-regulatory hormone response to hypoglycemia described in children (50). In the elderly, at least in those with insulintreated Type 2 diabetes, neurological symptoms predominate (51). This may be associated with an age-related change in subjective awareness and cognitive responses to hypoglycemia (52). Of more importance is the change in subjective awareness of hypoglycemia that most Type 1 diabetic patients describe over years. Most patients describe a change in symptoms, with an increasing dependence on neuroglycopenic symptoms, and some develop near-total inability to detect early hypoglycemia. Estimates of such hypoglycemia unawareness are quoted at approximately 25% of Type 1 patients (53–55). Unawareness is more common in people with long duration diabetes (53) and it is probably no coincidence that frequency of severe hypoglycemia likewise increases with disease duration. Because of the importance of autonomic activation in the defenses against hypoglycemia, one might assume that diabetic (or other) autonomic neuropathy would be

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associated with increased risk of asymptomatic and/or severe hypoglycemia. In fact, this has been difficult to prove, with early studies showing risk of severe hypoglycemic episodes judged prospectively or retrospectively to be strongly associated with counter-regulatory failure to induced hypoglycemia rather than with evidence of classical (i.e., cardiovascular) autonomic neuropathy (46). More recent studies show only minor differences in the ability to restore symptomatic and hormonal responses to induced hypoglycemia by scrupulous hypoglycemia avoidance in those with and without clinically evident autonomic neuropathy (56). Gastroparesis as a specific manifestation of autonomic neuropathy that alters food absorption and is often associated with unstable diabetes control and anecdotally with problems of severe recurrent hypoglycemia, although this has not been well documented in the literature. However, increased risk of severe hypoglycemia does occur in other disorders of food absorption, such as celiac disease (58). More commonly, the defects of counter-regulation are independent of established autonomic neuropathy or comorbidities, such as adrenal failure or hypopituitarism. The main cause of hypoglycemia unawareness seems to be a failure of glucose sensing.

MECHANISMS OF HYPOGLYCEMIA UNAWARENESS The glucose concentration for initiating the hormonal responses to hypoglycemia is significantly influenced by prior glycemic experience. Hormonal responses and subjective awareness of hypoglycemia have been shown to occur at higher glucose concentrations in poorly controlled diabetic patients than in non-diabetic people (15). The glucose concentration for initiation of hormonal and symptom responses is significantly lower after “improved” glycemic control in both Type 1 (Fig. 3) and insulintreated Type 2 diabetes, associated with lower HbA1c (59,60). Similar reductions in the glucose concentrations for hormonal responses and subjective awareness have also been induced artificially in healthy people and in people with diabetes, by experimental exposure to plasma glucose concentrations of between 2.7 and 3.9 mmol/L for 2 hours, and although the defects induced by the most mild of these preconditions were minor, those induced by the lower concentrations were very similar to those seen in patients with clinical problems recognizing their hypoglycemia (61–64). These defects are reversible, at least in part, by avoidance of exposure to blood glucose levels of <3 mmol/L (56,65–67). It is therefore suggested that impaired generation of symptomatic and protective responses to hypoglycemia seen in Type 1 diabetes may result from sustained intermittent exposure to low blood glucose concentrations as a result of imperfect insulin replacement regimens. Cryer has called this hypoglycemia associated automomic failure, or HAAF.

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Figure 3 Impaired counter-regulatory responses to acute experimental hypoglycemia. The figure shows the development of delayed and diminished adrenaline responses to a stepped reduction in arterialized plasma glucose applied by clamp technique in a small group of Type 1 diabetic subjects, before and after application of intensified insulin therapy. Source: Reprinted from Ref. 59.

The glucose concentration at the onset of detectable cortical dysfunction is more inflexible, with some change detectable at around 3 mmol/L in groups of patients irrespective of overall glycemic control, prior glycemic experience, and glucose concentration at the onset of subjective awareness (17,19,60,68). Such a change in the hierarchy of response during intensive therapy may contribute to the risk of severe hypoglycemia, as the patient may be effectively asymptomatic until blood glucose has fallen so low that cognitive impairment has rendered them incapable of normal behavior or self-treatment. Patients categorized as being hypoglycemia unaware have a three- to six-fold increase in risk of severe hypoglycemia, using the above definitions (69,70). THE SIZE OF THE PROBLEM We know that patients, and often relatives of patients, fear hypoglycemia as much as any other complication of diabetes, with the difference that the hypoglycemia is directly induced by medication (1). Few tools exist for measuring individual hypoglycemia experience in clinical practice. A recent attempt to define the severity of hypoglycemia in daily life has come from the Edmonton group in which a numerical value can be calculated for the “hypoglycemia burden” of an individual patient, based on frequency of episodes

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and the degree of disability and disruption caused by each (71). For the score to be calculated, patients recorded home blood test results every day for four weeks. For each hypoglycemic event (defined as a blood glucose test result of <3 mmol/ L), symptoms experienced, whether or not the episode was self-recognized, and what treatment was used was noted and points awarded according to the severity of the episode. The score was multiplied by 12 to give an annual score and additional points were included for recalled severe hypoglycemic episodes during the previous 12 months. Symptomatic episodes that were self-treated did not contribute to the score. This system for quantifying the burden of hypoglycemia for an individual patient is an important step towards the recognition of problematic hypoglycemia by clinicians involved in diabetes management and a useful tool for future studies of hypoglycemia. Meanwhile, quoted estimates of rates of severe hypoglycemia are greatly influenced by how it is defined and what denominators are used. In some studies, hypoglycemia rates may be high because many patients are experiencing a few episodes each or because a few patients are experiencing a very large number of hypoglycemic episodes. Clinically, both are important, but in very different ways. In prospective epidemiological studies, rates of severe hypoglycemia where the definition has been “hypoglycemia requiring intervention from another person” have been quoted ranging from 1.3 episodes per patient per year, affecting one third of all with Type 1 diabetes (72–74). In the DCCT, rates for patients on conventional treatment were 1.87 per patient year, increasing to 6.12 in the intensively treated group (3,5). Older studies defining severe hypoglycemia as that involving coma and/or the use of parenteral therapy suggested figures of 0.37 on conventional therapy, and 1.1 on intensified therapy (4). The potential for modern intensified therapy regimens to lower severe hypoglycemia rates, albeit from initially higher rates [e.g., 2.8 to 1.7 (74)], has been demonstrated. On average, a person with established Type 1 diabetes can expect to experience one severe hypoglycemic episode a year. Figures for early Type 2 diabetes are 100-fold lower [0.1-2.3 episodes per hundred patient years in the U.K. Prospective Diabetes Study (UKPDS) (75)].

ASSESSING INDIVIDUAL RISK Just as there is no universal tool for assessing the severity of an individual’s problem with hypoglycemia, there is no clear strategy for assessing an individual patient’s risk. Hypoglycemia unawareness is clearly an important factor, and at least two questionnaires have been published for defining an individual’s awareness status (69,70), both validated in terms of predicting increased severe hypoglycemia risk. Neither are in routine clinical use, and there are no accepted routine methods for identifying problematic hypoglycemia clinically.

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Apart from awareness status, we know that, in Type 1 diabetes, risk is increased by disease duration. For short duration disease, this has to do with the loss of all endogenous insulin secretion—C-peptide negativity is commonly associated with higher risk of severe hypoglycemia and residual C-peptide is considered protective (4). It may be that residual C-peptide is the main reason insulin-treated Type 2 patients have less problems with severe hypoglycemia than Type 1 patients, especially where insulin has been started relatively early in the course of the disease (75). However, in a population-based study where insulin will probably mostly have been started late in the disease progression, in keeping with clinical practice at the time, the call-out rate for emergency services because of severe hypoglycemia was not much lower in the insulin-treated Type 2 population than for those with Type 1 diabetes (76). C-peptide may provide protection simply because it indicates a residual endogenous insulin supply that can be turned off as blood glucose concentrations fall, allowing a relative reduction in the portal insulin concentrations seen by the liver. It is also likely that the effect of residual b-cell function on α-cell function in hypoglycemia is relevant. As yet, we do not have sufficient data to use C-peptide concentrations as a predictor of individual patient risk of severe hypoglycemia, and, indeed, most people with Type 1 diabetes of more than 5 years’ duration will be irretrievably C-peptide negative (31). For an individual, the main predictor of risk of severe hypoglycemia is the occurrence of such episodes in the past. This is separate from the documented increase in risk for a second severe hypoglycemia to occur within 24 hours of an initial episode, which may itself be related to acute hypoglycemia-induced counter-regulatory deficit making the second episode less symptomatic and/or a continuation of the cause of the initial hypoglycemia, such as a change in current insulin sensitivity (77). There is strong epidemiological evidence that people with a history of severe hypoglycemia, not necessarily in the immediate past, are at higher risk for subsequent episodes, presumably because of some unidentified personal characteristics of their endogenous and behavioral responses to any hypoglycemia and the likely co-existence of counter-regulatory impairment (4). However, again there is no easy definition of the amount of severe hypoglycemia required to describe an individual at high risk. Based on the proven increased risk of severe hypoglycemia in patients with unawareness, and on the evidence linking exposure to or avoidance of blood glucose concentrations <3 mmol/L to deficiency or restoration of protective hypoglycemia awareness, respectively, one should assume that any patient whose home blood glucose monitoring records show frequent values of <3 mmol/L is at high risk of a severe episode, especially if those episodes are asymptomatic. However, clinical experience suggests a small number of patients tolerate such glycemic experience without any severe hypoglycemia. A clinician would not be advised to make assumptions about an individual

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patient and clearly we urgently need to develop more robust ways of assessing individual risk. In the era of the genome there has naturally been interest in identifying genetic predispositions to more or less severe hypoglycemia. There have been controversial suggestions that the ACE genotype may be very influential. One group found that the DD genotype predicted a history of severe hypoglycemia (78) and later showed the D allele and high ACE activity to be associated with risk of patients defined by questionnaire responses as hypoglycemia unaware (79). The genotype was the major predictor of risk of severe hypoglycemia and may be associated with an increased tendency to cognitive impairment during any one episode of hypoglycemia (80). This has been controversial, because factors repeatedly associated with severe hypoglycemia risk in other populations (low HbA1c, negative C-peptide), failed to have any demonstrable influence in these Danish studies. A recent study from another group, published only in abstract, could not confirm their findings (81). ACE-inhibitors do not affect symptomatic or hormonal responses to induced hypoglycemia (82), and the data linking risk of hypoglycemia in diabetic patients to use of ACEinhibitors are not conclusive. If anything, they suggest increased risk with some ACE-inhibitors in conjunction with some hypoglycemic therapies, but remain limited to database investigations only (83,84). The search for a molecular risk identifier continues.

CURRENT STRATEGIES FOR RESTORING PROTECTION AGAINST SEVERE HYPOGLYCEMIA A patient presenting with problematic hypoglycemia, by which this author would include unexplained episodes of severe hypoglycemia but also episodes of hypoglycemia that cause social or professional difficulties even where self-treatment can be achieved, needs expert assessment. Predisposing factors for hypoglycemia include: conventional deficits of anti-insulin hormones, such as Addison’s disease or growth hormone deficiency; problems of insulin clearance such as hepatic and renal impairment hypothyroidism (as thyroxine enhances hepatic insulin clearance); problems of food absorption, such as co-incident malabsorptive disorders; and behavioral issues, such as failure to adjust insulin replacement adequately for exercise and alcohol ingestion, or even the use of recreational drugs. Some prescribed drugs increase the risk of hypoglycemia in insulin-treated patients, although this has rarely been seen as a clinical problem. Some of the predisposing conditions are more prevalent in Type 1 diabetes. These include autoimmune adrenal or thyroid failure, celiac disease, and gastroparesis. Screening for these is an important part of the assessment of the patient with problematic hypoglycemia (Table 1).

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Table 1

Amiel Biochemical Associations of Increased Hypoglycemia Risk

Diabetes related Complete insulin deficiency (C-peptide negativity) Use of some forms of intensified insulin therapy Lower HbA1c Increasing diabetes duration Other endocrine diseases Hypothyroidism Hypoadrenalism Growth hormone deficiency Hypopituitarism Reduced insulin clearance Hypothyroidism Renal failure Liver failure Disorders of food absorption Any cause of malabsorption or delayed absorption, especially: Celiac disease Gastroparesis Drugs and exercise Alcohol Unaccustomed exercise Other drugs, including recreational

Once comorbidities have been excluded (or treated), the physician needs to consider how best to avoid the perpetuation of hypoglycemia unawareness by hypoglycemia avoidance. The most common problem is inappropriate expectations of insulin replacement therapy. The Type 1 diabetic patient with problematic hypoglycemia is extremely likely to be entirely insulin deficient, and insulin replacement needs to be applied with care. The diabetologist needs always to keep in mind the pharmacodynamics of the insulin regimen the patient is using and be able to advise the appropriate behaviors needed to make the regimen safe. Good control with twice-daily mixed insulins, for example, is very hard to achieve unless the patient has a reproducible eating pattern, with predictable meals and between meal snacks, to which he/she is happy to conform (85). Even so, the use of conventional intermediate acting insulins given before the evening meal, as in a twice-daily mixed insulin regimen, has been clearly demonstrated to cause more hypoglycemia at night than using the same insulin preparation, albeit usually in a lower dose, at bedtime (86). Use of more flexible insulin regimens, in which background insulin replacement is completely separate from meal replacement (in contrast to older regimens where the tail of the meal insulin doses are used to provide a significant amount of background control), can provide improved glycemic control

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with a flexible lifestyle, but the patients need appropriate and accessible education to use these regimens successfully (6–8,87,88). While such programs have been repeatedly shown to be able to achieve improved average blood glucose without increase (or even with decrease) in severe hypoglycemia, it should be recognized that the ability to provide protection for patients who currently have problematic hypoglycemia has not been fully established (88). In research trials, insulin analogs have been shown to allow similar degrees of diabetic control with reduced frequency of hypoglycemia, especially at night, but in practice need to be used with the same care and understanding as any other insulin regimen (89–92). This is even more true of insulin infusion therapy (insulin pump therapy), which has a well-established ability to reduce the frequency of severe hypoglycemia (as illustrated in Fig. 4), assumed to be related at least in part to more physiological overnight insulin replacement (93–95). However, none of these strategies are completely successful and all patients on insulin therapy remain at risk.

PROBLEMS OF REVERSIBILITY

Events per 100 patient years

The demonstration that avoidance of blood glucose concentrations <3 mmol/L restores at least some of the defenses against hypoglycemia, particularly subjective awareness (64–67), might suggest that the problems of hypoglycemia unawareness and severe hypoglycemia are practically solved. Clinically this is very far from true. The most successful trials of restoration of hypoglycemia awareness have been in small numbers of patients with extreme input from the health professionals running the trials and also from

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Figure 4 The effect of insulin pump therapy on severe hypoglycemia rates in an experienced clinic. Source: From Ref. 77.

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the patients themselves, who, by virtue of becoming involved in trials, have already demonstrated a high level of interest and motivation. Multiple home blood glucose tests as well as multiple daily insulin administrations (or insulin pump therapy) are the rule. Clinical practice is full of examples of patients who either continue to experience hypoglycemia problems or, having achieved relief for a period of time, find their problems recur. Problems in achieving tight control without hypoglycemia include the difficulties inherent in adjusting insulin around frequent and often unpredictable changes in insulin sensitivity. Variations in degrees (and nature) of exercise and stress are recognized by patients, and have been shown in research to cause alterations in insulin needs, which are not always easily anticipated (96,97). Home blood glucose monitoring, even with modern devices, is painful and inconvenient and at best extremely intermittent, failing to offer patients enough information to observe the direction of glucose change. Training patients to a high level of understanding of both their insulin and of the physiology of hypoglycemia can be very helpful in restoring subjective awareness of hypoglycemia and reduces frequency of severe hypoglycemia by up to 50% but with no improvement in glycemic control, which in the quoted study was, at best, moderate (98). Nevertheless, restoring awareness is a very useful strategy, and the principles of the Blood Glucose Awareness Training education devised by Cox and colleagues have been adapted into many educational programs. Unless sponsored by psychologists, it is rarely offered in toto, and the benefits of piecemeal application are unproven. Other educational initiatives of proven efficacy that train patients to use insulin flexibly, to which allusion has already been made, have been shown to reduce average blood glucose concentrations with lowered HbA1c and no increase or even a reduction in severe hypoglycemia rate. None to date, however, have been proven to reduce severe hypoglycemia long-term in individuals who have already developed a problem. The psychological factors that contribute to hypoglycemia risk are poorly understood. Some patients with problematic hypoglycemia develop a fear of hypoglycemia that can manifest itself with varying degrees of anxiety symptoms, sometimes reaching the severity of a panic disorder or a phobia. For research purposes, such fear can be quantitated using the Fear of Hypoglycemia Scale, which can be used to measure the association between Fear of Hypoglycemia and diabetes outcomes (99). Polonsky found that fear of hypoglycemia was directly related to past experiences and associated with difficulty in differentiating symptoms of hypoglycemia from those of anxiety (100). Although much is known about restoring awareness of hypoglycemia by hypoglycemia avoidance, little is known about how best to achieve and sustain such avoidance over time. Some patients with recurrent severe hypoglycemia avoid situations that they perceive will provoke hypoglycemia, such as exercise, or

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overcompensate with deliberate hyperglycemia in situations that cannot be avoided, such as business meetings, to avoid potential humiliation and loss of control. This can result in high glycated hemoglobin concentrations predictive of high risk of long term vascular complications, but does not necessarily provide complete protection from severe hypoglycemia. Other patients manage their insulin and diet based on subjective appraisal of perceived symptoms rather than checking their blood glucoses. For other patients, there is enormous difficulty in changing behaviors the clinician thinks are contributing to risk, such as omitting food or taking excessive corrective doses of insulin if the blood glucose concentration is even slightly above the non-diabetic range. These patients may have near normal measures of average glycemia, but recurrent episodes of hypoglycemia associated with impaired cortical function. The learned fear of hyperglycemia-induced vascular complications and a dislike of between-meal snacking, extremely common among diabetic patients, may be associated with an unwillingness to experiment with or persist with advised dose reductions and lifestyle changes. A previous attribution of increased hypoglycemia risk with a specific insulin may make patients very reluctant to experiment with newer insulins or insulin regimens, even where these can be shown to offer potential specific advantage. The reluctance to snack between meals is explained by many patients as fear of weight gain. Although this is illogical, as the caloric consumption associated with hypoglycemia treatment is usually much greater than might be associated with controlled planned snacking, it can be a deeply held conviction. There are also issues around the use of food as a medication, rather than as something to be consumed at will. Whatever the reasons, many patients with problematic hypoglycemia do not snack routinely, even when using insulin regimens that logically require it. A study of patients presenting to emergency services with severe hypoglycemia showed that patients attributed 52% of episodes to inadequate food intake (101). Many patients with problematic hypoglycemia have long duration diabetes and it may be that reluctance for longstanding behavioral change is inevitable, as people’s practice will have developed over a lifetime. A surprising number of patients with hypoglycemia unawareness and episodes of severe hypoglycemia do not perceive their own hypoglycemia as a problem. To some extent, this may reflect the lack of subjective awareness of individual episodes, as people only perceive problems through experienced symptoms (102). Prevention of disastrous sequelae is by the intervention of others—it is only when talking to the families of the afflicted person that the magnitude of the problem becomes clear. Recurrent severe hypoglycemia can create enormous stresses within families. It follows that no assessment of a patient’s problems with hypoglycemia is complete without discussion with the patient’s family.

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The two coping strategies—minimization of risk of hypoglycemia by running high glucose concentrations, and failure to alter behaviors considered by medical advisors as contributing to the problem—result in very different clinical problems but may be different manifestations of the same underlying response to hypoglycemia experience. In psychology, avoidance and resistance can be interpreted as related responses, and, although the two scenarios represent different reactions to the problem, they are not mutually exclusive. Very few of these issues are formally documented or investigated, but it is clear from clinical experience that treating problematic hypoglycemia is not a simple matter of rectifying poor treatment regimens.

NEW TOOLS AND DEVICES From the foregoing, it follows that we need new strategies for diminishing risk of problematic hypoglycemia. Increasing use of home glucose monitoring and intensive education around the need for frequent dose calculations and adjustments may be helpful but require the physician to be aware of the nature of the problem. Hypoglycemia unawareness is not just found in patients. More recently, the availability of continuous glucose monitoring has been helpful at least in identifying problem times. These devices, which measure interstitial fluid glucose concentrations calibrated against conventional home capillary blood measurements, integrate a glucose concentration estimate every 5 minutes (13). Until very recently, the data have been downloaded after a period of use of a few days and decisions made then about the need for dose adjustments, based on perceived patterns in the data. These tools, again in trial reports, have been useful in improving glycemic control. The newest versions of these tools are able to give real-time data. It is too early to judge the impact of such devices on diabetes control in the clinical setting. There are concerns that people will have more information about their current blood glucose than they have the insulin regimens to deal with them. However, early anecdotal reports suggest that, after a short period of time, patients become accustomed to using the information on the trends in their glucose concentrations at least to be able to diminish hypoglycemia. The goal of glucose monitoring technology will ultimately be to link the glucose sensor to an insulin delivery device. Early reports of continuous intravenous glucose monitors linked to insulin delivery devices show some success but also difficulties based on the reactive nature of the insulin delivery command—as with standard in-patient sliding scales with intravenous insulin, the blood glucose changes and the change in insulin delivery follows. Electronic devices lack the cephalic phase of the physiological insulin response to eating. It is likely that these early difficulties will be

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susceptible to improved command algorithms. As yet there are no full publications of these methods in patients.

A NEW APPROACH It is axiomatic that what is needed for the insulin-dependent diabetic patient is a system for glucose-responsive insulin delivery. It is hoped that the mechanical solutions discussed above may in time be able to achieve this. But there are real problems still to be overcome. For current glucose sensor electrodes, which are inserted into the subcutaneous tissue, the most widely available on-line glucose monitoring devices require regular calibration against capillary blood glucose, have an effective life span of approximately 5 days and currently cost, in the United Kingdom, £45 each time. Not every day’s data are reliable—currently one has the option of deciding not to make a decision based on a set of recordings, and the user instructions suggest that blood glucose concentrations should be checked by conventional finger prick capillary blood glucose measurements before a treatment is performed (user instructions, GlucoWatch and Guardian on-line glucose monitors). There is a lag between changes in blood glucose and measured interstitial glucose, which may need to be factored in (103). If an on-line glucose sensing device is driving an insulin delivery device directly, the realization that an error has occurred may depend upon a significant deviation in blood glucose with possible clinical consequences. Much greater reliability and durability will be needed for closed loop systems to leave the research setting—accompanied ideally by further miniaturization, simplification of use, and reduction in cost. We need a therapy that can be available to all. The alternative approach is obviously to consider replacing the natural glucose responsive insulin delivery system of the pancreatic islet. The central principle of this system has been alluded to above: in a b-cell, insulin secretory rate is driven directly by the rate of intracellular glucose metabolism, itself controlled by the availability of glucose in the perfusing blood. In Type 1 diabetes, or late Type 2, the disease is caused by the loss of functioning b-cells. It is entirely logical to consider that the appropriate treatment for each disease is replacement of the missing cells.

TREATMENT OF DIABETES BY TRANSPLANTATION Diabetes is currently successfully treated by transplantation. Although whole organ pancreas transplant is most commonly still used in combination with renal replacement, the American Diabetes Association has reiterated its advice that whole organ pancreas transplant should be considered an option for the patient with severe recurrent hypoglycemia (104). Despite vast

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improvements in surgical technique and immunosuppression, whole organ pancreas transplant remains a major undertaking for the recipient, with a full success rate in the region of 85%. A successful whole organ transplant can render a diabetic patient insulin independent indefinitely, although as with any transplant there remains the risk of rejection and other complications of immunosuppression, as well as, for the Type 1 diabetic recipient, the risk of recurrence of the diabetic process in the new organ. It is of interest that successful whole organ pancreas transplantation does not completely obviate the occurrence of hypoglycemia (105). There have been several hypotheses to explain this, including systemic (as opposed to portal) insulin delivery in many of the procedures, and disruption of the normal innervation to the organ. Severe hypoglycemia in an insulinindependent organ recipient has not, however, been recorded. The islets constitute perhaps 2% of the whole pancreas mass. There is no abnormality of the exocrine pancreas in people with true Type 1 diabetes and so there is enormous redundancy in a whole pancreas transplant. The enzyme and alkaline secretions of the pancreas, unneeded by the patient, contribute most to the surgical risk of the procedure. Replacement of pancreatic islets needs to be developed as the logical next step. Donor islet transplantation is already a reality in some parts of the world (106). There are, however, major challenges to its widespread use, including the problems of donor supply, the loss of islets currently incurred both during isolation and after infusion into a recipient, and the lack of a sufficiently targeted immunosuppression regimen that will prevent both diabetes recurrence and transplant rejection.

ISLET TRANSPLANTATION AND HYPOGLYCEMIA Islet transplantation programs have multiplied substantially since the publication of the first series of 7 Type 1 diabetic patients rendered insulin independent for at least one year in June 2000 (107). Their success rates are variable and some have started and stopped again (108). Even the most successful programs have major limitations—they cannot accept patients with insulin resistance or who are over a certain body weight, as the chances of achieving insulin independence are dependent upon achieving an adequate islet mass in the recipient. Islets are lost in isolation and after administration, and so only people with small insulin requirements are eligible for the programs. Furthermore, the toxicity and expense of immunosuppression makes islet transplantation an option only for a patient very seriously at risk on current optimized therapy. It is inevitable but problematic that success of an islet program is currently judged solely on the ability to achieve insulin independence. While there is no doubt that that insulin independence must be the ultimate goal of

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the ongoing research, the present successful programs do have a very significant effect on hypoglycemia risk (72). In Edmonton, the selection criteria identify a cohort of patients with abnormally high hypoglycemia burden scores, compared to a standard Type 1 clinic population. That burden score falls to zero after transplantation (Fig. 5). Our own much more limited experience reflects this—even partial success in terms of insulin independence produces near total protection against severe hypoglycemia. The success of such programs strongly reinforces the belief that the ultimate solution for diabetes patients is replacement of the cells that make insulin directly in response to glucose availability within the body. Whether islets from cadaveric donors, cultured islets, or cells engineered to function as islets offer the best chance of success remains a question for the future.

CONCLUSIONS The discovery of insulin saved the lives of people with Type 1 diabetes. It is sobering to reflect that had insulin not been discovered, transplantation therapy for Type 1 diabetes may have advanced much more quickly and we would not now be discussing whether the best therapy for a failed organ system was not the replacement of that organ! The medical establishment needs to recognize the deficiencies of even our best insulin replacement regimens. It is essential to develop systems of

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Figure 5 The reduction in hypoglycemia burden after islet cell transplantation. Abbreviations: pre-Tx, patients selected for islet transplantation before procedure; post-Tx, patients after islet transplantation; T1DM, patients with Type 1 diabetes mellitus attending clinic. Source: Reproduced from Ref. 57.

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insulin delivery that are entirely driven by insulin need. There are different avenues that must be explored, including the development of mechanical systems. However, the extreme efficiency of the biological system for tying insulin secretion to insulin need is going to be difficult to beat, and cell therapy for diabetes must remain the best hope for the establishment of true normoglycemia for the future. Current programs of islet transplantation are not sufficiently effective to be considered a cure for diabetes. There are too many failures and too many complications for that. It is cause for celebration that we do at least have a system of both organ and islet transplantation that can provide relief for Type 1 diabetic patients whose lives are being destroyed by recurrent severe hypoglycemia. These patients are the pioneers around which we should be developing cell therapy for diabetes as a genuine cure.

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41. Cranston IC, Reed LJ, Marsden PK, Amiel SA. Changes in regional brain 18 F-fluorodeoxyglucose uptake at hypoglycemia in Type 1 diabetic men associated with hypoglycemia unawareness and counterregulatory failure. Diabetes 2001; 50:2329–36. 42. Teves D, Videen TO, Cryer PE, Powers WJ. Activation of human medial prefrontal cortex during autonomic responses to hypoglycemia. Proc Natl Acad Sci 2004; 101:6217–21. 43. Bingham EM, Dunn JT, Smith D, et al. Differential changes in brain glucose metabolism during hypoglycaemia accompany loss of hypoglycaemia awareness in men with type 1 diabetes mellitus. An [11C]-3-O-methyl-D-glucose PET study. Diabetologia 2005; 48:2080–9. 44. Dunn J, Marsden PK, Cranston IC, Reed LJ, Amiel SA. Activation of ventral striatum by hypoglycemia, with loss of amygdala pesponses in hypoglycemia unawareness. Diabetes 2006; 55(suppl 1):119. 45. Holstein A, Hammer C, Plaschke A, et al. Hormonal counterregulation during severe hypoglycaemia under everyday conditions in patients with type 1 and insulin-treated type 2 diabetes mellitus. Exp Clin Endocrinol Diabetes 2004; 112:429–34. 46. Ryder RE, Owens DR, Hayes TM, Ghatei MA, Bloom SR. Unawareness of hypoglycaemia and inadequate hypoglycaemic counterregulation: no causal relation with diabetic autonomic neuropathy. BMJ 1990; 301: 783–7. 47. White NH, Skor DA, Cryer PE, Levandoski LA, Beir DM, Santiago JV. Identification of type I diabetic patients at increased risk for hypoglycemia during intensive therapy. N Engl J Med 1983; 308:485–91. 48. Deary IJ, Hepburn DA, MecLeod KM, Frier BM. Partitioning the symptoms of hypoglycaemia using multi-sample confirmatory factor analysis. Diabetologia 1993; 36:771–7. 49. McCrimmon RJ, Gold AE, Deary IJ, Kelnar CJ, Frier BM. Symptoms of hypoglycemia in children with IDDM. Diabet Care 1995; 18:858–61. 50. Amiel SA, Simonson DC, Sherwin RS, Lauritano AA, Tamborlane WV. Exaggerated epinephrine responses to hypoglycemia in normal and insulindependent diabetic children. J Pediat 1987; 110: 832–7. 51. Jaap AJ, Jones GC, McCrimmon RJ, Deary IJ, Frier BM. Perceived symptoms of hypoglycaemia in elderly type 2 diabetic patients treated with insulin. Diabet Med 1998; 15:398–401. 52. Matyka K, Evans M, Lomas J, et al. Altered hierarchy of protective responses against severe hypoglycemia in normal aging in healthy men. Diabet Care 1997; 20: 135–41. 53. Hepburn DA, Patrick AW, Eadington DW, Ewing DJ, Frier BM. Unawareness of hypoglycaemia in insulin-treated diabetic patients: prevalence and relationship to autonomic neuropathy. Diabet Med 1990; 7:711–7. 54. Bolli G.B. Hypoglycaemia unawareness. Diabet Metabol 1997; 23:29–35. 55. Lagar I, Atvall S, Bloome G, Smith U. Altered recognition of hypoglycaemic symptoms in Type 1 diabetes during intensified control with continuous subcutaneous insulin infusion. Diabet Med 1986; 3:322–5.

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56. Fanelli C, Pampanelli S, Lalli C, et al. Long-term intensive therapy of IDDM patients with clinically overt autonomic neuropathy: effects on hypoglycemia awareness and counterregulation. Diabetes 1997; 46:1172–81. 57. Kong MF, Horowitz M. Diabetic gastroparesis. Diabet Med 2005; Suppl 4: 13–8. 58. Mohn A, Cerruto M, Lafusco D, et al. Celiac disease in children and adolescents with type I diabetes: importance of hypoglycemia. J Pediatr Gastroenterol Nutr 2001; 32:37–40. 59. Amiel SA, Sherwin RS, Simonson DC, Tamborlane WV. Effect of intensive insulin therapy on glycemic thresholds for counterregulatory hormone release. Diabetes 1988; 37: 901–7. 60. Korzon-Burakowska A, Hopkins D, Matyka K, et al. Effects of glycemic control on protective responses against hypoglycemia in type 2 diabetes mellitus. Diabet Care 1998; 21:282–91. 61. Davis SN, Shavers C, Mosqueda-Garcia R, Costa F. Effects of differing antecedent hypoglycemia on subsequent counterregulation in normal humans Diabetes 1997; 46:1328–35. 62. Heller S, Cryer PE. Reduced neuroendocrine and symptomatic responses to subsequent hypoglycemia after 1 episode of hypoglycemia in nondiabetic humans. Diabetes 1991; 40:223–6. 63. George E, Harris N, Bedford C, Macdonald IA, Hardisty CA, Heller SR. Prolonged but partial impairment of the hypoglycaemic physiological response following short-term hypoglycaemia in normal subjects. Diabetologia 1995; 38:1183–90. 64. Fanelli C, Pampanelli S, Calderone S, et al. Effects of recent, short-term hyperglycemia on responses to hypoglycemia in humans. Relevance to the pathogenesis of hypoglycemia unawareness and hyperglycemia-induced insulin resistance. Diabetes 1995; 44:513–9. 65. Cranston I, Lomas J, Maran A, Macdonald IA, Amiel SA. Restoration of hypoglycaemia awareness in patients with long-duration insulin-dependent diabetes. Lancet 1994; 344:283–7. 66. Dagogo Jack S, Rattaeasarn C, Cryer PE. Reversal of hypoglycemia unawareness, but not defective glucose counterregulation, in IDDM. Diabetes 1994; 43:1426–34. 67. Fanelli C, Pampanelli S, Epifano L, et al. Long-term recovery from unawareness, deficient counterregulation and lack of cognitive dysfunction during hypoglycaemia, following institution of rational, intensive insulin therapy in IDDM. Diabetologia 1994; 37:1265–76. 68. Widom B, Simonson DC. Glycemic control and neuropsychologic function during hypoglycemia in patients with insulin-dependent diabetes mellitus. Ann Intern Med 1990; 112:904–12. 69. Gold AE, MacLeod KM, Frier BM. Frequency of severe hypoglycaemia in patients with type 1 diabetes with impaired awareness of hypoglycaemia. Diabet Care 1994; 17: 697–703. 70. Clarke WL, Gonder-Frederick LA, Ricahrds FE, Cryer PE. Multifactorial origin of hypoglycemic symptom unawareness in IDDM. Association with

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84. Thamer M, Ray NF, Taylor T. Association between antihypertensive drug use and hypoglycemia: a case-control study of diabetic users of insulin or sulfonylureas. Clin Ther 1999; 21:1387–400. 85. Orre-Pettersson AC, Lindstrom T, Bergmark V, Arnqvist HJ. The snack is critical for the blood glucose profile during treatment with regular insulin preprandially. J Intern Med 1999; 245:41–5. 86. Fanelli CG, Pampanelli S, Porcellati F, Rossetti P, Brunetti P, Bolli GB. Administration of neutral protamine Hagedorn insulin at bedtime versus with dinner in type 1 diabetes mellitus to avoid nocturnal hypoglycemia and improve control. A randomized, controlled trial. Ann Intern Med 2002; 136: 504–14. 87. Mu¨hlhauser I, Jo¨rgens V, Berger M, et al. Bicentric evaluation of a teaching and treatment programme for type 1 (insulin-dependent) diabetic patients: improvement of metabolic control and other measures of diabetes care for up to 22 months. Diabetologia 1983; 25:470–6. 88. Amiel SA. Type 1 diabetes: treatment without tears. Diabetologia 2005; 48: 1963–4. 89. Ratner RE, Hirsch IB, Neifring JL, Garg SK, Mecca TE, Wilson CA, for the U.S. Study Group of Insulin Glargine in Type 1 Diabetes. Less hypoglycemia with insulin glargine in intensive insulin therapy for type 1 diabetes. Diabet Care 2000; 23:639–43. 90. Hermansen K, Madsbad S, Perrild H, Kristensen A, Axelsen M. Comparison of the soluble basal insulin analog insulin detemir with NPH insulin. Diabet Care 2001; 24:296–301. 91. Heller SR, Amiel SA, Mansell P, and the U.K. Lispro Study Group. Effect of the fast-acting insulin analog lispro on the risk of nocturnal hypoglycemia during intensified insulin therapy. Diabet Care 1999; 22:1607–11. 92. Brunelle BL, Llewelyn J, Anderson JH, Jr., Gale EAM, Koivisto VA. Metaanalysis of the effect of insulin lispro on severe hypoglycemia in patients with type 1 diabetes. Diabet Care 1998;21:1726–31. 93. Pickup J, Mattock M, Kerry S. Glycaemic control with continuous subcutaneous insulin infusion compared with intensive insulin injections in patients with type 1 diabetes: meta-analysis of randomised controlled trials. BMJ 2002; 324:705. 94. Bode BW, Steed RD, Davidson PC. Reduction in severe hypoglycaemia with long term subcutaneous insulin infusion in type 1 diabetes. Diabet Care 1996; 19:324–7. 95. Rodrigues IAS, Reid HA, Ismail K, Amiel SA. Indications and efficacy of continuous subcutaneous insulin infusion (CSII) therapy in Type 1 diabetes mellitus: a clinical audit in a specialist service. Diabetic Medicine 2005; 22: 842–9. 96. McMahon SK, Ferreira LD, Ratnam N, et al. Following afternoon exercise, glucose requirements are increased in a delayed and consistent manner in adolescents with type 1 diabetes. Diabetes 2005; 54Suppl:A27. 97. Wiesli P, Schmid C, Kerwer O, et al. Acute psychological stress affects glucose concentrations in patients with type 1 diabetes following food intake but not in the fasting state. Diabet Care 2005; 28:1910–5.

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98. Cox DJ, Gonder-Frederick L, Polonsky W, Schlundt D, Kovatchev B, Clarke W. Blood glucose awareness training (BGAT-2); long term benefits. Diabet Care 2001; 24:637–42. 99. Cox, D, Irvine, A, Gonder-Frederick, L, Nowacek, G, Butterfield, J. Fear of Hypoglycaemia: Quantification, validation and utilization. Diabet Care 1987; 10: 617–21. 100. Polonsky, W.H., Davis C.L., Jacobson A.M. & Anderson B.J. Correlates of hypoglycaemic fear in Type 1 and Type 2 DM. Health Psychology 1992; 11: 199–202. 101. Feher MD, Grout P, Kennerdy A, Elkeles RS, Touquet R. Hypoglycaemia in an inner-city accident and emergency department: a 12-month survey. Arch Emerg Med 1989; 6:183–8. 102. Leventhal, H., Diefenbach, M., & Leventhal, E.A. (1992) Illness cognition: using common sense to understand treatment adherence & affect cognition interactions. Cognitive Therapy & Research 1992; 16:143–63. 103. Boyne MS, Silver DM, Kaplan J, Saudek CD. Timing of changes in interstitial and venous blood glucose measured with a continuous subcutaneous glucose sensor. Diabetes 2003; 52:2790–4. 104. American Diabetes Association. Pancreas transplantation for patients with Type 1 diabetes. Diabet Care 2000; 23:117. 105. Esmatjes E, Flores L, Vidal M, et al. Hypoglycaemia after pancreas transplantation: usefulness of a continuous glucose monitoring system. Clin Transplant 2003; 17:534–8. 106. Shapiro AM, Ricordi C, Hering B. Edmonton’s islet success has indeed been replicated elsewhere. Lancet 2003; 9391:1242. 107. Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000; 343:230–8. 108. Rother KI, Harlan DM. Challenges facing islet transplantation for the treatment of type 1 diabetes mellitus. J Clin Invest 2004; 114:877–83

3 Patient Selection and Assessment: An Endocrinologist’s Perspective Peter A. Senior Division of Endocrinology, University of Alberta, Edmonton, Alberta, Canada

INTRODUCTION Clinical islet transplantation (CIT) and the prospect of a minimally invasive cure for Type 1 diabetes (T1DM) is an alluring prospect for patients and their families. However, the potential demand far outstrips the supply of donor pancreata, while recipients must take immunosuppression life-long. Thus it remains necessary to develop selection criteria to identify suitable candidates for CIT. The overall purpose of patient selection is to: 1. 2. 3. 4.

confirm the presence of indications for CIT exclude any remediable cause for, or alternative therapeutic approach to, the indication rule out any contraindication identify any comorbidity that might increase the risks of CIT or be exacerbated/accelerated by CIT

This approach ensures detailed assessment of the balance of risks and benefits for the individual and facilitates informed consent. Selection of candidates for CIT has the aim of maximizing benefit while minimizing risk. This is not an exact science since CIT is still a relatively novel treatment for T1DM, which continues to evolve, and the long-term effects remain unknown. It should be recognized that difficult

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choices will need to be made that may seem arbitrary to some, and that there may be tensions between the needs of the individual candidate versus those of the transplant program, as well as between short-term benefits versus long-term risks. This chapter addresses current indications and contraindications for CIT, focusing on islet-alone allotransplantation. An approach to patient assessment and selection is outlined from an endocrinologist’s perspective.

GOALS OF CLINICAL ISLET TRANSPLANTATION Patients (and their physicians) hope that CIT will lead to excellent metabolic control without risk of hypoglycemia, cause regression (or at least halt the progression) of microvascular complications, and prolong survival and improve quality of life without the need for insulin injections. It is important to have a realistic understanding of what CIT can achieve and differentiate that from the (as yet unproven) potential benefits. In most cases CIT can lead to insulin independence and excellent blood glucose control (1), resolution of hypoglycemia and stabilization of glycemia (2), as well as reduced fear of hypoglycemia (3). However, although Cpeptide production is sustained at 5 years, insulin independence is maintained in the longer term in only a minority (4). There is no clear evidence yet of a beneficial effect on micro- or macro-vascular complications, although some reports in islet after kidney transplant are promising (5,6). A short term worsening of retinopathy might be expected from the literature (7) and has been noted (8), along with a decline in GFR (9). Ryan et al. have reported on the risks and side effects of CIT (8).

INDICATIONS FOR CLINICAL ISLET TRANSPLANTATION Currently, the generally accepted indications for islet-alone transplantation are based on those described by the Edmonton group (10,11): T1DM complicated by severe hypoglycemia and hypoglycemia unawareness, and severe glycemic instability despite optimization of medical therapy. Although previously considered an indication, progressive microvascular complications would now be viewed as a caution. Islet after kidney transplantation (12) and simultaneous kidney-islet transplants (13) have been performed in some centers with good early results. The indications for transplantation may be somewhat broader, since these individuals are already committed to long-term immunosuppression, although the risks of rejection of a functioning kidney graft will have to be weighed. Similarly, the risk–benefit ratio for islet autotransplantation after

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pancreatectomy, where immunosuppression is not required, probably favors transplantation (14).

Type 1 Diabetes Generally, CIT-alone is restricted to T1DM subjects since they have an absolute b-cell deficiency and are insulin sensitive. Clinical islet allotransplantation could also be considered for individuals with diabetes secondary to pancreatectomy (for benign disease) if diabetes was sufficiently difficult to manage. It is conventional to confirm that C-peptide is absent (<0.1nmol/ L), both basally and after stimulation with arginine or a mixed meal test. Islet autoantibodies (GAD65, ICA, IAA) may also be measured, to confirm the autoimmune basis, and monitored post transplant, when an increase in titer could indicate autoimmune recurrence.

Hypoglycemia and Hypoglycemia Unawareness Intensification of glucose control is associated with increased risk of hypoglycemia (15) and will be experienced by most (if not all) individuals with T1DM. But how can potential CIT recipients with severe, recurrent hypoglycemia be identified? The perception of the severity and impact of hypoglycemia seems to vary between individuals, likely based on their previous experiences, social and occupational circumstances, and underlying personality. How individuals express this may in turn influence the endocrinologist’s perception of the severity of their hypoglycemia. Endocrinologists routinely assess hypoglycemia by considering the number and frequency of episodes, the need for third party assistance and the presence or absence of adrenergic or neuroglycopenic symptoms. Inappropriate matching of insulin doses to carbohydrate and exercise, inappropriate insulin adjustments, omitted meals, and poor injection techniques can all contribute and should be corrected. A tool to make an objective assessment of hypoglycemia has been developed that seeks to integrate these clinical factors into a scoring system (2). Using one month’s blood glucose records with a description of any episodes of hypoglycemia, a score (HYPO score) is calculated, with higher scores given to episodes with no warning symptoms, detected by others, and requiring third party assistance. The HYPO score is markedly elevated in islet transplant candidates compared with T1DM controls, improves following successful CIT, and correlates well with clinical assessment (2). The HYPO score is now routinely used in Edmonton’s CIT program with severe hypoglycemia defined as a HYPO score >90th percentile of the T1DM population.

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Loss of counter-regulatory hormones and hypoglycemia unawareness contribute to troublesome hypoglycemia. An optimized insulin regimen, perhaps using insulin analogues or continuous subcutaneous insulin infusion, ensuring strict avoidance of hypoglycemia can lead to improvements both in the frequency and severity of hypoglycemia but also to recovery of warning symptoms. The loss of counter-regulation or symptoms of hypoglycemia can be assessed objectively using hypoglycemic clamps, but this is time consuming and may not necessarily indicate the presence of recurrent or severe hypoglycemia impacting on quality of life.

Glycemic Lability Erratic blood glucose control may result from poor matching of insulin doses to carbohydrate intake and physical activity, or failure to adhere to dietary and lifestyle recommendations, and a number of psychosocial factors may contribute. Nevertheless, despite careful adherence, some T1DM subjects continue to experience wide, unpredictable swings in their blood glucose levels. Not surprisingly, glycemic lability often accompanies hypoglycemia. Previously, the mean amplitude of glycemic excursions (MAGE) was used as a tool to quantify lability (16). The MAGE is calculated from 7 blood glucose values per day collected over two days. It was difficult to be sure whether these two days were representative. The lability index (LI) (2), calculated from one month’s glucose records, has superseded the MAGE. Severe glycemic lability is now defined for Edmonton’s CIT program as an LI score >90th percentile of the general T1DM population. Continuous glucose monitoring systems (CGMS) using subcutaneous glucose sensors are another tool to assess glucose excursions. Usually, recordings are performed over a three-day period. The performance of CGMS in assessing lability has been explored in prospective islet transplant candidates (17) and to demonstrate stable glycemia after islet transplantation (18). Recurrent, frequent admissions for ketoacidosis, rather than an indication, should be viewed as a relative contraindication, or at least as a caution, to CIT. It raises questions about compliance and the adequacy of education and self-management skills.

. . . Despite Attempts to Optimize Medical Therapy Rather than a basis on which to exclude all but the most rigid and meticulous individuals, this caveat is to avoid transplanting individuals whose problems could be addressed perhaps relatively easily and probably more safely by conventional means rather than CIT.

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It behooves the endocrinologist to ensure that conventional therapies have been considered and explored before recommending islet transplantation. These would include carbohydrate counting, multiple daily injections, the use of long and short acting insulin analogues, and pump therapy. The utility and availability of such options will vary and be influenced by financial, economic, psychological, and cognitive factors as well as access to specialist support.

CONTRAINDICATIONS TO CLINICAL ISLET TRANSPLANTATION A number of contraindications are relevant to the endocrinologist. Some of these might be viewed as relative contraindications but will likely influence the ability to achieve insulin independence. Age Most CIT programs exclude children and the elderly. Defining an upper chronological age limit may appear arbitrary and raise ethical questions. Nevertheless, islets are a limited resource and the presence of comorbidities increases with age. For practical purposes an upper age limit seems to be a useful guideline. Transplantation of children is being considered (19) but will be easier to justify in those who are already on immunosuppressant drugs having received a previous solid organ or bone marrow transplant.

Obesity and Insulin Resistance There is some relationship between number of islets infused per kg body weight and insulin independence. Single donor success is more likely when preparations containing a large number of islets are infused into small recipients (20,21). Similarly, insulin independence may be more readily achieved in insulin sensitive individuals with low insulin requirements (21). In addition it will be difficult to provide a very large individual with an adequate islet mass without requiring multiple islet infusions. Since each infusion has procedural risks, which may increase with sequential procedures (22,23), this has safety implications. Improvements in islet isolation with bigger yields may help resolve this issue. Thus many programs exclude those with greater body weight (e.g., maximum of 90 kg), the obese (BMI>30), and those with high insulin requirements (>1 unit/kg/day). It is hoped that by minimizing the metabolic demands on transplanted islets better short and long-term outcomes will be achieved.

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Infection and Neoplasia Because long-term immunosuppression is associated with an increased risk of neoplasia and infection, evidence of current or previous infection with tuberculosis or hepatitis B or C should be sought to ensure adequate treatment. HIV or chronic infection with hepatitis B, and neoplasia, unless considered cured, would also be regarded as contraindications. Psychiatric Disease, Cognitive Impairment, and Non-Compliance Psychiatric illness characterized by psychosis or the inability to give informed consent because of cognitive impairment (or indeed for any other reason) should be contraindications to CIT. Previous or treated depression would not preclude CIT but should be weighed in the decision-making process. A similar approach would seem reasonable for personality disorders or certain personality traits, e.g., obsessive-compulsive tendencies, which seem over-represented in individuals seeking CIT. Non-compliance is hard to quantify but could jeopardize the chance of successful CIT. Care should be taken to document compliance as objectively as possible during the assessment process (completes glucose records as requested, has blood tests performed in a timely fashion, attends appointments, etc.) in case an applicant is declined on this basis. A psychosocial assessment is a helpful part of the routine assessment. A formal psychiatric assessment is probably required only in a minority of cases. Smoking, Alcohol, and Drugs Most transplant programs require candidates to abstain from smoking for a period (e.g., 6 months). Substance abuse (alcohol, prescription or other drugs) would generally be viewed as a contraindication to CIT. A psychological assessment of individuals who have successfully overcome previous addictions may be useful to determine their suitability for CIT. Long term therapy with drugs with significant interactions with immunosuppressants, e.g., phenytoin for seizure disorders, will need careful consideration. Consultation with a specialist may permit substitution with an alternative agent with less potential for interaction. DIABETES COMPLICATIONS AND OTHER COMORBIDITIES Micro- and macro-vascular complications of diabetes may be contraindications, or indicate the need for caution before proceeding, to CIT. Some complications or comorbidities may contribute to the indications for transplantation, and CIT may be unnecessary if these contributors are treatable. Moreover, the impact of CIT and immunosuppression on

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complications and comorbidities should be considered. Although CIT may be beneficial in the short term, the risk–benefit ratio may be unfavorable in the longer term if complications are accelerated by CIT or immunosuppression.

Retinopathy It is widely recognized that retinopathy may initially progress following rapid improvement in glycemic control (7). Thus for prospective CIT recipients it would seem prudent to ensure that those with severe nonproliferative or proliferative retinopathy have been assessed by an ophthalmologist, any necessary treatment be completed, and the retinopathy judged to be stable for a period prior to transplantation. Similarly, frequent surveillance (3 monthly) following CIT is recommended. Individuals with visual loss face a number of challenges following CIT, including the introduction of multiple new medications with frequent dose adjustments, regular lab work, and clinic visits, as well as ongoing frequent self-blood glucose monitoring. This would not be an insurmountable obstacle for those successfully living independently with this handicap. However, caution may be required in subjects with recent visual loss or with inadequate personal and social support.

Nephropathy Decisions regarding the wisdom of CIT for individuals with diabetic nephropathy are often challenging and illustrate the tension between shortterm benefits and potential long-term harm. Since normoglycemia following whole pancreas transplantation has been associated with the reversal of the histological changes of diabetic nephropathy (24), there has been optimism that nephropathy would improve following islet transplantation. Furthermore, the use of lower doses of nephrotoxic calcineurin inhibitors (e.g., tacrolimus and sirolimus), thought to be free of nephrotoxicity, was further grounds for optimism. However, recent data raise the possibility that sirolimus may not be free of adverse renal effects (25,26). Nephrotoxicity (probably due to calcineurin inhibitors) has been seen after CIT in individuals with established nephropathy and renal impairment (elevated serum creatinine) (1,11). Preliminary reports suggest GFR declines after CIT, and albuminuria progresses (normo- to micro-, or micro- to macroalbuminuria) in up to 40% (9). Heavy proteinuria has developed in a small number of subjects (personal observation). Care should be taken to identify subjects with, or at high risk for, nephropathy, or with renal impairment. GFR should be measured directly

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using a radionuclide method (e.g., clearance of DTPA or Cr-EDTA) or the clearance of Iohexol (Omnipaque± , Amersham Health, Princeton, New Jersey, U.S.A.), or estimated using an equation [e.g., MDRD (27) or Cockcroft-Gault (28) formulae], or from creatinine clearance. Direct measurements are preferable since estimates have not been validated in this population, which is particularly diverse—some individuals are hyperfiltering while others have significant renal impairment. Albuminuria and proteinuria should be measured and a timed urine collection is recommended (either timed overnight or 24 hour collection). Performing three collections will increase the precision of this assessment. Historical information regarding renal function and albuminuria (i.e., lab values) should be sought since it may permit an assessment of the rate of decline in GFR and may avoid underestimating the presence of, or risk for, nephropathy [since remission of even nephrotic range proteinuria has been reported (29)]. The latter point is important in T1DM subjects receiving ACE-inhibitors (ACE-I) or angiotensin two receptor antagonists (A2RA) who are currently normoalbuminuric or microalbuminuric. Such “normoalbuminuric” patients, who had previously been proteinuric (≥1 g/day), have encountered significant renal problems following CIT (personal observation). In those with longer diabetes duration (>10 years) a history of proliferative retinopathy or laser therapy may be a useful proxy. CIT should not be recommended for T1DM subjects with end-stage renal disease for whom simultaneous pancreas-kidney (SPK), or pancreas after kidney transplant would be considered the treatments of choice. Islet after kidney transplant may become an alternative in the future. For individuals not requiring kidney transplantation but with established diabetic nephropathy, precise recommendations are more difficult, particularly since the natural history of diabetic nephropathy is changing (30) and perhaps because of stricter metabolic and blood pressure control together with the widespread use of ACE-I and A2RA. Individuals with evidence of a progressive decline in their GFR who are likely to reach ESRD in a few years may be best advised to explore SPK (the current wait for CIT in Edmonton is approximately 2 years). In those with stable GFR and stable proteinuria the risks of progression of nephropathy and a decline in GFR should be discussed candidly. A complicating factor in this discussion is the risk that CIT will lead to sensitization to foreign antigens, making matching of a renal transplant more difficult. In some cases the risk of nephrotoxicity might be explored by a trial of immunosuppression. Currently CIT using sirolimus and tacrolimus is not recommended in subjects with GFR<40 ml/min/1.73 m2 or in those with macroproteinuria (>0.3 g/24 hr) in Edmonton’s CIT program. However, renal sparing protocols are being developed for patients with higher levels of proteinuria (0.3–1g/24 hr).

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Neuropathy Diabetic neuropathy may contribute to the indications for CIT (e.g., gastroparesis exacerbating lability and hypoglycemia) or be associated with increased risks following CIT, for example, in relation to premature cardiovascular mortality associated with autonomic neuropathy or difficulty with GI side effects of immunosuppression. Autonomic neuropathy may be the most important aspect of neuropathy for islet transplantation. It is an important risk factor for silent myocardial ischemia and cardiovascular mortality (31). Autonomic neuropathy is often under-recognized. Anecdotally, gastroparesis is probably the norm in most patients seeking CIT in Edmonton, although it is asymptomatic in the majority. Careful questioning may, however, reveal bloating after meals, altered bowel habit (constipation or diarrhea), erectile dysfunction, or decreased or inappropriate sweating. There may be a history of incomplete bladder emptying with a large post-void residual. Examination may reveal elevated resting heart rate or postural hypotension. Gastroparesis may contribute to hypoglycemia and lability. In those with symptoms of bloating after meals or vomiting, promotility agents should be considered if not already used. Symptomatic gastroparesis also raises concern about the ability to achieve and maintain stable therapeutic immunosuppression levels due to erratic absorption, particularly since nausea is one of the most common side effects encountered following CIT. Changes in bowel habit are a frequent side effect of immunosuppression and usually represent an exaggeration of pre-transplant gastrointestinal dysmotility. Earlier reports highlighted diarrhea, but constipation has emerged (since the transition from a liquid to tablet formulation of sirolimus) as an equally troublesome consequence for some. These GI side effects can be severe, requiring hospital admission for rehydration, and a laparotomy for a suspected bowel obstruction in one case. Neuropathy may further increase the risk of infection in individuals taking immunosuppressants. Those with peripheral neuropathy are at increased risk of foot ulcers, which will heal more slowly on sirolimus. Bladderemptying problems associated with autonomic dysfunction may predispose to urinary tract infections. A mild anemia is relatively common in CIT candidates and should prompt a search for an underlying cause. In the majority the anemia is normochromic or normocytic with no clear cause, although disordered erythropoietin secretion associated with autonomic neuropathy and nephropathy may contribute (32). The anemia is likely to worsen after transplantation as a result of bone marrow suppression related to sirolimus, trimethoprim-sulfamethoxazole, and ganciclovir (the latter two taken for pneumocystis carinii and cytomegalovirus prophylaxis, respectively). Treatment with recombinant erythropoietin may be required.

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Cardiovascular Disease and Risk Factors Cardiovascular disease is the leading cause of death in adults with T1DM (33) and with a functioning graft in solid organ transplantation. Although CIT is a minimally invasive procedure with low risk for periprocedural cardiovascular events, from a program perspective premature cardiovascular mortality with loss of a functioning graft is a concern. The identification of individuals with cardiovascular disease at high risk will have benefits for the individual as well as the transplant program. The optimal means to screen for cardiovascular disease in this population is not clear. Significant coronary stenoses are common (43%) in asymptomatic T1DM subjects being assessed for CIT (34). Positive myocardial perfusion imaging (MPI) is associated with increased mortality, but the vast majority of asymptomatic CIT candidates have negative MPI. However, a small number required revascularization for high-risk coronary lesions detected by angiography despite negative MPI. Nevertheless, angiography is expensive and time-consuming and has risks. Newer, non-invasive techniques assessing coronary artery calcification or using cardiac magnetic resonance imaging may be useful in the future. Cardiovascular risk factors, particularly hypertension and dyslipidemia, warrant attention. Many CIT candidates will benefit from medical therapy to control risk factors pre-CIT, particularly since asymptomatic coronary artery atherosclerosis is so common in this group. Immunosuppression, particularly with sirolimus, often causes elevation of blood pressure and lipids, necessitating increased lipid lowering or antihypertensive therapy following CIT. Antiplatelet Agents The widespread use of aspirin, either prescribed or over the counter, or other antiplatelet agents for cardioprotection may increase the risk of bleeding from the hepatic puncture. This risk could be reduced by the avoidance of antiplatelet agents prior to transplant. In low-risk individuals, aspirin could be discontinued while they are active on the waiting list. In high-risk individuals, aspirin could be discontinued when they reach, or approach, the top of the list. When antiplatelet agents cannot be discontinued or have been used in the two weeks prior to transplantation, an open procedure with cannulation of an omental vein, where control of bleeding can be assured, should be considered. Recent advances in techniques to seal the catheter tract, for example, using collagen “flour” (Avitene, Davol, Cranston RI), have been effective in reducing the incidence of bleeding after hepatic puncture. This reduction in risk may permit antiplatelet agents to be continued.

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Other Comorbid Conditions A number of other comorbid conditions should be considered. Autoimmune Disease Many CIT candidates will have coexisting autoimmune disorders, and it would seem sensible to ensure that therapy for existing conditions is optimized prior to transplantation. In a significant proportion, coexisting autoimmune disease may be as yet undiagnosed (35). Both Addison’s and celiac disease have the potential to exacerbate hypoglycemia and should be excluded. If detected, the indications for CIT should be reassessed after a period of therapy. Addison’s disease, if adequately treated, would not of itself be a contraindication to CIT. While avoidance of steroids is one factor contributing to recent success in CIT, physiological replacement doses are not expected to have an adverse impact on islet engraftment or function. Excessive steroid replacement, however, is likely to have a negative impact either directly, via islet toxicity, or indirectly, by reducing insulin sensitivity. Autoimmune thyroid disease is the most common comorbid autoimmune disease (35) and should also be sought. In those with anemia it would be sensible to exclude pernicious anemia. Hepatic Disorders Hepatic disease or disorders ought to be considered before intrahepatic islet transplantation. As examples, hemangiomata and portal hypertension may both increase the procedural risks of CIT. Unusual patterns of hepatic steatosis have been observed following CIT in up to 20% (36) and perhaps indicate a need for additional caution in individuals with underlying liver disease. AN APPROACH TO ASSESSMENT FOR ISLET TRANSPLANTATION The Transplant Endocrinologist’s Role—A Personal View In assessing individuals for CIT it is important to be aware of one’s own values and prejudices and to ensure these don’t unduly influence the assessment. The role of the endocrinologist as a member of the islet transplant program is not to take sole responsibility for deciding whether an individual should receive a transplant or not. Rather, by explaining clearly how CIT could help their individual situation and what risks are likely, the endocrinologist can help the individual make a wise decision as to whether or not CIT is appropriate for them. This role may differ from that of the referring endocrinologist who may act more in the role of patient advocate.

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I often indicate to potential recipients that my role during assessment is to explain all the bad news about islet transplantation. Most people already know what the good news is and are extremely optimistic and have high hopes for CIT. My aim is to ensure that their expectations are realistic and that both the benefits and costs of CIT are explained and described in a way which is meaningful and understandable. Sometimes guiding these decisions is straightforward: some individuals clearly do not have sufficient problems with hypoglycemia or lability to justify islet transplantation; others have such severe problems that islet transplantation is an obvious choice. In many, however, the situation is less clear: these individuals have indications for islet transplantation but whether these outweigh the risks is harder to judge, particularly since the long-term risks of CIT remain unclear. In these situations, the individual and their family should weigh the decision carefully. While I can provide information and guidance, I am cautious not to rule out transplantation for these “grey cases” since I cannot fully appreciate the impact diabetes has on that individual’s life. From a program perspective it is important to pick winners. There are a number of barriers to success in CIT. These may be least in individuals whose goal is to avoid hypoglycemia and who view insulin independence as a bonus; who are small, slim, and insulin sensitive with good diabetes control and excellent self-management skills; and who have realistic expectations, reliable psychosocial supports, and adequate financial resources. Nevertheless, many worthy and deserving CIT candidates do not have these advantages. CIT may be more challenging, but these latter individuals should not be judged ineligible. Efforts to ensure equitable access to CIT on the basis of clinical need and that the needs of the individual are not relegated to a position subordinate to those of the program should be maintained. The final decision regarding listing is best made by the CIT team in partnership with the applicant. Patient Expectations, Goals, and Aspirations It is often helpful to explore the individual’s current main concerns with respect to their diabetes and their reasons for seeking islet transplantation. Ultimately, the patient’s goals and expectations will have a major impact on their perception of the success of CIT. The stated goal for transplantation may not fully or accurately reflect the individual’s underlying goals, which may be unstated. This often reflects a desire, conscious or unconscious, to present goals that the assessor will find acceptable and legitimate. Prospective candidates approach CIT with a range of common goals. These include insulin independence; freedom from hypoglycemia (and fear of hypoglycemia), injections, or the restrictions of T1DM (testing, regular

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meals, etc.); stable blood sugars; good control; avoidance of micro- or macrovascular complications; increased life expectancy and enhanced quality of life; and, for some, a desire to further research efforts that will benefit future generations. Clearly many of these are interconnected. It can be helpful to explore which is the patient’s primary goal by asking which single outcome would make the risks of CIT worthwhile. CIT can deliver avoidance of hypoglycemia and stabilization of blood sugars most reliably, although not in all recipients. Thus, individuals identifying these as their key goals may be more likely to achieve an outcome they perceive as satisfactory. Unrealistic Expectations and Misconceptions Insulin Independence Only: Individuals whose main goal is insulin independence may be disappointed, particularly if they will view the resumption of insulin therapy as a failure on their part as an individual, as an ambassador for CIT, or of the program. In other cases, individuals identify insulin independence as their goal because it will lead to all of the other benefits but would not be disappointed if they were free of hypoglycemia but still taking some insulin. CIT: An Easier Option: Individuals who seek CIT as a less demanding alternative to insulin therapy, perhaps struggling with balancing busy schedules at work and home with professional, domestic, and parenting duties, may also encounter disappointment. CIT is not an easy option. In some cases, individuals will face all the demands and restrictions of a transplant recipient on life-long immunosuppression in addition to those of T1DM if still using multiple daily injections, albeit perhaps with smaller insulin doses and fewer problems with lability and hypoglycemia. CIT: The Last Resort: Individuals who perceive CIT as their only hope and their last resort may have unrealistic expectations or have attached more value to receiving an islet transplant than to what the transplant might achieve. The transplant itself is the goal rather than a means to an end. This can create a number of problems, most obviously if there are contraindications to transplantation or if CIT is unsuccessful but may also lead to low tolerance for adverse effects of immunosuppression and dissatisfaction (see post-transplant amnesia below). Assessment for transplantation is the first stage of a long process leading to listing, for some, and the waiting list may be long—so CIT is unlikely to be the short-term answer that these individuals might hope for.

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CIT: Doing it for Other People: In a small number of cases the driving force behind seeking transplantation has been family members—usually parents or partner. Managing severe unpredictable hypoglycemia may be particularly burdensome for them. The potential recipient may express a desire not to be a burden or to better fulfill their role in the family. Delays in the completion of blood glucose records or investigations may indicate that the individual is not fully committed to transplantation. Post-transplant Amnesia and Moving Goal Posts: Two phenomena encountered following transplantation that are common and may cause difficulty are worth considering. The first is of moving goal posts, which, though common, may not cause problems. Prior to transplantation the individual’s goals are to avoid frequent, severe hypoglycemia and have more stable blood glucose control. However, within a short time following transplantation, there is disquiet if post-prandial glucose values exceed 7.0 mmol/L (126 mg/dl). In other situations, with the same stated pre-transplant goals, insulin-independence or freedom from self bloodglucose monitoring can emerge as new or underlying (though previously unstated) goals and have the potential to lead to conflict with the transplant team. The phenomenon of post-transplant amnesia is related and reflects the tendency to view current problems as the most important and pressing. This might best be illustrated by two case studies. Case Study 1. A 28-year-old received two islet transplants for severe hypoglycemia with hypoglycemia unawareness. This individual was insulin independent for three years, tolerated the immunosuppressants well, and suffered no major side effects. Insulin therapy was resumed because of a decline in islet function, although C-peptide production continued. There was some minor hypoglycemia related to exercise after the resumption of insulin. A year later, the recipient expressed a desire to withdraw immunosuppression because of side effects and a perceived lack of benefit from CIT. The chart was reviewed with the patient, examining the initial application to the program and pre-transplant blood glucose records. The recipient was reminded of the severe problems with hypoglycemia prior to CIT and the major impact on their lifestyle. Realizing they were indeed still benefiting from CIT, although no longer insulin independent, the recipient decided to continue immunosuppression. Case Study 2. A 33-year-old recipient of two transplants for hypoglycemia and hypoglycemia unawareness was insulin

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independent. However, this individual was troubled by side effects of mouth ulcers, diarrhea and fatigue. The fatigue interfered with this individual’s ability to work and fulfill the roles of breadwinner and parent. These current difficulties dominated the recipient’s view of the success of CIT, and the previous difficulties with hypoglycemia were forgotten or ignored. Despite insulin independence, the recipient requested withdrawal of immunosuppression. Fortunately, changes in the immunosuppressive regimen resulted in a significant amelioration of the side effects and the recipient continues to benefit from CIT. The Benefits of Hindsight Despite efforts to explore the motivations for CIT and ensure preparation for the demands of CIT and long-term immunosuppression, it may only become clear that some people are unable to put up with these demands in retrospect, particularly if CIT is only partially successful or side effects are more severe than average. It is hoped that this situation can be avoided by clear explanations of the risks and frequency of side effects delivered in a meaningful way. Nevertheless, recipients frequently remark that they only really understood the severity of some side effects with personal experience.

Social, Financial, and Psychological Considerations CIT is associated with a number of further demands that ought to be considered prior to listing. In Canada, many transplant recipients spend a substantial period in Edmonton after transplantation (6–12 weeks on average). For those from other parts of the country, this may require separation from friends and family and absence from work. Absence of usual support networks can be difficult, since the peri-transplant period is challenging emotionally and physically. Loneliness, boredom, and anxiety can all be encountered. Absence from work and the costs of travel, accommodations, and drugs are important factors that may present financial barriers to islet transplantation. The relative importance of these factors will vary in different parts of the world depending on health care and social security systems as well as geographical factors and individual circumstances. Loss of income as a result of work absences or restricted duties because of side effects from immunosuppressants can prove a significant strain for those who are the main or sole source of household income. Consideration and planning for these aspects of transplantation is extremely important. Arrangements for child care, accommodations, travel at short notice if called for transplantation, adequate financial resources for travel and subsistence, and adequate coverage for drugs should be made as

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early as possible, and in the case of drug reimbursement, ahead of listing. A sympathetic employer will make attending for regular blood tests and follow-up visits much easier. Discussions with primary care physicians and other physicians providing diabetes care regarding their willingness to participate in posttransplant care should take place ahead of listing. This is particularly important for patients living at a distance from the transplant center (especially if the patient lives in a region with a different medical jurisdiction, e.g., a different province in Canada) or where referral to the program was not initiated by the individual’s usual physician/ endocrinologist.

Clinical Assessment As indicated earlier, the main focus of the clinical assessment is to ensure an individual has indications for CIT that cannot be resolved by conventional therapy and to rule out any contraindications or comorbidity that would make CIT unsafe. An outline of features in the history and physical examination that should be considered is presented in Table 1 and a list of suggested investigations and additional assessments in Table 2.

The Waiting List and Reassessment The length of time spent waiting for transplantation will depend on a large number of factors, e.g., blood group, organ supply, recipient weight, and the presence of alloreactive antibodies. In most cases, the wait will not be insubstantial. A strategy for reviewing listed individuals at regular intervals is necessary. Individuals on the waiting list should be instructed to notify the transplant program of any changes in their health status or medication. Temporary suspension from the active list may be necessary in some cases. Potential indications include foot ulceration, intercurrent infection, myocardial infarction, active retinopathy, or a surgical procedure. In addition, non-medical circumstances, such as holidays abroad or bereavement, may necessitate placing the candidate “on hold.” In Edmonton, individuals on the wait list attend for review annually. Essentially, this is a somewhat abbreviated version of the initial assessment consisting of a history and physical and some lab investigations (blood count, routine biochemistry, urinary albumin excretion, and an assessment of GFR, plus additional tests tailored to the individual). A major focus of the review is to ensure that individuals continue to have indications for islet transplantation and have not developed contraindications. The development or progression of microvascular complications or comorbidity is not (Text continues on page 76.)

Neuropathy/ ulceration Macrovascular disease

Nephropathy

Complications Retinopathy

HYPO score and LI

Therapy Hypoglycemia Frequency Symptoms 3rd party assistance Blood glucose monitoring Blood glucose patterns

(Continued)

Recent retinal exam? Previous laser / vitrectomy / cataract surgery? Any visual impairment: acuity/fields? Few patients are aware of their albuminuria status or whether ACE-inhibition was introduced because of persistent microalbuminuria. Painful, peripheral neuropathy may be noted. Previous ulceration or amputations are important. Cardiovascular, cerebrovascular, or peripheral vascular disease

Excessive variability? Are adjustments of insulin doses and treatment of hypoglycemia appropriate? Any pattern of hypoglycemia? Calculated from one month’s self blood glucose records (best completed prior to assessment) See Ref. 2

Timing, predictability, relationship to exercise, alcohol, or other factors Presence or absence of adrenergic and neuroglycopenic symptoms Frequency of severe hypo, admissions, ER visits, or paramedic assistance in the last year Frequency and consistency

A review of the initial presentation and diabetes duration, previous control, and episodes of ketoacidosis Current and previous insulin regimens, types of insulin frequency, and route of delivery

Comments

An Outline Approach to the Clinical Assessment of Individuals Seeking Clinical Islet Transplantation

Diabetes Diabetes diagnosis

History

Table 1

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Foot exam Injection sites

Autonomic function Retinopathy

Physical examination Cardiovascular

Family history Systems enquiry

Abnormal pulses in major vessels or bruits may suggest the presence of atherosclerosis, requiring further evaluation. Lying and standing blood pressure and pulse Document current degree of retinopathy and rule out presence of retinopathy requiring therapy Vascular disease, neuropathy, ulceration, “at-risk” feet For lipohypertrophy or lipoatrophy

Occupation, shift work, physical activity. Tobacco, alcohol use. Family, social, and financial situation. Health care / drug coverage. Autoimmunity, cardiovascular disease, neoplasia Cardiovascular symptoms, particularly atypical symptoms of CAD, e.g., exertional dyspnea as an angina equivalent Symptoms of autonomic neuropathy, including abnormal sweating, erectile dysfunction, bloating after meals, GI dysmotility (including gastroparesis), and orthostatic hypotension

Both medical and surgical Potential interactions with immunosuppressants? ACE-I masking microalbuminuria?

Comments

An Outline Approach to the Clinical Assessment of Individuals Seeking Clinical Islet Transplantation (Continued )

Past medical history Drug therapy and allergies Social history

History

Table 1

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Table 2

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Suggested Investigations and Additional Assessments

Lab tests Complete blood count Clotting studies Blood and tissue typing Alloreactive antibodies Electrolytes, urea, creatinine Liver function tests Bone biochemistry Fasting lipid profile HbA1c C-peptide TSH 9am cortisol Antitransglutaminase antibody

Comments

Basal and stimulated [see Ensuret (Abbott, Saint-Laurent, Que´bec, Canada)/arginine test below]

Albumin excretion rate

To rule out the presence of undiagnosed comorbid autoimmune disease that could contribute to hypoglycemia. An ACTH stimulation test should be performed where the 9am cortisol is equivocal. Can be calculated from a 24hr or timed overnight urine collection. A review of historical data regarding albuminuria is recommended, particularly in individuals receiving ACE-I.

Other investigations

Comments

ECG CXR USS abdomen and portal vein doppler

To rule out hemangiomata or abnormal portal venous flow

Mantoux Viral serology Glomerular Filtration

Rate Coronary Assessment Ensure/arginine test Autonomic function tests Gastric emptying study

HIV, Hepatitis B and C, EBV, CMV Nuclear techniques (99Tc-DTPA or 51Cr-EDTA) or Iohexol (Omnipaque± , Amersham Health, Princeton, New Jersey, U.S.A.) clearance are the gold standard. Creatinine clearance or estimates (e.g., MDRD) may be adequate as screening tests. Exercise stress testing, myocardial perfusion imaging, and angiography are performed in Edmonton. Permit measurement of stimulated C-peptide. Arginine avoids the hyperglycemia associated with Ensure.

When clinically indicated (Continued)

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Senior Suggested Investigations and Additional Assessments (Continued )

Other assessments Psychosocial Dietitian Ophthalmology Dental

Comments Identify any psychological or social barriers to transplantation Ensure adequate medical-nutrition therapy Ensure adequate treatment of any diabetic retinopathy Rule out potential source of sepsis

uncommon. This may necessitate suspension from the active list or may influence the risk/benefit ratio for CIT. Another focus is to update the candidate on developments and progress in islet transplantation, particularly in terms of CIT outcomes and drug side effects that may be substantial since the field is so new. Overall, this will permit a reassessment of the risks and benefits of islet transplantation and ensure that the patient is confident that they are still happy to proceed with islet transplantation and remain on the list. In some individuals the original indications for CIT may be less clear. Problems with hypoglycemia may subside in some people. Reassessment of the HYPO score and LI is particularly helpful in these cases by giving an objective and quantifiable measure that can be compared with historical values. In other cases, the original indications may be even more marked and this may permit some prioritization of the wait list. The decision to remove an individual from the wait list can be a difficult one. Their substantial investment of time and effort means some feel they have earned the right to undergo CIT. However, if it is clear that an individual no longer has indications for islet transplantation or the risks exceed potential benefit, it is not appropriate for them to remain listed. In some cases removal from the wait list has been at the candidate’s request. Some individuals having coped with diabetes for a number of years on the wait list feel their need for CIT is no longer as pressing as they had thought previously. Others find the uncertainty of waiting has a deleterious impact on their quality of life, while others are no longer sure that CIT will be able to deliver in the long term what they had been hoping for, or may feel that the side effects of drugs are more than they are prepared to endure.

CONCLUSIONS Recent advances mean that islet transplantation is now a realistic clinical option for selected individuals with T1DM. When successful it can deliver freedom from hypoglycemia, stable glycemia, and euglycemia. Shortage of donor pancreata, procedural and immunosuppressant-related risks, as well

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as uncertainty about long-term efficacy with respect to glycemia and microvascular complications suggest that CIT should be restricted to a small and highly selected group of patients. Hopefully, future developments, enhancing the performance of CIT and reducing its risks, will permit the benefits of CIT to be expanded to a broader range of patients with T1DM. REFERENCES 1.

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Ryan EA, Lakey JR, Paty BW, et al. Successful islet transplantation: continued insulin reserve provides long-term glycemic control. Diabetes 2002; 51(7):2148–57. Ryan EA, Shandro T, Green K, et al. Assessment of the severity of hypoglycemia and glycemic lability in Type 1 diabetic subjects undergoing islet transplantation. Diabetes 2004; 53(4):955–62. Johnson JA, Kotovych M, Ryan EA, Shapiro AM. Reduced fear of hypoglycemia in successful islet transplantation. Diabetes Care 2004; 27(2): 624–5. Ryan EA, Paty BW, Senior PA, et al. Five-year follow-up after clinical islet transplantation. Diabetes 2005; 54(7):2060–9. Fiorina P, Folli F, Zerbini G, et al. Islet transplantation is associated with improvement of renal function among uremic patients with type I diabetes mellitus and kidney transplants. J Am Soc Nephrol 2003; 14(8):2150–8. Fiorina P, Folli F, Maffi P, et al. Islet transplantation improves vascular diabetic complications in patients with diabetes who underwent kidney transplantation: a comparison between kidney-pancreas and kidney-alone transplantation. Transplantation 2003; 75(8):1296–301. The Diabetes control and complications Trial and Research Group. Early worsening of diabetic retinopathy in the Diabetes Control and Complications trial. Arch Ophthalmol 1998; 116(7):874–86. Ryan EA, Paty BW, Senior PA, Shapiro AM. Risks and side effects of islet transplantation. Curr Diab Rep 2004; 4(4):304–9. Senior PA, Zeman M, Paty BW, Shapiro AM, Ryan EA. Renal outcomes after clinical islet allotransplantation at the University Of Alberta - 4 year follow up. Diabetes 2004; 53(Supp 2):A69. Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000; 343(4):230–8. Ryan EA, Lakey JR, Rajotte RV, et al. Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol. Diabetes 2001; 50(4):710–9. Bertuzzi F, Grohovaz F, Maffi P, et al. Successful [correction of Succesful] transplantation of human islets in recipients bearing a kidney graft. Diabetologia 2002; 45(1):77–84. Lehmann R, Weber M, Berthold P, et al. Successful simultaneous islet-kidney transplantation using a steroid-free immunosuppression: two-year follow-up. Am J Transplant 2004; 4(7):1117–23.

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14. Watkins JG, Krebs A, Rossi RL. Pancreatic autotransplantation in chronic pancreatitis. World J Surg 2003; 27(11):1235–40. 15. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. The Diabetes Control and Complications Trial Research Group. N Engl J Med 1993; 329(14):977–86. 16. Service FJ, Molnar GD, Rosevear JW, Ackerman E, Gatewood LC, Taylor WF. Mean amplitude of glycemic excursions, a measure of diabetic instability. Diabetes 1970; 19(9):644–55. 17. Paty BW, Ryan EA, Senior PA, McDonald C, Shapiro AM. The continuous glucose monitor in the assessment of glycemic ability and hypoglycemic risk in islet transplant recipients. Diabetes 2004; 52(Supp 1):A98. 18. Kessler L, Passemard R, Oberholzer J, et al. Reduction of blood glucose variability in type 1 diabetic patients treated by pancreatic islet transplantation: interest of continuous glucose monitoring. Diabetes Care 2002; 25 (12):2256–62. 19. Hathout E, Lakey J, Shapiro J. Islet transplant: an option for childhood diabetes? Arch Dis Child 2003; 88(7):591–4. 20. Shapiro AM, Ricordi C. Unraveling the secrets of single donor success in islet transplantation. Am J Transplant 2004; 4(3):295–8. 21. Hering BJ, Kandaswamy R, Ansite JD, et al. Single-donor, marginal-dose islet transplantation in patients with type 1 diabetes. JAMA 2005; 293(7):830–5. 22. Casey JJ, Lakey JR, Ryan EA, et al. Portal venous pressure changes after sequential clinical islet transplantation. Transplantation 2002; 74(7):913–5. 23. Rafael E, Ryan EA, Paty BW, et al. Changes in liver enzymes after clinical islet transplantation. Transplantation 2003; 76(9):1280–4. 24. Fioretto P, Steffes MW, Sutherland DE, Goetz FC, Mauer M. Reversal of lesions of diabetic nephropathy after pancreas transplantation. N Engl J Med 1998; 339(2):69–75. 25. Fervenza FC, Fitzpatrick PM, Mertz J, et al. Acute rapamycin nephrotoxicity in native kidneys of patients with chronic glomerulopathies. Nephrol Dial Transplant 2004; 19(5):1288–92. 26. Dittrich E, Schmaldienst S, Soleiman A, Horl WH, Pohanka E. Rapamycinassociated post-transplantation glomerulonephritis and its remission after reintroduction of calcineurin-inhibitor therapy. Transpl Int 2004; 17(4): 215–20. 27. Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med 1999; 130(6):461–70. 28. Cockcroft DW, Gault MH. Prediction of creatinine clearance from serum creatinine. Nephron 1976; 16(1):31–41. 29. Hovind P, Rossing P, Tarnow L, Toft H, Parving J, Parving HH. Remission of nephrotic-range albuminuria in type 1 diabetic patients. Diabetes Care 2001; 24(11):1972–77. 30. Hovind P, Tarnow L, Rossing K, et al. Decreasing incidence of severe diabetic microangiopathy in type 1 diabetes. Diabetes Care 2003; 26(4):1258–64.

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Vinik AI, Maser RE, Mitchell BD, Freeman R. Diabetic autonomic neuropathy. Diabetes Care 2003; 26(5):1553–79. Thomas S, Rampersad M. Anaemia in diabetes. Acta Diabetol 2004; 41(Suppl 1:S13–7. Laing SP, Swerdlow AJ, Slater SD, et al. The British Diabetic Association Cohort Study, II: cause-specific mortality in patients with insulin-treated diabetes mellitus. Diabet Med 1999; 16(6):466–71. Senior PA, Welsh RC, McDonald CG, Paty BW, Shapiro AM, Ryan EA. Coronary artery disease is common in nonuremic, asymptomatic type 1 diabetic islet transplant candidates. Diabetes Care 2005; 28(4):866–72. Davyduke TM, Cholin SL, McDonald C, et al. Prevalence of known and undiagnosed autoimmune disease in islet transplant candidates. Diabetes 2003;52(Supp 1):A572. Bhargava R, Senior PA, Ackerman TE, et al. Prevalence of hepatic steatosis after islet transplantation and its relation to graft function. Diabetes 2004; 53 (5):1311–17.

4 The Surgical Aspects of Pancreas Procurement for Pancreatic Islet Transplantation Neal R. Barshes, Timothy C. Lee, Ian W. Udell, Christine A. O’Mahony, F. Charles Brunicardi, and John A. Goss Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, U.S.A.

A. M. James Shapiro Department of Surgery, Walter C. Mackenzie Health Center, University of Alberta, Edmonton, Alberta, Canada

INTRODUCTION Techniques for the procurement of the whole organ pancreas for use in whole or segmental pancreas transplantation were first described more than 20 years ago (1–6). Although many of the clinical and experimental studies of pancreatic islet transplantation have utilized the same procurement techniques as those used in whole pancreas transplantation, the use of modified techniques designed especially for pancreatic islet transplantation may result in higher rates of successful pancreatic islet isolation (7–9). These techniques include modification of the preservation solution used, the technique of preservation during transport of the pancreas, and certain modifications in surgical technique. University of Wisconsin (UW) solution (Viaspan, Barr Laboratories, Pomona, New York, U.S.A.) is thought to provide protection to ischemic tissues by providing a similar electrolyte composition to that found in intracellular fluid. In doing so, it inhibits the efflux of intracellular potassium from cells and is referred to as an “intracellular solution.” UW

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solution has been the standard preservation solution for whole pancreas procurement over the past 15 years (6,10,11). Cold storage of the pancreas in UW does allow for the successful isolation of pancreatic islets (12–14), but recently other solutions and combinations of solutions have been evaluated for use in pancreatic islet transplantation. One such solution is histidine-tryptophan-ketoglutarate (HTK) solution (Dr. Franz Ko¨hler, Chemie GmbH, Alsbach-Ha¨hnlein, Germany; also Custodial, Pharmapal, Ltd., Athens, Greece). Originally developed as a cardioplegia solution in the 1970s (15), HTK solution has been used in whole pancreas transplantation with increasing frequency. HTK solution utilizes the acid-buffering capacity of histidine to minimize ischemic injury to cells (16). In contrast to the “intracellular”-type electrolyte composure of UW solution, HTK has a low potassium concentration and is considered an “extracellular” solution. Advantages of HTK solution include a viscosity similar to that of water, allowing for higher flow rates and lower pressure during infusion (17) and allowing effective perfusion to occur by gravity alone (16). Although HTK is approximately half the price of UW solution per liter and does not require a filter to be used (14), larger volumes are typically required to provide adequate preservation (17). Studies comparing UW and HTK in experimental whole pancreas transplantation have suggested that the use of these two solutions results in equivalent graft function (18,19) and integrity of the pancreatic islets (20). A recent clinical study that compared whole pancreata infused with HTK to historical control pancreata infused with UW solution found no difference in post-transplant graft function (14). A non-randomized series of 33 whole pancreas transplants performed at the University of Pittsburgh also showed no difference between UW and HTK in graft function or post-transplant complication rate (17). At this time, however, no clinical study has compared the effectiveness of HTK to UW or other solutions in preservation of the human pancreas for use in pancreatic islet transplantation. Celsior (SangStat Medical, Menlo Park, California, U.S.A.) is also a low viscosity, “extracellular”-type preservation solution. Studies of segmental pancreas autotransplantation in Landrace pigs have demonstrated that Celsior provides adequate pancreas preservation for at least 24 hours (21,22). Another study of allogeneic heterotopic pancreaticoduodenal transplantation in Gottingen minipigs demonstrated a lower partial pressure of oxygen, more edema, and higher levels of endothelin-1 after transplantation of grafts stored in Celsior as compared to grafts stored in UW solution. No difference in graft blood flow was found between the two groups (23). Only one clinical study of Celsior use in whole pancreas transplantation has been published to date. This study, a prospective randomized trial of 105 whole pancreas transplants performed at the University of Pisa, showed that the use of UW and Celsior produced

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equivalent post-transplant graft function and graft survival (13). No experimental or clinical studies have evaluated the efficacy of Celsior as a preservation solution for pancreata used in pancreatic islet transplantation. Another advance in the procurement of whole pancreata for use in islet transplantation is the introduction of the “two-layer technique” of pancreas preservation (24–30). This technique employs perfluorocarbon (PFC), a synthetic chemical solution with a hydrocarbon base that is effective in transporting oxygen from areas of high partial pressure to areas of low partial pressure. In contrast to hemoglobin, however, the ability of PFC to carry oxygen is not significantly affected by acidosis, alkalosis, or low temperatures (28). By bubbling oxygen through the PFC solution during storage, the stored pancreas is provided with oxygen levels that are adequate to minimize cold ischemic injury. In addition, direct phosphorylation of adenosine provided by the UW solution facilitates the production of adenosine triphosphate (ATP) by the stored pancreas, helping to maintain cell membranes during cold ischemia. The two-layer technique appears to be more effective than UW solution alone in preserving the viability of the pancreatic islets during cold storage (31). Because of the effectiveness in preservation, the two-layer technique has allowed for pancreata to be shipped to distant organ isolation centers (8,32) and may also decrease the costs associated with jet travel often required to ship pancreata to procurement centers expeditiously (33). Optimal pancreas preservation for islet transplantation is addressed in detail in the following chapter. The donor selection criteria and the technique of pancreas procurement for pancreatic islet transplantation differ significantly from the criteria and techniques used in pancreas procurement for whole pancreas transplantation. Such differences are important, as donor selection and procurement of the pancreas in pancreatic islet transplantation are important to both maximizing the likelihood of success of pancreatic islet transplants as well as decreasing the time and money spent on procurements that ultimately result in unsuccessful pancreatic islet isolations. The remainder of this chapter further outlines important aspects of donor selection, specialized equipment needed, and the surgical aspects of pancreas procurement for pancreatic islet transplantation.

TECHNIQUE Selection of Donors to Optimize Pancreatic Islet Yield Selection of optimal donors is the first step in maximizing the pancreatic islet yield that results from pancreas procurement. Clinical studies in the mid-1990s examined the yield and success rate of pancreatic islet procurements using human pancreata. Several predictors of high pancreatic islet

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yield and/or good post-transplant pancreatic islet function have been identified by these studies (Table 1). Among the best studied is donor age. Several studies have demonstrated a better pancreatic islet yield with donors age >15–20 years of age as compared to younger patients. This has been attributed to the inherent difficulty in separating the islets from the surrounding exocrine tissue in younger pancreata (12,34). The poor pancreatic islet yield from donors greater than 50 or 60 years of age that has been demonstrated by some studies has been attributed to pancreatic fibrosis (34,35), but other studies have found that donors older than 50 years of age can also produce good pancreatic islet yields (12). Many centers performing clinical pancreatic islet transplants since 2000 appear to have used donors that were at least 30 years of age (36–38), and a recent series of pancreatic islet transplants using single donors from the University of Minnesota have further restricted eligibility for organ donation to patients less than 50 years old (39). Thus all prospective donors age 18þ should be considered for pancreas procurement for pancreatic islet transplantation, but those 20–50 years of age may provide the optimal yield. The degree of fat accumulation in the donor pancreas is another important consideration. Fatty pancreata not only contain a higher absolute number of pancreatic islets than non-fatty pancreata but are also associated with a higher percentage of recovered islets (31). As such, body mass index (BMI) directly correlates with pancreatic islet yield (12,40–42). Recent series from the Baylor College of Medicine, University of Pennsylvania, and the National Institute of Health transplanted pancreatic islets from donors with a BMI of at least 21 kg/m2 (8,36,37), while the University of Minnesota single donor series used the more selective criterion of BMI >27 kg/m2 (39). A normal donor blood glucose level has also been shown to be associated with a better pancreatic islet yield than donors with hyperglycemia (12,34,35), though often it is not clear if donor hyperglycemia represents occult glucose intolerance, a metabolic response to brain death, or a secondary effect of glucocorticoids given for head trauma. Hyperglycemia in a trauma patient with no known history of hyperglycemia

Table 1

Donor Selection Criteria That May Help to Optimize Pancreatic Islet Yield

Donor age >20 and possibly <50 years old (12,34,35,40,41) Increased body mass index (minimum of >21) (12,35,40,41) Donor normoglycemia (<200 mg/dL) (12,35,40) Absence of hypotension or cardiac arrest (12,40) Traumatic cause of death (12,35)

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or overt diabetes may be further evaluated by obtaining a hemoglobin A1C level; results that are within the normal limit of <6% suggest that the donor has had no pre-existing problems with glucose metabolism. Patients with a known history of Type 1 or Type 2 diabetes mellitus should not be considered as prospective pancreas donors (43). Donors who have not experienced cardiac arrest or hypotension are obviously preferable to those that have (12,40). Ten minutes of cardiac arrest should be the upper limit allowed when considering a prospective donor for pancreas procurement for use in pancreatic islet transplantation. In addition, the length of hospitalization prior to procurement is negatively correlated with pancreatic islet yield, a relationship attributed to a decreased ability of the pancreas to expand when infused with collagenase solution. This correlation may be the effect of poor nutrition on prospective organ donors (35). Donor brain death due to trauma has been associated with improved pancreatic islet yields versus donor brain death due to intracranial hemorrhage (12,34), but this association may be confounded by the younger aged patients that are typically found in the former group (34). Finally, septicemia, malignancies other than brain tumors, and other contraindications that typically preclude the harvesting of other solid organs should also be used as exclusion criteria for pancreas procurement for pancreatic islet transplantation. Many experimental studies of both whole pancreas transplants (44,45) and pancreatic islet transplants (46) have suggested that administering corticosteroids to the prospective organ donor may improve the function of the transplanted pancreatic islets, but no clinical evidence supports this practice in pancreatic islet transplantation. In contrast to life-saving transplants such as liver or heart, pancreatic islet transplants are not curative. At many transplant centers the procedure is considered experimental, and thus many transplant clinicians are very conservative in exposing prospective pancreatic islet recipients to risk of blood-borne infection. Serological testing of prospective donors for pathogens such as human immunodeficiency viruses 1 and 2, hepatitis B and C viruses, human T-lymphotrophic virus, and cytomegalovirus is standard in most organ procurement organizations, but serology results may be confounded if the prospective organ donor has received moderate or large volumes of crystalloid solution and/or blood products. Prospective donors should therefore be excluded from consideration in pancreas donation for pancreatic islet transplantation if, during the 48 hours preceding serological testing, either: (1) the total volume of crystalloids and colloids exceeds the prospective donor’s plasma volume (estimated as donor weight in kilograms divided by 0.025 ml/kg); or (2) the total fluid volume given (i.e., blood products, crystalloid, and colloid) exceeds the prospective donor’s blood volume (estimated in ml as donor weight in kg divided by 0.015) (47).

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Table 2

Additional Equipment Needed to Optimize Pancreas Procurement for Pancreatic Islet Transplantation Not Typically Required for Standard Multi-Organ Procurements

Wire mesh screen (800–1,000 µm diameter) Perfluorochemical (PFC; Fluoromed, LP, Round Rock, Texas, U.S.A.) Nalgene jar (Nalge Nunc Intl., Rochester, New York, U.S.A.) Sterile arterial line tubing Intravenous cannula Additional sterile isolation bags, sterile ice slush, labels, University of Wisconsin solution

Preparation Prior to Procurement Prior to departure to procure a pancreas for use in pancreatic islet transplantation, additional equipment not found in the typical operating room must be gathered. This additional equipment is listed in Table 2. The regional organ procurement organization should also be notified regarding the need for additional equipment. As would be expected, pancreata with short cold ischemia times (<6–8 hours) provide better yields than pancreata with longer cold ischemia times (12,34,35,40,41). Likewise, minimizing warm ischemia time will improve the likelihood of success (40,41). Two previous studies have also demonstrated that local procurement teams have greater success than distant procurement teams, although the reason for this is unclear (12,35). Thus, transportation to and from the pancreatic islet isolation center should be arranged prior to harvesting to minimize cold ischemia. Upon arrival at the operating room, the surgeon should confirm the diagnosis of brain death, the serology results, and the ABO blood group of the donor. A signed form documenting informed consent for the organ harvesting procedure should also be present in the chart. The patient should be positioned and prepared in a fashion similar to that of typical multiorgan harvesting procedures. Additional preparatory steps are needed: first, a nasogastric tube should be placed prior to the operation to decompress the stomach. 300mL of a mixture of 20% povidone-iodine and normal saline is infused into the duodenum before the laparotomy is begun, then pulled back into the stomach. Next, a container should be prepared on the back table to allow the pancreas to be preserved using the two-layer method immediately after procurement. A sterilized clear plastic Nalgene jar (Nalge Nunc Intl., Rochester, New York, U.S.A.) should be placed within a larger plastic basin containing ice slush. Approximately 333 ml of perfluorochemical (PFC; Fluoromed, LP, Round Rock, Texas, U.S.A.) should first be added to the container. Approximately 400 ml University of Wisconsin solution is slowly poured on top of the PFC. A difference in the density of the two solutions

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will maintain an interface between them (Fig. 1). The oxygen content of the PFC solution should then be maximized by bubbling oxygen into the solution for at least 30 minutes. This bubbling may be begun when the surgeon is procuring the pancreas and is best achieved by connecting a pressurized O2 tank to nasal cannula tubing, which can then be connected to a sterilized arterial line tubing. A pair of hemostats can assure that the cannula and distal end of the tubing stay properly placed below the PFCUW solution interface and deep into the PFC solution (Fig. 2). The valve of the oxygen tank should be loosened just enough to provide gentle bubbling of oxygen into the PFC solution (typically a rate of about 2 L/min).

Exposure for Multi-Organ Harvesting Skin preparation (topical antimicrobial) solution should be applied between the donor’s chin and anterior thigh extending as far laterally as possible. The abdomen and chest are entered via a midline incision from suprasternal notch to pubis that is typical of multi-organ harvests. If the liver is to be

Figure 1 Schematic diagram demonstrating the two-layer method for preserving the whole pancreas for use in pancreatic islet transplantation.

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Figure 2 Sterilized IV tubing providing bubbling oxygen to the PFC layer in the Nalgene jar (Nalge Nunc Intl., Rochester, New York, U.S.A.). Hemostats may be used to keep the tubing raising to the surface. The Nalgene jar should also be surrounded by ice slush to maintain the preservation solution at 4 ˚ C.

procured as well, dissection of the liver should precede the dissection of the pancreas.

Dissection of Pancreas Prior to Cross-Clamping First, the crura of the diaphragm should be incised to allow for mobilization of the supraceliac aorta proximal to the celiac trunk. Once this segment is adequately mobilized, umbilical tape should be placed around the aorta. Then, the lesser sac is opened by dividing the gastrocolic ligament (Fig. 3). Any attachments between the posterior surface of the stomach and the anterior surface of the pancreas should be divided with sharp dissection. Next, the stomach is retracted in the cephalad direction, and the short gastric arteries and veins are ligated and divided to completely free the

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Coronary vein

Liver

Pancreas

Gallbladder

Duodenum

Figure 3

Open the lesser sac.

stomach from the spleen. As the spleen is mobilized the lienocolic and splenophrenic ligaments are divided. Further dissection should completely free the spleen from all attachments except those medial attachments between the spleen and the tail of the pancreas. At this point, the spleen can easily be lifted and retracted further towards the midline (7,48–50). The colon should also be mobilized from the hepatic to the splenic flexure (48). If the kidneys will also be retrieved for transplantation, the surgeon may also consider mobilizing the entire left colon to allow for the identification and mobilization of the kidney and left ureter. At this point the gross appearance of the pancreas can be assessed. Ideally, the pancreas should appear well-perfused and without any evidence of venous congestion or trauma. The presence of peripancreatic fat is acceptable. The duodenum and proximal pancreas should next be mobilized by performing a Kocher maneuver (49). If not already done, the porta hepatis should be dissected. The gastroduodenal artery should be left patent during this dissection. The middle colic vessels are doubly ligated and divided, but manipulation of all arterial inflow to the pancreas should be avoided in an attempt to preserve blood flow to the pancreas (Fig. 4). The first and fourth portions of the duodenum are divided with a GIA linear cutting stapler (EndoGIA, U.S. Surgical, Norwalk, Connecticut, U.S.A.). The spleen and tail of the pancreas are then lifted and brought towards midline to facilitate dissection of the posterior pancreas from the retroperitoneum. This is done carefully to minimize arterial vasospasm and vascular injury (Figs. 5 and 6). Injury to the pancreas during the procurement process may lead to difficulties in perfusing the pancreas with collagenase during the pancreatic islet isolation process. The superior mesenteric artery, the gastroduodenal artery, and the splenic artery should be left intact to avoid vasospasm/vascular injury and

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Figure 4

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Staple the duodenum.

minimize ischemia to the pancreas. Likewise, no vessel loops should be placed around these vessels. The aorta should be cannulated immediately proximal to the infrarenal aortic bifurcation in the standard fashion. The venous cannula should be inserted into the inferior mesenteric vein carefully to prevent the tip of the cannula from advancing proximally into the splenic or portal veins; this assures that the venous drainage of the pancreas is not impaired (7).

Spleen

Figure 5

Bring spleen to midline.

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Spleen

Pancreas

Figure 6

Bring spleen to midline.

Perfusion and Cross-Clamping and Retrieval Approximately three liters of UW solution is infused through the distal aortic cannula to provide in situ flush for the pancreas. We maintain a 24 inch height differential between the arterial and venous cannula to allow for venous outflow and avoid venous congestion of the pancreas. Sterile ice slush should be placed both anterior and posterior to the pancreas during the infusion of preservation solution (Fig. 7). The pancreas should ideally be the first organ removed after crossclamping. The common bile duct may be divided just proximal to its intrapancreatic portion. A Carrel patch that includes the origin of the celiac

Figure 7

Embed pancreas in iced saline slush (front and back).

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Duodenum

Pancreas capsule intact

Spleen

Figure 8

Remove the pancreas.

axis and superior mesenteric artery is not necessary; the length of these vessels should be preserved for the cadaveric liver allograft. Likewise, the portal vein should be divided in a fashion that will leave as much length as possible for the liver allograft. The pancreas is removed en bloc with the 2nd, 3rd, and 4th portions of the duodenum as well as the spleen (Fig. 8). Back-Table Preparation and Cold Storage After removal, the pancreas is placed in a bowl with UW solution ice slush (approximately 4 ˚ ) on the back table (Fig. 9). The spleen, duodenum, and

Figure 9

Keep cold.

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peripancreatic fat may be removed from the pancreas ex vivo at this point or may alternatively be removed at the pancreatic islet isolation center. When the back-table preparation is complete, the pancreas is then transferred to the Nalgene jar for transportation to the pancreatic islet isolation facility. Insertion of a sterilized 800–1,000 µm diameter wire mesh screen cut to fit securely within the jar ensures that the organ remains at the UW/PFC interface (Fig. 1). SUMMARY Several important differences exist between pancreas procurement for whole pancreas transplantation and pancreas procurement for pancreatic islet transplantation. Optimizing the yield of pancreatic islets begins with selecting good donors. Specifically, the chances of success may be maximized by selecting donors that are between 20 and 50 years of age, have a high body mass index, have maintained euglycemia (blood glucose level of <200 mg/dL (11.1 mmol/L), have experienced no or very brief periods of hypotension/ cardiac arrest, and have received no or few blood products. Specialized equipment not required for typical multi-organ transplant harvests should be gathered well before procurement of the pancreas for pancreatic islet transplantation. This equipment includes a sterilized 800–1,000 µm diameter wire mesh screen, perfluorochemical (PFC) solution, an extra Nalgene jar, additional UW solution, and ice slush. Additional equipment such as arterial line tubing and oxygen cannulae needed for the “two-layer” technique of pancreas preservation are typically available in most operating rooms. Although the technique of pancreatectomy resembles that used in pancreas procurement for whole pancreas transplantation, some important differences exist when pancreas procurement is undertaken for pancreatic islet transplantation (Table 3). In particular, manipulation of the pancreas Table 3 General Surgical Principles in the Procurement of a Whole Pancreas for Use in Pancreatic Islet Transplantation Minimize manipulation of the pancreas during all phases of procurement. Keep the pancreatic capsule intact. Maximize blood flow to the pancreas, keeping the superior mesenteric artery, the gastroduodenal artery, and the splenic artery patent. Prevent venous congestion of the pancreas during infusion of University of Wisconsin (UW) solution by keeping a 40 cm height differential between the arterial and venous cannulae. Minimize warm ischemia to the pancreas by packing ice slush both anterior and posterior to pancreas and doing all ex vivo work with pancreas submerged in 4 ˚ C UW solution. Utilize a local procurement team when possible.

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should be minimized. The surgeon should also extend every possible effort to maintain the capsule of the pancreas intact, as an intact capsule facilitates later distention of the pancreas with collagenase and increases the pancreatic islet yield (41). The blood flow to the pancreas should be maintained prior to cross-clamping of the donor aorta by keeping the superior mesenteric artery, the gastroduodenal artery, and the splenic artery patent. Finally, venous congestion of the pancreas during the infusion of UW solution should be avoided by keeping a height differential of about 40 cm between the arterial and venous cannulae.

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13. Boggi U, Vistoli F, DelChiaro M , et al. Pancreas preservation with University of Wisconsin and Celsior solutions: a single-center, prospective, randomized pilot study. Transplantation 2004; 77:1186–1190. 14. Fridell JA, Agarwal A, Milgrom ML, Goggins WC, Murdock P, Pescovitz MD. Comparison of histidine-tryptophan-ketoglutarate solution and University of Wisconsin solution for organ preservation in clinical pancreas transplantation. Transplantation 2004; 77:1304–1306. 15. Bretschneider HJ. Myocardial protection. Thorac Cardiovasc Surg 1980; 28: 295–302. 16. Chan SC, Liu CL, Lo CM, Fan ST. Applicability of histidine-tryptophanketoglutarate solution in right lobe adult-to-adult live donor liver transplantation. Liver Transpl 2004; 10:1415–1421. 17. Potdar S, Malek S, Eghtesad B, et al. Initial experience using histidinetryptophan-ketoglutarate solution in clinical pancreas transplantation. Clin Transplant 2004; 18:661–665. 18. Hesse UJ, Troisi R, Jacobs B, et al. Cold preservation of the porcine pancreas with histidine-tryptophan-ketoglutarate solution. Transplantation 1998; 66: 1137–1141. 19. Leonhardt U, Tytko A, Exner B, et al. The effect of different solutions for organ preservation on immediate postischemic pancreatic function in vitro. Transplantation 1993; 55:11–14. 20. Troisi R, Meester D, VanDen BC , et al. Functional and structural integrity of porcine pancreatic grafts subjected to a period of warm ischemia and cold preservation with histidine-tryptophan-ketoglutarate (custodiol) or University of Wisconsin solution. Transplantation 2003; 75:1793–1799. 21. Baldan N, Rigotti P, Furian L, et al. Pancreas preservation with Celsior solution in a pig autotransplantation model: comparative study with University of Wisconsin solution. Transplant Proc 2001; 33:873–875. 22. Baldan N, Parise P, Furian L, et al. Swine pancreas preservation with Celsior solution. Transplant Proc 2000; 32:29–31. 23. Uhlmann D, Armann B, Ludwig S, et al. Comparison of Celsior and UW solution in experimental pancreas preservation. J Surg Res 2002; 105: 173–180. 24. Tsujimura T, Kuroda Y, Avila JG, et al. Influence of pancreas preservation on human islet isolation outcomes: impact of the two-layer method. Transplantation 2004; 78:96–100. 25. Tsujimura T, Kuroda Y, Churchill TA, et al. Short-term storage of the ischemically damaged human pancreas by the two-layer method prior to islet isolation. Cell Transplant 2004; 13:67–73. 26. Ricordi C, Fraker C, Szust J, et al. Improved human islet isolation outcome from marginal donors following addition of oxygenated perfluorocarbon to the cold-storage solution. Transplantation 2003; 75:1524–1527. 27. Tsujimura T, Kuroda Y, Kin T, et al. Human islet transplantation from pancreases with prolonged cold ischemia using additional preservation by the two-layer (UW solution/perfluorochemical) cold-storage method. Transplantation 2002; 74:1687–1691.

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28. Lakey JR, Tsujimura T, Shapiro AM, Kuroda Y. Preservation of the human pancreas before islet isolation using a two-layer (UW solution-perfluorochemical) cold storage method. Transplantation 2002; 74:1809–1811. 29. Kuroda Y, Tanioka Y, Morita A, et al. Protective effect of preservation of canine pancreas by the two-layer (University of Wisconsin solution/ perfluorochemical) method against rewarming ischemic injury during implantation. Transplantation 1994; 57:658–661. 30. Kuroda Y, Kawamura T, Suzuki Y, Fujiwara H, Yamamoto K, Saitoh Y. A new, simple method for cold storage of the pancreas using perfluorochemical. Transplantation 1988; 46:457–460. 31. Lakey JR. Pancreas Donor Criteria. Presentation at the 4th Human Islet Isolation and Transplantation Techniques (HITT) Training Congress. 2003. 32. Goss JA, Schock AP, Brunicardi FC, et al. Achievement of insulin independence in three consecutive type-1 diabetic patients via pancreatic islet transplantation using islets isolated at a remote islet isolation center. Transplantation 2002; 74:1761–1766. 33. Hering BJ, Matsumoto I, Sawada T, et al. Impact of two-layer pancreas preservation on islet isolation and transplantation. Transplantation 2002; 74: 1813–1816. 34. Zeng Y, Torre MA, Karrison T, Thistlethwaite JR. The correlation between donor characteristics and the success of human islet isolation. Transplantation 1994; 57:954–958. 35. Benhamou PY, Watt PC, Mullen Y, et al. Human islet isolation in 104 consecutive cases. Factors affecting isolation success. Transplantation 1994; 57:1804–1810. 36. Markmann JF, Deng S, Huang X, et al. Insulin independence following isolated islet transplantation and single islet infusions. Ann Surg 2003; 237: 741–749. 37. Hirshberg B, Rother KI, Digon BJ, III, et al. Benefits and risks of solitary islet transplantation for type 1 diabetes using steroid-sparing immunosuppression: the National Institutes of Health experience. Diabetes Care 2003; 26: 3288–3295. 38. Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000; 343:230–238. 39. Hering BJ, Kandaswamy R, Ansite JD, et al. Single-donor, marginal-dose islet transplantation in patients with type 1 diabetes. JAMA 2005; 293:830–835. 40. Toso C, Oberholzer J, Ris F, et al. Factors affecting human islet of Langerhans isolation yields. Transplant Proc 2002; 34:826–827. 41. Brandhorst D, Hering BJ, Brandhorst H, Federlin K, Bretzel RG. Influence of donor data and organ procurement on human islet isolation. Transplant Proc 1994; 26:592–593. 42. Matsumoto I, Sawada T, Nakano M, et al. Improvement in islet yield from obese donors for human islet transplants. Transplantation 2004; 78:880–885. 43. Deng S, Vatamaniuk M, Huang X, et al. Structural and functional abnormalities in the islets isolated from type 2 diabetic subjects. Diabetes 2004; 53:624–632.

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44. Tersigni R, Toledo-Pereyra LH, Najarian JS. Effects of methylprednisolone, glucagon, and allopurinol in the protection of pancreaticoduodenal allografts perfused for twenty-four hours. Surgery 1975; 78:599–607. 45. Kyriakides GK, Arora VK, Lifton J, Nuttall FQ, Miller J. Porcine pancreatic transplants. II. Allotransplantation of duct ligated segments. J Surg Res 1976; 20:461–466. 46. Toledo-Pereyra LH, Zammit M, Cromwell PW, Malcom SE. Improvement of islet cell transplant survival with reduced number of islets cells after donor pretreatment with methylprednisolone and glucagon. J Surg Res 1980; 29: 302–308. 47. University of Miami Diabetes Research Institute. Information for pancreatic islet transplant coordinators. 2003. 48. Brayman KL, Sutherland DER, Najarian JS. Pancreatic transplantation. In: Trede M, Carter DC, editors. Surgery of the Pancreas. New York: Churchill Livingstone, 1993. 49. Sutherland DER, Ascher NL. Whole pancreas donation from a cadaver. In: Simmons RL, Finch ME, Ascher NL, Najarian JS, editors. Manual of Vascular Access, Organ Donation and Transplantation. New York: SpringerVerlag, 1984. 50. Toledo-Pereyra LH. Pancreas harvesting and preservation techniques. In: Toledo-Pereyra LH, editor. Pancreas Transplantation. Boston: Kluwer Academic Publishers, 2005.

5 Pancreas Preservation for Islet Isolation Mohammadreza Mirbolooki and Jonathan R. T. Lakey Department of Surgery, Walter C. Mackenzie Health Center, University of Alberta, Edmonton, Alberta, Canada

INTRODUCTION The evolution of clinical islet transplantation has made islet allografting a practical treatment for patients with Type 1 diabetes. However, in its present form, it can benefit less than 0.5% of all affected patients (1). Offsetting this success is the increasing discrepancy between the availability of, and demand for, transplantable islets. Pancreas preservation method has been reported as a major factor affecting the quality of islets for transplantation (2). Suppression of cellular metabolism by hypothermia is the main method being used to preserve pancreas. During organ removal, blood supply is necessarily interrupted and should be replaced with an appropriate hypothermic preservation solution. The composition of this solution is critical to make the hypothermic storage tolerable maintaining pancreatic viability. During the last three decades, several solutions have been used for hypothermic preservation of pancreas. These solutions have been designed to address biological and physiological requirements for survival in a low-temperature environment. They differ in basic ionic composition, which can reflect either intracellular (rich in Kþ), including standard University of Wisconsin (UW), Custodiol HTK (Dr. F. Ko¨hler Chemie, Alsboch-Ha¨hnlein, Germany), Los Angeles preservation solution (LAP-1), and EuroCollins solution; or extracellular environment (rich in Naþ) including Celsior solution (Stag Stat Medical Corp., Menlopark, California, U.S.A.). Some of these solutions have been modified by adding nutrients, antioxidants, and anti-apoptotic agents to improve the current outcomes of islet isolation. In this chapter, we 99

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first discuss the principles of pancreas preservation and then review the advantages and disadvantages of different solutions.

METABOLIC CHANGES DURING HYPOTHERMIA The principle of cell preservation is ingrained in the observation that life processes are temperature-dependent chemical reactions, whose sum is the metabolism. Since the 1950s, hypothermia has been known to provide considerable protection against ischemic damage (3). Kinetic predictions indicate that most enzymes of normothermic animals will show a 1.5- to 2fold decrease in metabolic activity for every 10 ˚ C decrease in temperature (4). A decrease in temperature from 37 to 4 ˚ C will decrease the metabolism by 12-fold (5). Consequently, the preferred method for long-term organ preservation is through reduced temperature. Hypothermia does not stop metabolism but slows biochemical reactions. However, underlying deleterious effects of low-temperature preservation may counteract the obvious benefits of extended hypothermic preservation. It is generally thought that the best range of temperatures for cold preservation is at 0–4 ˚ C. This is based on the premise that the water content of a cell would freeze below 0 ˚ C and thus the cellular structure would be destroyed (6). There have been few studies that have specifically addressed the subject of the optimal temperature for short-term organ preservation. Belzer et al. showed that the viability of cultured kidney tubule cells was highest at 6 ˚ C (7). Creatinine clearance post-transplant was significantly improved if the rabbit’s kidney was perfusion-preserved at 8 ˚ C rather than at 0 ˚ C (8). Continuous liver perfusion at 10 ˚ C for 24 hrs showed significantly higher tissue adenine nucleotide levels than those following continuous perfusion at 4 ˚ C (9). We previously showed that both the yield and function of islets obtained from pancreata stored at 7–10 ˚ C was superior to those obtained from pancreata stored at 4 ˚ C (10). However, Inui et al. found that
0.6 ˚ C is superior to 4 ˚ C for cold islet preservation (11). In their study, both the static challenge and the stimulation index were significantly higher in islet cells stored at –0.6 ˚ C as compared to those at 4 ˚ C. A temperature of approximately 4 ˚ C remains the most feasible temperature for hypothermic pancreas preservation. Physiologically, cells are immersed in an extracellular solution high in sodium and low in potassium. This is maintained by the Na-K ATPase pump, which uses adenosine triphosphate (ATP) derived from oxidative phosphorylation in mitochondria. Hypothermic preservation reduces the activity of the membrane ion exchange mechanisms, specifically the Na-K ATPase system, which consumes one third of the total cell energy at body temperature (12). Temperature reduction leads to an intracellular accumulation of sodium, leading to an osmotic increase in cell water content, or

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so-called “cold swelling” (13). Cold-induced acceleration of Na–H exchange is an additional route for sodium influx (14). If cold-induced accumulation of sodium is unchecked, the rise in cytosolic sodium will ultimately lead to membrane depolarization, opening of voltage-dependent Ca2þ channels, rapid influx of calcium, and initiation of membrane phospholipid hydrolysis (Fig. 1) (15). Once initiated, the pathological series of effects leading to necrotic cell death during hypothermia is largely uncontrollable and analogous to irreversible membrane injury. To solve this problem, colloids are added to preservation solution to match osmotic pressure within the intracellular compartment and compensate the tendency to cell swelling. Impermeants that remain within the extracellular compartment are also used for this purpose and are either saccharides, including raffinose, lactobionate, mannitol, and glucose, or anions such as phosphate, sulfate, citrate, and gluconate. METABOLIC CHANGES IN HYPOXIA Hypoxia exists when oxygen delivery does not meet the demands of the pancreas. During pancreas procurement, blood supply and hence oxygen is

Figure 1 ATP turnover of cells as a function of time exposed to anoxia or hypothermia. Abbreviations: Mito, mitochondria; ER, endoplasmic reticulum. Source: Ref. 16.

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necessarily interrupted. Therefore, cells cannot continue to meet the energy demands of active ion-transporting systems leading to similar consequences to hypothermia (Fig. 1) (16). In early stages of ischemia, cellular ATP demands tend to remain constant. This leads to an energetic deficit that can only be made up for by activation of anaerobic ATP supply pathways. Anaerobic ATP production cannot sustain the pre-existing energy demands because of the rapid depletion of fermentable substrate together with the accumulation of deleterious end-products (e.g., lactate and Hþ). Mismatch balance between supply and demand of ATP eventually results in further suppression of mitochondrial oxidative phosphorylation and, consequently, necrosis and apoptosis (17). Moreover, accumulation of lactic acid due to anaerobic glycolysis results in tissue acidosis, which is deleterious to normal cell function. During ischemia, in addition to the usual pathway for lactic acid production, lactate dehydrogenase (LDH) converts pyruvate to lactic acid. The high concentrations of lactic acid not only injure cells directly but can also activate macrophages, leading to cytokine production and the initiation of an inflammatory response (18). To prevent acidosis, hydrogen ion buffers are used in pancreas preservation solutions, including potassium phosphate, sodium bicarbonate, magnesium sulfate, and histidine. When blood flow and the supply of oxygen is re-established, the availability of oxygen to tissues that have accumulated anaerobic metabolites leads to the production of harmful oxygen free radicals. The production of free radicals occurs via the hypoxanthine–xanthine oxidase reaction and can contribute to cell injury by participating in lipid peroxidation (Fig. 2). These radicals can cross-link membrane proteins, cleave peptide bonds, alter the function of glycosaminoglycans, and promote DNA disruption. In addition, prolonged ischemia can deplete the tissue of protective antioxidants. Administering exogenous antioxidants like glutathione has long been known to play an important role in protecting the ischemic tissue from reperfusion injury. Several preservation fluids

Figure 2

Mechanism of ischemia reperfusion injury.

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include glutathione as a specific additive to limit oxygen free radical injury (19). Reduced glutathione combines with reactive oxygen species and free radicals to minimize oxidative injury to tissues. Other antioxidants and free radical scavengers used in various preservation fluids include superoxide dismutase, allopurinol, prostaglandin synthesis inhibitors, and vitamin E (a lipid soluble antioxidant).

CURRENT PRESERVATION SOLUTIONS EuroCollins Solution In the early 1970s, a hypothermic hyperosmolar solution with an ionic composition broadly comparable with intracellular fluid, but without colloid additives (Table 1), was introduced. This solution has been shown to successfully to preserve canine kidneys for over 48 hours (20). The first Collins solutions had high concentrations of potassium, magnesium sulfate, and glucose. To increase the osmolality of Collins solution, the glucose concentration was raised [EuroCollins solution (EC)]. However, the use of glucose to maintain increased extracellular osmolality proved less than optimal for two reasons. First, long-term preservation of organs is associated with increased glucose permeability. The gradual diffusion of glucose into the intracellular space diminishes osmolality of the solution and cannot be relied upon to prevent cell edema (21). Secondly, glucose is the initial substrate for anaerobic glycolysis, which takes place during hypothermia. If glucose is available in large amounts, it may exacerbate tissue acidosis. Later, EC solution became a standard solution for cold storage of kidney (22). Thereafter, several papers reported that pancreas preservation could be improved in vitro by protection with alternative solutions as compared to EC solution. In 1990, a study compared the preservation of porcine pancreas by the standard EC solution with that by the cardioplegic histidine-tryptophan-ketoglutarate solution (HTK). Protection with HTK significantly improved pancreas preservation after 24 hr ischemia by lowering lactate content in the reperfusate and increasing arteriovenous flow rate and pancreatic oxygen consumption (23). Three years later, the same group showed that immediate post-ischemic function was similar in HTK and UW preserved pancreata (24). During reperfusion with a constant arterial pressure, the arteriovenous flow rate and oxygen consumption were significantly higher for HTK and UW than for EC. The lowest lactate content in the reperfusate was found after HTK protection. Amylase in the venous effluent was significantly lower for HTK or UW protection than for EC. In 2003, preservation of pancreas was assessed by X-ray microanalysis and showed the best calcium retain (indicates differences in preservation of secretory granules) in UW and least in EC (25). EC is thus currently

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Table 1

Mirbolooki and Lakey Components and Their Concentrations of Each Solutiona UW

Membrane stabilizers Lactobionate Raffinose Manitol HES Tryptophane Glucose Buffers Phosphate Histidine HCo3Energy substrates Magnesium Adenosine Ketoglutarate FRIs and ORSSs Gluthation Allopurinol SOD Nicotinamide Glutamate Electrolytes Naþ Kþ Caþ Sulphate Chloride Hydroxide Osmolality PH Additives Insulin Penicillin Dexamethasone

HTK

100 30 30

LAP-1

Celsior

100

80

30

60

EC

50 g/L 2 182 25

15 198

57.5 30 10

5 5

4

5

13

1 3 1 30  103 U/L 5 20 30 120

15 10 0.015

5

30 120 5

28 100 320 7.4

100 15 0.25

310 7.4

10 115 15

28 100 320 7.3

340 7.3

355 7.4

100 U/L 8 mg/L

a All units are mmol/L unless otherwise stated. Abbreviations: EC, EuroCollins solution; FRI, free radicals inhibitors; HES, hydroxyethyl starch; HTK, histidine-tryptophan-ketoglutarate solution; LAP-1, Los Angeles preservation solution; ORS, oxygen reactive species scavengers; SOD, superoxide dismutase; UW, University of Wisconsin solution.

considered inferior to other preservation solutions such as UW and HTK and is no longer employed for clinical pancreas preservation (26). It is, however, used as a preservation solution during pancreas cannulation in islet isolation procedures (27).

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Histidine-Tryptophan-Ketoglutarate Solution (Bretschneider) HTK solution, developed in the 1970s by Bretschneider as a cardioplegia solution (28), is being used increasingly for both kidney (29) and liver (30) transplantation. HTK contains less potassium and sodium and a strong histidine buffer that increases the osmotic effect of mannitol, which is also included in this solution. Tryptophan is added as a membrane stabilizer, and ketoglutarate is added as a metabolism substrate (Table 1). HTK solution has been used successfully in experimental pancreas transplantation with results comparable to those of UW solution (31). Using this solution showed that minimal periods of warm ischemia and cold preservation had little impact on graft viability. Although there has been some concern about cold preservation of pancreatic grafts beyond 24 hr, in a clinical study it has been shown that in patients with demonstrated cold ischemic times no longer than 15 hr, the only significant difference between UW and HTK solutions was the greater volume of HTK solution used. This is an anticipated finding because HTK is less viscous than UW solution. Despite the greater volume of solution used, HTK preservation remained less costly than UW solution with a similar clinical result (32). Pig models also showed that it is possible to flush and store porcine pancreatic grafts for 24 hr with HTK or UW solution and to transplant these grafts with good metabolic function, suggesting that cold storage with HTK solution is a safe preservation method even for pancreatic grafts (33). Cold preservation with HTK and UW is feasible for 48 hr; however, the success rate is equally reduced with HTK and UW solution. Cold storage for 72 hr in either HTK or UW solution results in uniform graft failure (34). There are several centers that currently use HTK as their routine pancreas preservation solution.

University of Wisconsin Solution Research efforts by one of the pioneers of organ preservation, Folkert O. Belzer, and his colleague, James H. Southard, resulted in a development of a preservation solution in the late 1980s based on five philosophies (4). They developed UW solution containing impermeants (raffinose, lactobionate) to minimize hypothermic induced cell swelling, buffers (phosphate) to prevent intracellular acidosis, colloid (hydroxyethyl starch) to prevent the expansion of interstitial space during flush-out period, free radical inhibitors and scavengers (glutathione, allopurinol) to prevent injury from oxygen free radicals during ischemia and after reperfusion, and energy precursors (Mgþ, adenosine) for energy metabolism during reperfusion period. They also added vasoactive agents and hormones (steroid, insulin) and penicillin as an antibiotic agent (Table 1). In 1990, it was demonstrated that the UW solution is superior to commonly used solutions for storage of the pancreas prior to islet isolation in rodent models and human tissue up to 20 hours

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after procurement with respect to both the number of islets recovered, and the in vitro functional integrity of those islets (35). However, modified UW solutions showed better results in some studies. As an example, the high-Naþ-histidine solution has been reported to be superior to standard UW solution for rat pancreas preservation. This is probably due to the buffer, histidine, which prevented the acidosis of ischemic tissue during the period of preservation. This solution had higher sodium content and lower potassium content. Adenosine, insulin, hydroxyethyl starch, and dexamethasone, which are components of the UW solution, were not present in this modified solution. Functional success rates in diabetic rats receiving pancreata that had been preserved in high-Naþ-histidine and in high-Naþlactobionate solutions at 4 ˚ C were 100% after 48-hr preservation. By contrast, standard UW solution gave only a 44% success rate after 48-hr preservation (36). Further studies suggested that, although a colloid may not be essential for short-term preservation of kidney and liver, for consistently successful 48-hr preservation of the pancreas, hydroxyethyl starch is an important component of the UW solution (37). However, replacement of hydroxyethylstarch by dextran-40 in UW solution has also shown successful 72-hr preservation of the canine pancreas (38). UW was originally formulated for preservation of the pancreas. However, it was quickly adopted by many centers as an excellent solution for the cold preservation of liver and kidney as well. The introduction of UW solution into clinical practice allowed the safe and extended preservation of the pancreas for up to 30 hr with good graft function (39). Soon, it became the standard against which to judge any new preservation solution intended to be used in pancreas transplantation. Two-Layer Method In the late 1980s, Kuroda et al. developed a two-layer cold storage method (TLM) using perfluorocarbon (PFC) and UW solution for whole-pancreas preservation (40). PFC is a hyperoxygen carrier designed to release oxygen into the surrounding tissue more effectively. PFC differs from hemoglobin preparations in that it is a synthetic compound formed on a liquid hydrocarbon base. In contrast to hemoglobin, oxygen is not chemically bound to the PFC carrier. PFC takes up and releases oxygen following Henry’s linear law, on the basis of the partial pressure of the gas, rather than Barcroft’s sigmoid curve described for hemoglobin (Fig. 3)(41). Unlike hemoglobin, acidosis, alkalosis, and temperature seem to have no or little effect on the oxygen delivery of PFC, allowing this compound to be used effectively during cold storage of organs. As PFC is immiscible with aqueous systems and has a high density, it is clearly separated from UW. The two-layer (UW solution–PFC) cold storage method continuously supplies oxygen to a pancreas during preservation and reduces cold ischemic

Pancreas Preservation for Islet Isolation

Figure 3

107

Oxygen diffusion in perfluorocarbon as compared to hemoglobin.

injury (42). Furthermore, canine pancreata subjected to 90 min of warm ischemia were resuscitated during preservation by the two-layer method at 4 ˚ C for 24 to 48 hours (43). One of the mechanisms of this method is to augment ATP, which maintains cellular integrity and controls ischemic cell swelling. The endogenous substrate for ATP synthesis is downregulated during ischemia. However, during the preservation by the two-layer method, ATP is synthesized within the ischemically damaged pancreas by means of the direct phosphorylation of adenosine contained in the UW solution (44). Because ATP is an essential source of energy to repair damaged cells, it is likely that ATP regeneration plays a key role in restoration of the ischemically damaged pancreas during preservation. Hiraoka et al. (45) achieved insulin independence in Type 1 diabetic patients after single-donor islet transplantation using less than 8 hours of cold storage with TLM and new immunosuppression protocols. Also, Ricordi et al. (46) demonstrated significantly improved islet recovery from marginal “older” donors by using TLM. Although the experimental numbers were not large, Matsumoto et al. (47) showed significantly improved islet recovery by using TLM after 6 to 8 hours of cold storage in UW solution with short and prolonged total cold storage time. Recently, Brandhost et al. presented compelling data that oxygenated perfluorocarbon can be used in a one-layer method (OLM) with comparable outcomes to TLM preservation solution (48). After a warm ischemia period of 12 to 15 minutes, pig pancreata were preserved for 7 hours either by OLM (PFC alone, n ¼ 8) or by TLM (PFC þ UW, n ¼ 10)

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and compared to unstored pancreata (n ¼ 6). Pancreata were stored in 600 ml PFC pre-oxygenated for 10 minutes with 100% oxygen at a flow rate of 2 L/min with flow rate of 300 ml/min maintained during preservation. Additional 300 ml UW solution was used for TLM. The authors reported that OLM was significantly superior to TLM in isolation index (calculated as the ratio between IEQ and islet number as an estimate of islet fragmentation) and islet insulin content, but not in pre- and postpurification islet yield, intra-islet ATP content, and final graft function. The article then concluded that the presence of a second layer of UW solution is not essential during oxygenation of the PFC-immersed pancreas, and that their data should be considered for the preservation of human pancreas. However, the article did not address several issues, including confounding factors, control groups, PFC oxygen saturation, and core pancreatic tissue oxygen consumption that may affect this conclusion (49). In a recently published paper, pO2 was measured using fiber optic sensors in the core of porcine pancreatic tissue preserved with TLM in media saturated with 100% oxygen. Experimental measurements verified that pO2 is virtually zero in the core of a 1 cm-thick pancreatic piece preserved with the TLM (50). Accordingly, no beneficial effect of TLM on islet isolation and transplantation outcomes was observed in our recent study (51). Los Angeles Preservation Solution 1 Dr. Kenmochi and his colleagues developed a new cold preservation solution designated Los Angeles preservation solution no. 1 (LAP-1) in 1998 (52). They believed that the major reasons for failure of islet grafts to maintain normoglycemia in patients with diabetes were insufficient islet number and quality. Therefore, they attempted to develop an isolation method that yields high quality islets in larger numbers, paying special attention to the vulnerability of islets to oxidative and mechanical stress. The solution consisted of lactobionate Kþ and D-mannitol as impermeants to inhibit cell swelling by maintaining extracellular osmotic pressure (Dmannitol also is known to have scavenging activity for hydroxy radicals to which b-cells are highly susceptible) (53); KH2PO4 as a component of the buffering system; superoxide dismutase (SOD) which would provide protection against hydroxy radicals released by leukocytes and other cells contained in the pancreas; and nicotinamide, which would enter islet cells that may have been damaged during the isolation process, protect them from further damage, and support their recovery after being transplanted (Table 1). Islets preserved in this solution showed a tendency towards higher insulin content and stimulation index than those preserved in UW solution, although differences were not statistically significant. However, use of a two-step digestion process and LAP-1 cold preservation solution significantly improved the quantity and quality of islets isolated from human

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pancreas (54). Since there have been no recent publications to support the data of Kenmochi et al., further research is required to clarify the efficacy of this solution in comparison to others.

Celsior Solution Celsior is a recently developed extracellular-type, low-viscosity preservation solution specifically designed for heart transplantation (55) and already used in clinical liver (56) and kidney (57) transplantation. Its composition reflects the putative shortcomings of UW: hydroxyethyl starch was omitted, the potassium concentration was decreased, calcium was included, and the magnesium concentration was increased. Histidine was added to improve buffering, offsetting the removal of phosphate buffer as well as scavenging for toxic free radicals, and mannitol replaced raffinose. Moreover, the reduced glutathione present in Celsior is among the most effective antioxidants available for clinical use (Table 1). Three studies compared UW and Celsior solutions for pancreas transplantation in animals and humans but reported conflicting results. Baldan et al. (58), using an autotransplantation model, found that Celsior was an effective alternative to UW for pancreas procurement, whereas Uhlmann et al. (59), using an allotransplantation model, reported that the use of Celsior was associated with increased ischemia-reperfusion (I/R) injury when compared with UW. In a human based study, 105 consecutive procurements were randomized to graft preservation with UW (n ¼ 53) solution or Celsior (n ¼ 52) solution. Mean cold ischemia times were 11.0 ± 2.1 h for UW compared with 10.8 ± 1.8 hr for Celsior. Within the range of cold ischemia time reported in this study, UW and Celsior solutions have similar safety profiles for pancreas preservation (19). The different compositions of UW and Celsior solutions might lead to an increased susceptibility of Celsior-preserved grafts to post-reperfusion edema because of the higher chloride and lower lactobionate concentrations and the lack of hydroxyethyl starch. UW solution is now considered the standard preservation solution for liver, kidney, and pancreas transplantation; however, the rapidly oxidized glutathione in shelf-stored UW solution definitely lacks the protective effect of preventing oxidative injury related to post-reperfusion free radical production provided by oxygen radical scavengers in Celsior.

CONCLUSIONS Although UW solution has proven to be very effective for vascularized pancreas preservation, organ storage in UW solution before islet isolation, for even short periods (less than 4 hours), appears to have a profoundly negative

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impact on islet yield and clinical outcomes. Refined procurement and preservation techniques would allow better allocation of pancreata among islet isolation facilities and organ transplant centers, according to bestmatched clinical need, and more efficient use of limited resources. It seems that all existing preservation solutions, despite major differences in constituents, have broadly similar effects on islets. Perfluorochemicalbased preservation (TLM) is the only method available that supplies oxygen and metabolic substrates to the pancreas during preservation, thereby maintaining cellular integrity and reducing ischemic cell swelling (60). However, one of the potential drawbacks of PFCs (other than cost) is their relative immiscible nature and high specific gravity (1.93 g/ml), which may result in physical injury attributed to forcing a delicate tissue under the surface of a dense immiscible liquid. Although the dependence of perfluorocarbons on Henry’s Law of partial pressures allows the potential for increased oxygen availability, this fact of oxygen delivery also limits the effective use of perfluorocarbons to situations when the partial pressure of oxygen is supranormal. Compelling evidence that PFC alone or in combination with UW overcomes the problems associated with human pancreas preservation before islet isolation is lacking and difficult to obtain. Reliable methods for the detection of ischemically damaged tissue as well as specific markers predictive of successful clinical outcomes are also lacking. Future research should be focused on deeper understanding of the underlying cellular pathways related to hypothermic preservation, facilitating hypothesis-driven evidence-based development of truly optimized preservation methods.

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24. Leonhardt U, Tytko A, Exner B, et al. The effect of different solutions for organ preservation on immediate postischemic pancreatic function in vitro. Transplantation. 1993; 55(1):11–4. 25. Kozlova I, Roomans GM. Preservation of pancreas tissue during cold storage assessed by X-ray microanalysis. Am J Transplant. 2003; 3(6):697–707. 26. Sutherland DE, Gruessner AC, Gruessner RW. Pancreas transplantation: a review. Transplant Proc 1998; 30(5):1940–3. 27. Lakey JR, Kobayashi N, Shapiro AMJ, Ricordi C, Okitsu T. Current human islet isolation protocol. Medical Review Co. Ltd: Tokyo, 2004. 28. Bretschneider HJ. Myocardial protection. Thorac Cardiovasc Surg 1980; 28 (5):295–302. 29. de Boer J, De Meester J, Smits JM, et al. Eurotransplant randomized multicenter kidney graft preservation study comparing HTK with UW and Euro-Collins. Transpl Int 1999; 12(6):447–53. 30. Hatano E, Kiuchi T, Tanaka A, et al. Hepatic preservation with histidinetryptophan-ketoglutarate solution in living-related and cadaveric liver transplantation. Clin Sci (Lond) 1997; 93(1):81–8. 31. Troisi R, Meester D, Regaert B, et al. Physiologic and metabolic results of pancreatic cold storage with Histidine-Tryptophan-Ketoglutarate-HTK solution (Custodiol) in the porcine autotransplantation model. Transpl Int 2000; 13(2):98–105. 32. Fridell JA, Agarwal A, Milgrom ML, Goggins WC, Murdock P, Pescovitz MD. Comparison of histidine-tryptophan-ketoglutarate solution and University of Wisconsin solution for organ preservation in clinical pancreas transplantation. Transplantation 2004; 77(8):1304–6. 33. Hesse UJ, Troisi R, Jacobs B, Berrevoet F, De Laere S, de Hemptinne B. Pancreas preservation with HTK solution in the pig. Transplant Proc 1997; 29 (8):3522–3. 34. Hesse UJ, Troisi R, Jacobs B, et al. Cold preservation of the porcine pancreas with histidine-tryptophan-ketoglutarate solution. Transplantation 1998; 66(9): 1137–41. 35. Kneteman NM, DeGroot TJ, Warnock GL, Rajotte RV. The evaluation of solutions for pancreas preservation prior to islet isolation. Horm Metab Res Suppl 1990; 25:4–9. 36. Urushihara T, Sumimoto R, Sumimoto K, et al. Prolonged rat pancreas preservation using a solution with the combination of histidine and lactobionate. Transpl Int 1992; 5(Suppl 1):S336–9. 37. Ploeg RJ, Boudjema K, Marsh D, et al. The importance of a colloid in canine pancreas preservation. Transplantation 1992; 53(4):735–41. 38. Morel P, Moss A, Schlumpf R, et al. 72-hour preservation of the canine pancreas: successful replacement of hydroxyethylstarch by dextran-40 in UW solution. Transplant Proc 1992; 24(3):791–4. 39. Stratta RJ, Taylor RJ, Gill IS. Pancreas transplantation: a managed cure approach to diabetes. Curr Probl Surg 1996; 33(9):709–808. 40. Kuroda Y, Kawamura T, Suzuki Y, Fujiwara H, Yamamoto K, Saitoh Y. A new, simple method for cold storage of the pancreas using perfluorochemical. Transplantation 1988; 46(3):457–60.

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41. Riess JG. Fluorocarbon-based in vivo oxygen transport and delivery systems. Vox Sang 1991; 61(4):225–39. 42. Kuroda Y, Fujino Y, Morita A, Tanioka Y, Ku Y, Saitoh Y. Oxygenation of the human pancreas during preservation by a two-layer (University of Wisconsin solution/perfluorochemical) cold-storage method. Transplantation 1992; 54(3):561–2. 43. Kuroda Y, Tanioka Y, Morita A, et al. The possibility of restoration of human pancreas function during preservation by the two-layer (University of Wisconsin solution/perfluorochemical) method following normothermic ischemia. Transplantation 1994; 57(2):282–5. 44. Kuroda Y, Hiraoka K, Tanioka Y, et al. Role of adenosine in preservation by the two-layer method of ischemically damaged canine pancreas. Transplantation 1994; 57(7):1017–20. 45. Hiraoka K, Trexler A, Eckman E, et al. Successful pancreas preservation before islet isolation by the simplified two-layer cold storage method. Transplant Proc 2001; 33(1–2):952–3. 46. Ricordi C, Fraker C, Szust J, et al. Improved human islet isolation outcome from marginal donors following addition of oxygenated perfluorocarbon to the cold-storage solution. Transplantation 2003; 75(9):1524–7. 47. Matsumoto S, Qualley SA, Goel S, et al. Effect of the two-layer (University of Wisconsin solution-perfluorochemical plus O2) method of pancreas preservation on human islet isolation, as assessed by the Edmonton Isolation Protocol. Transplantation 2002; 74(10):1414–9. 48. Brandhorst D, Iken M, Brendel MD, Bretzel RG, Brandhorst H. Successful pancreas preservation by a perfluorocarbon-based one-layer method for subsequent pig islet isolation. Transplantation 2005; 79:433. 49. Mirbolooki M, Lakey JR. Oxygen and pancreas preservation. Transplantation. 2006; 81(3):492. 50. Papas KK, Hering BJ, Gunther L, Rappel MJ, Colton CK, Avgoustiniatos ES. Pancreas oxygenation is limited during preservation with the two-layer method. Transplant Proc 2005; 37(8):3501–4. 51. Kin T, Mirbolooki M, Salehi P, et al. Islet isolation and transplantation outcomes of pancreas preserved with University of Wisconsin solution versus two-layer method using pre-oxygenated perfluorocarbon. Transplantation 2006; 82(10):1286–90 52. Kenmochi T, Miyamoto M, Sasaki H, et al. LAP-1 cold preservation solution for isolation of high-quality human pancreatic islets. Pancreas 1998; 17(4): 367–77. 53. Kehrer JP. Free radicals as mediators of tissue injury and disease. Crit Rev Toxicol 1993; 23(1):21–48. 54. Kenmochi T, Miyamoto M, Une S, et al. Improved quality and yield of islets isolated from human pancreata using a two-step digestion method. Pancreas 2000; 20(2):184–90. 55. Vega JD, Ochsner JL, Jeevanandam V, et al. A multicenter, randomized, controlled trial of Celsior for flush and hypothermic storage of cardiac allografts. Ann Thorac Surg 2001; 71(5):1442–7.

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56. Cavallari A, Cillo U, Nardo B, et al. A multicenter pilot prospective study comparing Celsior and University of Wisconsin preserving solutions for use in liver transplantation. Liver Transpl 2003; 9(8):814–21. 57. Baldan N, Toffano M, Cadrobbi R, et al. Kidney preservation in pigs using celsior, a new organ preservation solution. Transplant Proc 1997; 29(8): 3539–40. 58. Baldan N, Rigotti P, Furian L, et al. Pancreas preservation with Celsior solution in a pig autotransplantation model: comparative study with University of Wisconsin solution. Transplant Proc 2001; 33(1–2):873–5. 59. Uhlmann D, Armann B, Ludwig S, et al. Comparison of Celsior and UW solution in experimental pancreas preservation. J Surg Res 2002; 105(2): 173–80. 60. Lakey JR, Tsujimura T, Shapiro AM, Kuroda Y. Preservation of the human pancreas before islet isolation using a two-layer (UW solutionperfluorochemical) cold storage method. Transplantation 2002; 74(12): 1809–11.

6 Aspects and Challenges of Islet Isolation Mohammadreza Mirbolooki, Jonathan R. T. Lakey, Tatsuya Kin, Travis Murdoch, and A. M. James Shapiro Department of Surgery, Walter C. Mackenzie Health Center, University of Alberta, Edmonton, Alberta, Canada

INTRODUCTION The discovery of insulin by Banting and Best in 1922 transformed diabetes from a fatal disease into a chronic incurable illness with major comorbidities and premature death (1). It is well established that tight control of blood glucose is essential to the prevention of microvascular complications. However, even aggressive insulin therapy is unable to control transient variations in blood glucose. Chronic hyperglycemia and peripheral hyperinsulinemia are believed to accelerate diabetic microangiopathy (2). Hence, b-cell replacement has been believed to be the only treatment that reestablishes and maintains long-term glucose homeostasis with near-perfect feedback controls (3). Pancreas transplantation has been the standard therapy for Type 1 diabetes with established or imminent end-stage renal disease for several years (4). The procedure is technically demanding and continues to have significant perioperative mortality and morbidity despite refined surgical techniques, effective immunosuppression modalities, antiviral prophylaxis, and post-transplant monitoring (5). In contrast, islet transplantation, with its reduced antigen load, technical simplicity, and low morbidity, has the potential to prevent chronic transplantation complications, while providing physiologic glucose control.

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Transplanting pieces or extracts of pancreas in patients with diabetes was performed over 100 years ago (6); however, accelerated developments and improved understanding of the issues that face clinical islet transplantation during the last 30 years have led this simple concept to a successful treatment for diabetes. After implantation in patients with Type 1 diabetes, the treatment can provide near perfect, moment-to-moment control of blood glucose, far more effectively than injected insulin. The hope is that, with tighter glucose control, the long-term complications of diabetes may be avoided. Since cells are injected percutaneously into the liver via the portal vein under radiographic guidance, and the transplant procedure avoids major surgery, islet transplantation offers the benefits of whole pancreas transplantation but with less risk. Achieving good islet isolation is one of the most important factors in achieving successful islet transplantation. Islet isolation involves the extraction of islets of Langerhans from organ donors through complex digestion and purification processes. In the following sections, the challenges of the extraction of islets, arguably the most complicated and difficult procedure involved in islet transplantation, are reviewed.

HISTORY OF ISLET ISOLATION Watson Williams performed the first clinical attempt to transplant the islet cells in 1893. He implanted fragments of freshly slaughtered sheep’s pancreas in the subcutaneous tissues of a 13-year-old boy dying of diabetic ketoacidosis. There was temporary improvement in glucosuria before his death three days later (6). In 1902, Ssobolew proposed transplanting only the endocrine tissue, but he had no practical means to separate the islets from the acinar tissue. Consequently, this approach would lie in a neardormant state for more than 60 years (7,8). Prior to the late 1960s, the harvesting of islets for morphological and physiological studies required the painstaking microdissection of rodent pancreata (7). In 1965, the first major development in islet isolation occurred when Moskalewski introduced a mechanical and enzymatic method of dispersing guinea pig pancreatic tissue with collagenase, a fermentation product derived from Clostridium histolyticum (9). In 1967, Lacy and Kostianovsky could disperse minced rodent pancreatic tissue into fragments from which large numbers of islets could then be separated. They distended the pancreas with a balanced salt solution via the pancreatic duct. They then chopped it into small fragments and mechanically agitated with bacterial collagenase enzyme (10). In 1970, Younoszai et al. demonstrated some amelioration of glucosuria and glycemia in diabetic rats by intraperitoneal implantation of allografted islets (8). The first reports of successful islet transplantation in rats with chemically-induced diabetes were published in

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1972 by Ballinger and Lacy (11). Efforts to improve tissue digestion and increase islet yields, however, would be hampered by crude bacterial enzyme preparations and technical obstacles, thus hindering islet transplantation research for almost 30 years. Methods would eventually be developed that enabled the isolation of islets from dogs, pigs, primates, and humans (12). Several methods of dissociating pancreatic tissue have been attempted, including tissue maceration, counter-rotational blades, and Velcro (13,14,15). However, the shear forces created by these methods resulted in excessive islet fragmentation. Gray et al. described a less traumatic method whereby human islets could be separated from the undigested fibrous capsule by gently teasing the gland apart, shaking the tissue with forceps, and then passing the partially collagenase-digested tissue through a series of differently sized needles until the islets were free from the exocrine tissue (16). Ductal collagenase delivery, whether by direct injection (12) or continuous perfusion (Fig. 1) (17), cleaves the connective tissue matrix more readily than any method previously described, although inadvertent islet enzyme

Figure 1 Pancreas digestion. (A) Distention of the pancreas with temperature- and pressure-controlled perfusion. (B) Dissociation by enzymatic and mechanical processes. Source: From Ref. 103.

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penetration still produces significant islet destruction (18). Nonetheless, it was possible to successfully isolate islets from dog (19), pig (20), and monkey (21). Using an automated re-circulating perfusion apparatus based on technology originally described by Horaguchi and Merrell (22), Lakey et al. demonstrated that retrograde intraductal LiberaseTM (Roche, Indianapolis, Indiana, U.S.A.) delivery produced superior islet recovery and islet survival when compared to syringe loading (23). The present-day approach to clinical islet isolation is based on techniques developed in large animal models (24). The technologies currently used in clinical islet cell isolation are based on an automated method that was first introduced in 1988 (25). Using this method, Scharp et al. showed that it was possible to reverse diabetes and obtain insulin independence after human transplantation of islets in 1990 (26).

DONOR SELECTION FOR ISLET ISOLATION Many factors still limit the successful clinical application of islet isolation. Donor factors have proven to be critical determinants of islet isolation results (27). Critical factors in the multi-organ cadaveric donors have been identified as donor age, body mass index (BMI), cause of death, prolonged hypotensive episodes, procurement team, hyperglycemia, frequency and duration of cardiac arrest, and increased duration of cold storage before islet isolation (Fig. 2) (28). The suitability of pancreata from overweight and obese or aged donors has remained controversial. We previously reported that the use of donors with a BMI of more than 25 kg/m2 resulted in a significantly higher islet yield and an increased likelihood of islet isolation success. Brandhorst

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et al. (29) showed that use of lean (BMI < 21) donors was associated with significantly lower islet yields, as compared with normal (BMI, 21–24) and overweight or obese (BMI ≥24) donors. However, Benhamou et al. (30) showed that a lower donor BMI tended to result in better islet recovery and viability, and Zeng et al. (31) reported that obese donors resulted in the worst islet yield, as compared with non-obese donors. It is known that as the degree of obesity or age increases [coinciding with gradually increasing circulating free fatty acid (FFA) levels], after a period of adaptation and glucose intolerance, incidence of Type 2 diabetes increases markedly (32). Accordingly, we hypothesized that the correlation between donor age and BMI with islet yield may not be linear. We found that selecting donors from among those with BMI 28–32 kg/m2 and age 32–64 yrs has 3.5 (CI 95% 1.6–7.9) times likelihood of islet isolation success in comparison to donors without these criteria. We identified donor age and BMI as factors that were positively correlated with isolation success. However, not all organs from obese or older donors will result in a good pancreatic islet yield (33). Recently, we defined a scoring system that has proven to be effective in assessing potential isolation outcome. Combination of donor quality and pancreas quality is given a numerical score from zero to 100 for use in determining the quality of a pancreas for islet isolation (Fig. 3). By analyzing overall pancreas score, a standardized decision can be made on

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Figure 3 (A) The donor variables assessment form. (B) The pancreas physical properties assessment form. Source: From Ref. 34.

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whether to accept or decline the pancreas. Another benefit of the scoring system is that it is a quick and efficient way to monitor and evaluate trends in the quality of donor organs (34). Besides the scoring system, there are absolute donor rejection criteria including clinical or active viral hepatitis (A, B, or C), acquired immune deficiency syndrome (AIDS) or human immunodeficiency virus (HIV) seropositivity, human T-lymphocyte virus (HTLV) type I/II, active viral encephalitis or encephalitis with unknown origin, Creutzfeldt-Jakob disease, rabies, treatment for active tuberculosis, septicemia, dementia, malignancy, diabetes mellitus Type 1 or 2, and serious illness of unknown etiology. Donors with a history of high-risk behaviors are also excluded. CHALLENGES IN PANCREAS DIGESTION The next major advancement in islet isolation technology was in 1988 when Ricordi et al. introduced a tissue dissociation chamber (25). Several stainless steel marbles are added into the shaking chamber, which also contains the pancreas pieces. A stainless steel 500 µm screen is inserted and the system is sealed. The Liberase-HITM solution (Roche) collected from the perfusion unit is added into the dissociation system (Fig. 4, 5) (35). A major obstacle to successful human pancreatic dissociation has been the low enzymatic activity of the bacterial collagenase preparations. The introduction of Liberase HI has helped to eliminate some of the lot-to-lot and intra-lot variability of enzyme effectiveness, and the need for pre-isolation screening. Liberase digestion consistently yields large numbers of islets without compromising functional viability and has become the “gold standard” for islet isolation (36). The chamber is shaken gently vertically with 2 to 3 cm

Figure 4

Continuous palsatile perfusion of enzyme in a perfusion unit.

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Ricordi’s dissociation chamber.

of deflection (100 times/min) to agitate the pancreas. The shaking continues until dithizone-stained free islets are seen in the samples taken at 2-minute intervals. When the integrity and number of islets are acceptable, the enzyme is diluted and cooled. A new method has been reported recently, in which the pancreas remains uncut and is kept intact during collagenase intraductal injection. A large filtration chamber to accommodate whole pancreas, low concentration of collagenase (Liberase HI) for digestion, and large plastic containers for large-scale islet purification are used to improve islet isolation outcome (37). A major obstacle to successful human and canine pancreatic dissociation has been the low enzymatic activity of the bacterial collagenase preparations. Endogenous proteases and their respective inhibitors within donor pancreas have critical roles in the islet isolation process due to their effects on collagenase proteolysis, digestion times, islet yield, and functional viability. Endogenous pancreatic enzyme activity of the donor pancreas increases during the digestion phase. High trypsin levels are associated with poor islet yields and adverse viability and functional outcomes (38). Trypsin is believed to act through the proteolysis of collagenase (39). PefablocTM [4-(2-aminoethyl)-benzene sulfonyl fluoride, hydrochloride] (Roche Molecular Biochemicals, Mannheim, Germany), a broad-spectrum serineprotease inhibitor, has been used successfully in pig and human islet isolations (40). We have previously shown that Pefabloc supplementation during the isolation phase can improve islet recovery from human pancreata with prolonged cold ischemia times (41). There was no significant difference in the enzymatic activity digestion time with or without Pefabloc, suggesting that other non-serine proteases or other mechanisms may be altering

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collagenase activity (42). Recently, significantly higher islet yield in porcine islet isolation has been reported by trypsin inhibition but reduced collagenase inhibition, in comparison to UW solution, with a new designed “M-Kyoto” solution containing a cytoprotective against stress (trehalose) with different Naþ and Kþ concentrations and osmolality in addition to a trypsin inhibitor (ulinastatin) (43). The optimal combination of enzymes necessary to maximize the isolation of large numbers of high-quality islets has yet to be determined. The slightest amount of hydration of the Liberase during storage can reduce enzyme function (44). This hydration activates the proteases, which then degrade the higher molecular weight collagenase, resulting in poor yields, viability, and functional outcomes. We are currently evaluating the extent of degradation of collagenase that occurs during storage. Human islet isolation outcomes remain highly variable despite considerable efforts to manufacture highly purified and standardized collagenase blends. The heterogeneity of collagenase preparations and the immense variability between human donor pancreata continue to hamper a process that is inherently difficult to control (45). It has been clarified that the ratio between collagenase class I (ccI) and class II (ccII) is of significant relevance for releasing of islets from pancreatic tissue and optimizing islet yield and viability (46). The variability in collagenase blends has been considered as the most important determinant of the success or failure in isolated islet yields, and this variation in potency has been observed between, and even within, lots of Liberase HI (47). Recently, Hughes et al. reported that collagen VI is a major component of the islet-exocrine interface of the adult pancreas, the content being more than double that of collagen I or IV. Their results may facilitate the design of new collagenases, targeting major substrates such as collagen VI in order to improve clinical islet isolation (48). A better understanding of the characteristics and specific activities of each component in the collagenase blends will allow more specific and selective cleavage of the islets from the surrounding extra cellular matrix. CHALLENGES IN ISLET PURIFICATION Currently, the purification of islets from exocrine tissue is performed by continuous Ficoll gradients using a refrigerated COBE 2991 cell processor (49). It is believed that there are several advantages to transplanting highly purified islets, including improved engraftment, increased safety, and reduced graft immunogenicity (50). Despite overwhelming success in animal models, implantation of unpurified human pancreatic preparations (which may contain greater than 90% exocrine tissue) has been plagued with serious complications: wedge splenic infarction, splenic capsular tear, disseminated intravascular coagulation (DIC), systemic hypertension, portal vein thrombosis, sequelae of portal hypertension including bleeding

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esophageal varices, hepatic infarction, liver failure, and even death. This increase in portal pressure is believed to be the direct result of embolization of large volumes of unpurified tissue into the liver (51). The aforementioned studies demonstrated that transplantation of dispersed human pancreatic tissue was unsafe, suggesting that some form of purification was necessary to decrease the complications, improve islet engraftment, and reduce graft immunogenicity. In 1989, Lakey et al. developed a method for large-scale purification of human islets suitable for safe transplantation (52). Originally designed to process bone marrow and to remove the cryoprotectant from banked blood, the COBE 2991 cell processor permitted rapid, large volume Ficoll gradient processing of a single pancreas within a sterile, self-contained disposable system. Unfortunately, this method still produces significant b-cell stress as demonstrated by zymogen degranulation and loss of insulin content (53). The most common method of islet purification remains density gradient centrifugation (54). Density-dependent elutriation or isopyknic separation of tissue separates individual cells as they migrate and settle within the density gradient according to their specific gravity. Lacy and Kostianovsky were able to separate rodent islets from digested exocrine tissue by differential density elutriation using discontinuous sucrose gradients although the islets were unresponsive to hyperglycemic challenge in vitro (55). This observation was more likely the result of hyperosmolar injury from cellular and islet dehydration rather than dissociation-induced trauma. The replacement of sucrose with Ficoll, a high molecular weight polymer of sucrose (40 kD), permitted the recovery of functionally viable islets (56,57). When Ficoll powder is dissolved in EuroCollins (EC) solution (Euro-Ficoll), hypertonic exposure of the exocrine tissue reduces cell swelling and enhances the islet/exocrine density differential, thereby improving islet recovery (58). Other continuous and non-continuous density gradients have been tested with varying degrees of success: bovine serum albumin (BSA), dextran, hypaque-Ficoll, metrizamide, percoll, and sodium diatrizoate (59,60). The inability to produce consistent highly purified human islet preparations has hindered the development of islet transplantation as a realistic treatment option for patients with Type 1 diabetes (61). Gores et al. have suggested that until specific tolerance protocols are a reality, more effort should be directed at modifying the host’s immune response while using impure preparations to maximize islet yield (62). Crude or partially purified pancreatic homogenates have been used to maximize islet engraftment mass (63,64). Thrombotic complications are believed to be secondary to the thromboplastins released from the digested exocrine tissue. Mehigan et al. found that the addition of heparin and aprotinin (Trasylol) to the tissue preparation at the time of transplantation could ameliorate the risk of DIC (65). We have demonstrated that highly purified islet preparations,

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small packed cell volumes (PCV) <10 ml (preferably <5 ml), graded lowdose heparinization, and careful monitoring of portal pressure during islet infusion reduces the risk of portal vein thrombosis and its sequelae (66). The introduction of low-endotoxin Liberase may also be critical in minimizing the acute risk of physiological perturbations associated with infusion of nonpurified islet preparations (67). Attempts to purify islets with nylon mesh sieves, sedimentation at unit gravity, centrifugal elutriation, and isokinetic gradient centrifugation (68,69) have been unsuccessful due to the minimal size difference between the islets (average diameter about 150 µm) and exocrine tissue. The purification of islets with magnetic microspheres coated with antiislet or cytotoxic anti-acinar monoclonal antibodies (MAbs) is a unique concept that has the potential for large-scale purification (70,71). Photothermolysis of specifically targeted acinar tissue permits the recovery of functionally viable islets (72). Selective destruction of exocrine tissue by antibody-mediated radiosensitization is based on the premise that islets are less radiosensitive (blood glucose remains normal following radiation of the canine pancreas) than exocrine tissue. Treatment of unpurified graft with 5000 rad increases the insulin/amylase ratio (index for graft purity) after 24 hours culture (73). Another approach exploits the tenfold osmotic permeability difference between the exocrine and endocrine tissues (74). A 30-second exposure of the pancreatic digest to a hypotonic solution selectively lyses the exocrine tissue without damaging the islets. Other methods not specifically discussed herein include cryopreservation, antiacinar cytotoxic antibodies, tissue culture, florescence-activating cell sorting, and cell sorting by simple filtration (75,76). Large animal studies with nonpurified islet grafts suggest that it should be possible to treat multiple recipients from a single pancreas (77).

CHALLENGES IN ISLET ASSESSMENT Islet Yield The determination of islet mass is important for the normalization of islet experiments in the laboratory and for the precise dosing of islets for transplantation. The common microscopic analysis is based on individual islet sizing, calculation of the frequency distribution, and conversion into islet equivalents (IEQ), which is the volume of a spherical islet with a diameter of 150 µm (Fig. 6). However, islets are of irregular form, which makes this determination user dependent, and the analysis irreproducible once the original sample is discarded. Recently, Lembert et al. showed that areal density measurements allow a rapid and reproducible estimation of IEQ without counting individual islets. It can be performed in a single step analysis without computer programming and is valuable for online

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determinations of islet yield preceding transplantation (78). An improved method of islet volume determination using digital image analysis (DIA) has also been developed to remove operator bias and automate the islet counting process. It was found that volumes determined by DIA correlated more closely with insulin content and DNA content than did conventionally

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determined volumes. Quantification of isolated islet tissue volume using DIA has been shown to be rapid, consistent, and objective. In the laboratory, use of this method as the standard for islet volume measurement will allow more meaningful comparison of experimental results between centers. In the clinic, its use will allow more accurate dosing of transplanted tissue (79).

Islet Viability One of the most important factors in clinical islet transplantation is isolation of a great number of islets with good viability. Current functional assessment of isolated islets includes static incubation and perifusion tests of glucose-stimulated insulin secretion, which are used retrospectively, take a good deal of time, and require various apparatuses. For clinical islet transplantation, viability assessments of isolated islets must be simple, rapid, sensitive, and prospective. Fluorescein diacetate (FDA) causes live cells to fluoresce green under blue light excitation (490 nm), and ethidium bromide (EB) causes dead cells to fluoresce red. Discrimination of living from dead islets by insulin secretion correlated well with viability as determined by FDA/EB staining. The FDA/EB assay prospectively and easily provides a rapid, accurate, and objective measurement of the proportion of living cells and dead cells in isolated islets for clinical islet transplantation (80). Double staining with fluorescein diacetate and propidium iodide (FDA/PI) is the current international standard to determine islet viability. However, a study by our group that evaluated the SYTO-13/ethidium bromide (SYTO/EB) and FDA/PI techniques suggests that FDA/PI staining may overestimate islet viability and demonstrates consistently elevated values when compared to SYTO/EB. The discrepancies found between FDA/PI scoring and visual quality, compared with alternative stains, suggests that the FDA/PI stain may not be the optimal approach to assess islet viability (81).

CHALLENGES IN ISLET CULTURE After isolation from a donor pancreas, human islets may either be transplanted immediately, or may be cultured for a period of time beforehand. Culturing islets before transplantation has several advantages over immediate transplantation. First, short-term tissue culture may reduce the immunogenicity of islets (82). Kuttler et al. found that long-term culture reduced CD45þ leukocytes in islets (83). Endothelial cells, which are thought to play a role in graft rejection (84), may be reduced in cultured islets (85). Islet culture may facilitate islet purification if media composition is designed to maintain islets while suppressing exocrine survival. Furthermore, islet transplantation is currently restricted to a small number

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of centers, and the ability to maintain islets over a period of days allows time for distant patients to travel to the center for the transplant procedure. Culture facilitates the option for shipment of islets between centers, providing an opportunity to regionalize the complex islet isolation procedure within relatively few cGMP facilities. In addition, culture permits maintenance of islets while pre-transplantation quality control tests are undertaken (86). However, the culture of islets has been in the past problematic and inconsistent in terms of islet survival and functional viability. As well as being subject to necrotic cell death (87,88,89), islets tend to lose their ability to respond to high glucose challenge after long-term culture (90). There are certain basic requirements of pre-transplantation islet culture media. It must have fully defined constituents that are determined to be of low risk to the patient. For instance, the addition of fetal calf serum (FCS) to culture is extremely beneficial to islets (91). Yet because of the risks associated with transplantation of xenoproteins, FCS is no longer being used for the culture of human islets intended for transplantation into immunosuppressed patients. Holmes et al. examined the culture of rat, porcine, and human islets in commercial media, including RPMI 1640 (11 mM glucose), RPMI 1640 (2.2 mM glucose), Dulbecco’s modified Eagle’s medium, Medium 199, CMRL 1066, Iscove’s modified Eagles medium, Waymouth’s medium, Serotec serum-free medium, and Ham’s F-12 (92). After further testing with the three best media, they found that human islets cultured in CMRL 1066 had a significantly higher stimulation index than those cultured in the other culture media. Fraga et al. used CMRL 1066 when comparing serum-free and serum supplemented media, and were able to maintain islets for 2 months with 65% recovery (90). CMRL 1066 was originally designed for use with fibroblasts and kidney epithelial cells (93). Determining an optimal concentration of glucose is extremely important for the maintenance of islets in culture, as both high and low concentrations of glucose can have harmful effects on islets. While studies with rodent islets have revealed an optimal concentration of around 10 or 11 mM glucose, studies directly pertaining to human islet culture have determined that the most advantageous concentration of glucose for human islets lies around 5.5 mM (94). Several groups have examined the benefit of a few different vitamins on islets. The most significantly studied is nicotinamide, a derivative of the B vitamin niacin, which is non-toxic (95), and has very potent impact in improving islet viability and function. It has been shown to prevent necrosis in islets. Ascorbic acid transiently inhibits b-cell depolarization (96), and thus may have a modulatory role in glucose-sensing. Vitamin C is thought to interfere with lipid-soluble molecules of Vitamin E, and thus regenerate oxidated Vitamin E, restoring its anti-oxidative functions (97). When islets

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are preincubated in a medium containing α-Tocopherol (vitamin E), it improves islets’ resistance to NO (98). α-Tocopherol limits the propagation cycle of lipid peroxidation induced by iron and ascorbate (99). Vitamin D3 may help preserve porcine neonatal islets, due to its antioxidant effect (100). There are many other supplements that may be important to islet culture; islet cells, like most eukaryotic cells, require a complex environment to survive. Islet culture may be complicated by possible residual presence of proteases from the digestion phase of islet isolation (101); these enzymes can degrade the collagen matrix of the islet, causing fragmentation and death. The addition of protease inhibitors to culture media may combat this possible detrimental effect. Improving methods and supplementation of islet culture provides a unique opportunity to learn how we can further augment islet mass for transplantation as a means of increasing success with single donor transplants. While supplementation of islet culture is beneficial, its use may have limitations. The mimicry of the physiological state with supplements is not perfect; while islets are usually in culture for up to 72 hours (89), they can survive post-transplantation in a patient over 18 years (102). It is important to apply previous research to improve culture conditions for islets in a clinical environment, but perhaps only innovative culture techniques will be sufficient to realize the full potential of clinical islet culture.

CONCLUSION Clinical outcomes are influenced by numerous variables in the isolation process and pre-transplant culture. Many technical challenges in these procedures must be addressed if clinical islet transplantation is to improve. Cooperation between organ transplant centers, the procurement team, and the isolation laboratory is crucial to ensure that available cadaveric pancreata are referred appropriately and expediently. Islet yields remain quite variable (typically 25% to 75% of the potential islet mass). Moreover, clinical results vary considerably across centers in spite of comprehensive efforts to standardize isolation/purification procedures and establish strict quality control criteria in accordance with World Health Organization (WHO) Good Manufacturing Practice (GMP) guidelines. The production of high-quality islets is expensive, labor-intensive, and time-consuming. Because the process has a steep learning curve and has yet to be standardized, the technology would be best served by establishing centralized processing facilities and regional networks dedicated to recruiting potential recipients and procuring and transplanting organs. Efforts to manufacture high-yield preparations suitable for clinical transplantation have been challenging. Numerous methods have been attempted,

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including continuous or discontinuous density (isopyknic) gradients, magnetic microspheres coated with islet or cytotoxic anti-acinar monoclonal antibodies, photothermolysis of exocrine tissue by antibody-mediated radiosensitization, exploitation of the osmotic permeability differential between the exocrine and endocrine tissues, florescence-activating cell sorting, cryopreservation, tissue culture, and cell sorting by simple filtration. Despite efforts to manufacture highly purified and standardized collagenase blends, the heterogeneity of the preparations, quality and nature of donor pancreata, and prolonged cold ischemia times hamper a process that is inherently difficult to control. One solution may be to determine the acinar, ductal, and endocrine elements of the donor pancreas using sophisticated genetic or phenotypic and molecular assays and then prepare an enzyme cocktail incorporating specific recombinant collagenase enzymes tailored to each donor pancreas. Such individualized processing may be required to consistently maximize the islet yield and viability for transplantation.

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13. Alderson D, Kneteman NM, Scharp DW. The isolation of purified human islets of Langerhans. Transplant Proc 1987; 19(1 Pt 2):916–917. 14. Krestchner GJ, Sutherland DE, Matas AJ, Steffes MW, Najarian JS. The dispersed pancreas: transplantation without islet purification in totally pancreatectomized dogs. Diabetologia 1977; 13(5):495. 15. Lacy PE, Lacy ET, Finke EH, Yasunami Y. An improved method for the isolation of islets from the beef pancreas. Diabetes 1982; 31(suppl 4):109. 16. Gray DWR, McShane P, Grant A, Morris PJ. A method for isolation of islets of Langerhans from the human pancreas. Diabetes 1984; 33(11):1055. 17. Warnock GL, Kneteman NM, Evans MG, Dabbs KD, Rajotte RV. Comparison of automated and manual methods for islet isolation. Can J Surg 1990; 33(5):368. 18. van Suylichem PT, Wolters GH, van Schilfgaarde R. Peri-insular presence of collagenase during islet isolation procedures. J Surg Res 1992; 53(5):502. 19. Warnock GL, Cattral MS, Rajotte RV. Normoglycemia after implantation of purified islet cells in dogs. Can J Surg. 1988; 31(6):421. 20. Ricordi C, Finke EH, Lacy PE. A method for the mass isolation of islets from the adult pig pancreas. Diabetes 1986; 35(6):649. 21. Gray DW, Warnock GL, Sutton R, Peters M, McShane P, Morris PJ. Successful autotransplantation of isolated islets of Langerhans in the cynomolgus monkey. Br J Surg 1986; 73(10):850. 22. Horaguchi A, Merrell RC. Preparation of viable islet cells from dogs by a new method. Diabetes 1981; 30(5):455. 23. Ao Z, Lakey JR, Rajotte RV, Warnock GL. Collagenase digestion of canine pancreas by gentle automated dissociation in combination with ductal perfusion optimizes mass recovery of islets. Transplant Proc 1992; 24(6):2787. 24. Lacy PE. Pancreatic islet cell transplant. Mt Sinai J Med 1994; 61(1):23–31. 25. Ricordi C, Lacy PE, Finke EH, Olack BJ, Scharp DW. Automated method for isolation of human pancreatic islets. Diabetes 1988; 37(4):413–20. 26. Scharp DW, Lacy PE, Santiago JV, et al. Insulin independence after islet transplantation into type I diabetic patient. Diabetes 1990; 39(4):515–8. 27. Toso C, Oberholzer J, Ris F, et al. Factors affecting human islet of Langerhans isolation yields. Transplant Proc 2002; 34(3):826–7. 28. Lakey JR, Warnock GL, Rajotte RV, et al. Variables in organ donors that affect the recovery of human islets of Langerhans. Transplantation 1996; 61 (7):1047–53. 29. Brandhorst H, Brandhorst D, Hering BJ, Federlin K, Bretzel RG. Body mass index of pancreatic donors: a decisive factor for human islet isolation. Exp Clin Endocrinol Diabetes 1995; 103(Suppl 2):23–26. 30. Benhamou PY, Watt PC, Mullen Y, et al. Human islet isolation in 104 consecutive cases. Factors affecting isolation success. Transplantation 1994; 57 (12):1804–10. 31. Zeng Y, Torre MA, Karrison T, Thistlethwaite JR. The correlation between donor characteristics and the success of human islet isolation. Transplantation 1994; 57(6):954–8. 32. Lingohr MK, Buettner R, Rhodes CJ. Pancreatic b-cell growth and survival— a role in obesity-linked type 2 diabetes? Trends Mol Med 2002; 8(8):375–84.

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33. Mirbolooki M, Kin T, Mcghee-Wilson D, et al. Improvement the islet yield in human pancreatic islet isolation by considering donor obesity and age. CST Annual Meeting, Banff, Canada, 2005. 34. O’Gorman D, Kin T, Murdoch T, et al. The standardization of pancreatic donors for islet isolations. Transplantation 2005; 80(6):801–6. 35. Lakey JRT, Kobayashi N, Shapiro AMJ, Ricordi C, Okitsu T. (eds) Current Human Islet Isolation Protocol. Medical Review Co., Ltd: Chuo-ku, Osaka, Japan, 2004:10. 36. Lakey JR, Cavanagh TJ, Zieger MA, Wright M. Evaluation of a purified enzyme blend for the recovery and function of canine pancreatic islets. Cell Transplant 1998; 7(4):365–72. 37. Yonekawa Y, Matsumoto S, Okitsu T, et al. Effective islet isolation method with extremely high islet yields from adult pigs. Cell Transplant 2005; 14(10): 757–62. 38. Bai RX, Fujimori K, Koja S. Effect of prophylactic administration of trypsin inhibitors in porcine pancreas islet isolation. Transplant Proc 1998; 30(2)349. 39. Heiser A. Isolation of porcine pancreatic islets: low trypsin activity during the isolation procedure guarantees reproducible high islet yields. J Clin Lab Anal 1994; 8(6):407. 40. Basir I, van der Burg MP, Scheringa M, Tons A, Bouwman E. Improved outcome of pig islet isolation by Pefabloc inhibition of trypsin. Transplant Proc. 1997; 29(4):1939–41. 41. Lakey JR, Helms LM, Kin T, et al. Serine-protease inhibition during islet isolation increases islet yield from human pancreases with prolonged ischemia. Transplantation 2001; 72(4):565. 42. Rose NL, Palcic MM, Helms LM, Lakey JR. Evaluation of Pefabloc as a serine protease inhibitor during human-islet isolation. Transplantation 2003; 75(4):462. 43. Noguchi H, Ueda M, Nakai Y, et al. Modified Two-Layer Preservation Method (M-Kyoto/PFC) Improves Islet Yields in Islet Isolation. Am J Transplant 2006; 6(3):496–504. 44. Johnson PR, White SA, London NJ. Collagenase and human islet isolation. Cell Transplant 1996; 5(4):437. 45. Wolters GH, Vos-Scheperkeuter GH, Lin HC, van Schilfgaarde R. Different roles of class I and class II clostridium histolyticum collagenase in rat pancreatic islet isolation. Diabetes 1995 Feb; 44(2):227–33. 46. Brandhorst D, Huettler S, Alt A, et al. Adjustment of the ratio between collagenase class II and I improves islet isolation outcome. Transplant Proc 2005; 37(8):3450–1. 47. Barnett MJ, Zhai X, LeGatt DF, Cheng SB, Shapiro AM, Lakey JR. Quantitative assessment of collagenase blends for human islet isolation. Transplantation 2005; 80(6):723–8. 48. Hughes SJ, Clark A, McShane P, Contractor HH, Gray DW, Johnson PR. Characterisation of collagen VI within the islet-exocrine interface of the human pancreas: implications for clinical islet isolation? Transplantation 2006; 81(3):423–6. 49. Ricordi C. In: Methods in cell transplantation. Ricordi C, Ed. RG Landes: Austin TX, 1995: 99–112.

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50. Gores PF, Sutherland DE. Pancreatic islet transplantation: is purification necessary? Am J Surg 1993; 166(5):538–42. 51. Casey JJ, Lakey JR, Ryan EA, et al. Portal venous pressure changes after sequential clinical islet transplantation. Transplantation 2002; 74(7):913–5. 52. Lake SP, Bassett PD, Larkins A, et al. Large-scale purification of human islets utilizing discontinuous albumin gradient on IBM 2991 cell separator. Diabetes 1989; 38(Suppl 1):143–5. 53. Pearson TC, Alexander DZ, Winn KJ, Linsley PS, Lowry RP, Larsen CP. Transplantation tolerance induced by CTLA4-Ig. Transplantation 1994; 57 (12):1701–6. 54. Ricordi C, Rastellini C. Automated method for pancreatic islet separation. In: Ricordi C ed. Methods in islet implantation. RG Landes: Austin, TX 1995: 433. 55. Lindall A, Steffes M, Sorenson R. Immunoassayable insulin content of subcellular fractions of rat islets. Endocrinology 1969; 85(2):218. 56. Scharp DW, Kemp CB, Knight MJ, Ballinger WF, Lacy PE. The use of Ficoll in the preparation of viable islets of Langerhans from the rat pancreas. Transplantation 1973; 16(6):686. 57. Nash JR, Horlor M, Bell PR. The use of ficoll in the separation of viable islets of Langerhans from the rat pancreas. Transplantation 1976; 22(4):411. 58. Lakey JR, Cavanagh TJ, Zieger MA. A prospective comparison of discontinuous EuroFicoll and EuroDextran gradients for islet purification. Cell Transplant 1998; 7(5):479. 59. Tze WI, Wong FC, Tingle AJ. The use of hypaque-ficoll in the isolation of pancreatic islets in rats. Transplantation 1976; 22(2):201. 60. Lake SP, Anderson J, Chamberlain J, Gardner SJ, Bell PR, James RF. Bovine serum albumin density gradient isolation of rat pancreatic islets. Transplantation 1987; 43(6):805. 61. Shapiro AMJ, Lakey JRT, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000; 343(4):230. 62. Gores PF, Najarian JS, Stephanian E, Lloveras JJ, Kelley SL, Sutherland DE. Transplantation of unpurified islets from single donors with 15-deoxyspergualin. Transplant Proc 1994; 26(2):574. 63. Wahoff DC, Papalois BE, Najarian JS, et al. Autologous islet transplantation to prevent diabetes after pancreatic resection. Ann Surg 1995; 222(4):562. 64. Kretschmer GJ, Sutherland DE, Matas AJ, Cain TL, Najarian JS. Autotransplantation of pancreatic islets without separation of exocrine and endocrine tissue in totally pancreatectomized dogs. Surgery 1977; 82(1):74. 65. Mehigan DG, Bell WR, Zuidema GD, Eggleston JC, Cameron JL. Disseminated intravascular coagulation and portal hypertension following pancreatic islet autotransplantation. Ann Surg 1980; 191(3):287. 66. Froberg MK, Leone JP, Jessurun J, Sutherland DE. Fatal disseminated intravascular coagulation after autologous islet transplantation. Hum Pathol 1997; 28(11):1295. 67. Gill JF, Chambers LL, Baurley JL, et al. Safety testing of Liberase, a purified enzyme blend for human islet isolation. Transplant Proc 1995; 27(6): 3276.

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68. Cavanagh TJ, Dwulet FE, Fetterhoff TJ, et al. Collagenase selection. In: Lanza RP, Chick, WL, eds. Pancreatic islet transplantation volume I: procurement of pancreatic islets. Landes: Austin, TX, 1994: 39. 69. Hering BJ, Gramberg D, Ernst E, et al. Isokinetic gradients: a new approach to reduce islet graft immunogenicity. Transplant Proc 1993; 25(1 Pt 2):959. 70. Fujioka T, Terasaki PI, Heintz R, et al. Rapid purification of islets using magnetic microspheres coated with anti-acinar cell monoclonal antibodies. Transplantation 1990; 49(2):404. 71. Davies JE, James RF, London NJ, Robertson GS. Optimization of the magnetic field used for immunomagnetic islet purification. Transplantation 1995; 59(5):767. 72. Brunicardi FC, Oh Y, Shevlin L, et al. Laser destruction of human nonislet pancreatic tissue. Transplant Proc 1994; 26(6):3354. 73. Nason RW, Rajotte RV, Procyshyn AW, et al. Purification of pancreatic islet cell grafts with radiation. Transplant Proc 1986; 18:174. 74. Liu C, McGann LE, Gao D, Haag BW, Critser JK. Osmotic separation of pancreatic exocrine cells from crude islet cell preparations. Cell Transplant 1996; 5(1):31. 75. Jiao L, Gray DW, Gohde W, Flynn GJ, Morris PJ. In vitro staining of islets of Langerhans for fluorescence activated cell sorting. Transplantation 1991; 52(3):450. 76. Salvalaggio PR, Deng S, Ariyan CE, et al. Islet filtration: a simple and rapid new purification procedure that avoids ficoll and improves islet mass and function. Transplantation 2002; 74(6):877. 77. Payne WD, Sutherland DE, Matas AJ, Gorecki P, Najarian JS. DL-ethionine treatment of adult pancreatic donors. Amelioration of diabetes in multiple recipients with tissue from a single donor. Ann Surg 1979; 189(2):248. 78. Lembert N, Wesche J, Petersen P, Doser M, Becker HD, Ammon HP. Areal density measurement is a convenient method for the determination of porcine islet equivalents without counting and sizing individual islets. Cell Transplant 2003; 12(1):33–41. 79. Stegemann JP, O’Neil JJ, Nicholson DT, Mullon CJ. Improved assessment of isolated islet tissue volume using digital image analysis. Cell Transplant 1998; 7(5):469–78. 80. Miyamoto M, Morimoto Y, Nozawa Y, Balamurugan AN, Xu B, Inoue K. Establishment of fluorescein diacetate and ethidium bromide (FDAEB) assay for quality assessment of isolated islets. Cell Transplant 2000; 9(5):681–6. 81. Barnett MJ, McGhee-Wilson D, Shapiro AM, Lakey JR. Variation in human islet viability based on different membrane integrity stains. Cell Transplant 2004; 13(5):481–8. 82. Stein E, Mullen Y, Benhamou Py, et al. Reduction in immunogenicity of human islets by 24˚C culture. Trans Proc 1994; 26:755. 83. Kuttker B, Hartmann B, Wanka H. Long-term culture of islets abrogates cytokine-induced or lymphocyte induced increase of antigen expression on bcells. Transplantation 2002; 74:440–445. 84. Rose ML. Endothelial cells as antigen-presenting cells. Role in human transplant rejection. Cell Mol Sci 1998; 54:965–978. 85. de Graaff MPA, Wolters GHL, van Schiffgaarde R. Endothelial cells in pancreatic islets and the effect of culture. Transplant Proc 1994; 26(6):1171.

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86. Bretzl RG, Alejandro R, Hering BJ, van Suylichem PTR, Ricordi C. Clinical islet transplantation: Guidelines for islet quality control. Transplant Proc 1994; 26:388. 87. Andersson A. Isolated mouse pancreatic islets in culture: Effects of serum and different culture media on the insulin production of the islets. Diabetologia 1978; 14:397–404. 88. Ilieva A, Yuan S, Wang RN, Agapitos D, Hill DJ, Rosenberg L. Pancreatic islet cell survival following islet isolation: the role of cellular interactions in the pancreas. J Endocrinol 1999; 161:357–64. 89. Brandhorst D, Brandhorst H, Hering BJ, Bretzel RG. Long-term survival, morphology and in vitro function of isolated pig islets under different culture conditions. Transplantation 1997; 67:1533–41. 90. Fraga DW, Sabec O, Hathaway DK, Gaber AO. A comparison of media supplement methods for the extended culture of human islet tissue. Transplantation 1998; 65:1060–6. 91. Ling Z, Hannaert JC, Pipeleers D. Effect of nutrients, hormones and serum on survival of rat islet b-cells in culture. Diabetologia 1994; 37:15–21. 92. Holmes MA, Clayton HA, Chadwick DR, Bell PRF, London NJM, James RFL. Functional studies of rat, porcine, and human pancreatic islets cultured in ten commercially available media. Transplantation 1995; 60:854–60. 93. Moore GE, Gerner RE, Franklin HA. Culture of normal human leukocytes. JAMA 1967; 199:519–24. 94. Eizirik DL, Korbutt GS, Hellerstrom C. Prolonged exposure of human pancreatic islets to high glucose concentrations in vitro impairs the b-cell function. J Clin Invest 1992; 90:1263–68. 95. Petley A, Macklin B, Renwick AG, Wilkin TJ. The pharmacokinetics of nicotinamide in humans and rodents. Diabetes 1995; 44:152–5. 96. Bergsten P, Moura AS, Atwater I, Levine M. Ascorbic acid and insulin secretion in pancreatic islets. J Biol Chem 1994; 269:1041–5. 97. Winter DT, Eich T, Jahr H, Brendel MD, Bretzel RG. Influence of antioxidant therapy on islet graft survival. Transplant Proc 2002; 34:2366–8. 98. Volker B, Gross-Eick A, Bellmann K, Ju¨rgen R, Kold H. Suppression of nitric oxide in islet cells by α-tocopherol. FEBS Letters 1995; 364:259–63. 99. Wagner BA, Buettner GR, Burns CP. Vitamin E slows the rate of free radicalmediated lipid peroxidation in cells. Arch Biochem Bio-phys 1996; 334(2): 261–7. 100. Luca G, Nastruzzi C, Basta G, et al. Effects of anti-oxidizing vitamins on in vitro cultured porcine neonatal pancreatic islet cells. Diabetes Nutr Metab 2000; 13:301–7. 101. Brandhorst H, Brandhorst D, Hesse F, et al. Successful human islet isolation utilizing recombinant collagenase. Diabetes 2003; 52:1143–6. 102. Kendall DM, Teuscher AU, Robertson RP. Defective glucagon secretion during sustained hypoglycemia following successful islet allo- and autotransplantation in humans. Diabetes 1997; 46:23–7. 103. Lakey JRT. Current Human Islet Isolation Protocol. Medical Review Co Ltd.: Japan, 2004:10.

7 Percutaneous Portal Vein Access: Radiological Aspects Richard J. Owen Department of Radiology, University of Alberta, Edmonton, Alberta, Canada1

INTRODUCTION The portal vein has emerged as the favored site for pancreatic islet cell transplant (ICT) based on ease of technical access combined with cumulative data indicating that this route provides the safest and most durable access for transplantation (1). Early work involved surgical techniques for exposing the vein (2) and to this day laparoscopic mesenteric vein exposure remains an option in cases where percutaneous access is not possible or desired (3). However, the preferred approach at most centers is percutaneous access under imaging guidance (4,5). The first reports of image-guided access to the portal vein for the purpose of islet transplantation were described by Weimar and colleagues, who used a combination of CT and fluoroscopy (6). More recently a combination of ultrasound and fluoroscopic guidance has been used, and in the largest series to date this technique is used almost exclusively (7). One of the keys to the success of ICT has been the routine use of a percutaneous transhepatic approach for infusion of the isolated islets, and minimal morbidity of the procedure is very important for the ongoing success of an ICT program. CHOICE OF ACCESS From a radiological perspective, embolization into the portal vein makes good sense; there is a large vascular bed available for distribution, the Illustrations by Dawne Colwell

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anatomy is favorable, and previous reports of portal vein procedures confirm a proven safety record (8). Selective transhepatic catheterization of the portal vein was first used as a diagnostic tool and as access to treat bleeding esophageal varices (9). Subsequently, the technique has been and is still used to induce hypertrophy of the anticipated liver remnant prior to major liver resection. This technique has a complication rate of less than 5% (8), supporting the belief that this offers a safe route for islet cell transplantation. We also know that the liver has a dual blood supply and that portal venous supply is adequate to maintain hepatic viability following hepatic artery embolization with particulate or chemo-embolic agents in patients with hepatic tumors (10,11). Finally, percutaneous access to the liver for transhepatic cholangiograms, biliary drainage procedures, and liver biopsy is widely practiced with a proven safety record. Catheter placement within the portal vein can be carried out percutaneously via a transhepatic route, which is the preferred mode, but a transjugular transhepatic route could also be utilized. This latter technique does have the potential advantage of avoiding a capsular puncture. However, the procedure is cumbersome and time consuming and reports on transjugular intrahepatic portosystemic shunts indicate that there are still significant risks of biliary, capsular, and extrahepatic portal vein puncture (12,13). This route would remain an option if other routes have failed. IMAGING EQUIPMENT The procedure is usually carried out in a dedicated angiography room with a fixed C-arm—this is the optimal arrangement. An adequate portable C-arm would also suffice. Ultrasound with color duplex capability is recommended, although the procedure can be carried out with fluoroscopic guidance alone (7). ULTRASOUND The use of ultrasound has a low incidence of complications (14) and is associated with a reduced incidence of biliary punctures (4) and a reduced number of punctures of the liver capsule (7,14), which shortens procedure time. The recommended portal vein entry site is a 2nd or 3rd order branch. Punctures closer to the hilum run an increased risk of hepatic artery or bile duct puncture, and more superficial punctures result in a shorter parenchymal track, which is more difficult to accurately embolize. PRE-PROCEDURE REQUIREMENTS AND CONTRAINDICATIONS Contraindications as for biliary punctures should be observed and include sepsis, biliary dilatation, overlying infection, and coagulopathy. The PT/INR

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should be <1.5 and platelet count normal. Antiplatelet agents such as aspirin, clopidogrel (Plavix), and dipyridamole should be discontinued a suitable interval prior to the procedure to allow normal platelet function; in the case of aspirin this means 7 days prior to the ICT. Sterile technique should be practiced at all time.

PROCEDURE Sedation is recommended; intravenous midazolam and fentanyl and oxygen via nasal cannula would be a suitable choice. The patient is positioned supine on the procedure table and the point of hepatic puncture (anterior or mid axillary line) determined using ultrasound, fluoroscopy, or a combination thereof. Aseptic technique is essential, and the subcutaneous tissues and hepatic capsule are infiltrated with local anesthetic. A 22 gauge (0.022 inch) Chiba needle is then advanced into a branch of the right portal vein under fluoroscopic or ultrasound guidance (Fig. 1). An 18 gauge (0.018 inch) guidewire is then advanced through the needle into the main portal vein. Acute angles both at the capsular puncture point and at the portal vein entry site should be avoided, as they may lead to catheter kinking during respiratory excursions. Exchange using a standard NEFF percutaneous access set (Cook Canada, Inc., Stouffville, Ontario, Canada) or with a micropuncture set can be done at this stage and a 4- or 5-F angio catheter advanced into the portal vein. Use of the smallest catheter possible is recommended, although lumen size should not be <0.035 inch; islet cell clumps measure up to 500 µm. The stiffened micropuncture set (Cook Canada, Inc., Stouffville, Ontario, Canada) has a central metallic stiffener, a 3-F inner dilator (accepting an 0.018 inch guidewire), and an outer 4-F

Figure 1 Diagrammatic representation of the procedural setup. Percutaneous portal vein puncture under ultrasound guidance.

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Figure 2 The stiffened micropuncture set (Cook Canada, Inc., Stouffville, Ontario, Canada) with a central metallic stiffener, a 3-F inner dilator (accepting a 0.018 inch guidewire), and an outer 4-F dilator accepting a 0.038 inch (965 µm).

dilator accepting a 0.038 inch (965 µm) guidewire. This was specifically designed for percutaneous islet cell transplants (Fig. 2). The tip of the cannula is positioned within the main portal vein just proximal to the confluence, free flow is confirmed, and a portal venogram with 10–20 mls contrast carried out to confirm normal anatomy (Figs. 3 and 4). We recommend that the volume of radiographic contrast be kept to a minimum both before and after islet cell infusion in order to minimize hepatocyte and transplanted islet cell exposure. A baseline portal venous pressure (mmHg) should be recorded at this stage using an indirect pressure transducer.

ISLET CELL INFUSION The sterile islet preparation is then infused using gravity with intermittent portal pressure recordings (Fig. 5). We record portal pressure after 5 ml of packed cell volume (PCV) and after every subsequent 1 ml of PCV. Preparations with a higher PCV are generally less pure with a higher proportion of non-cellular stroma and are thought to pose a greater risk of inducing thrombosis. Heparin is added to the islet infusion at 70 units per Kg recipient body weight. The procedure is terminated if portal pressure is greater than 20 mmHg at the outset or rises to double the baseline or to

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Portal venogram with catheter tip proximal to portal confluence.

greater than 22 mmHg during the procedure. One of the feared complications of the procedure is branch or, worse still, main portal vein thrombosis; both are reported (5,15) and may be a cause of excess morbidity and even mortality. For this reason both the use of heparin and careful observation of

Figure 4 Diagrammatic representation of the 4-F catheter lying in the portal vein, tip at the portal confluence.

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Diagrammatic representation of islet infusion set–up.

portal pressures are obligatory. The advent of rising portal pressure indicates a reduction of the available portal venous bed and may lead to stasis and thrombosis. Following the islet infusion procedure the catheter is withdrawn into the parenchymal tract and embolization carried out.

PARENCHYMAL TRACK EMBOLIZATION With the advent of 4-F delivery systems, embolization of the hepatic tract was not initially deemed necessary (5). However, when further data revealed a significant bleeding risk, Edmonton initiated routine tract embolization and now consider bleeding an avoidable complication “provided the intraparenchymal liver tract is sealed effectively” (7). Several other centers both at the time and subsequently (4,16) have reported that effective sealing of the hepatic tract is essential. Many embolization techniques have been used: stainless steel or platinum embolization coils, Tisseal (Baxter Corp., Mississauga, Ontario, Canada), gelatin-sponge (Gelfoam, Pharmacia & Upjohn, Mississauga, Ontario, Canada), thrombin-saturated Gelfoam collagen paste (17), and Avitene paste (MedChem Products, Woburn, Massachussets, U.S.A.). The ideal material would: effectively seal the tract, not interfere with subsequent imaging or interventions, have a satisfactory safety profile, be able to be accurately placed, be bioabsorbable, and, finally, be clearly visible to the operator. The favored material in use in our center that fulfills these criteria is Avitene paste, a microfibrillar collagen hemostat made from flour. The material (Figs. 6 and 7) is mixed to a paste with 3 ml of radiographic contrast and 3 ml of 0.9% saline. This forms a thick paste, which can be loaded into 1 ml syringes (Figs. 8 and 9). The 4-F infusion catheter is then withdrawn under fluoroscopic guidance until the tip is lying

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Setup for Avitene paste.

in the parenchymal tract. The Avitene paste, which is clearly visible, is then injected through the catheter as it is withdrawn, “lacing” the tract from portal vein puncture site out to the liver margin (Figs. 10 and 11). No complications from this procedure have been recorded at our center in the 20 procedures in which we have used this product.

PROCEDURE-RELATED COMPLICATIONS Complications of ICT do occur and can be categorized into major and minor. Major complications relate to portal vein thrombosis (PVT), branch

Figure 7

Mixing the paste.

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Loading the 1 ml syringe.

PVT, and bleeding complications. A single case of main PVT has been reported in the literature (15). Branch PVT has been reported on several occasions, with cumulative data from the 6 largest series recording 9 branch PVTs in 405 procedures, giving an incidence of 2.2% (6,7,14,16–20). Data from animal models have shown that occlusion of the vascular bed progressively increases the vascular resistance, and this will eventually lead to elevation of the portal venous pressure (21). Increased flow in the hepatic artery and reduced flow in the portal vein has been documented following embolization of the portal vein with particulate emboli; this is explained by simple fluid mechanics (11). Islet cell clumps act as particulate emboli and

Figure 9

Paste consistency.

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Tract obliteration using Avitene paste.

may occlude arterioles up to 500 micrometers in diameter. A significant pressure elevation in the portal vein following islet embolization has also been demonstrated in relation to the number of islets transplanted, the packed cell volume, and number of procedures per patient (22). We allow a maximum of 10 ml of packed cell volume and prefer to have less than 5 ml. Hemorrhagic complications have been reported in 29/405 procedures giving an incidence of 7.2% (6,7,14,16–20). Hemorrhagic complications have noticeably fallen in incidence since the advent of universal track

Figure 11

Parenchymal tract outline by radio-opaque Avitene paste.

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embolization, now considered obligatory. One hemothorax is reported in the literature (14). Minor complications include biliary or gall bladder puncture, vasovagal reactions, nausea or vomiting, hiccups from diaphragmatic irritation, and reactions to opiates and benzodiazepines. Conversion to general anesthetic is also reported (5) when acute discomfort during the procedure, triggered by distension of the gall bladder, led to completion of the procedure under a general anesthetic. POST-PROCEDURAL MANAGEMENT AND FOLLOW-UP Patients are managed post-procedure according to standard protocol for post-hepatic interventions with bed rest in a right recumbent position and close observation for 4 hours. Ultrasound of the liver with doppler interrogation of the portal vein is carried out within 24 hours of transplantation. The post-procedure diabetic status is monitored closely. SUBSEQUENT TRANSPLANTS AND SECONDARY INTERVENTIONS Limitations of islet yield from donors mean that only in a minority are enough islets isolated to allow insulin independence from a single procedure. Supplemental transplants are therefore required in most cases to provide an adequate islet engraftment mass as previously described (23). Previous ICT does not limit subsequent procedures, and the previous parenchymal tract is rarely visible. In cases where a prior track embolization with metallic coils has been carried out, these may be visible as echogenic foci on ultrasound but rarely would they interfere with the procedure. Secondary interventions could include arteriovenous fistula embolization (18) or, potentially, bleeding point embolization. CONCLUSION The percutaneous route for intraportal infusion of isolated pancreatic islets in the hands of an experienced interventional radiologist is a straightforward and safe procedure that is well within the capability of all dedicated interventionists.

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Warnock GL, Kneteman NM, Ryan EA, Seelis REA, Rabinovitch A, Rajotte RV. Normoglycaemia after transplantation of freshly isolated and cryopreserved pancreatic islets in type 1 diabetes. Diabetologia 1991; 34:55–58.

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Johnson PR, White SA, Robertson GS et al. Pancreatic islet autotransplantation combined with total pancreatectomy for the treatment of chronic pancreatitis-the Leicester experience. J Mol Med 1999; 77(1):130–132. Gaber AO, Chamsuddin A, Fraga D, Fisher J, Lo A. Insulin independence achieved using the transmesenteric approach to the portal vein for islet transplantation. Transplantation 2004; 77(2):309–311. Goss JA, Soltes G, Goodpastor SE, et al. Pancreatic islet transplantation: the radiographic approach. Transplantation 2003; 76(1):199–203. Owen RJ, Ryan EA, O’Kelly K, et al. Percutaneous transhepatic pancreatic islet cell transplantation in type 1 diabetes mellitus: Radiologic aspects. Radiology 2003; 229(1):165–170. Weimar B, Rauber K, Brendel MD, Bretzel RG, Rau WS. Percutaneous transhepatic catheterization of the portal vein: A combined CT and fluoroscopyguided technique. Cardiovasc Intervent Radiol 1999; 22(4):342–344. Villiger P, Ryan EA, Owen R, et al. Prevention of bleeding after islet transplantation: lessons learned from a multivariate analysis of 132 cases at a single institution. Am J Transplant 2005; 5(12):2992–8. Abdalla EK, Hicks ME, Vauthey JN. Portal vein embolization: rationale, technique and future prospects. Br J Surg 2001; 88(7):165–175. Viamonte M Jr, LePage J, Lunderquist A, et al. Selective catheterization of the portal vein and its tributaries. Preliminary report. Radiology 1975; 114(2): 457–460. Van Beers B, Roche A, Cauquil P, Jamart J, Pariente D, Ajavon Y. Transcatheter arterial chemotherapy using doxyrubicin, iodized oil and gelfoam embolization in hepatocellular carcinoma. Acta Radiologica 1989; 30(4):415–418. Kito Y, Nagino M, Nimura Y. Doppler sonography of hepatic arterial blood flow velocity after percutaneous transhepatic portal vein embolization. Am J Roentgenol 2001; 176(4):909–912. Owen RJT, Rose JDG. Endovascular treatment of a portal vein tear during TIPSS. CVIR 2000; 23(2):230–232. Luca A, D’Amico G, La Galla R, Midiri M, Morabito A, Pegliaro L. TIPS for prevention of recurrent bleeding in patients with cirrhosis: meta-analysis of randomized clinical trials. Radiology 1999; 212(2):411–421. Venturini M, Angeli E, Maffi P, et al. Technique, complications, and therapeutic efficacy of percutaneous transplantation of human pancreatic islet cells in type 1 diabetes: the role of US. Radiol 2005; 234(2):617–24. Brennan DC, Shannon MB, Koch MJ, Polonsky KS, Desai N, Shapiro J. Portal vein thrombosis complicating islet transplantation in a recipient with the Factor V Leiden mutation. Transplantation 2004; 78(1):172–3. Hering BJ, Kandaswamy R, Ansite JD, et al. Single-donor marginal-dose islet transplantation in patients with type 1 diabetes. JAMA 2005; 293(7):830–835. Froud T, Yrizarry JM, Alejandro R, Ricordi C. Use of D-STAT to prevent bleeding following percutaneous transhepatic intraportal islet transplantation. Cell transplant 2004; 13(1):55–59. Bucher P, Mathe Z, Bosco D, et al. Morbidity associated with intraportal islet transplantation. Transplant Proc 2004; 36(4):1119–20.

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Barshes NR, Lee T, Goodpasture S, et al. Achievement of insulin independence via pancreatic islet transplantation using a remote isolation centre: a first year review. Transplant Proc 2004; 35(4):1127–1129. Hirshberg B, Rother KI, Digon BJ, et al. Benefits and risks of solitary islet transplantation for type 1 diabetes using steroid sparing immunosuppression: The National Institutes of Health experience. Diabetes care 2003; 26(12): 3288–3295. Schenck E, Nelson JA, Starr FL, Coldwell D. Animal model of portal hypertension with observations regarding the relationship between portal flow and pressure. Invest Radiol 1993; 28(5):442–445. Casey JJ, Lakey JRT, Ryan EA, et al. Portal venous pressure changes following sequential clinical islet transplantation. Transplantation 2002; 74(7): 913–915. Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppression regimen. N Engl J Med 2000; 343(4):230–238.

20.

21.

22.

23.

8 Care of the Islet Transplant Recipient: Immunosuppressive Management and Complications Raquel N. Faradji Department of Medicine, University of Miami Leonard M. Miller School of Medicine, and Clinical Islet Transplant Program, Cell Transplant Center, Diabetes Research Institute, Miami, Florida, U.S.A.

Pablo Cure University of Miami Leonard M. Miller School of Medicine, and Clinical Islet Transplant Program, Cell Transplant Center, Diabetes Research Institute, Miami, Florida, U.S.A.

Camillo Ricordi DeWitt Daughtry Family Department of Surgery, University of Miami Leonard M. Miller School of Medicine, and Clinical Islet Transplant Program, Cell Transplant Center, Diabetes Research Institute, Miami, Florida, U.S.A.

Rodolfo Alejandro Department of Medicine, University of Miami Leonard M. Miller School of Medicine, and Clinical Islet Transplant Program, Cell Transplant Center, Diabetes Research Institute, Miami, Florida, U.S.A.

IMMUNOSUPPRESSIVE MANAGEMENT Before 2000, less than 10% of islet allograft recipients with Type 1 diabetes mellitus were insulin independent one year after transplantation and durable long term graft survival was not a rule (1–3). Improvements in isolation techniques, the use of newer induction therapies, and more potent maintenance immunosuppressive protocols have allowed longer allograft survival, leading to better long-term metabolic control in the absence of severe hypoglycemia and improvement in overall quality of life (4–7). Long-term immunosuppression helps to prevent graft loss, but it can often be associated with worsening of pre-existing conditions or new complications (8–10). Close monitoring of immunosuppressive trough levels is a key factor for 147

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islet graft preservation. Adjustments to immunosuppressive drug doses are often necessary in order to decrease toxicity and side effects (11,12). Current immunosuppressive protocols for islet transplantation alone are based on a glucocorticoid-free regimen that includes induction therapy with monoclonal antibody against interleukin-2 receptor [daclizumab (Zenapax; Hoffman-La Ruche, Inc., Nutley, New Jersey, U.S.A.)] and maintenance immunosuppression with sirolimus (Rapamune; Wyeth Pharmaceuticals, Philadelphia, Pennsylvania, U.S.A.) and tacrolimus (Prograf; Astellas Pharma US, Inc., Deerfield, Illinois, U.S.A.) known as the Edmonton Protocol (Table 1 and Fig. 1) (2,6). In some occasions, due to drug toxicity, mycophenolate mofetil or mycophenolate sodium may be introduced as maintenance therapy instead of tacrolimus or sirolimus (8,13). Management of maintenance immunosuppressive drugs (sirolimus and tacrolimus) is one of the most important factors in the prevention of graft rejection and the minimization of side effects and drug toxicity. Follow-up, including immunosuppressive trough levels, complete blood counts, and basic metabolic parameters (electrolytes, liver function tests, magnesium, calcium, and phosphorus), should be done twice a week for the 1st month, every week for the 2nd month, every two weeks until the 4th month, and then monthly thereafter, unless more frequent testing is necessary based on clinical judgment. This is done to identify and monitor the incidence of side effects such as neutropenia, anemia, electrolyte imbalances, etc. (Table 2) associated with the different therapies. Toxicity assessments should also be done monthly in order to identify more subjective side effects (i.e., memory loss, tremor) that can interfere with the patient’s normal daily functions affecting their quality of life. Newer and more potent alternatives to the standard induction therapies used by the Edmonton Protocol have arisen in the past few years (Table 1). The main goals have been to prevent acute rejection, to favor better early engraftment, and to improve long-term survival of the islets. Current alternatives, such as induction therapies with the humanized monoclonal antibody alemtuzumab (Campath 1H; Genzyme Corp., Cambridge, Massachusetts, U.S.A.), a potent lymphocyte-depleting agent that is being used in islet transplantation, have demonstrated similar results to the Edmonton Protocol at an early follow-up. Other protocols based on induction therapy with rabbit anti-thymocyte globulin (ATG, Thymoglobulin; Genzyme Corp.), used recently by the Minnesota group, showed remarkable results on a series of recipients of single donor islet transplantation (15). Standard Induction Therapy Daclizumab Mechanism of Action: Daclizumab (Zenapax) is a humanized monoclonal antibody that acts by blocking the α-chain of the interleukin-

1 mg/kg IV 2hr prior to IT

NA

1mg/kg IV on IT day

1mg/kg IV on IT day

Miami

Miami 2 (Campath)

Edmonton (standard)

Minnesota (standard)

1 mg/kg IV q 2 weeks
4 doses

1 mg/kg IV
4 doses

1 mg/kg IV
4 doses. Then 1 dose per month for 1 year NA

Anti-inflammatory ATG Other doses

0.5 mg/kg IV day 2

NA

NA

NA

Campath-H1 Initial dose

1.5 mg/kg IV days 0 to +2c

NA

NA

NA

TNF-alfa blocker Other doses

b

Requires Pre-medication: acetaminophen, diphenhydramine, and methylprednisolone Requires Pre-medication: acetaminophen, diphenhydramine c Requires Pre-medication: acetaminophen, diphenhydramine, and pentoxifilline d Only used in 10 subjects.

a

Induction Zenapax Initial dose

Enbrel 50 mg IV

NA

20 mg IV on day –1a

NA

Initial dose

25 mg s.c. on days 3, 6 & 10

NA

20 mg IV on IT dayb

NA

Other doses

Current Immunosuppressive Protocols Commonly Used in Islet Transplant (IT) Recipients

Trials

Table 1

Infliximab 10 mg/kg on IT dayd NA

Enbrel 50 mg IV

Enbrel 50 mg IV

Initial dose

(Continued)

NA

25 mg s.c. twice per week for 2 weeks None

25 mg s.c. twice per week for 2 weeks

Other doses

Care of the Islet Transplant Recipient 149

7–10 5–15

12–15

5–15

0.1 mg/kg p.o. 0.1 mg/kg p.o. 0.1 mg/kg p.o.

1 mg p.o. b.i.d. 2–4 mg p.o. b.i.d 2–4 mg p.o. b.i.d.

1 mg p.o. b.i.d.

Maintenance

2–4 mg p.o. b.i.d. 2–4 mg p.o. b.i.d 2–4 mg p.o. b.i.d.

2–4 mg p.o. b.i.d.

Subsequent

Tacrolimus

f

MMF is introduced in replacement of tacrolimus at 3 months after transplantation. MMF is introduced in replacement of tacrolimus at 1 month after transplantation. g MMF can be introduced only if needed based on clinical judgment and side effects from other IS drugs.

e

8–10

10–12

0.1 mg/kg p.o.

8–12

1st 3 Mo

12–15

Subsequent

>3 Mo

Levels (ng/ml)

0.2 mg/kg p.o. on day 1 0.2 mg/kg p.o. 0.2 mg/kg p.o. 0.2 mg/kg p.o. on day 2

Sirolimus

3–6

3–6

4–6

4–6

1st 3Mo

3–6

3–6

4–6

4–6

>3 Mo

Levels (ng/ml)

Current Immunosuppressive Protocols Commonly Used in Islet Transplant (IT) Recipients (Continued)

Initial dose

Table 1

750–1000 mg p.o. b.i.d.f 750–1000 mg p.o. b.i.d.. 750–1000 mg p.o. b.i.d.e

750–1000 mg p.o. b.i.d.g

Initial/subsequent

MMF

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Sirolimus * Tacrolimus* Induction Agent

Etanercept: 50mg i.v. day of Tx, then 25mg s.c. twice a week for 2 weeks

Islet Isolation

–3 –2 –1 0

5

11

Islet culture Islet Infusion

Days

3

Months

Sirolimus: 12–15 ng/ml (1st3 mo) 10–12 ng/ml (>3mo) Tacrolimus: 4-6 ng/ml

*Tacrolimus and Sirolimus can be substituted by MMF in case of drug toxicity.

Figure 1 tation.

Flow chart of current immunosuppressive protocols for islet transplan-

2 (CD25þ) receptor located on the surface of the activated lymphocytes, therefore inhibiting T-cell proliferation. It has been widely used in transplantation where it shows decreased rejection rates without increasing the incidence of infection (16). This induction therapy was a particularity of the Edmonton Protocol for islet transplantation alone (2), and its success led other centers to start using similar induction therapies, obtaining comparable results at 1 year follow-up (6). Dose: Daclizumab 1 mg/kg of body weight is given intravenously (IV) 2 hours prior to the transplant. Then 1 mg/kg IV is given every 14 days for a total of 5 doses post-transplant (2). Induction therapy is repeated with subsequent islet transplants, if more than one transplant is given. Due to concerns of graft dysfunction following reappearance of CD25þ cells in peripheral blood, daclizumab infusions are continued monthly for the first year and bi-monthly for the second year in the Miami islet transplantation alone protocol (6). The beneficial effect of these long-term daclizumab infusions remains unclear. Side Effects: In trials using daclizumab to prevent acute rejection in kidney transplant patients, the administration of this medication was not associated with any immediate side effect. There was no difference in

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Table 2

Frequency of Adverse Events Related to Each Immunosuppressive Medication Frequency High frequency (61% or greater)

Medium frequency (11% to 60%)

Adverse events Mouth ulcers similar, but often larger, than cold sores Fever Infection Gastrointestinal problems such as abdominal pain, diarrhea, nausea, and vomiting Bronchitis and pneumonia Burning sensation of the hands and feet, tremors, insomnia, headaches, cardiomegaly, hyperglycemia, hyperkalemia, hypertension, increased risk of infection, tachycardia, and anxiety Increased risk of infection, hypercholesterolemia, hypertriglyceridemia, hypertension, and wound healing delay Local skin rash at site of injection, abdominal pain, gastroesophageal reflux, indigestion, bloating, upper and lower extremities edema, tremors, headaches, dizziness, oliguria, dysuria, kidney damage, chest pain, fever, pain, fatigue, hypertension, hypotension, shortness of breath, coughing, pleural effusion, poor wound healing, acne, insomnia, tachycardia, hypercoagulation, bleeding, edema, hyperglycemia, severe allergic reaction Injection site reactions such as redness, pain or swelling, upper respiratory infection, sinusitis, headaches, and nausea No known adverse events in this category

Medication Sirolimus (Rapamune) ATG (Thymoglobulin) Etanercept (Enbrel) MMF (Cellcept) Tacrolimus (Prograf) MMF (Cellcept) Tacrolimus (Prograf)

Sirolimus (Rapamune)

Daclizumab (Zenapax)

Etanercept (Enbrel)

ATG (Thymoglobulin) (Continued)

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Table 2

Frequency of Adverse Events Related to Each Immunosuppressive Medication (Continued ) Frequency Low frequency (10% or less)

Adverse events Development of cancer especially lymphomas and skin cancer, leukopenia and very rarely fatal lung disease Development of cancer especially lymphomas, kidney damage, diabetes Increased risk of cancer (lymphoma), kidney damage, lung problems Constipation, nausea, diarrhea, vomiting Nerve damage resulting in burning, tickling, pricking or tingling in the legs, muscle tremors, blurred vision, loss of bladder and/or bowel control and partial or complete paralysis (neurological problems usually resolve partially or completely after discontinuing the drug), in very rare cases serious infection and death have occurred, cancer, immune-system mediated multi-organ damage, anemia Dizziness, chills, leukopenia, headache, abdominal pain, diarrhea, hypertension, nausea, thrombocytopenia, edema of the upper and lower extremities, shortness of breath, weakness, hyperkalemia, tachycardia, fatigue, and infection

Medication MMF (Cellcept)

Tacrolimus (Prograf)

Sirolimus (Rapamune)

Daclizumab (Zenapax) Etanercept (Enbrel)

ATG (Thymoglobulin)

Abbreviations: ATG, anti-thymocyte globulin; MMF, mycophenolate mofetil.

adverse events between the daclizumab and the placebo group (16). In the Miami experience no severe adverse effects have been encountered (Table 2). Alternative Induction Therapies Alemtuzumab (Campath 1H) Mechanism of Action: Alemtuzumab (Campath 1H) is a monoclonal antibody that binds to the CD52þ portion of the cell surface of B and T

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lymphocytes, monocytes, macrophages, NK cells, and a subpopulation of granulocytes, causing antibody-mediated lysis. Alemtuzumab is thus recognized as a potent lymphocyte-depleting agent. Some islet transplant centers are currently using immunosuppressive protocols based on this medication as an alternative way of decreasing acute rejection rates and improving long-term graft survival. Dose: Alemtuzumab is given IV at an initial dose of 20 mg over 3 hours on day–1, followed by a second IV dose of 20 mg on the day of transplant. Pre-medication with diphenhydramine (50 mg IV), acetaminophen (650 mg p.o.) and methylprednisolone (125 mg IV) is given 30 minutes prior to the first dose of alemtuzumab. For the second dose, the same premedication regimen is given, excluding methylprednisolone. Side Effects: The most common side effects of alemtuzumab include infusion-related reactions characterized by fever, chills, rigors, nausea, vomiting, hypotension, and skin rash. These usually occur with the initial dose and can be prevented or lessened with the pre-medication regimen described above. There was no apparent increase in incidence of infectious complications in 31 renal transplant patients who received alemtuzumab as induction therapy when compared to other immunosuppressive regimens (18). Other trials using alemtuzumab for treatment of patients with multiple sclerosis have revealed a high incidence of Graves’ disease (19). This complication has not been described in patients who received alemtuzumab for other indications (lymphoid malignancies, renal transplant recipients). Monitoring of thyroid function should be a standard test every 6 months as well as in the event of symptomatology suggesting Graves’ disease. Three cases of idiopathic thrombocytopenic purpura have been reported in patients with multiple sclerosis treated with high dose alemtuzumab. One resulted in fatality (20,21). Other possible side effects include: peripheral edema, headache, dysthesias, dizziness, rash, urticaria, pruritus, anorexia, diarrhea, abdominal pain, stomatitis/mucositis, leukopenia, severe anemia, weakness, myalgia, dyspnea, cough, bronchitis/pneumonitis, pharyngitis, infections, and diaphoresis. Rabbit Anti-Thymocyte Globulin Mechanism of Action: Rabbit anti-thymocyte globulin (ATG, Thymoglobulin) is an anti-thymocyte polyclonal antibody that has been among the most potent immunosuppressive agents used in organ transplantation for many years (22). It induces a rapid and profound lymphocytopenia that can be attributed to several mechanisms of action that are still not fully understood. The possible mechanisms by which Thymoglobulin can cause immunosuppression in vivo include: T-cell clearance from the circulation and modulation of T-cell activation, homing,

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and cytotoxic activities (23). In patients treated with this agent, T-cell depletion is usually observed within a day of initiating Thymoglobulin therapy (24). It has been used as an induction agent in kidney and pancreas transplant recipients to prevent acute rejection (25,26). More recently, it was used with success in single donor marginal mass islet transplantation in combination with daclizumab (15). Dose: ATG 6 mg/kg is given IV for a total of five doses, on days –2, –1, 0, þ1, and þ2. The dose is increased gradually as follows: 0.5 mg/kg on day –2, 1.0 mg/kg on day –1, and 1.5 mg/kg on days 0, þ1, and þ2. The first dose should be administered over 12 hours and subsequent doses may be administered over 6 hours. Patients should be pre-medicated with diphenhydramine, acetaminophen and methyprednisolone, in order to decrease peri-infusion side effects. It is preferable to give ATG via central line. If no central access is obtained it can be given peripherally, diluting it in normal saline with heparin and hydrocortisone (27). Side Effects: Adverse reactions include hypertension, peripheral edema, tachycardia, chills, fever, headache, pain, malaise, rash, hyperkalemia, abdominal pain, diarrhea, nausea, leukopenia, thrombocytopenia, weakness, dyspnea, and systemic infections. Anaphylactic reactions and serum sickness have also been reported with the use of ATG.

Maintenance Immunosuppression Sirolimus Mechanism of Action: Sirolimus (Rapamune), discovered from a soil sample collected in Easter Island also known as Rapa Nui (28,29), is a macrocyclic lactone isolated from Streptomyces hygroscopicus characterized by potent immunosuppressive activity that blocks T- and B-cell activation by cytokines. Following entry into the cytoplasm, sirolimus binds to the FK binding protein (FKBP) and the complex presumably modulates the activity of the mammalian target of rapamycin (mTOR) (30), inhibiting interleukin-2 mediated signal transduction. This results in cell cycle arrest in the G1-S phase. Sirolimus blocks T- and B-cell activation by cytokines, which prevents cell-cycle progression and proliferation, in contrast to tacrolimus and cyclosporine (CyA) (Neoral, Novartis Pharmaceuticals Corp., East Hanover, New Jersey, U.S.A.), which inhibit cytokine production (31). In addition to its immunosuppressive effects, sirolimus has also shown anti-proliferative effects in terms of malignancy (anti-cancer), angiogenesis (anti-vascularization), and cellular proliferation (anti-regeneration), the latter two having potential effects on the islet graft. Furthermore, sirolimus can inhibit fibroblast growth factors required for tissue repair, which can result in impaired wound healing (32).

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Dose: Sirolimus can be given pre-transplant on day –1 or on the day of transplant, at a single oral dose of 0.2 mg/kg, followed by 0.1 to 0.15 mg/kg per day thereafter. Daily dose is adjusted to maintain a whole blood 24 hr trough target level of 10–15 ng/ml for the first 3 months and 8–12 ng/ml thereafter, as tolerated. If a patient experiences severe neutropenia (absolute neutrophil count <500 cells/mm3), sirolimus dose can be reduced based on protocol management of neutropenia. Adjustments on sirolimus dose to a given blood trough target level can be made based on the following equation (11): Predicted SRL level ¼ Old SRL level x [new SRL dose / old SRL dose] Side Effects: The principal adverse reactions associated with the administration of sirolimus are: mucosal ulcers (oral and gastrointestinal) (33), dyslipidemia, hypertension, skin rash, and acne, as well as facial and leg edema. In addition, deterioration of kidney function has been observed in transplanted patients receiving sirolimus in combination with tacrolimus (13). There have been some cases of interstitial pneumonitis, some fatal, with no other identified etiology in patients receiving sirolimus. This condition has resolved in some cases when the medication was discontinued or the dose was reduced (34). There has been one reported case of sirolimusinduced pneumonitis in an islet transplant recipient (35). There may also be an increased risk for infection and the development of lymphoma. Other possible side effects include: chest pain, fever, headache, insomnia, hypophosphatemia, hypokalemia, abdominal pain, nausea, vomiting, diarrhea, constipation, dyspepsia, weight gain, urinary tract infection (UTI), anemia, thrombocytopenia, leukopenia, arthralgia, weakness, back pain, increased serum creatinine, dyspnea, upper respiratory infection, and pharyngitis. Tacrolimus Mechanism of Action: Tacrolimus (Prograf) (FK506) is a macrolactam with potent immunosuppressive activity. It belongs to the family of calcineurin inhibitors. It binds to the intracellular protein, FKBP-12, creating a complex (FK:FKBP), which constitutes the active drug. The complex targets the calcium-regulated phosphatase calcineurin, inhibiting its function, and thus preventing cytokine transcription and T-lymphocyte activation. Dose: Tacrolimus is started at 1 mg orally b.i.d. and subsequently adjusted to achieve 12 hr trough levels of 4–6 ng/ml. Side Effects: Tacrolimus, similar to other calcineurin inhibitors, has been associated with nephrotoxicity, neurotoxicity, and diabetogenesis (36). It can cause an increased risk for infection. There is also the possibility of developing lymphoma. Lymphomas have developed in patients receiving

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tacrolimus and other forms of immunosuppressive therapy after transplantation, though no causal relationship has been established. Additional side effects associated with tacrolimus include a burning sensation in the hands or feet, hand tremors, insomnia, headache, mild nausea, abdominal cramps, and diarrhea. Other side effects such as chest pain, hypertension, dizziness, pruritus, rash, hyperglycemia, hyperkalemia, hyperlipidemia, hypomagnesemia, hypophosphatemia, constipation, dyspepsia, anemia, leukopenia, thrombocytopenia, arthralgia, back pain, weakness, paresthesia, abnormal kidney function, UTI, high blood urea nitrogen (BUN), atelectasis, and increased cough have been noted in some patients treated with tacrolimus. Cyclosporine, USP Mechanism of Action: Cyclosporine (CyA) (Neoral) is a calcineurin inhibitor known to be a potent immunosuppressive agent that has been extensively used in solid organ transplantation. Its main action results from specific and reversible inhibition of T-lymphocytes in the G0- and G1-phase of the cell cycle. T-helper cells are the main target, but it can also inhibit Tsuppressor cells. Lymphokine production and release and interleukin-2 can be also inhibited by this drug. CyA has been used as maintenance immunosuppressive therapy in islet after kidney and simultaneous islet-kidney recipients (37–39). Currently there are no studies in islet transplantation alone using this drug as a maintenance immunosuppressive therapy, but due to its similar mechanism of action it can be used instead of tacrolimus if clinically indicated. Dose: CyA is administered orally at an initial dose of 6 mg/kg/d divided in two doses per day. No results have been published regarding trough levels with maintenance immunosuppressive regimen based on sirolimus and CyA for islet transplantation alone after the Edmonton era. Side Effects: The most common adverse reactions of CyA therapy are renal dysfunction, tremor, hirsutism, hypertension, and gingival hyperplasia. Other side effects include: headache, edema, hypertrichosis, hypertriglyceridemia, female reproductive disorders, nausea, diarrhea, dyspepsia, infection, and allergic reactions. Mycophenolate Mofetil Mechanism of Action: Mycophenolate mofetil (MMF) (Cellcept; Hoffman-La Roche, Inc.) is an immunosuppressive drug that acts by interfering with the de novo synthesis of purines via inhibition of the inosine monophosphate dehydrogenase, therefore inhibiting T- and B-cell proliferation. It has been used as an alternative therapy to tacrolimus or sirolimus due to a lesser incidence of nephrotoxic side effects (9,15).

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Dose: MMF should be used orally at a dose of 500–1500 mg b.i.d. as a replacement for tacrolimus or sirolimus (40). Due to high intra- and interpatient variability, MMF is given based on a fixed dose according to patient’s tolerability, instead of target levels. Side Effects: The primary adverse reactions associated with the administration of MMF include diarrhea, leukopenia, sepsis, and vomiting. There is also evidence of higher frequency of infections. Lymphoproliferative disease and/or lymphoma developed in 0.4–1% of patients receiving MMF (2 g or 3 g daily) with other immunosuppressive agents in controlled clinical trials of renal, cardiac, and hepatic transplant patients followed for at least 1 year. Non-melanoma skin carcinomas occurred in 1.6–4.2% of patients; other types of malignancy occurred in 0.7–2.1% of patients. In patients that do not tolerate the MMF increase due to severe diarrhea or leukopenia, the dose can be increased at a slower pace or kept at a lower, more tolerable dosage. If the patient cannot tolerate the medication at all (severe gastrointestinal side effects, i.e., nausea or diarrhea) the patient can be switched to an enteric-coated form (Myfortic ; Novartis Pharmaceuticals Corp.) that might decrease or alleviate gastrointestinal symptoms. Severe neutropenia is one of the most common side effects in patients taking MMF; if absolute neutrophil count is <500 cell/mm3, the MMF dose can be decreased and the patient should be treated according to the protocol management for neutropenia. Mycophenolate Sodium Mycophenolate sodium (Myfortic) may be used as a replacement for MMF. Mycophenolate sodium will be dosed at 360–720 mg orally twice a day (180 mg of mycophenolate sodium are equivalent to 250 mg of MMF). In some patients, mycophenolate sodium has the advantage of causing fewer gastrointestinal side effects than MMF.

Concomitant Medications: Anti-inflammatory Therapy Etanercept Mechanism of Action: Etanercept (Enbrel; Immunex Corp., Thousand Oaks, California, U.S.A.) is a soluble form of the tumor necrosis factor α (TNF-α) receptor, which binds specifically to TNF-α and blocks its interaction with the cell surface TNF-α receptors, preventing TNF-α mediated cellular response (41). TNF-α is a naturally occurring cytokine involved in normal inflammatory and immune responses. Due to the antiinflammatory and immunomodulating properties of etanercept, it has been used in patients with rheumatoid arthritis, ankylosing spondylitis, and

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psoriasis. It has been shown that TNF-α is cytotoxic to human islet b-cells (42), therefore its blockage can represent an important addition to the current induction therapies, improving early islet engraftment and subsequent outcome. Dose: Etanercept is given IV at a dose of 50 mg, 1 hour prior to transplant, followed by 25 mg subcutaneously twice a week for 2 weeks. The IV infusion of etanercept has not been approved by the FDA, but it is used to prevent TNF-α mediated cytokine release in the early post-transplant period. To date, in the Miami experience, no severe adverse effects have been observed using the IV administration route. Side effects: Most common adverse effects seen in patients treated with etanercept are: headaches, local site reaction, respiratory tract infection, rhinitis, infection, and positive ANA and DNAds antibodies. Other side effects seen in a lower percentage of cases include: dizziness, rash, abdominal pain, dyspepsia, nausea, vomiting, weakness, pharyngitis, respiratory disorder, sinusitis, and cough. Other important and rare but potentially serious reactions include: allergies, angioedema, aplastic anemia, neutropenia, pancytopenia, thrombocytopenia, cerebral ischemia, myocardial ischemia and infarction, heart failure, coagulopathy, demyelinating disorders, depression, fever, flushing, infection (serious), intestinal perforation, leukopenia, lupus-like syndrome, lymphadenopathy, malignancies (including lymphoma), membranous glomerulopathy, hyper-/hypotension, pancreatitis, polymyositis, pulmonary disease, and weight gain.

Anticoagulation In order to decrease the incidence of portal vein thrombosis, anticoagulation with IV heparin or subcutaneous low molecular weight heparin (Lovenox) can be given. Prophylactic Therapies Pneumocystis carinii Prophylaxis Trimethoprim-Sulfamethoxazole: Trimethoprim-Sulfamethoxazole (Bactrim SS; Hoffman-La Roche, Inc.) is the most effective prophylactic therapy against Pneumocystis carinii pneumonia (PCP) in immunosuppressed patients (43). It is given orally at a dose of 80 mg/400 mg q.d. starting at day of transplant and for the duration of immunosuppression. There are however, alternative prophylactic agents for PCP in patients with sulfa allergies or neutropenia. These include

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Dapsone: 100 mg orally twice a week. It is worth noting that dapsone can cause hemolysis and has been associated with falsely low A1c levels, independent of blood glucose control (44). Atovaquone (Mepron):

1500 mg orally daily.

Pentamidine Isethionate (Pentam): Given by inhalation at a dose of 300 mg in 6 ml sterile water every 4 weeks. Cytomegalovirus Prophylaxis Due to the risk of the novo (R) or reactivation (Rþ) of cytomegalovirus (CMV) infection in the early post-transplant period (when patients are highly immunosuppressed), ganciclovir 1,000 mg t.i.d. or valganciclovir 900 mg q.d. can be given orally starting on day –1 and for 90 days after transplantation, irrespective of CMV status of donors and/or recipients. Both regimens have shown similar efficacy in preventing CMV in solid organ transplant recipients (45). Adjuvant Therapies Some known antioxidants and other vitamins are used in order to improve engraftment of the transplanted islets. Pentoxifilline (Trental; Aventis Pharmaceuticals, Bridgewater, New Jersey, U.S.A.) is one of the antioxidants commonly used in the early post-transplant period. It can be given orally at a dose of 400 mg three times per day for one week and up to 3 months after transplantation. Side effects are mild and the majority of patients tolerate this medication well. Other antioxidants and vitamins that are used frequently include: vitamin C 1,000 mg tablets p.o. daily, vitamin E 800 I.U. p.o. daily†, vitamin A 25,000 I.U. p.o. daily‡, and vitamin B6 100 mg p.o. daily. Drug Metabolism and Interactions Drug interactions play an important role in the follow-up and treatment of patients after islet transplantation. Most of the maintenance immunosuppressive drugs used in islet transplantation are metabolized by the Cytochrome P-450 enzymes. These are recognized as important factors in the biosynthesis and degradation of endogenous compounds such as steroids, lipids, and vitamins and in the metabolism of many chemicals †

Due to the possible risk of increased all-cause mortality with long-term, high-dose vitamin E (46), we no longer recommend the dose of 800 I.U. p.o. daily. A dose of 400 I.U. p.o. daily may be more appropriate. ‡ Due to the possible increased risk of hepatoxicity (47) fractures and (48) with long-term, high-dose vitamin A intake, we no longer recommend the dose of 25,000 I.U. p.o. daily. A dose of 4,000–5,000 I.U. p.o. daily may be more appropriate.

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present in the diet and environment, as well as medications (49). Therefore, the number of drugs and/or compounds with which these immunosuppressive drugs might interact is extensive (Table 3). The CYP3A is probably the most important of all drug-metabolizing enzymes, accounting for nearly 50 percent of the Cytochrome P-450 enzymes (50). In addition, the immunosuppressive drugs are known to be substrates for p-glycoprotein which can affect the rate of drug absorption (51). In the case of sirolimus and tacrolimus, they are specifically metabolized by the CYP3A4 iso-enzyme from the P-450 family located in the liver and in the enterocytes of the small intestine. Compounds that reduce CYP3A4-mediated metabolism in the wall of the gut, such as grapefruit juice, can increase levels of sirolimus and tacrolimus and therefore should be avoided. Macrolides, with the exception of azithromycin, can also increase the levels of both sirolimus and tacrolimus. In patients where strong inhibitors or inducers of CYP3A4 are indicated, alternative therapeutic agents with less potential for inhibition or induction of CYP3A4 should be considered. Close drug monitoring, including trough levels of immunosuppression, is highly recommended when a new agent with possible interactions is introduced. All health care professionals involved in the care of an islet transplant recipient should discuss with the transplant team before any new drug is introduced. CONCLUSIONS Effective immunosuppressive management is one of the most important factors in the prevention of acute graft rejection and for the improvement of long-term graft survival. Drug toxicity and adverse events that arise from the use of these drugs can become a major limitation for their use, affecting the transplant recipient’s health and quality of life. Close monitoring of maintenance immunosuppressive drugs trough levels and monthly toxicity assessments are currently an important part of the follow-up and care of the transplant recipients. Dose adjustments and pharmacological therapy are often necessary in order to treat the most common side effects. In addition, changes in immunosuppressive therapies after transplantation have become a more standard practice when indicated (dermatotoxicity, neurotoxicity, or nephrotoxicity) than in the past. Newer immunosuppressive agents with more specific and limited mechanisms of action will help to decrease the widespread range of side effects and to minimize complications and organ toxicity. IMMUNOSUPPRESSIVE THERAPY-RELATED COMPLICATIONS Upon starting immunosuppressive therapy, both the patient and the transplant team are faced with risks, such as the worsening of pre-existing medical diseases, as well as the presentation of new complications such as

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Table 3

List of Most Common Agents that Can Alter the Drug Metabolism of Sirolimus and Tacrolimus Increase levels Antibiotics Fluoroquinolones Gatifloxacin Moxifloxacin Macrolides Clarithromycin Erythromycin Antifungal agents Clotrimazole Fluconazole Itraconazole Ketoconazole Voriconazole Antivirals Delavirdine Nelfinavir Ritonavir Antidepressants Nefazadone Calcium channel blockers Diltiazem Nicardipine Verapamil H2 inhibitors Cimetidine Immunosuppressants Cyclosporine Steroids Methylprednisolone (>250 mg/dL) Others Bromocriptine Cisapride Danazol Grapefruit Metoclopramide

Decrease levels Antibiotics Nafcillin Rifabutin Rifampin Rifapentine Antivirals Efavirenz Nevirapine Anticonvulsivants Carbamazepine Phenobarbital Phenytoin Others St. John’s wort

hyperlipidemia, hypertension, opportunistic infections, cancer, and other systemic diseases secondary to immunosuppression (52). The full extent of immunosuppressive related complications in the field of islet transplantation is not fully known, given that this field, and its success, is relatively young (2,3,6,8–10,53). In this section, the most common immunosuppressive related complications that have been observed in islet transplant recipients over

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the last five years will be reviewed (Table 4) (8–10,15,53). Most will be related to the experience of the Edmonton Protocol of immunosuppression. Common complications are usually mild to moderate in severity, such as diarrhea and sirolimus-induced mouth ulcers (77–89%) (8,9). Occasionally uncommon severe or life-threatening conditions may present, such as pneumonia, meningitis, or encephalitis (8–10,35). Elevation in Liver Function Tests There is a significant elevation in liver function tests, both alanine-amino transferase (ALT) and aspartate-amino transferase (AST), which peaks in the first week post-islet transplant and normalizes in most subjects by 30 to 40 days (8,54). This transaminitis (54,55) is believed to be secondary to the intraportal islet infusion, and therefore not an immunosuppressive related complication, per se. Elevation of liver function tests can also be seen in relationship to the use of sirolimus and tacrolimus (54,56). Hematological Abnormalities Anemia Anemia is commonly seen after islet transplantation (96–100% of patients) (8–10,53). Anemia can be both normocytic, normochromic, and/or from iron deficiency. The main contributory factor for anemia in post-islet transplant patients is the sirolimus and tacrolimus bone marrow immunosuppressive effects (57,58), compounded by the significant amount of blood drawn in the peri-transplant period for research and monitoring purposes. The authors recommend performing baseline iron studies (iron, ferritin, total iron binding capacity) pre-transplant and repeating them if anemia is present. Most patients will require iron supplementation. Few patients (8%) have required treatment with erythropoietin-α for the management of the anemia (9). Leukopenia Depending on the timing and immunosuppressive regimen used, the severity and type of leukopenia will differ. Using the Edmonton Protocol of immunosuppression, Miami has observed leukopenia in 100% of the patients, National Cancer Institute (NCI) Criteria grade 2 (2000–3000/µl) in 62% and grade 3 (1000–2000/µl) in 38% of cases (8). This resolved without treatment in the majority of patients after 3 months post-transplant. Grade 4 neutropenia (<500/µl) was observed in 23% (6/26) of the patients. The immunosuppressive drugs sirolimus and MMF are common causes of neutropenia. The prophylactic antibiotics, trimethoprim-sulfamethoxazole and valganciclovir are common culprits associated with neutropenia. Lowering the dose of both types of drugs can assist in the resolution of the neutropenia. Nevertheless,

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Table 4

Adverse Events by Frequency of Appearance in 26 Recipients of Islet Transplantation from April 2000 to June 2004: Miami Experience Grade 1–3 Frequency High frequency (61% or greater)

Medium frequency (11% to 60%)

Grade 4

Adverse event

No. pts.

%

No. pts.

%

Increased ALT Increased AST Leukopenia Menstrual irregularitiesa Anemia Hypophosphatemia Increased LDL Oral ulcers Diarrhea Hypomagnesemia Upper respiratory infections Thrombocytopenia Ovarian cysts Insomnia Acne Dysfunctional uterine bleeding Vaginal infection Edema Headache Increased alkaline phosphatase Increased bilirubin Vomiting Nausea Increased serum creatinine Fatigue Tremor Neutropenia Bronchitis Bacterial skin infection Folliculitis Depression Urinary tract infections Eczema Myalgia Tendonitis Anxiety Arthralgia Dental infection Fungal skin infection Memory loss Pneumonia

26 26 26 15 25 25 20 20 18 18 18 16 9 14 14 8 7 12 12 12 12 12 11 10 8 7 — 6 6 6 5 5 4 4 4 3 3 3 3 3 3

100 100 100 100 96 96 77 77 69 69 69 62 60 54 54 53 47 46 46 46 46 46 42 38 31 27 — 23 23 23 19 19 15 15 15 12 12 12 12 12 12

— — — — — — — — — — — — — — — — — — — — — — — — — — 6 — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — — — — — — — 23 — — — — — — — — — — — — — —

(Continued)

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Table 4

Adverse Events by Frequency of Appearance in 26 Recipients of Islet Transplantation from April 2000 to June 2004: Miami Experience (Continued ) Grade 1–3 Frequency

Adverse event

No. pts.

Low frequency (10% or less)

Alopecia areata Dizziness Hypertension Urticariform rash Procedure related Procedure related bleeding Abdominal hernia Subacute cholecystitis Aseptic meningitis Aspiration pneumonia CMV disease Febrile neutropenia Hypereosinophilic syndrome Influenza infection Parvovirus B19 infection Retinal central vein occlusion Tractional retinal detachment Skin squamous cell Ca

2 2 2 2 2 1 1 1 1 1 1 1 1 — — 1 1

Grade 4

%

No. pts.

%

8 8 8 8

— — — —

— — — —

8 4 4 4 4 4 4 4 4 — — 4 4

1 — — — — — — — — 1 1 — —

4 — — — — — — — — 4 4 — —

a

Percentage from total number of females (n=15). Abbreviations: ALT, alanine-amino transferase; AST, aspartate-amino transferase; CMV, cytomegalovirus; LDL, low-density lipoprotein.

CMV disease should be ruled out as another possible underlying cause of leukopenia. In case of an absolute neutrophil count (ANC) below 1000/µl in a febrile patient, or a count below 500/µl in a febrile or afebrile patient, treatment with granulocyte colony stimulating factor (GCSF-Neupogen; Amgen, Inc., Thousand Oaks, California, U.S.A.; 5 µg/kg body weight, subcutaneous injection) until neutrophil count reaches 1000/µl, is warranted (59,60). This has resulted in resolution of neutropenia (8,53). In case of fever and an ANC below 500/µl the patient should be hospitalized with the diagnosis of neutropenic fever, placed on neutropenic precautions, and treated with IV antibiotics. In the Miami experience there has only been one such case (1/26) (8). Expected grade 4 lymphopenia can be seen with induction regimens based on alemtuzumab and anti-thymoglobulin (15,61), and can last anywhere from 6 to 12 months. Thrombocytopenia Thrombocytopenia is a known side effect of sirolimus (62). Most Miami patients (62%) have presented a transient and mild form of

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thrombocytopenia in the first three months post-transplant (8). Thrombocytopenia can be seen with alemtuzumab use and as described in the previous section it can present as immune thrombocytopenic purpura (ITP). Thrombocytopenia can also be seen with ATG. Oral Ulcers Sirolimus interferes with wound healing, contributing to ulcer formation in the oral mucosa (63). Mouth ulcers are the most common side effect encountered in islet transplant recipients (8–10,15; 53). They are usually small, white lesions, either flat or plaque-like, with a reddish contour and variable depth (Fig. 2). They are most common in the first three months

Figure 2 Aphthous ulcers located on the lower lip and tongue, respectively, of two different islet transplant recipients. Both resolved satisfactorily after treatment.

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post-transplant when sirolimus dose is at its highest. Treatment with simple antiseptic measurements (antibacterial mouthwash) and oral triamcinolone (1% dental paste) is usually successful (8,9). Viral cultures for CMV, Epstein-Barr virus (EBV), and herpes simplex virus (HSV) are usually negative, and are not recommended unless there is a relatively high index of clinical suspicion (multiple ulcers, preceded by vesicles associated with fever). The ulcers are commonly uncomfortable and can be painful, resulting in limited food intake. It is rare for the lesions to be severe enough to require surgical debridement and/or hospitalization (9). Sirolimus dose reduction or a switch from sirolimus to MMF have been required on rare occasions. Gastrointestinal Disorders Nausea, vomiting, and diarrhea are common complications seen in islet transplant recipients (approximately 50%) (8–10) and are common side effects of sirolimus, tacrolimus, and MMF. These symptoms, especially if associated with pre-existing autonomic neuropathy (gastroparesis and enteropathy), can interfere with achieving adequate levels of immunosuppression (9). An infectious cause for diarrhea must be ruled out. Stools should be sent for fecal leukocytes, fecal occult blood, bacterial culture (including E. coli), ova, and parasites (including Cryptosporidium, Isospora, Microsporidia, and Cyclospora). Clostridium difficile toxin is particularly important to test for, since the patients are chronically taking trimethoprim-sulfamethoxazole for PCP prophylaxis. If studies are negative, symptomatic treatment or immunosuppressive drug reduction can be implemented. If diarrhea persists, or if it is associated with blood loss, the patient should have a colonoscopy, since ischemic or CMV colitis (Fig. 3) may be the culprit (64,65).

Figure 3 Pictures of a colonoscopy showing lesions compatible with cytomegalovirus colitis in an islet transplant recipient. Terminal ileum shows ileitis with mild erythema, edema with several tiny erosions of the mucosa (A). Colitis and ulceration of the transverse (B) and ascending (C) colon showing scattered erosions, erythema, and edema of the mucosa. Arrows indicate ulcerative lesions.

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Neurological Disorders The reporting of tacrolimus-associated neurological abnormalities is variable. The Edmonton group has reported fine tremor on physical examination, as well as the subjective perception of tremulousness not evident on examination, to be a common side effect on islet transplant recipients. Two patients developed benign essential tremor of moderate severity associated with gait disturbance, which improved after alteration of immunosuppressive medications (9). In the Miami experience, insomnia (54%), headaches (46%), tremors (27%), anxiety (12%), and short-term memory impairment (12%) were found to be common (8). Two patients developed severe tacrolimus neurotoxicity, requiring conversion to MMF with resolution of symptoms (8). No information is available for long term diabetic neuropathy outcomes post-islet transplant (66). Dyslipidemia Sirolimus can cause an increase in lipid levels (52,67). Most patients have an increase in LDL levels (>100 mg/dl / 3 mmol/L), requiring the introduction of lipid-lowering therapy in ∼60% of cases and an increase in pre-existing therapy in ∼20% of the cases to obtain LDL levels of ∼70 mg/dl/2.1 mmol/L or lower (6,8–10). Statins are the preferred agents used to treat the dyslipidemia, although on occasion (i.e., statin-related myositis), ezetimibe (Zetia  ; Merck Schering-Plough Pharmaceuticals, North Wales, Pennsylvania, U.S.A.) has been used. Since lipid levels are usually monitored monthly post-transplant, there has been no delay in initiation of therapy. It is possible that this is the reason for the low incidence of hypertriglyceridemia seen in islet transplantation, as opposed to solid organ transplant series (8,10,52,67,68). Other explanations could be related to the patients’ characteristics, such as lower body mass index (BMI) without preexisting insulin resistance, absence of pre-existing kidney failure, or avoidance of cyclosporine and/or steroids in islet transplant recipients. Renal Disorders Hypertension The underlying pathophysiological mechanisms of calcineurin inhibitorinduced hypertension include enhanced sympathetic nervous system activity, renal vasoconstriction, and sodium and water retention (52,69). These are reported to be more frequent in patients treated with cyclosporine than with tacrolimus therapy (52,70). In the Miami experience, two patients that were on antihypertensive medications continued with their therapy, and one patient developed hypertension post-transplant requiring therapy (6,8). In

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the Edmonton experience, 36% of the patients pre-transplant were not on any anti-hypertensive medication, and 6% were on more than one medication for hypertension. Post-transplant, the respective percentages at 5 years are 15% and 42% (9). Edema Intermittent peripheral edema is common (∼40%) in islet transplant recipients (8–10). This is usually mild, not requiring therapy. In moderate cases, support stockings and diuretics can be used for symptomatic relief (8). In some cases, periorbital, facial, upper extremity, or unilateral edema can occur. Sirolimus conversion to MMF has been required for resolution of severe edema in some cases (9,10). Alterations in Kidney Function The Edmonton group has seen a significant increase in serum creatinine at 5 years post-transplant when compared to pre-transplant (p < 0.001) and 1 year post-transplant (p < 0.01); and a non-significant trend towards a decrease in creatinine clearance with time (9). In Miami, there has been a nonsignificant trend towards increased serum creatinine with time (8). There has also been a case of tacrolimus-induced nephrotoxicity with an increase in serum creatinine to 2 mg/dl (180 µmol/L) and a decrease in creatinine clearance from 81.5 ml/min to 48.0 ± 4.0 ml/min (n ¼ 3 measurements). The patient was successfully converted from tacrolimus to MMF with improvement in serum creatinine (1.1 mg/dl, 100 µmol/L) and creatinine clearance (70 ml/min) (8). Electrolyte Disorders Hypomagnesemia and hypophosphatemia are common side effects of calcineurin inhibitors (52) and have been observed in islet transplantation (8). The significance on bone mineral density of these abnormalities is not yet known. Replacement with oral supplements is required if severe deficiency is demonstrated by serum electrolyte measurements. Proteinuria Progression from normoalbuminuria (<30 mg/24 hrs) to macroalbuminuria (>300 mg/24 hrs) has been observed in 2 of 12 (17%), and 3 of 30 (10%) patients, in the Miami and Edmonton experience, respectively. Progression from microalbuminuria (30–300 mg/24 hrs) to macroalbuminuria has been reported in 3 of 4 (75%), and 5 of 11 (45%) patients in the Miami and

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Edmonton experience respectively (6,9). Froud et al. reported proteinuria (>150 mg/24 hrs to <1 g/24 hrs) in all islet transplant recipients (n ¼ 16) at a mean of 15 ± 10 months post-transplant (6). Senior et al. has also reported 3 cases of proteinuria (2–3 g/24 hrs) which resolved after conversion from sirolimus to MMF (13). Sirolimus-induced proteinuria appears to be secondary to an increase in glomerular filtration rate (71) and a renal tubular mechanism (72). The long-term implications of this proteinuria on kidney function and diabetic nephropathy are not known. The use of angiotensin converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs) is recommended in these situations (71). Skin Manifestations Most patients developed skin manifestations, consisting of acne (50–54%) (8,9), folliculitis (23%), eczema (12%), urticariform rash (8%), and alopecia areata (8%) (8). Acne is a common side effect of sirolimus. Eczema is most likely secondary to tacrolimus (73). Squamous cell carcinoma has been reported on one occasion (8). Infectious Diseases The risk of infection in transplant recipients is determined by the overall immunosuppression, exposure to pathogens, and the virulence of the organism in question (74). In the first three months, when immunosuppression is maximal, nosocomial bacterial and fungal infections increase in solid organ recipients (Fig. 4) (52,74). Since islet transplant recipients have a very short hospital stay, a significant increase in the nosocomial bacterial infections has not been reported to date. In the Miami experience, thus far there has been one case of parvovirus infection and one case of aspiration pneumonia within the first three months post-transplant (8). The risk of opportunistic infections such as PCP, mycobacterium, opportunistic fungi, and CMV and other viruses is usually higher in the first 4 months posttransplant. The authors use trimethoprim-sulfamethoxazole for PCP prophylaxis for the duration of immunosuppression and, to date, no PCP has been reported in islet transplant recipients. Valgancyclovir or ganciclovir prophylaxis is given in the first three months after each transplant for CMV prophylaxis. In solid organ recipients, after four months post-transplant, the risks of infection are similar to the general population (52,74). In islet transplantation, immunosuppression levels are kept higher than in solid organ transplantation. There are not yet sufficient data and follow-up has been too short to determine if the risk of infections is higher in islet transplant recipients when compared to non-transplanted individuals with Type 1 diabetes. Given the high risk of infections in immunosuppressed

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Figure 4 Most common infections after organ transplantation based on the time of occurrence. Source: Ref. 74.

patients, surveillance and reporting of infections as adverse events are important (75). Cytomegalovirus CMV is one of the most frequent viral infections encountered in patients after solid organ transplantation. It can be acquired during the early posttransplant period, due to transmission from a CMV positive donor or reactivation of pre-existing disease (CMV-positive recipient) (74,76). CMV can also appear in the late post-transplant period (more than six months) due to the previously noted causes or due to community-acquired infection, in the form of CMV syndrome, infection, or disease (colitis or retinitis). In islet transplantation the probability of transmission from donor tissue is low, even though the ratio of CMV positive donors to negative Type 1 diabetes recipients is higher (77). CMV syndrome can present with unexplained low grade fever associated with malaise and laboratory abnormalities such as leukopenia, atypical lymphocytosis, thrombocytopenia, and mild hepatitis (65,74). Assessment of CMV DNA by polymerase chain reaction is the main test to determine the presence of the virus in

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patients where CMV is suspected. Prophylactic therapy is given after each islet transplant (See section on CMV prophylactic therapy). Treatment for CMV disease is based on intravenous CMV hyper-immune globulin and/or valgancyclovir or ganciclovir. In the Edmonton series, two CMV seroconversions have been reported, without overt disease (9). In the Miami experience, one case of seroconversion (6.4 months after a CMV donor positive islet infusion) and one case of CMV colitis (15 months after the last CMV donor positive islet infusion) have been observed (Fig. 3). Community-acquired disease was the presumed cause in the latter case (65). Other Infections Over a mean follow up period of 22 ± 11 months, in 26 patients, upper respiratory infections were the most frequent adverse event seen in Miami (69% of individuals). Other infections consisted of bronchitis (23%), skin bacterial infections (23%), urinary tract infections (19%), dental infections (12%), fungal skin infections (12%), and pneumonia (12%). Other rare infections seen included one case of aspiration pneumonia, one case of parvovirus, and one case of suspected adenoviral encephalitis. In these three incidences, immunosuppression withdrawal was required with subsequent graft loss. Complete recovery without sequelae was observed in all three. One case of presumed fungal pneumonia requiring discontinuation of sirolimus has been reported (9).

Alterations of the Female Reproductive System Cure et al. (78) reported menstrual cycle alterations and ovarian cysts in islet transplant recipients. All females with history of regular menses (n ¼ 6) became irregular after transplantation, and 43% of the female population presented with clinically significant ovarian cysts between 1 and 21 months post-transplant. Treatment with oral progestin or oral contraceptive preparations has been partially successful when treating the menstrual alterations and ovarian cysts (78). In few occasions (n ¼ 2) the cysts required surgical treatment. Surveillance with pelvic ultrasound and gynecological evaluation pre- and post-transplant is recommended, at least yearly, given the high frequency of these alterations. Weight Loss Weight loss is common after islet transplantation. The most likely cause is the disappearance of hypoglycemic events, resulting in a decreased need for frequent snacking (8,53). Other causes may include the nausea, vomiting, and diarrhea associated with the immunosuppressive regimen.

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Malignancy Squamous cell carcinomas are the most frequent skin cancers in transplant recipients. They are mainly related to sun exposure, and the risk is increased with immunosuppression (79). In Miami, only one case of squamous cell carcinoma has been reported (8). One case of papillary thyroid carcinoma has been reported in islet transplantation alone in the Edmonton experience (9). There may be an increased risk for the development of lymphoma in islet transplant recipients. To date, no post-transplant lymphoproliferative disorder has been reported. Retinopathy The Edmonton group has reported deterioration of eye disease in 4/62 (6%) subjects (9,66), with the need for photocoagulation or vitrectomy within 5 months of transplant. Hafiz et al. (8) has reported progression of stable proliferative retinopathy in 3/26 (12%) patients. These patients presented with pre-retinal bleeds (n ¼ 2) and tractional retinal detachment (n ¼ 1). The early increase in retinopathy is believed to be secondary to the acute improvement in glycemic control seen after islet transplantation. This is similar to what was observed in the intensive treatment arm during the first year in the Diabetes Control and Complications Trial (80), as well as in kidney-pancreas transplant recipients (81). Pre-transplant ophthalmologic evaluation with retinal photography to assess stability of retinal disease should be undertaken before transplantation. Follow-up three months after transplantation and regularly thereafter is recommended to detect disease progression with early laser photocoagulation performed if necessary. CONCLUSIONS All the patients treated under the Edmonton Protocol of immunosuppression have had adverse events (Table 4). Most of them have been mild, selflimiting, and easily treated. However, some have required urgent medical attention. Patient education and frequent monitoring have been extremely important in timely detection and treatment of these events. To date, no post-transplant lymphoproliferative disorder or graft-versus-host disease has been reported. There has been a low frequency of procedure-related complications in the Miami experience. To date, out of 74 procedures, we have only observed three bleeds (one severe, requiring transfusion) and three pleural effusions. No portal vein thrombosis has been observed in the Miami experience. One of the main goals in the field of islet transplantation is to achieve tolerance. This would eventually allow immunosuppressive drug-free regimens and, therefore, fewer adverse events. With current and future

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progress in human pharmacogenomics, drugs that are target specific and have fewer side effects will be found and will be available for clinical use in place of the ones presented in this chapter. Until this is achieved we will continue to use these drugs. A multidisciplinary team approach expert in the management of immunosuppression and related complications is necessary in order to offer optimal care to islet transplant recipients. ACKNOWLEDGMENTS This work has been supported by grants From: The National Institutes of Health, The National Institute of Diabetes and Digestive and Kidney Diseases (RQ1 DK52802), The National Center for Research Resources (GCRCM01RR16587; U42RRQ166Q3), the Juvenile Diabetes Research Foundation International (4-2004-361), the State of Florida, and the Diabetes Research Institute Foundation. The authors are indebted to Elizabeth Meyer, who helped in the preparation and editing of this manuscript. REFERENCES 1.

Brendel MD HB, Schultz AO, Bretzel RG. International Islet Transplant Registry Newsletter 1999; 9:1–20. 2. Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000; 343:230–238. 3. Ricordi C. Islet transplantation: a brave new world. Diabetes 2003; 52: 1595–1603. 4. Ricordi C, Strom TB. Clinical islet transplantation: advances and immunological challenges. Nat Rev Immunol 2004; 4:259–268. 5. Poggioli R, Faradji RN, Ponte G, et al. Quality of life after islet transplantation. Am J Transplant 2006; 6:371–378. 6. Froud T, Ricordi C, Baidal DA, et al. Islet transplantation in type 1 diabetes mellitus using cultured islets and steroid-free immunosuppression: Miami experience. Am J Transplant 2005; 5:2037–2046 7. Ryan EA, Lakey JR, Paty BW, et al. Successful islet transplantation: continued insulin reserve provides long-term glycemic control. Diabetes 2002; 51:2148–2157. 8. Hafiz MM, Faradji RN, Froud T, et al. Immunosuppression and procedurerelated complications in 26 patients with type 1 diabetes mellitus receiving allogeneic islet cell transplantation. Transplantation 2005; 80:1718–1728. 9. Ryan EA, Paty BW, Senior PA, et al. Five-year follow-up after clinical islet transplantation. Diabetes 2005; 54:2060–2069. 10. Ryan EA, Paty BW, Senior PA, Shapiro AM. Risks and side effects of islet transplantation. Curr Diab Rep 2004; 4:304–309. 11. Cattaneo D, Merlini S, Pellegrino M, et al. Therapeutic drug monitoring of sirolimus: effect of concomitant immunosuppressive therapy and optimization of drug dosing. Am J Transplant 2004; 4:1345–1351.

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12. Ragette R, Kamler M, Weinreich G, Teschler H, Jakob H. Tacrolimus pharmacokinetics in lung transplantation: new strategies for monitoring. J Heart Lung Transplant 2005; 24:1315–1319. 13. Senior PA, Paty BW, Cockfield SM, Ryan EA, Shapiro AM. Proteinuria developing after clinical islet transplantation resolves with sirolimus withdrawal and increased tacrolimus dosing. Am J Transplant 2005; 5:2318–2323. 14. Baidal DA, Denham M, Faradji R, et al. Campath induction and sirolimustacrolimus/MMF maintenance in islet transplantation. Am J Transplant 2007; 7 (Suppl 2):204–205. 15. Hering B, Kandaswamy R, Ansite J, et al. Single-donor, marginal-dose islet transplantation in patients with type 1 diabetes. JAMA 2005; 293:830–835. 16. Hershberger RE, Starling RC, Eisen HJ, et al. Daclizumab to prevent rejection after cardiac transplantation. N Engl J Med 2005; 352:2705–2713. 17. Vincenti F, Kinkman R, Light S, et al. Interleukin-2-receptor blockade with daclizumab to prevent acute rejection in renal transplantation. N Engl J Med 1998; 338:161–165. 18. Calne R, Moffatt SD, Friend PJ, et al. Campath IH allows low-dose cyclosporine monotherapy in 31 cadaveric renal allograft recipients. Transplantation 1999; 68:1613–1616 19. Coles AJ, Wing M, Smith S, et al. Pulsed monoclonal antibody treatment and autoimmune thyroid disease in multiple sclerosis. Lancet 1999; 354:1691–1695. 20. Administration USFaD: Alert for Health Care Professional Alemtuzumab. 2005. 21. Genzyme: Genzyme and Schering AG Announce Interim Results from Trial of Campath for Multiple Sclerosis. 2005. 22. Bonnefoy-Berard N, Revillard JP. Mechanisms of immunosuppression induced by antithymocyte globulins and OKT3. J Heart Lung Transplant 1996; 15:435–442. 23. Genzyme C: Thymoglobulin Package Insert. 2005. 24. Gaber AO, First MR, Tesi RJ, et al. Results of the double-blind, randomized, multicenter, phase III clinical trial of Thymoglobulin versus Atgam in the treatment of acute graft rejection episodes after renal transplantation. Transplantation 1998; 66:29–37. 25. Uslu A, Nart A, Coker I, et al. Two-day induction with thymoglobulin in kidney transplantation: risks and benefits. Transplant Proc 2004; 36:76–79. 26. Eason JD, Blazek J, Mason A, Nair S, Loss GE. Steroid-free immunosuppression through thymoglobulin induction in liver transplantation. Transplant Proc 2001; 33:1470–1471. 27. Marvin MR, Droogan C, Sawinski D, Cohen DJ, Hardy MA. Administration of rabbit antithymocyte globulin (thymoglobulin) in ambulatory renaltransplant patients. Transplantation 2003; 75:488–489. 28. Vezina C, Kudelski A, Sehgal SN. Rapamycin (AY-22,989), a new antifungal antibiotic. I. Taxonomy of the producing streptomycete and isolation of the active principle. J Antibiot (Tokyo) 1975; 28:721–726. 29. Sehgal SN, Baker H, Vezina C. Rapamycin (AY-22,989), a new antifungal antibiotic. II. Fermentation, isolation and characterization. J Antibiot (Tokyo) 1975; 28:727–732.

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30. Hardinger KL, Koch MJ, Brennan DC. Current and future immunosuppressive strategies in renal transplantation. Pharmacotherapy 2004; 24:1159–1176. 31. Kelly PA, Gruber SA, Behbod F, Kahan BD. Sirolimus, a new, potent immunosuppressive agent. Pharmacotherapy 1997; 17:1148–1156. 32. Taylor AL, Watson CJ, Bradley JA. Immunosuppressive agents in solid organ transplantation: Mechanisms of action and therapeutic efficacy. Crit Rev Oncol Hematol 2005; 56:23–46. 33. Molinari M, Al-Saif F, Ryan EA, et al. Sirolimus-induced ulceration of the small bowel in islet transplant recipients: report of two cases. Am J Transplant 2005; 5:2799–2804. 34. Morelon E, Stern M, Israel-Biet D, et al. Characteristics of sirolimusassociated interstitial pneumonitis in renal transplant patients. Transplantation 2001; 72:787–790. 35. Digon BJ, 3rd, Rother KI, Hirshberg B, Harlan DM. Sirolimus-induced interstitial pneumonitis in an islet transplant recipient. Diabetes Care 2003; 26: 3191. 36. Gruessner RW. Tacrolimus in pancreas transplantation: a multicenter analysis. Tacrolimus Pancreas Transplant Study Group. Clin Transplant 1997; 11:299–312. 37. Warnock GL, Kneteman NM, Ryan EA, et al. Continued function of pancreatic islets after transplantation in type I diabetes. Lancet 1989; 2:570–572. 38. Warnock GL, Kneteman NM, Ryan EA, Rabinovitch A, Rajotte RV. Longterm follow-up after transplantation of insulin-producing pancreatic islets into patients with type 1 (insulin-dependent) diabetes mellitus. Diabetologia 1992; 35:89–95. 39. Alejandro R, Lehmann R, Ricordi C, et al. Long-term function (6 years) of islet allografts in type 1 diabetes. Diabetes 1997; 46:1983–1989. 40. Jacobson PA, Green KG, Hering BJ. Mycophenolate mofetil in islet cell transplant: variable pharmacokinetics but good correlation between total and unbound concentrations. J Clin Pharmacol 2005; 45:901–909. 41. Tyring S, Gottlieb A, Papp K, et al. Etanercept and clinical outcomes, fatigue, and depression in psoriasis: double-blind placebo-controlled randomised phase III trial. Lancet 2006; 367:29–35. 42. Rabinovitch A SW, Rajotte RV, Warnock GL. Cytotoxic effects of cytokines on human pancreatic islet cells in monolayer culture. J Clin Endocrinol Metab 1990; 71:152–156. 43. Kovacs JA, Masur H. Prophylaxis against opportunistic infections in patients with human immunodeficiency virus infection. N Engl J Med 2000; 342: 1416–1429. 44. Serratrice J, Granel B, Swiader L, et al. Interference of dapsone in HbA1c monitoring of a diabetic patient with polychondritis. Diabetes Metab 2002; 28: 508–509. 45. Park JM, Lake KD, Arenas JD, Fontana RJ. Efficacy and safety of low-dose valganciclovir in the prevention of cytomegalovirus disease in adult liver transplant recipients. Liver Transpl 2006; 12:112–116. 46. Miller ER, 3rd, Pastor-Barriuso R, Dalal D, Riemersma RA, Appel LJ, Guallar E. Meta-analysis: high-dosage vitamin E supplementation may increase all-cause mortality. Ann Intern Med 2005; 142:37–46.

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65. Cure P, Pileggi A, Faradji RN, et al. Cytomegalovirus infection in a recipient of solitary allogeneic islets. Am J Transplant, 2006; 6 (5 pt 1):1089–90. 66. Ryan EA, Bigam D, Shapiro AM. Current indications for pancreas or islet transplant. Diabetes Obes Metab 2006; 8:1–7. 67. Trotter JF, Wachs ME, Trouillot TE, et al. Dyslipidemia during sirolimus therapy in liver transplant recipients occurs with concomitant cyclosporine but not tacrolimus. Liver Transpl 2001; 7:401–408. 68. Groth CG BL, Morales JMet al. Sirolimus (rapamycin)-based therapy in human renal transplantation: similar efficacy and different toxicity compared with cyclosporine. Sirolimus European Renal Transplant Study Group [see comments]. Transplantation 1999; 67:1036–1042. 69. Textor SC, Taler SJ, Canzanello VJ, Schwartz L, Augustine JE. Posttransplantation hypertension related to calcineurin inhibitors. Liver Transpl 2000; 6: 521–530. 70. Devlin J, Williams R, Neuhaus P, et al. Renal complications and development of hypertension in the European study of FK 506 and cyclosporin in primary liver transplant recipients. Transpl Int 1994; 7 (Suppl 1):S22–26. 71. Saurina A, Campistol JM, Piera C, et al. Conversion from calcineurin inhibitors to sirolimus in chronic allograft dysfunction: changes in glomerular haemodynamics and proteinuria. Nephrol Dial Transplant 2006; 21:488–493. 72. Straathof-Galema L, Wetzels JF, Dijkman HB, Steenbergen EJ, Hilbrands LB. Sirolimus-associated heavy proteinuria in a renal transplant recipient: evidence for a tubular mechanism. Am J Transplant 2006; 6:429–433. 73. Ponte GM, Baidal DA, Romanell P, et al. Resolution of severe atopic dermatitis after tacrolimus withdrawl. Cell Transplant 2007; 16 (1):23–30. 74. Fishman JA, Rubin RH. Infection in organ-transplant recipients. N Engl J Med 1998; 338:1741–1751. 75. Humar A, Michaels M. American Society of Transplantation. Recommendations for Screening, Monitoring and Reporting of Infectious Complications in Immunosuppression Trials in Recipients of Organ Transplantation. American Journal of Transplantation 2006; 6:262–274. 76. Rubin RH. Impact of cytomegalovirus infection on organ transplant recipients. Rev Infect Dis 1990; 12 (Suppl 7):S754–766. 77. Hafiz MM, Poggioli R, Caulfield A, et al. Cytomegalovirus prevalence and transmission after islet allograft transplant in patients with type 1 diabetes mellitus. Am J Transplant 2004; 4:1697–1702. 78. Cure P, Pileggi A, Froud T, et al. Alterations of the female reproductive system in recipients of islet grafts. Transplantation 2004; 78:1576–1581. 79. Euvrard S, Kanitakis J, Claudy A. Skin cancers after organ transplantation. N Engl J Med 2003; 348:1681–1691. 80. DCCT Research Group: The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993; 329:977–986. 81. Chow VC, Pai RP, Chapman JR, et al. Diabetic retinopathy after combined kidney-pancreas transplantation. Clin Transplant 1999; 13:356–362.

9 Islet Graft Monitoring and Imaging Christian Toso Department of Surgery, University of Alberta Hospital, Edmonton, Alberta, Canada, and Department of Surgery, University of Geneva Hospital, Geneva, Switzerland

Thierry Berney Department of Surgery, University of Geneva Hospital, Geneva, Switzerland

INTRODUCTION In recent years, islet transplantation results have improved significantly, with an insulin independence rate of 60–80% at one year (1,2,3). However, patients following successful islet transplantation have b-cell function of only 20% of that of healthy individuals, even though they received islets from more than one donor (3). This low rate of islet engraftment is the consequence of a loss of endocrine cells, which results from multiple insults (Fig. 1). Damage to islets during isolation or at the time of injection, non-specific inflammatory reactions, and activation of the coagulation cascade play an early role after transplantation, while allo- and autoimmunity have a more delayed action. This progressive exhaustion of the islets should be better understood in order to be able to impact on it. As such, better monitoring of the islet graft appears to be an absolute requirement. Currently, clinical islet graft monitoring is mainly based on the assessment of islet function and has a low accuracy. Therefore, several groups have focused their attention on finding ways to refine either islet mass or rejection monitoring. Current studies have taken two novel approaches to address this problem: measurement of islet rejection markers in serum and imaging of islets after transplantation.

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50 %

Poor primary implantation: damage during isolation and transplant, IBMIR, nonspecific inflammatory activation Progressive isletloss: allo-, auto-immunity activation

Insulin requirement Time after transplant (years)

Figure 1 Potential evolution of islet loss after transplant. Abbreviation: IBMIR, instant blood-mediated inflammatory reaction.

MONITORING OF ISLET FUNCTION Current clinical monitoring is based on islet function (Table 1) (4). It includes monitoring serum levels of glucose, C-peptide and insulin, HbA1c and fructosamine, monitoring of exogeneous insulin requirements, and of number of hypoglycemic episodes requiring outside help or not. These

Table 1

Islet Graft Function Monitoring

Overall function

Glucose stability

Hypoglycemic events Stimulation tests

Serum glucose Serum insulin Serum C-peptide HbA1c, fructosamine Insulin requirement (U/Kg) Number of hypoglycemias Secretory Unit of Islet Transplant Objects (SUITO) Beta score Mean Amplitude of Glycemic Excursions (MAGE) Lability index Continuous Glucose Monitoring Systems (CGMS) HYPO score Continuous Glucose Monitoring Systems (CGMS) Arginine stimulation test Glucagon test Mixed meal stimulation test Oral Glucose Tolerance Test (OGTT) IV Glucose Tolerance Test (IVGTT)

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parameters are recorded at each visit, and, based on them, islet grafts can be classified as being fully (insulin-independence), partially (on insulin and detectable C-peptide), or not functioning (no detectable C-peptide). While C-peptide levels vary greatly according to blood glucose, the Secretory Unit of Islet Transplant Objects (SUITO) has been developed recently. It computes both blood glucose and C-peptide and can be calculated as: 1500 x fasting C-peptide in ng/dl/ (fasting blood glucose in mg/dl-63) (5). However, these ways of qualifying islet function are imprecise, as a patient may be off insulin and yet have high glucose values with elevated HbA1c. The Beta score was introduced to take all these parameters into account. It is based on fasting blood glucose, HbA1c, daily insulin requirements, use of oral hypoglycemic agent, and stimulated C-peptide (Table 2) (6). It rates islet function from 0 to 8: the higher the score, the better the islet function.

Assessment of Glycemic Variability and Burden of Hypoglycemia Monitoring can be refined by the calculation of other scores assessing blood glucose stability and the occurrence of hypoglycemia. The MAGE (Mean Amplitude of Glycemic Excursions) index reflects blood glucose stability. It can be calculated from 14 blood glucose values taken over 48 hours, by computing the mean of all blood glucose increases or decreases measured during the study period. Excursions of less than one standard deviation are excluded. Control individuals have a MAGE index between 1.0 and 3.3 mmol/L, while patients with unstable Type 1 diabetes can have values of up to 15 mmol/L (7,8). The limitations of this score are linked to the fact that it samples two days only and periods of unstable glycemia can be missed. On the other hand, it is easy to obtain and can be performed often, thus decreasing the risk of such a bias. The only side effect for the patient is that some of them have to perform more self blood glucose monitoring than usual. MAGE has been widely used in islet transplant recipients (1,9,10). The Lability Index (LI) also assesses blood glucose stability. It has been introduced more recently by the Edmonton group. It has been tested

Table 2

Beta Score to Assess Islet Graft Function

Blood glucose (mmol/L) HbA1c (%) Daily insulin (units/Kg) or OHA Stimulated C-peptide (nmol/L) Abbreviation: OHA, oral hypoglycemic agent.

2

1

0

≤5.5 ≤6.1 None ≥0.3

5.6–6.9 6.2–6.9 0.01–0.24 and/or OHA 0.1–0.29

≥7 ≥7 ≥0.25 <0.1

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on a large group of patients, and correlates better with the clinical assessment of lability than the MAGE. It is, however, more cumbersome and requires self-monitored glucose values assessed over four weeks (8). The following equation has to be computed for each week: LI ¼ Σ[(Glucn
Glucnþ1)2/(hnþ1
hn)], where “Gluc” (in mmol/L) is the nth reading of the week taken at time hn (rounded to the nearest hour). The LI can then be calculated as the mean value of the four weeks. Most patients with Type 1 diabetes have LI between 0 and 400 mM2/h*week (median 223 mM2/h*week) (8). The burden of hypoglycemia can be assessed by the HYPO score. This combines detailed assessment of blood glucose values recorded over a fourweek period with the patient’s self-reported episodes over the previous year (8). More points are awarded for asymptomatic hypoglycemia or if outside help has been required. The more severe the problem of hypoglycemia, the higher the score. A score ≥433 is indicative of problematic hypoglycemia, and a score ≥1,047 is of very serious concern. Most patients with Type 1 diabetes have HYPO scores between 40 and 450 (median 143), while patients on the waiting list for islet transplant have significantly higher scores. The complexity of this score limits its regular clinical use. Blood glucose stability and the occurrence of hypoglycemia can further be assessed by Continuous Glucose Monitoring Systems (CGMS) profiling. This requires placement of a probe in the subcutaneous tissue, which is removed at the end of the record. Several centers have been using such devices (11,12,13), which measures mean glucose levels every 5 minutes continuously over several days. It has not entered routine clinical practice for the monitoring of islet grafts to date. Assessment of Stimulated Insulin / C-Peptide Secretion Monitoring can be improved by measuring the amount of insulin produced by the islets after stimulation (14). These methods have been established as sensitive measures of islet mass. Several tests can be performed. The arginine stimulation test is probably the test most often used. It can be easily performed without significant side effects over a short period of time (15). Serum insulin is usually measured
10, 0, 2, 3, 4, 5, 7, and 10 minutes from IV injection of 5 g arginine over 30 sec in the fasting state (10). In Geneva, this test is performed at 3, 6, 9, and 12 months post-transplant and twice yearly thereafter. The area under the curve for insulin (AUC) can be computed as the area under the curve above the mean of –10 and 0 minute values. It reflects islet mass. The acute insulin response (AIR) can be calculated as the mean of the three highest values between 2 and 5 minutes minus the mean of values at -10 and 0 minutes. In healthy volunteers, mean AUC was 183±57 mU.min/l and mean AIR 31.5 ± 9.5 mU/l (Fig. 2)(10). In many centers, this test has replaced the glucagon stimulation test (insulin

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60 50 40 30 20 10 0 T-10

T0

T2

T3 T4 Time (min)

T5

T7

T10

Figure 2 Arginine test: mean (± standard deviation) insulin levels in 7 healthy individuals (3 females, 4 males; mean age 38 ± 9 years) after injection of 5 g arginine IV.

secretion after injection of 1 mg of glucagon IV), which is associated with side effects in the form of nausea and vomiting (16). The mixed meal stimulation test can also be easily performed. It provides simple information about islet function. After an overnight fast, blood glucose, C-peptide and insulin levels are measured prior to and 90 minutes after consuming a standard meal (Ensure HP; Abbott, Abbott Park, Illinois, U.S.A.), containing 391 kcal with 8.5 g fat, 44 g carbohydrate, and 17 g protein (2). Control subjects usually have C-peptides rising up to 1–1.5 nmol/l. Similarly, the oral glucose tolerance test (OGTT) is performed after an overnight fast with blood samples drawn before and at 30, 60, 90, and 120 minutes after ingesting 75 g of oral glucose. The OGTT is the only metabolic stimulation test included in the ADA definition of impaired glucose tolerance and diabetes (17). The intravenous glucose tolerance test (IVGTT) is among the tests providing the most information, but it is also quite cumbersome to perform. It is performed in the fasting state using 50% dextrose, 300 mg/kg of body weight, given over 1 minute after two baseline samples (
10 and 0 min) for glucose, insulin, and C-peptide have been taken. Sampling is then usually at 3, 4, 5, 7, 10, 15, 20, 25, and 30 minutes, with 0 being the start of the infusion (2). Some groups perform more frequent sampling (18,19). This test allows calculation of the acute insulin response to glucose based on the mean of the insulin level at 3, 4, and 5 minutes minus the mean basal insulin level. Glucose disposal rate (KG) is calculated as the slope of the natural log of the glucose values from 10–30 min. It reflects insulin resistance. KG values <
1.0 are considered normal. The areas under the curve for insulin and C-peptide are defined as the area under the curve over baseline from time 0–30 minutes.

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Some caveats regarding the interpretation of these tests have to be raised. Islet recipients may have varying degrees of impaired kidney function, which can impact on C-peptide excretion and thus prolong halflife. In addition, C-peptide secretion is highly influenced by timing, blood glucose, and the use of exogenous insulin, especially in patients with partial islet function, who have recommenced insulin injections. Furthermore, one must be aware that there is a great deal of variability between laboratories in the techniques used for each test. This can lead to inconsistent interpretation. Overall, these tests are all more or less refined ways to assess islet graft function. Such monitoring is mandatory for clinical studies and for followup of patients. The down side of these tests is that abnormality can only be detected once this has resulted in impaired graft function and thus potentially too late to initiate any salvage therapy. Novel approaches are therefore required to detect early harmful events impacting on islets.

ISLET GRAFT BIOPSY Graft biopsy is the best way to detect rejection. It is widely used for all solid organs. However, in the case of islet transplantation, the islet/hepatocyte ratio is very low, and percutaneous needle biopsies have relatively low probability of including islets. Such biopsies have thus not entered routine clinical practice. In order to have a more accessible site for biopsy, it has been proposed to transplant some of the islets in the forearm, as a sentinel graft (20). While initially appearing logical, this approach is limited by the fact that implantation and survival of islets are site-dependent and islets implanted at two different sites will likely not behave the same way. In the specific case of combined organ transplant, rejection is known to usually affect both organs at the same time. Recipients of combined islet/ kidney transplants can thus be treated according to rejection detected in the kidney.

MOLECULAR MONITORING In a preliminary study, the University of Geneva group demonstrated that circulating mRNA for insulin can be detected by reverse transcription polymerase chain reaction (RT-PCR) immediately after islet transplantation (21). This reflects early islet damage during the engraftment period, with systemic release of b-cells. Circulating mRNA was observed over a longer period in patients treated with steroid-containing immunosuppression. In a recent report, increased circulating insulin mRNA predicted the occurrence of subsequent event signaling islet damage (increase in the amount of

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injected exogeneous insulin, decrease in C-peptide levels) (22). This assay is indicative of b-cell shedding in general and is not specific to allorejection. Islet damage could also be the result of autoimmune destruction, nonspecific inflammatory mechanisms, or loss of function through progressive exhaustion. Specificity for early detection of immune-mediated toxicity may be enhanced by combining RT-PCR for insulin with analysis of expression of cytotoxic lymphocyte genes such as granzyme B, perforin, and Fasligand. This combination has been tested in non-human primate and in human recipients of islet transplants (23,24). Further clinical validation is, however, required to determine sensitivity and specificity for prediction of early islet rejection.

ISLET GRAFT IMAGING Several workshops on “Imaging of pancreatic b-cell in health and disease” have emphasized the importance and existing limitations of transplanted islet imaging (25). Three modalities have demonstrated potential for clinical application in the near future: optical imaging, magnetic resonance imaging (MRI), and positron-emission tomography (PET). Optical imaging has a high sensitivity and can detect as few as 50 islets. Penetration through tissue is only a few centimeters (26) and, while very useful in various animal models, this technique will likely never be applicable to humans (27). We will therefore focus on the two other modalities. In contrast to optical imaging, MRI is suitable for and easily applied within the clinical setting. The first attempts at islet graft MRI studied surrogate markers of islet engraftment. Islet recipients demonstrate signs of focal steatosis around the portal spaces. It is thought that this is directly induced as a paracrine effect of transplant insulin secretion, and it appears to be more prominent in patients with maintained islet graft function (28,29). Further MRI studies used super-paramagnetic iron oxide (SPIO) particles, which are widely used clinically as contrast agents, especially for liver imaging. Two groups have used these particles to label islets prior to transplantation in pre-clinical models. After intraportal infusion, labeled islets could be identified within the liver of rats and appeared as hypointense spots on T2-weighted MR images. The signal remained stable within the liver and allowed imaging of islets for several months after syngeneic transplantation (30,31). In the case of allotransplantation in the absence of immunosuppression, MR signal became undetectable 3 weeks after transplantation (32,33). SPIO labeling does not negatively affect islets (viability, in vitro glucose-induced insulin release, function after transplantation)(30,32) and appears very promising for possible clinical

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application. A preliminary study, conducted at the University of Geneva, demonstrated the feasibility of MR monitoring of labeled islet grafts in humans (34). However, some challenges still remain to be resolved. Analysis of the images requires further optimization. Iron particles induce a disturbance of the magnetic field, and the related image is larger than the particle itself. As a consequence, two similar spots on an image can represent differing numbers of iron particles. Furthermore and most importantly, correlation of clinical outcome with imaging changes must be confirmed. The critical issue will be to determine whether a decrease of signal can be detected early enough in the loss of islets to enable successful intervention, preventing further loss of graft function. Other MR compatible contrast agents have also been studied. Lipophilic Gd3þ complexes, which bind to the cell membrane and are able to label islets ex vivo have been designed by Zheng et al. (35). Uptake of manganese, an MR-enhancing agent, by glucose-activated b-cells has been observed and proposed as a method for functional islet graft imaging (36). Another strategy is to identify inflammatory cells around the islets, which are likely to increase in the presence of allo- or autoimmune activation (37). These methods have not yet been validated in the setting of islet transplantation. The sensitivity of PET is higher than MRI, and it allows accurate quantification of signal. PET-compatible tracers can be used to label islets ex vivo, prior to transplant, or, if specific enough, they can be injected intravenously after transplantation. Ex vivo labeling of islets prior to clinical intraportal transplantation has been attempted with 2-[18F]fluoro-2-deoxy-D-glucose (FDG). Islets could be detected for the first 6 hours after transplant only (Fig. 3)(38). The same strategy was used with similar results in the pig (39). Limiting factors for long-term assessment were the short half-life of the bþ-emitting radionucleotides (110 minutes for 18F) and the rapid efflux of tracer from the cell. However, this technique could be of some value in studying the fate of islets very early after transplant. PET imaging can also be performed later after transplant, but this option requires highly specific tracers. Considering the very low mass of islet grafts, it has been suggested that a useful probe should be retained at least 1000 times more efficiently by the islets than by the surrounding tissue (40,41). This is especially challenging considering that numerous tracers are metabolized by the liver, inducing high background noise. Several b-cell–specific antibodies have been studied (42). Employing an anti-IC2 monoclonal antibody modified with a radioisotope chelator, decreased accumulation of the probe in mice with streptozocin-induced diabetes has been demonstrated. While analysis of b-cell mass in mouse pancreas in vivo has been reported, it is unclear whether these antibodies could be used for clinical imaging of native pancreas or islet

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Figure 3 PET imaging of rats in control (A) or one hour after syngeneic intraportal transplantation of 2-[18F]fluoro-2-deoxy-D-glucose (FDG)-labeled islets (B). In controls, FDG is eliminated with urine (images of kidneys and bladder); in transplanted rats, it is mainly located in the liver.

transplants (43). Anti-ganglioside monoclonal antibodies, tested by radioimmunoscintigraphy demonstrated less specificity for b-cells (44). Although expected to be potential candidates, glibenclamide, tolbutamide, serotonin, L-DOPA, dopamine, nicotinamide, and fluorodithizone all had low specificities for b-cells when tested in vitro (45). [11C] Dihydrotetrabenazine (DTBZ) is a radio-ligand currently used in clinical imaging of the brain. It binds specifically to vesicular monoamine transporter 2 (VMAT2), a transporter found specifically in the brain and b-cells. Longitudinal PET imaging of the native pancreas in BB rats, during spontaneous development of diabetes, demonstrated a decline of signal, reflecting the decreasing b-cell mass (46). This technique appears promising, but still needs to be replicated in the islet transplant setting given the ubiquitously high uptake by the liver.

CONCLUSION Outcomes after islet transplantation have improved over the years in a stepwise fashion. One significant step was the development of techniques allowing large-scale isolation of islets by Camillo Ricordi et al. Another was the introduction of the Edmonton Protocol with insulin-independence achieved in most patients. While most patients can currently discontinue insulin initially, most of them have to return to smaller amounts of exogenous insulin treatment over time. Nevertheless, a degree of islet function is still maintained in the long

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term, and is often sufficient to protect against hypoglycemia. This progressive exhaustion of the islets must be better understood in order to be able to impact on it. This requirement can be viewed as the current hurdle in the field. To surmount this, improved monitoring of the islet graft appears to be an absolute requirement. Current follow-up is based on the assessment of islet function by metabolic tests, which can only detect the loss of a large amount of cells, often too late to attempt any salvage treatment. Islet imaging appears to be a promising way to monitor graft mass and function over time. Many centers are concentrating their efforts on developing novel imaging strategies, with meaningful clinical translation envisaged in the near future.

REFERENCES 1.

Ryan EA, Paty BW, Senior PA, et al. Five-year follow-up after clinical islet transplantation. Diabetes 2005; 54:2060–2069. 2. Ryan EA, Lakey R, Paty BW, et al. Successful islet transplantation: continued insulin reserve provides long-term glycemic control. Diabetes 2002; 51: 2148–57. 3. Ryan EA, Lakey JR, Rajotte RV, et al.Clinical outcomes and insulin secretion after islettransplantation with the Edmonton protocol Diabetes 2001; 50:710–9. 4. Pileggi A, Ricordi C, Alessiani M, Inverardi L. Factors influencing islet of Langerhans graft function and monitoring. Clinica Chimica Acta 2001; 310: 3–16. 5. Matsumoto S, Yamada Y, Okitsu T, et al. Simple evaluation of engraftment by secretory unit of islet transplant objects for living donor and cadaveric donor fresh or cultured islet transplantation. Transplant Proc 2005; 37: 3435–3437. 6. Ryan EA, Paty BW, Senior PA, Lakey JRT, Bigam D,and Shapiro AMJ. Beta-score, an assessment of b-cell function after islet transplantation. Diabetes Care 2005; 28:343–347. 7. Service FJ, Molnar GD, Rosevear JW, Ackerman E, Gatewood LC, and Taylor WF. Mean amplitude of glycemic excursions, a measure of diabetic instability. Diabetes 1970; 19:644–655. 8. Ryan EA, Shandro T, Green K, et al. Assessment of the severity of hypoglycemia and glycemic lability in type 1 diabetic subjects undergoing islet transplantation. Diabetes 2004; 53:955–962. 9. Froud T, Ricordi C, Baidal DA, et al. Islet transplantation in type 1 diabetes mellitus using cultured islets and steroid-free immunosupression: Miami experience. Am J Transplant 2005; 5:2037–2046. 10. Toso C, Baertschiger R, Morel P, et al. Sequential kidney/islet transplantation: efficacy and safety assessment of a steroid-free immunosuppression protocol. Am J Transplant 2006; 6:1049–1058. 11. Kessler L, Passemard R, Benhamou PY, et al.Reduction of blood glucose variability by islet transplantation: Interest of continuous glucose monitoring. Diabetes Care 2002; 25:2256–62.

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12. Geiger MC, Ferreira JV, Hafiz MM, et al. Evaluation of metabolic control using a continuous subcutaneous glucose monitoring system in patients with type 1 diabetes mellitus who achieved insulin independence after islet cell transplant. Cell Transplant 2005; 14:77–84. 13. Paty BW, Senior PA, Lakey JR, Shapiro AM, and Ryan EA. Assessment of glycemic control after islet transplantation using the continuous glucose monitor in insulin-independent versus insulin-requiring type 1 diabetes subjects. Diabetes Technol Ther 2006; 8:165–173. 14. Shapiro AM, Hao EG, Lakey JR, et al. Novel approaches toward early diagnosis of islet allograft rejection. Transplantation 2001; 71:1709–18. 15. Dupre J, Curtis JD, Unger RH, Waddell RW, and Beck JC. Effects of secretin, pancreozymin, or gastrin on the response of the endocrine pancreas to administration of glucose or arginine in man. J Clin Invest 1969; 48:745–757. 16. Samols E, Marri G, and Marks V. Promotion of insulinsecretion by glucagon. Lancet 1965; 28:415–416. 17. Expert committee on the diagnosis and classification of diabetes mellitus. Report of the expert committee on the diagnosis and classification of diabetes mellitus. DiabetesCare 2003; 26:S5–S20. 18. Clausen JO, Borch-Johnsen K, Ibsen H, et al.Insulin sensitivity index, acute insulin response, and glucose effectiveness in a population-based sample of 380 young healthy Caucasians: analysis of the impact of gender, body fat, physical fitness, and life-style factors. J Clin Invest 1996; 98:1195–1209. 19. Rickels MR, Schutta MH, Markmann JF, Barker CF, Naji A, and Teff KL. Beta-cell function following human islet transplantation for type 1diabetes. Diabetes 2005; 54:100–106. 20. Stegall MD. Monitoring human islet allografts using a forearm biopsy site. Ann Transplant 1997; 2:8–11. 21. Ritz-Laser B, Oberholzer J, Toso C, et al.Molecular detection of circulating bcells after islettransplantation. Diabetes 2002; 51:557–61. 22. Berney T, Mamin A, Shapiro AMJ, et al.Detection of insulin mRNA in the peripheral blood after human islet transplantation predicts deterioration of metabolic control. Am J Transplant 2006; 6(7):1704–1711. 23. Han D, Xu X, Pastori RL, Ricordi C, and Kenyon NS. Elevation of cytotoxic lymphocyte gene expression is predictive of islet allograft rejection in nonhuman primates. Diabetes 2002; 51:562–566. 24. Han D, Xu X, Baidal D, et al. Assessment of cytotoxic lymphocyte gene expression in the peripheral blood of human islet allograft recipients. Elevation precedes clinical evidence of rejection.Diabetes 2004; 53:2281–2290. 25. Paty BW, Bonner-Weir S, Laughlin MR, McEwan AJ, and Shapiro AMJ. Toward development of imaging modalities for islets after transplantation: insights from the national institute of health workshop on b-cell imaging. Transplantation 2004; 77:1133–7. 26. Fowler M, Virostko J, Chen Z, et al. Assessment of pancreatic islet mass after islet transplantation using in vivo bioluminscence imaging. Transplantation 2005; 15:768–776. 27. Massoud TF and Gambhir ,SS. Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genomics 2003; 17:545–580.

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28. Markmann JF, Rosen M, Siegelman ES, et al.Magnetic resonance-defined periportal steatosis following intraportal islet transplantation: a functional footprint of islet survival? Diabetes 2003; 52:1591–1594. 29. Bhargava R, Senior PA, Ackerman TE, Ryan EA, Paty BW, Lakey JR, and Shapiro AM. Prevalence of hepatic steatosis after islet transplantation and its relation to graftfunction. Diabetes 2004; 53:1311–1317. 30. Jirak D, Kriz J, Herynek V, et al. MRI of transplanted pancreatic islets. Magn Reson Med 2004; 52:1228–1233. 31. Evgenov NV, Medarova Z, Dai G, Bonner-Weir S,and Moore A. In vivo imaging of islet transplantation. Nat Med 2006; 12:144–148. 32. Kriz J, Jirak D, Girman P, et al. Magnetic resonance imaging of pancreatic islets in tolerance and rejection. Transplantation 2005; 80:1596–1603. 33. Evgenov NV, Medrova Z, Pratt J, et al. In vivo imaging of immune rejection in transplanted pancreatic islets. Diabetes 2006; 55:2419–2428. 34. Toso C, Vallee JP, Morel P, et al. Clinical magnetic resonance imaging of allogeneic islet grafts after iron-labeling.Am J Transplant 2006; 6:452. 35. Zheng Q, Dai H, Merritt ME, Malloy C, Pan CY, Li WH. A new class of macrocyclic lanthanide complexes for cell labeling and magnetic resonance imaging applications. J Am Chem Soc 2005; 127:16178–16188. 36. Gimi B, Leoni L, Oberholzer J, et al.Functional MR microimaging of pancratic b-cell activation. Cell Transplant 2006; 15:195–203. 37. Moore A, Grimm J, Han B,and Santamaria P. Tracking the recruitment of diabetogenic CD8þ T-cells to the pancreas in real time. Diabetes 2004; 53: 1459–1466. 38. Toso C, Zaidi H, Morel P, et al. Positron-emission tomography imaging of early events after transplantation of islets of Langerhans. Transplantation 2005; 79:353–355. 39. Lundgren T, Eich T, Eriksson O, et al.Imaging porcine allo-islet transplantation using positron emission tomography (PET). Am J Transplant 2006; 6:990. 40. Sweet IR, Cook DL, Lernmark A, Greenbau CJ, Krohn KA. Non-invasive imaging of b-cell mass: a quantitative analysis. Diabetes Technol Ther 2004; 6: 652–659. 41. Toso C, Zaidi H, Morel P, et al. Assessment of 18F-FDG-leukocyte imaging to monitor rejection after pancreatic islet transplantation. Transplant Proc 2006; 38(9):3033-4. 42. Hampe CS, Wallen AR, Schlossen M, Ziegler M, Sweet IR. Quantitative evaluation of a monoclonal antibody and its fragment as potential markers for pancreatic b-cell mass. Exp Clin Endocrinol Diabetes 2005; 113:381–387. 43. Moore A, Bonner-Weir S, Weissleder R. Noninvasive in vivo measurement of b-cell mass in mouse model of diabetes. Diabetes 2001; 50:2231–2236. 44. Ladriere L, Malaisse-Lagae F, Alejandro R, Malaisse WJ. Pancreatic fate of a (125) I-labelled mouse monoclonal antibody directed against pancreatic B-cell surface ganglioside(s) in control and diabetic rats.Cell Biochem Funct 2001; 19:107–115.

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45. Sweet IR, Cook DL, Lernmark A, et al. Systematic screening of potential Beta-cell imaging agents. Biochemical and Biophysical Research Communication 2004; 314:976–83. 46. Souza F, Simpson N, Raffo A, et al. Longitudinal noninvasive PET-based bcell mass estimates in a spontaneous diabetes rat model. J Clin Invest 2006; 116:1506–1513.

10 Metabolic Measures of Islet Function and Mass After Islet Transplantation R. Paul Robertson Pacific Northwest Research Institute and the Departments of Medicine and Pharmacology, University of Washington, Seattle, Washington, U.S.A.

INTRODUCTION The use of laboratory animals to examine the function and fate of pancreatic islets transplanted into various sites (liver, spleen, kidney, testes, brain, peritoneal cavity, omentum) has provided interesting and important information. Such studies are very invasive and typically involve histologic examination of the transplanted islets for examination of function and structure. These approaches are obviously not possible in human recipients, except in the case of organ biopsy. Thus, ascertainment of function and mass after islet transplantation is a more daunting task in humans and, of necessity, one that relies on more indirect measures. Chief among these are conventional metabolic tests that can be conducted safely without compromising the recipient or the transplanted islets. Recent data from the Edmonton series underscore the need to gain a deeper understanding of the consequences of intrahepatic islet transplantation on the fate of the transplanted islet. This research area gathered great momentum in the year 2000 with a report from the Edmonton group of 100% success in a small series of recipients (1). Follow-up results continued to indicate excellent results with 80% of the patients remaining insulin independent after 1 year (2). By five years of follow-up, the Edmonton group reported that 80% of 66 recipients were C-peptide positive with average HbA1c levels of approximately 7%. However, they also provided the sobering news that 80% of these patients were once again using insulin, albeit 193

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at reduced amounts compared to pre-transplant dosages (3). These metabolic outcomes five years after transplantation raise the critical issue of the mechanism(s) of the functional decline in transplanted islets. Two major questions are whether the loss of b-cell function is due to a decrease in b-cell mass, to b-cells that have become dysfunctional, or both. This chapter begins by briefly reviewing normal islet physiology. Next, a description is provided of the metabolic tests that have been performed in human islet recipients and their results. Finally, a strategy for maximizing the clinical information that can be derived from metabolic tests is recommended.

ISLET PHYSIOLOGY Islets of Langerhans are scattered throughout the pancreas and comprise roughly 2–3% of total pancreatic mass. In humans, islets are fairly evenly distributed throughout all portions of the pancreas. Each islet is comprised of roughly 2,000 cells, which includes b, α, ∆, and PP cells. The b-cell synthesizes and releases insulin in response to its principal agonist, glucose. It also responds to other agonists such as oral hypoglycemic agents, amino acids, and b adrenergic stimulation. Insulin secretion is inhibited by epinephrine, somatostatin, and prostaglandin E2. Once released from the islet, insulin travels through the hepatic portal and systemic circulation to reach the liver, muscles, and fat cells where it exerts its primary metabolic actions. Insulin decreases glucose production by the liver and increases glucose uptake by muscle and fat cells. The α-cell synthesizes and secretes glucagon, primarily in response to hypoglycemia, although amino acids can also stimulate α-cell function. During hypoglycemia, glucagon secretion provides the primary counterregulatory hormonal response by traveling via the hepatic portal vein to the liver where it stimulates glycogenolysis and hepatic glucose production. Glucagon secretion is inhibited by hyperglycemia and by somatostatin. The ∆-cell synthesizes and secretes somatostatin, a response that is enhanced by high glucose levels. Since somatostatin inhibits both insulin and glucagon secretion, it is often purported to be a paracrine regulator of the neighboring α- and b-cells. The PP cell synthesizes and secretes pancreatic polypeptide, primarily in response to hypoglycemia and secretin. No function in humans for this hormone has been established.

ISLET FUNCTION AFTER TRANSPLANTATION: WHAT IS KNOWN IN HUMANS? Compared with the vast amount of published information describing islet function after islet transplantation in experimental animals, much less has

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been published from studies of human recipients. The majority of the reports from studies in humans have only limited metabolic information, such as levels of blood glucose, C-peptide, and HbA1c. More recently, however, research groups have published information from studies using several approaches to assessing b-cell function and mass, as well as glucagon secretion. The results of these studies will be described separately for autoislet and alloislet transplantation.

Metabolic Studies of Autoislet Recipients Beginning in the 1980s at the University of Minnesota, non-diabetic patients with unrelenting, chronic, painful pancreatitis were managed by complete pancreatectomy and autoislet transplantation (4). To prevent diabetes, unpurified islets were isolated from the resected pancreas within two hours and returned to the patient via the hepatic portal circulation. By 1995 it was estimated that 74% of patients were insulin-independent for greater than two years after transplantation if they received greater than 300,000 islets (5). Another large series from Leicester, U.K., also reported success using this procedure, although with a lesser degree of insulin independence two years post-transplant (6). Pyzdrowski et al. reported that intrahepatic autoislet transplantation of as few as 265,000 islets can maintain normoglycemia and provide normally timed insulin secretion in response to intravenous glucose or arginine (7). They also observed that glucagon responses to arginine were intact, but absent in response to insulin-induced hypoglycemia. In one patient, glucagon responses to intravenous arginine were measured in different sites simultaneously. Both insulin and glucagon increments appeared first in the hepatic vein followed by the splenic artery followed by the portal vein. This sequence of appearance, which is opposite to that which happens when the two hormones are secreted by the native pancreas, provided metabolic validation that the intrahepatic islets were metabolically functional. The reason for the lack of glucagon responsiveness to hypoglycemia was not clear and at odds with the observation that glucagon immunostaining was positive in a liver biopsy specimen from this patient. In later studies of this group of patients, Teucher et al. reported results from the metabolic test of glucose potentiation of arginine-induced insulin secretion (GPAIS), which was performed in eight autoislet recipients and matched, healthy controls (8). Four of the subjects were studied both pre- and post-transplant. GPAIS involves a pulse of intravenous arginine followed by a glucose infusion to produce hyperglycemia, followed by a second pulse of arginine 60 minutes after the onset of the glucose infusion (Fig. 1). Experiments in animal models have

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Figure 1 Example of the method of glucose potentiation of arginine-induced insulin secretion.

successfully related the magnitude of the potentiated arginine response to b-cell mass (9). In the study of human autoislet recipients, all subjects were insulin-independent and normoglycemic; however, acute insulin responses to arginine, glucose, and GPAIS were significantly reduced (8). Importantly, the magnitudes of all three responses showed significant linear correlations with the mass of islets transplanted (Fig. 2). Data from hemipancreatectomized donors (who were assumed to have 500,000 islets) and control subjects (who were assumed to have 1,000,000 islets) generally agreed with the regression lines. The significant correlation between islet cell mass and metabolic responses in the autoislet recipients is even more striking when one considers that variable periods of time had elapsed since islet transplantation. The average post-transplant time was three years but the range extended from one to nine years. The fact that the data from the patients who had been transplanted for the greater periods of time did not depart from the linearity of the relationship between the number of islets transplanted and the metabolic measures implies that recipients of autoislet transplantation do not undergo marked loss of islets posttransplant or generate new islets to take the place of islets that have undergone apoptosis. These results are in marked contrast to the five-year

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Figure 2 Correlation between number of islets transplanted and AIRargMax derived from GPAIS as a measure of insulin secretory reserve in eight patients with chronic pancreatitis who underwent pancreatectomy and intrahepatic autoislet transplantation (small filled squares). Control data from hemipancreatectomized donors and normal control subjects are also shown. Source: From Ref. 8.

data from the Edmonton alloislet recipients mentioned above. Robertson et al. (10) reported six autoislet recipients who were followed for up to 13 years after intrahepatic islet transplantation of 290,000 to 678,000 islets. As a group these patients maintained stable insulin secretory reserve over time, although the insulin responses to glucose tended to decrease in three patients. Glucose disappearance rates after intravenous glucose injection correlated significantly with the number of islets originally transplanted. Kendall et al. (11) evaluated α-cell function in successful recipients of intrahepatic auto- and alloislet transplantation. They used the hypoglycemic, hyperinsulinemic clamp to assess glucagon responses during hypoglycemia. Glucagon responses to hypoglycemia, but not arginine, were absent in autoislet recipients, which confirmed the initial observation by Pyzdrowski et al. (7). Glucagon responses to hypoglycemia in two alloislet recipients were also absent.

Alloislet Transplantation Success rates with alloislet transplantation of Type 1 diabetic subjects were very disappointing until the year 2000. Yet, in 1997 Alejandro et al. reported long-term function of islet allografts in Type 1 diabetic recipients for up to six years (12). Nonetheless, of the approximately 300 allografts

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that had been reported prior to 2000, the success rate was less than 10%. In the year 2000, Shapiro et al. reported from Edmonton, Alberta that seven of seven patients had maintained insulin-independence after transplantation of intrahepatic alloislets an average of one year earlier (1). Glucose tolerance tests and mixed meal tests were performed that indicated some of the subjects had impaired glucose tolerance, but they all had normal HbA1c levels. In a follow-up publication in 2001, Ryan et al. reported that the area under the curve of insulin secretion during intravenous glucose tolerance tests correlated significantly with the number of islets transplanted intrahepatically (2). In the most recent publication from this group in 2002, both the acute insulin response to intravenous arginine and the area under the curve of insulin during intravenous glucose tolerance testing correlated significantly with the number of islet equivalents transplanted (13). At about the same time Luzi et al. examined a group of 45 diabetic patients who had undergone alloislet transplantation and concluded that restoration of approximately 60% of endogenous insulin secretion is capable of normalizing alterations in protein and lipid metabolism (14). These studies involved infusion of radioactive glucose and leucine both in the post-absorptive state and during a hyperinsulinemic clamp. The use of intravenous arginine to assess islet function has also been used in two more recent publications. In the first of these, Hering et al. examined b-cell function in five of eight Type 1 diabetic patients who became insulin independent for greater than one year following islet transplantation from a single donor source (15). They reported that acute insulin responses and acute C-peptide responses to arginine and to glucose were present but with magnitudes on average of approximately 50% of control data. However, no attempt to correlate the magnitude of the responses to the number of islets transplanted was reported. In another article by Rickels et al., C-peptide responses to intravenous glucose and intravenous arginine were found to be subnormal in five patients who had received an average of approximately 12,000 islet equivalents (16). They observed that the greater impairment was to intravenous glucose, which correlates well with the fact that the fasting glucose level for the group at 12 months was greater than 115 mg/dl (17). This group also performed the GPAIS test in five insulin-independent subjects and demonstrated significant impairments in the glucose-potentiation slope and the maximal response to arginine. A highly significant correlation was observed between fasting plasma glucose levels and the insulin response to arginine during a glucose infusion. Notably, these authors found no significant correlations of the number of islets transplanted with the various measures of b-cell function which led them to conclude that the islet equivalent per kilogram, which is an estimate of transplanted islet mass, may overestimate the number of islets actually surviving intrahepatic transplantation.

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Alpha cell function in successful recipients of intrahepatic alloislet transplantation was assessed by Paty et al. who used the stepped hypoglycemic clamp (18). They observed the anticipated decline in circulating C-peptide levels during the clamp but saw no glucagon response in seven subjects. This confirms the initial observation by Kendall et al. (11) that glucagon responsivity in alloislet recipients during hypoglycemia is not restored by intrahepatic islet transplantation. Paty et al. also observed that epinephrine responses and symptom responses during hypoglycemia were not significantly different than those found in a group of Type 1 diabetic subjects who had not been transplanted (18). This indicated that despite provision of prolonged insulin independence and nearly normal glycemic control in patients with long-standing Type 1 diabetes, hormonal counter-regulation and symptom recognition of hypoglycemia were not improved by intrahepatic islet transplantation. The possible explanation for the lack of improvement in glucagon secretion was provided previously by a study of Gupta et al. (19). These investigators demonstrated intact glucagon responses to hypoglycemia in pancreatectomized dogs whose islets had been placed into the peritoneal cavity, but not in dogs that had their islets placed intrahepatically. Thus, the failure for islet transplantation to restore glucagon responses to hypoglycemia is a transplant site-determined problem. As yet, no mechanism has been identified to explain this loss of glucagon secretion from intrahepatic islets. However, a reasonable hypothesis is that elevated intrahepatic glucose flux, especially during hypoglycemia and glycogenolysis, may be overriding the stimulus that intrahepatic α-cells receive from systemic hypoglycemia (20).

RECOMMENDED CLINICAL MONITORING FOR ISLET TRANSPLANT RECIPIENTS It seems clear that metabolic measures more sophisticated than levels of glucose, C-peptide, and HbA1c are required for islet recipients because islet transplantation provides a full spectrum of results that ranges from success to partial success to failure. Patients who were initially considered successes over time have become partial successes as they return to insulin-based management to optimally control their glucose levels. Carefully controlled metabolic monitoring on a longitudinal basis is very likely to provide insights into the mechanisms whereby islets progressively fail. Issues such as potentially glucotoxic effects of chronically impaired glucose tolerance as well as changes in insulin resistance are important phenomena to assess. A suggested strategy for recipient follow-up is shown in Table 1.

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Robertson Recommended Metabolic Tests for Islet Transplant Recipients

Test Fasting glucose 2 hr. postprandial glucose AIRglu AIRarg AIRargMAX Insulin sensitivity Counterregulatory hormonal responses to hypoglycemia

Method Home glucose monitor Home glucose monitor IV glucose pulse IV arginine pulse GPAIS Hyperinsulinemic, euglycemic clamp Stepped hypoglycemic clamp

Frequency Weekly Weekly Yearly Yearly Yearly Yearly Yearly

REFERENCES 1.

Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen [see comments]. N Engl J Med 2000; 343:230–238. 2. Ryan EA, Lakey JR, Rajotte RV, et al. Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol. Diabetes 2001; 50:710–719. 3. Ryan EA. Presentation at 5th Annual Rachmiel Levine Symposium. Los Angeles, CA: Oct 6–9, 2004. 4. Najarian JS, Sutherland DE, Baumgartner D, et al. Total or near total pancreatectomy and islet autotransplantation for treatment of chronic pancreatitis. Ann Surg 1980; 192:526–542. 5. Wahoff DC, Papalois BE, Najarian JS, et al. Autologous islet transplantation to prevent diabetes after pancreatic resection. Ann Surg 1995; 222:562–575; discussion 575–569. 6. Clayton HA, Davies JE, Pollard CA, White SA, Musto PP, Dennison AR. Pancreatectomy with islet autotransplantation for the treatment of severe chronic pancreatitis: the first 40 patients at the Leicester General Hospital. Transplantation 2003; 76:92–98. 7. Pyzdrowski KL, Kendall DM, Halter JB, Nakhleh RE, Sutherland DE, Robertson RP. Preserved insulin secretion and insulin independence in recipients of islet autografts [see comments]. N Engl J Med 1992; 327:220–226. 8. Teuscher AU, Kendall DM, Smets YF, Leone JP, Sutherland DE, Robertson RP. Successful islet autotransplantation in humans: functional insulin secretory reserve as an estimate of surviving islet cell mass. Diabetes 1998; 47:324–330. 9. Ward WK, Wallum BJ, Beard JC, Taborsky GJ, Jr., Porte D, Jr. Reduction of glycemic potentiation. Sensitive indicator of b-cell loss in partially pancreatectomized dogs. Diabetes 1988; 37:723–729. 10. Robertson RP, Lanz KJ, Sutherland DE, Kendall DM. Prevention of diabetes for up to 13 years by autoislet transplantation after pancreatectomy for chronic pancreatitis. Diabetes 2001; 50:47–50.

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11. Kendall DM, Teuscher AU, Robertson RP. Defective glucagon secretion during sustained hypoglycemia following successful islet allo- and autotransplantation in humans. Diabetes 1997; 46:23–27. 12. Alejandro R, Lehmann R, Ricordi C, et al. Long-term function (6 years) of islet allografts in type 1 diabetes. Diabetes 1997; 46:1983–1989. 13. Ryan EA, Lakey JR, Paty BW, et al. Successful islet transplantation: continued insulin reserve provides long-term glycemic control. Diabetes 2002; 51:2148–2157. 14. Luzi L, Perseghin G, Brendel MD, et al. Metabolic effects of restoring partial b-cell function after islet allotransplantation in type 1 diabetic patients. Diabetes 2001; 50:277–282. 15. Hering BJ, Kandaswamy R, Ansite JD, et al. Single-donor, marginal-dose islet transplantation in patients with type 1 diabetes. Jama 2005; 293:830–835. 16. Rickels MR, Schutta MH, Markmann JF, Barker CF, Naji A, Teff KL. BetaCell function following human islet transplantation for type 1 diabetes. Diabetes 2005; 54:100–106. 17. Brunzell JD, Robertson RP, Lerner RL, et al. Relationships between fasting plasma glucose levels and insulin secretion during intravenous glucose tolerance tests. J Clin Endocrinol Metab 1976; 42:222–229. 18. Paty BW, Ryan EA, Shapiro AM, Lakey JR, Robertson RP. Intrahepatic islet transplantation in type 1 diabetic patients does not restore hypoglycemic hormonal counterregulation or symptom recognition after insulin independence. Diabetes 2002; 51:3428–3434. 19. Gupta V, Wahoff DC, Rooney DP, et al. The defective glucagon response from transplanted intrahepatic pancreatic islets during hypoglycemia is transplantation site-determined. Diabetes 1997; 46:28–33. 20. Robertson RP. Islet transplantation as a treatment for diabetes – a work in progress. N Engl J Med 2004; 350:694–705.

11 Challenges in Setting Up a New Islet Transplant Program Paul R. V. Johnson Oxford Islet Transplant Program, Nuffield Department of Surgery, University of Oxford, and Department of Pediatric Surgery, John Radcliffe Hospital, Oxford, U.K.

INTRODUCTION The recent success of clinical islet transplantation resulting from the Edmonton Protocol has led to a considerable expansion of islet transplant centers worldwide (1). Indeed, in 2005, islet transplants were performed in 76 centers around the world, compared with a total of 46 centers during the whole of the 1990s (2). However, looking back over the last five years a number of important lessons can be learned. This chapter aims to highlight some of the challenges that currently exist in setting up a new islet transplant program and to discuss ways in which these challenges might be overcome in the future.

EXPERIMENTAL CONTEXT OF ISLET TRANSPLANTATION Despite considerable clinical success, islet transplantation is still considered in most countries to be experimental rather than a routine treatment. This is in stark contrast to whole pancreas transplantation, and this perception underlies many of the challenges that new islet transplant programs face. This particularly affects the funding streams available to such programs, but also influences the way that this field is perceived within the management of many hospitals, the infrastructure available to such programs, and the interrelationships with other key specialties. Certainly the improved clinical outcomes of islet transplantation have improved many aspects of this, but it 203

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will still be a few years before islet transplantation benefits from the same core government funding of other more established treatments. Indeed, in the United Kingdom at present, the quid pro quo arrangements between the National Health Service and the Universities that have benefited many clinical research programs in previous decades and that have greatly helped advance the fields of islet isolation and islet transplantation, are now much less available as clinical funding becomes increasingly stretched and budgets become directly linked to individual clinical services. In addition, although the pure laboratory research projects associated with islet transplantation are supported by major grant bodies, the funding for the translational clinical islet transplant program often falls outside the remit of basic research grants (3). Currently therefore, many islet transplant programs largely depend on funding from charitable sources or benefactors. This type of funding is unpredictable and takes significant input of time and resources. Funding is an integral part of any islet program and competition for funds one of the major challenges facing any group planning to set up a new islet program. COMPONENTS OF AN ISLET TRANSPLANT PROGRAM A successful islet transplant program comprises a number of different components. Each component must be established in a new program and each presents different challenges. The four main components to be considered in this chapter are: pancreas procurement, human islet isolation, the clinical islet transplants themselves, and the associated research agenda. The practical aspects of each of these have been outlined in previous chapters of this book, but the specific logistical challenges of each component will now be addressed.

Pancreas Procurement It is now well recognized that optimal islet isolation is dependent on optimal retrieval and transport of the donor pancreas (4,5). However, meeting this requirement is often challenging both practically and financially, particularly for a new stand-alone islet transplant program. There are four main models of pancreas procurement to consider when setting up a new program, the precise logistics of which clearly vary between different countries and different centers. First, those islet transplant centers that also have a whole pancreas transplant program can benefit from a combined program for pancreas procurement, with the islet program benefiting from the inherent infrastructure of the pancreas transplant team. This enables resources to be pooled and also provides a high quality retrieval service 24 hours a day throughout the year. On a broader level, it can also facilitate a combined approach to patient selection for whole pancreas and islet

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transplants, respectively. However, issues of pancreatic allocation can be a problem in this situation (see below). Second, some islet teams establish a close collaboration with a single regional multi-organ retrieval team. This ensures optimal retrieval, but may have significant financial implications for the islet team and also limits retrieval to those being attended by the particular retrieval team. Third, other islet teams rely on pancreata being retrieved from a number of different retrieval teams (often at great distances away from the islet center) with no direct input into the retrieval process. This can ensure a large number of pancreata being referred into the islet program, but can result in variability in standards of pancreatic retrieval and hence pancreas quality, as well as significant expense in retrieval/ transport costs. Clear communication and education of each retrieval team is paramount for this model. Finally, a few islet transplant teams have their own pancreas retrieval teams who specifically retrieve pancreata from the local retrieval zone for the islet program. Although this may be ideal for controlling the procurement and transport variables, it is also the most challenging both logistically and financially, as the whole procurement team is the responsibility of the islet program. In addition, the islet team in this situation is often the lowest in the “pecking order” at the multi-organ retrieval, and has little control over how the pancreas is managed and preserved during the removal of the other intra-abdominal organs. It is clear that all these models of procurement have advantages and disadvantages, and the choice for any new islet program is clearly often based on local variables. However, the overriding challenge that impacts on all models of procurement is that of pancreas allocation, both in terms of allocation to whole pancreas programs versus islet programs, and in terms of the problems of allocation of organs for research purposes. Whole Pancreas vs. Islet Transplant Allocation It is widely stated that the ideal pancreatic donor for each modality is different with little overlap (6,7). Indeed, the current literature suggests that the ideal donor for pancreatic islet isolation is a female of high body mass index aged over 50 years, whereas such an organ would fulfill the exclusion criteria for whole pancreas programs (8,9). However, it is important to remember that this discrepancy is based on the practical limitations of current islet isolation procedures, rather than physiological considerations. Indeed, the currently perceived ideal donor for islet isolation also fulfills many of the predisposing features for Type 2 diabetes mellitus and may even in part account for the late islet graft failures being encountered! Clearly, until current isolation techniques are optimized to enable islets to be efficiently isolated from younger donors, the differences in donor selection will continue. However, as islet isolation groups strive to improve islet isolation techniques and these advances become realized, competition for

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pancreata may be an increasing challenge for islet groups and one that must be addressed at a national level in each participating country (10). Allocation of Donor Organs for Research As has already been discussed, one of the challenges of islet transplantation is the perception that it is an experimental treatment. One of the areas in which this is most highlighted is pancreas allocation (11). Although the logistics vary from country to country, consent for donor organs for research has become increasingly difficult to obtain. This partly reflects the overall shortage of suitable organ donors, but has been compounded by several highly publicized cases in which organs were retained for research (12). In the United Kingdom, for instance, pancreas procurement for islet isolation is now consented under the “research section” of the consent form whether the islets are being used for clinical transplantation or used purely for basic islet physiology research. This has had a significant impact on consent rates and hence numbers of pancreata donated for islet transplantation. There are about 1000 organ donors in the United Kingdom per year and about 60 whole pancreas transplants performed. Of the other 940 potentially available donor pancreata, only about 100 are retrieved for islet isolation. Although the reasons for this include a shortage of suitable surgical retrieval teams in some regions, a significant number are not retrieved as a result of a failure to obtain consent. In an attempt to address this, a national database has recently been established for pancreas referrals for islet transplantation. Although this is funded by a charitable organization and run by the islet transplant groups themselves, it is integrally linked to the national U.K. pancreas-for-islets donor referral service and (it is anticipated) will help address this important issue. A number of other countries have similar arrangements. The frustrating paradox is that the “research” label will only be lifted once islet transplantation has been proven to be as clinically successful as its whole organ counterpart. However, for this to be achieved, sufficient numbers of optimal donor pancreata need to be routinely available for islet grafts. Islet Isolation Islet isolation is one of the essential components of islet transplantation. Indeed, all islet transplant programs are dependent on the isolation of sufficient numbers of viable human islets. Although islet isolation is integrally linked to pancreas procurement, the logistical and practical challenges of islet isolation per se are numerous. Traditionally islet isolation has been performed within an islet isolation facility integral to each islet transplant program. There are now a number of reasons why this may not necessarily be the best model (13).

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These include the technical challenges of human islet isolation, the recently introduced requirements to isolate human islets within a Clean Room Facility, and the number of personnel required to maintain a viable islet isolation rota. Careful consideration should be given to these challenges before initiating any new islet transplant program. Technical Challenges of Islet Isolation As has already been outlined in Chapter 6, islet isolation is a technically demanding procedure with many different factors influencing successful outcome. Indeed, even in the leading islet isolation centers, only 50% of pancreata processed currently result in sufficient numbers of viable islets for transplantation. Frequently described as “an art rather than a science,” islet isolation requires considerable expertise and experience. As such, it is not possible to set up a new islet isolation facility and expect staff unfamiliar with the techniques to rapidly be able to successfully isolate islets. This is highlighted by one of the important findings in the interim results of the Immune Tolerance Network multi-center trial of clinical islet transplantation, in which clinical success was demonstrated to correlate most closely with those centers that had established islet isolation laboratories. From the point of view of concentrating expertise and ensuring ongoing maximal experience of islet isolation techniques, therefore, it can strongly be argued that the establishment of regional islet isolation facilities providing islets for a network of satellite islet transplant centers makes considerable sense. Several such networks have been established worldwide and the GRAGIL and NORDIC networks in particular have reported considerable clinical success using this approach (14,15). The successful approach established in Miami is addressed in detail in Chapter 14. Requirement for Clean Room Facility If a strong case could be made for centralizing islet facilities on the basis of consolidating technical expertise, the recent introduction of legislation in many countries dictating that human islet isolation be performed in purpose-built Clean Room Facilities strengthens the case even further (16). Not only are these facilities extremely expensive to build, but also the ongoing running costs and continual monitoring and detailed operating procedures required are both costly and labor intensive. The challenge for a new islet transplant program, therefore, is to decide whether to build and fund such a facility or whether to team up with an already established islet isolation facility as part of a network. If a new Clean Room facility is required, clearly there are numerous design features that are essential. It is particularly helpful to visit different facilities worldwide at the design stage and also to have close liaisons with

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specific laboratories to avoid design errors. In addition, there are a few practical suggestions to recommend, based on the recent design and build of our purpose-built facility in Oxford (Fig. 1). First, as this field is relatively new and evolving all the time, it is wise to plan a new facility with any envisaged future accreditation requirements in mind. Any facility based purely on today’s requirements may well need upgrading in a relatively short time. This means anticipating future capacity as well as, as far as budget allows, ensuring that the facility over-fulfills current legal requirements. Second, in order to minimize the number of personnel required to enter the actual isolation room and yet at the same time ensure training of new personnel in isolation techniques, it is beneficial to have the facility centered around a central glasspaneled control room or office enabling direct visibility and communication into the facility. In our own facility we additionally have camera links from the flow hoods and microscopes directly into our office, which enables the whole islet isolation process to be fully observed and monitored from the Grade C non-sterile office. This has proved a very useful feature. Third, a state-of-the-art islet facility requires a suite of rooms with clearly defined directional room-to-room flow for both equipment and personnel. Such designs can be potentially wasteful in terms of corridor space and clearly this can have ongoing rental cost implications if rental is calculated per square

Figure 1 Diabetes Research and Wellness Foundataion Islet Isolation Facility in Oxford, U.K.

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foot. Careful attention to detail with regard to room adjacencies can have huge implications for the final total square area of the facility. Number of Personnel Required The final argument for centralizing islet isolation facilities is to ensure sufficient numbers of experienced personnel to be able to isolate islets each day of the year. This has previously been achieved in most centers by having a small, dedicated team that was continually available on a voluntary basis. However, recent changes in employment laws in a number of countries have resulted in significant constraints on the number of continual hours that employees are permitted to work. As a result, considerably more personnel are now required to maintain a legal and viable ‘on call’ rota. For this to be achieved with experienced personnel, it makes considerable sense for these personnel to be centralized within few centers. Clinical Islet Transplantation In contrast to the technical challenges of human islet isolation, the technical challenges of clinical islet transplantation seem comparatively small. However, there are considerable challenges in setting up a new, successful clinical islet transplant program. These include establishing a multidisciplinary team, recruiting suitable patients into the program, and obtaining relevant ethical approval. Establishing a Multidisciplinary Islet Transplant Team Islet transplantation overlaps a number of different medical specialties, and, therefore, establishing a coherent, functional, multidisciplinary team representing all related disciplines is essential. In addition to the islet isolation team discussed above, the islet transplant team should include transplant surgeons (with a primary interest in the pancreas and wide experience with immunosuppressive management); diabetologists (ideally with a principal interest in Type 1 diabetes and hypoglycemic unawareness, and also previous experience with managing whole pancreas and/or islet transplant patients); interventional radiologists with expertise in percutaneous transhepatic access (17,18); transplant coordinators (particularly important in the current era where ‘competition’ exists for pancreas procurement); diabetes and transplant nurse specialists; hepatobiliary surgeons fully involved in the program in the event of liver or portal vein complications and in cases where the percutaneous method is contraindicated or fails and a laparoscopic approach is required; nephrologists, if an islet after kidney program is being incorporated; and transplant

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immunologists involved in graft monitoring and in the in development of novel immunosuppressants and immune tolerance strategies. It is also prudent to have pediatric involvement within the overall team, as it must not be forgotten that the ultimate aim of islet transplantation is to be able to transplant in children soon after diagnosis. This goal will be reached more readily if those managing children are involved in helping to establish both the clinical and research strategies from the outset. Clearly the exact structure and hierarchy of the team will vary from center to center, but the overall requirement for a broad team of differing expertise is important for all programs. Such a team often crosses over traditional institutional boundaries and the challenge for the team leader is to keep the team together and to ensure that everyone focuses on the same agenda! Good communication and an environment of transparent collaboration are vital for this process. It is observed that many islet transplant teams, unlike their whole pancreas counterparts, are comprised of members that are involved in many other clinical areas. The success of the original Edmonton Protocol reminds us what can be achieved when a team focuses exclusively on islet transplantation. Ethical Approval Although this is an essential part of any new clinical procedure, this aspect is deliberately included as a separate point to remind new programs to apply for this early. As these applications are becoming increasingly demanding and time-consuming in most countries, it can be immensely frustrating to have all components of a new program established but to still be waiting for ethical approval before patient recruitment can be commenced. Patient Recruitment For a new islet transplant program to be viable, it is important that sufficient numbers of suitable patients are recruited. Indeed, a careful assessment of potential numbers of islet recipients should be made prior to embarking on any program. This is particularly important when deciding whether to justify an integral islet isolation facility. To enable this assessment, a program must decide whether it plans to transplant only islet-alone (IA) patients or whether to transplant also/only islet-after-kidney (IAK) or simultaneous islet and kidney (SIK) patients. This will depend on local arrangements, but it is important to decide this in order to be able not only to establish the necessary resources and expertise, but also to establish patient referral pathways. It has been our own observation and that of other colleagues that as newer insulin regimes are being developed, the number of referrals for islet transplantation with the indication of hypoglycemia unawareness has significantly reduced. In our own program, like many other

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groups, we are now including patients with progressive complications despite maximal medical treatment as well as potential IAK recipients. Good communication should be established with the diabetologists and nephrologists within all the referring hospitals and the different members of their teams. For many, islet transplantation will be a new procedure and it is often helpful to present this work at the relevant multidisciplinary meetings as part of the process. Where successful whole pancreas transplant programs are already established, it is important to educate on the relative benefits of islet transplantation and to have colleagues fully supportive of this newer technique. Although this may be time consuming, it is time well spent! Transplant and diabetes nurse specialists play a vital role in every stage of patient recruitment and provide invaluable links between different specialties. In our own program, this group of professionals has played a key role in managing the patient recruitment as well as establishing islet transplantation education to different health care groups. As soon as ethical approval is obtained, patient work-up should be undertaken in parallel to the establishment of the other components of a new program. It should not be underestimated how long this pretransplant assessment takes for each patient. It must also be remembered that only 1 in 10 referrals to most major islet programs are finally placed onto the waiting list. Initiating multidisciplinary islet transplant clinics is beneficial from the outset. They not only help to establish a team approach, but also facilitate a more efficient service for the patient and greater momentum for recruitment. Many centers have found the use of a clinical research fellow helpful in coordinating such clinics, but this should not obviate the involvement of senior clinicians throughout the recruitment process. Islet Transplant Procedure A detailed discussion of the actual islet transplant procedure is outside the scope of this chapter. However, there are a number of logistical points that should be considered when first establishing a program. First, it is essential to incorporate a team of interventional radiologists fully into the program. Such a team has to be available at all times, especially if the islets are being transplanted immediately after isolation rather than as a more planned procedure after a period of culture. In addition, in the initial stages of a new program, the basis for involvement may be as part a research collaboration rather than a fully remunerated clinical service, and as such, the radiologists need to have the same enthusiasm for developing the islet transplant program as the team leaders! The radiologists must not only be familiar with the percutaneous transhepatic approach (18), but also be aware of some of the unique challenges presented by islet infusion. It is beneficial for them to establish

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links with the interventional radiologists in established islet transplant centers, and preferably to visit such a center before starting. This also provides a useful resource for advice and collaborative research once the program has become established. The second point is the decision whether to transplant fresh islets immediately after islet isolation or whether to incorporate a period of culture first. This clearly depends on a number of different factors and is discussed elsewhere in this book. Certainly, incorporating a period of islet culture makes the logistics of planning the islet transplant procedure much easier. However, centers first establishing islet isolation may elect not to incorporate this step initially in order to avoid an additional variable. The third point is a logistical warning to ensure that patients of all blood groups are recruited onto the waiting list at the start of the program. This is important not only to enable the full range of pancreata to be procured for the program and thus establish a routine rather than selective retrieval service, but also to avoid the situation of having a waiting list made up primarily of potential recipients of blood group O. In the Edmonton series the average wait for blood group O patients was 2 years. This wait is further exacerbated if there is a local whole pancreas program. Finally, there is the logistical problem of receiving organs matched to a patient but that even in the best centers worldwide have up to a 50% chance of not yielding enough islets for transplantation. Such uncertainty poses a real dilemma as to when to call the patient in to the hospital to ensure minimal disappointment for them and yet have them maximally prepared for a timely transplant. This problem is greatly reduced when islets are transplanted electively after a period of culture.

Associated Research Program In addition to the fact that islet transplantation has only been made possible by good, basic research, there are several reasons why islet transplant programs have been traditionally associated with strong research programs and why this should continue. First, it is clear that even in the post-Edmonton era, in many countries islet transplantation is still an experimental treatment, and at present programs largely fall under the auspices of university research departments. As such, basic research into different aspects of islet isolation, islet transplantation, and islet biology are an integral component of most programs’ raison d’etre. This has also provided sufficient personnel for the islet isolation rotas, many of whom conduct research for higher degrees while being ‘on call’ for islet isolation. The main reason for maintaining a strong research emphasis within each program is that we still have a long way to go before islet transplantation has achieved its goal. The success of the Edmonton Protocol has been a major step forward. However, overall it has only

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optimized all the steps that were previously being used (albeit very successfully) rather than making any major new discovery. It is so important that basic scientific research remains integral to clinical islet programs to enable the field to continually be advanced. For this to be achieved, any new islet transplant program must combine clinical expertise with strong scientific endeavor. Each center must determine its research strengths and focus on them, but purely developing a clinical service without a clear research agenda will miss important opportunities to develop the field. In addition, islet programs have unique opportunities for collaborative research between different centers, and these opportunities should be explored as soon as a credible program has been established. CONCLUSIONS Establishing an islet transplant program presents unique challenges not seen with whole organ transplantation. The islet isolation phase in particular adds logistical, financial, and personnel issues that must be considered carefully before embarking on such a program. One of the keys to a successful program is establishing and maintaining a cohesive, multidisciplinary team that covers all aspects of islet transplantation. This includes incorporating a strong research program from the outset, not only to facilitate some of the practical challenges of the present, but more importantly to enable islet transplantation to progress and for new discoveries to be made so that the ultimate aim of transplanting islets into children with newly diagnosed diabetes can be realized. REFERENCES 1.

2. 3. 4.

5.

6. 7.

Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000; 343(4):230. Brendel MD, Hering BJ, Schultz AO, Bretzel RG. International Islet Transplant Registry. http://www.med.uni-giessen.de/itr. (Accessed, 2005). NIH funds centers to study islet transplant. J Investig Med 2004; 52(8):497. Kessler L, Bucher P, Milliat-Guittard L, et al. Influence of islet transportation on pancreatic islet allotransplantation in type 1 diabetic patients within the Swiss-French GRAGIL network. Transplantation 2004; 77(8):1301. Ricordi C, Inverardi L, Kenyon NS, Goss J, Bertuzzi F, Alejandro R. Requirements for success in clinical islet transplantation. Transplantation 2005; 79(10):1298. Berney T, Buhler LH, Morel P. Pancreas allocation in the era of islet transplantation. Transpl Int 2005; 18(7):763. Briones RM, Miranda JM, Mellado-Gil JM, et al. Differential analysis of donor characteristics for pancreas and islet transplantation. Transplant Proc 2006; 38(8):2579.

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Brandhorst D, Hering BJ, Brandhorst H, Federlin K, Bretzel RG. Influence of donor data and organ procurement on human islet isolation. Transplant Proc 1994; 26(2):592. Lakey JR, Warnock GL, Rajotte RV, et al. Variables in organ donors that affect the recovery of human islets of Langerhans. Transplantation 1996; 61 (7):1047. Ris F, Toso C, Veith FU, Majno P, Morel P, Oberholzer J. Are criteria for islet and pancreas donors sufficiently different to minimize competition? Am J Transplant 2004; 4(5):763. Ridgway DM, White SA, Kimber RM, Nicholson ML. Current practices of donor pancreas allocation in the UK: future implications for pancreas and islet transplantation. Transpl Int 2005; 18(7):828. Hunter M. Alder Hey report condemns doctors, management, and coroner. BMJ 2001; 322(7281):255. Goss JA, Goodpastor SE, Brunicardi FC, et al. Development of a human pancreatic islet-transplant program through a collaborative relationship with a remote islet-isolation center. Transplantation 2004; 77(3):462. Benhamou PY, Oberholzer J, Toso C, et al. Human islet transplantation network for the treatment of Type I diabetes: first data from the Swiss-French GRAGIL consortium (1999–2000). Groupe de Recherche Rhin Rhjne Alpes Geneve pour la transplantation d’Ilots de Langerhans. Diabetologia 2001; 44 (7):859. Rydgard KJ, Song Z, Foss A, et al. Procurement of human pancreases for islet isolation-the initiation of a Scandinavian collaborative network. Transplant Proc 2001; 33(4):2538. Rastellini C, Braun M, Cicalese L, Benedetti E. Construction of an optimal facility for clinical pancreatic islet isolation. Transplant Proc 2001; 33(7–8): 3524. Neeman Z, Hirshberg B, Harlan D, Wood BJ. Radiologic aspects of islet cell transplantation. Curr Diab Rep 2006; 6(4):310. Goss JA, Soltes G, Goodpastor SE, et al. Pancreatic islet transplantation: the radiographic approach. Transplantation 2003; 76(1):199.

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12 Key Factors to Consider in Setting Up Clinical Trials in Islet Cell Transplantation: A Nursing Coordinator’s Perspective Barbara S. DiMercurio Adult General Clinical Research Center, University of Colorado Health and Science Center, Denver, Colorado, U.S.A.

INTRODUCTION In a complex, health-oriented society such as ours that is increasingly receptive to patients’ concerns related to the cost, quality, availability, and accessibility of health care, it is of paramount importance to clearly define the future direction of research in islet cell transplantation. Clinical islet transplantation is changing rapidly and at an unpredictable pace. Globally, numbers of health care centers setting up clinical trials in islet cell transplantation continue to grow. One of the major focuses of current and future clinical trials in islet cell transplantation is the implementation of optimally designed outcome trials that will provide a foundation for truly evidence-based clinical practice in the community. Practice guidelines informed by robust research findings will become benchmarks for costeffective, highest quality clinical practice. In addition, islet researchers are visibly involved at an international level, participating in policy making, providing testimony at Congressional hearings, and lobbying for funding. This chapter is focused on the key factors necessary for setting up safe and effective clinical trials in islet cell transplantation. These are outlined in Table 1. A practical guide is provided for new and more established centers. 215

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Table 1

Key Factors to Consider in Setting Up Clinical Trials in Islet Cell Transplantation Center infrastructure Personnel Budget Space Project management Protocol development Training by experienced islet cell transplantation centers United Network of Organ Sharing (UNOS) and local organ procurement organization (OPO) Recruitment Consenting Data management Standard operating procedures Regulatory Investigation new drug application Institutional review board (Ethics Committee) Data safety monitoring board

Specific requirements for those working in the United States are addressed, although all underlying themes are pertinent worldwide.

CENTER INFRASTRUCTURE Establishment of a solid infrastructure prior to commencement of clinical trials in islet cell transplantation is of utmost importance. A reliable and sound infrastructure will ensure the development of successful clinical trials in islet cell transplantation. Basic elements of a successful islet cell transplantation clinical trials program include adequate personnel, sufficient funding to support the research and clinical demand, and adequate space to grow.

Personnel To be successful, islet cell transplantation centers must have personnel with all the necessary skills, knowledge, and motivation to set up effective clinical trials in this field. Since islet cell transplantation in so highly specialized, proper skills and knowledge must be gained from centers with established experience and expertise. Although it is essential that all personnel work closely together, they can be divided into two broad categories: clinical personnel and islet cell isolation personnel. Figures 1 and 2 outline recommended clinical and islet cell isolation personnel.

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Interventional radiologist Administrative support Principal investigator

Research transplant coordinator

Regulatory specialist

Figure 1

Data entry personnel

Social worker

Clinical Personnel.

Budget Clinical trials in islet cell transplantation currently remain experimental. Islet cell transplantation is not approved as an insurance-reimbursable procedure in the United States. Most institutions thus receive their funding from some form of government, institution, industry, or private research source. The status of islet transplantation is different in other countries; however, for example, in Canada clinical islet transplantation has been funded as part of routine transplant care since 2001. The current initiatives of the clinical islet transplant consortium (National Institutes of Health Funded initiative) through a series of Phase II and III clinical trials will

Islet technologist QA/QC

Regulatory specialist

Laboratory technicians

Laboratory director

Resident or fellow

Figure 2

Islet isolation personnel.

Data entry personnel

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hopefully render a Biological License and approval of islet transplantation in the United States. Until such time, the funding for experimental islet transplant programs remains critically dependent on external grant funding. An adequate budget is the lifeline to a successful islet cell transplantation program. Sufficient funds are needed to cover infrastructure costs, equipment, supplies, reagents, personnel salaries, organ acquisition, and patient care cost including medication and immune monitoring.

Space Provision of adequate space is one of the hardest obstacles to overcome for most institutions beginning clinical trials in islet cell transplantation. The clinical management team requires dedicated office space for personnel, clinic space to screen and conduct study-related activities, interventional radiology space to administer the islet cell infusion, and an inpatient or observational unit to observe the subject after infusion. In the United States, the islet isolation processing facility must meet all cGMP requirements outlined in 21 Code of Federal Regulations (CFR) 211 and 606. Institutions that do not have their own processing facility may arrange for islets to be shipped for islet cell transplantation from a nearby cGMP approved facility.

PROJECT MANAGEMENT Setting up clinical trials in islet cell transplantation takes superb project management skills. A knowledgeable and skilled project manager understands the process of planning, organizing, and implementing clinical trials in accordance with all regulatory requirements. The project manager may be the principal investigator and/or research transplant coordinator. In most islet cell transplantation centers, the principal investigator delegates project management responsibilities to the research transplant coordinator. He/she must have the ability to visualize the research project as a whole. Effective communication with all personnel and disciplines involved in the development of clinical trials in islet cell transplantation is essential. In early phases of development, frequent team meetings should occur to identify who will accomplish which phases of the program development. Once the infrastructure is in place, the project manager should primarily focus on protocol development, organizing training from an experienced islet cell transplantation center, initiating communications with the United Network of Organ Sharing (UNOS) and the local organ procurement organization (OPO), recruitment, evaluation, data management, development of standard operating procedures, and regulatory compliance (see section entitled “Regulatory Issues”).

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Protocol Development The principal investigator (PI) is the person who takes full responsibility for the clinical trial. Protocol design and development is primarily the role of the PI. This role includes monitoring the conduct of the protocol, monitoring the enrollment of subjects to guarantee eligibility criteria have been met, monitoring the protocol to ensure study related therapies are administered safely, monitoring the outcome of subjects, and ensuring that the data are collected promptly and accurately. Table 2 lists some of the key elements that should be included in an islet cell transplantation protocol. The development of a successful islet cell transplantation protocol is both labor- and resource-intensive. It is imperative that the design of the protocol is not self-limiting. In the early stages of protocol development, logistical aspects and feasibility of subject compliance should be considered. The less labor-intensive the protocol design is for the subject, the greater the likelihood of compliance with the protocol.

Training by Experienced Islet Cell Transplantation Centers Centers all over the world have been investigating islet cell transplantation for over a decade. Until recently, the long-term success rate for insulin independence has been limited (1). In July 2000, researchers at the University of Alberta in Edmonton, Canada reported a dramatic improvement in successful islet cell transplantation in seven consecutive patients (2). Internationally, researchers have been in collaboration with Edmonton, Canada to learn their techniques and review their procedures as a hallmark for islet cell isolation and medical management, specifically the regimen for immunosuppressive therapy. Collaboration with experienced islet cell transplantation centers is paramount in the development of a successful program. It is highly recommended that all new centers conducting clinical trials in islet cell transplantation gain their initial experience from centers that have a wellestablished and successful program. Both the clinical management team and the islet cell isolation team should participate in this training.

UNOS and Local OPO In 1984, the U.S. Congress, under the National Organ Transplant Act (NOTA), established the Organ Procurement and Transplantation Network (OPTN). The OPTN contract was awarded to UNOS September 30, 1986. UNOS is a private, nonprofit organization. The national, computerized waiting list of transplant candidates is updated and maintained by UNOS (3).

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Table 2

Key Elements That Should Be Included in an Islet Cell Transplantation Protocol Title page Table of contents Background information and scientific rationale Description of investigational product Method of preparation Isolation of pancreas Enzymatic digestion Purification of islets Preparation of purified islets Product testing Quality control testing Reagents used during islet cell preparation Summary of pre-clinical and clinical experience Summary of known and potential risks and benefits Study medications Portal vein cannulation Transplant of allogeneic tissue Risks of renal dysfunction Immunosuppressive agents Other risks Assessment of compliance Study objectives Primary and secondary objectives Study design Description of study design Primary and secondary end-points Description of study population Screening, randomization, and enrollment Description of study treatments and dosage regimen and labeling Islet infusion(s) Immunosuppressive therapy Duration of subject participation and follow-up Description of stopping rules Data collection Selection and withdraw of subjects Subject inclusion criteria Subject exclusion criteria Subject withdrawal criteria Donor organ inclusion criteria Donor organ exclusion criteria Study therapy Description and justification of dosage and route of islet cells Study medications Immunosuppressive therapy Concomitant medications (Continued)

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Table 2

Key Elements That Should Be Included in an Islet Cell Transplantation Protocol (Continued )

Study therapy Prophylactic medications Accountability of investigational product(s) Treatment of subjects Number and timing of transplants Treatment administration and follow-up Screening assessments Pre-transplant baseline testing Day of transplant Immediate post-transplant activities Long-term post-transplant monitoring Medications permitted and not permitted before and during trial Assessment of safety and efficacy Statistical criteria Access of source data and documents Quality control and quality assurance Ethical issues Data handling and record keeping References

The United States has been divided into eleven geographic regions to facilitate expedited organ allocation. Within these regions are local OPOs. The local OPOs are responsible for approaching families for organ donation, medically evaluating the potential donors, coordinating recovery, preservation, and transportation of organs, and educating society on the need for organ donation. Currently, there are 255 transplant centers registered with UNOS. Of the 255 transplant centers, 30 are registered as islet cell transplantation centers (4). These 30 registered centers have a total of 303 candidates listed for islet cell transplantation (5). Table 3 outlines the breakdown of islet cell transplantation OPOs. All new centers in the United States setting up islet cell transplantation programs are required to register with UNOS. Contact your local OPO for a detailed policy and procedure for pancreas allocation in your region.

Recruitment During the protocol development phase a detailed recruitment plan should be established. This recruitment plan should outline the method of advertisement, any prequalification questionnaires, a standard operating procedure (SOP) for communications with the prospective subjects, and a

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Table 3 Region 5 5 8 2 3 3 7 10 1 7 11 2 9 2 11 4 4 11 6 7

DiMercurio Breakdown of Islet Cell Transplantation Centers Organ procurement organization California Transplant Donor Network (CADN) OneLegacy (CAOP) Donor Alliance (CORS) Washington Regional Transplant Consortium (DCTC) Life Alliance Organ Recovery Agency (FLMP) LifeLink of Georgia (GALL) Gift of Hope Organ & Tissue Donor Network (ILIP) Indiana Organ Procurement Organization (INOP) New England Organ Bank (MAOB) LifeSource Upper Midwest Organ Procurement Organization (MNOP) Lifeshare of the Carolinas (NCCM) New Jersey Organ and Tissue Sharing Network (NJTO) New York Organ Donor Network ( NYRT) Gift of Life Donor Program (PADV) Mid-South Transplant Foundation (TNMS) LifeGift Organ Donation Center (TXGC) Southwest Transplant Alliance (TXSB) LifeNet (VATB) LifeCenter Northwest Donor Network (WALC) Organ Procurement Organization at the University of Wisconsin (WIUW)

plan for how special efforts will be implemented to recruit women and minority groups. Methods of advertisement may include institutional websites, local or national newspaper advertisements, protocol flyers, and by a referring endocrinologist or primary care physician. Most institutions require approval by the local Institutional Review Board (IRB) or Independent Ethics Committee (IEC) prior to implementing the recruitment plan. Recruitment advertisements should include a brief description of the study, major inclusion and exclusion criteria, and whether compensation is offered. The development of a prequalification questionnaire is a key element in the recruitment process. Since islet cell transplantation is currently not an insurance reimbursable therapy, the development of a detailed prequalification questionnaire may help defer screening failures. Deferring screening failures will allow internal resources to be utilized more effectively as well as decreasing the budgetary demand during the screening process. Developing an SOP for communication with prospective subjects is essential. This SOP should contain information regarding a primary phone line for incoming and outgoing communication with subjects and referring physicians, template letters for subjects who do not meet the prequalification questionnaire inclusion criteria, and template letters for subjects who

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meet the prequalification questionnaire inclusion criteria. Effective communication between subjects and staff will ensure a smooth recruitment process.

Consenting In clinical trials, informed consent means that a person who is capable of making a decision, or his or her authorized legal representative, has freely and voluntarily agreed to participate in an experimental study. All human subjects who participate in islet cell transplantation clinical trials must sign an informed consent prior to participating in the study. Prospective islet cell transplantation recipients must have sufficient time to decide whether to participate in the study. These recipients must be fully informed of any potential benefits and risks of participation and by no means coerced into participating. The language of the informed consent should be written at eighth grade school level (reading age of 13 years). Every protocol consent document should contain eight key elements and six optional elements as defined in 21 CFR 56.25 and shown in Table 4 (6).

Data Management Data management is one of the most important areas in clinical research. The ability to collect, store, manipulate, analyze, retrieve, and publish data is critical in the development of standardization throughout islet cell transplantation centers. Developing an instrument or method with acceptable validity and reliability for data collection is a key element in overall data management. In islet cell transplantation trials, data should be recorded from both the clinical components of the study and from the islet isolation laboratory. The development of case report forms (CRFs) is considered the best mechanism to capture the required administrative, research, and regulatory data for clinical trials in islet cell transplantation. The quality of the data collected is directly affected by the design of the CRFs. Therefore, both the clinical and islet cell isolation laboratory CRFs should be reviewed by the end-user for consistency and clarity. Vigilantly collecting and transcribing the data to the CRFs from the beginning of the study will maximize accuracy and make it easier to analyze the data at a later date. Internal audits should be established to ensure that the integrity of the data is being met. It is recommended that internal audits be conducted monthly by an independent staff member (individual who did not enter the data). All discrepancies should be reviewed with the principal investigator. An announced or unannounced external audit by the Food and Drug Administration (FDA) or sponsor of the clinical trial may occur. Both

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Table 4

DiMercurio Key Elements of Informed Consent

1.

A statement that the study involves research, an explanation of the purposes of the research and the expected duration of the subject’s participation, a description of the procedures to be followed, and identification of any procedures that are experimental 2. A description of any reasonably foreseeable risks or discomforts to the subject 3. A description of any benefits to the subject or to others which may reasonably be expected from the research 4. A disclosure of appropriate alternative procedures or courses of treatment, if any, that might be advantageous to the subject 5. A statement describing the extent, if any, to which confidentiality of records identifying the subject will be maintained 6. For research involving more than minimal risk, an explanation as to whether any compensation and an explanation as to whether any medical treatments are available if injury occurs and, if so, what they consist of, or where further information may be obtained 7. An explanation of whom to contact for answers to pertinent questions about the research and research subjects’ rights, and whom to contact in the event of a research-related injury to the subject 8. A statement that participation is voluntary, refusal to participate will involve no penalty or loss of benefits to which the subject is otherwise entitled, and the subject may discontinue participation at any time without penalty or loss of benefits to which the subject is otherwise entitled When appropriate, one or more of the following elements of information shall also be provided to each subject: 1. A statement that the particular treatment or procedure may involve risks to the subject (or to the embryo or fetus, if the subject is or may become pregnant) which are currently unforeseeable 2. Anticipated circumstances under which the subject’s participation may be terminated by the investigator without regard to the subject’s consent 3. Any additional costs to the subject that may result from participation in the research 4. The consequences of a subject’s decision to withdraw from the research and procedures for orderly termination of participation by the subject 5. A statement that significant new findings developed during the course of the research which may relate to the subject’s willingness to continue participation will be provided to the subject 6. The approximate number of subjects involved in the study

internal and external audits consist of verifying accurate transfer of the raw data directly from the medical record to the case report forms.

Standard Operating Procedures The development of SOPs enables clinical trials to be conducted safely, efficiently, and reliably. An SOP should be kept short and simple and in

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a convenient location; otherwise, staff will not refer to them when needed. Some examples of SOPs needed for islet transplantation trials are screening, treatment, follow-up, donor acceptance, isolation procedures, and product release testing. REGULATORY ISSUES Regulatory agencies such as the FDA have a primary goal of enhancing the public’s ability to secure adequate health care through the regulation of medical products and food. Therefore, allogeneic pancreatic islets for transplantation are regulated by the FDA in the United States. Allogeneic pancreatic islets are regulated as biological products subject to licensing under Section 351 of the Public Health Service Act (PHS Act), 42 USC 262. They also meet the definition of “drug” in the Federal Food, Drug, and Cosmetic Act (FD&C Act), 21 USC 321(g), and are thus subject to certain requirements of the FD&C Act (7). Regulatory compliance is essential in the development and implementation of clinical trials in islet cell transplantation. Some of the important regulatory bodies involved in these clinical trials include the FDA, Institutional Review Board (IRB), and the Data Safety Monitoring Board (DSMB). Investigational New Drug Application Prior to the initiation of clinical trials in islet cell transplantation, an Investigational New Drug (IND) application should be submitted for review by the FDA. To prevent delays in the approval process, it is imperative that the applicant stays in contact with the agency to address any questions or concerns related to the chemistry, manufacturing, and controls (CMC) section or the clinical protocol. Authorization is required prior to the initiation of these clinical trials in accordance with 21 CFR 312.40 (8). Institutions outside the United States should check with their health authorities for specific regulations regarding islet cell transplantation. Table 5 outlines the content and format for an IND submission as described in 21 CFR 312.23 (8). Detailed instructions and information on IND regulations, required forms, and how to submit an IND can be obtained from the FDA website (See Ref. 9). Institutional Review Board (Independent Ethics Committee) As defined in 21 CFR 56, IRB means any board, committee, or other group formally designated by an institution to review, to approve the initiation of, and to conduct periodic review of, biomedical research involving human

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DiMercurio Investigational New Drug Application Content and Format

A sponsor who intends to conduct a clinical investigation subject to this part shall submit an Investigational New Drug application (IND) including, in the following order: 1. Cover sheet (Form FDA-1571) 2. Table of contents 3. Introductory statement 4. General investigational plan 5. Investigator’s brochure 6. Clinical protocols 7. Chemistry, manufacturing, and control information 8. Pharmacology and toxicology information 9. Previous human experience 10. Additional information Additional documents should be included in the application: 1. Statement of investigator (Form FDA 1572) 2. Informed consent of human subjects (21 CFR 50, Subpart B) 3. Disclosure: financial interests and arrangements of clinical investigators (Form FDA 3455)

subjects. The primary purpose of such review is to assure the protection of the rights and welfare of the human subjects. An IRB shall review and have authority to approve, require modifications in (to secure approval), or disapprove all research activities covered by these regulations (10). The FDA and the Department of Health and Human Services (DHHS) regulations require all clinical research trials involving islet cell transplantation to undergo review by an IRB also known as an Institutional or Independent Ethics Committee (IEC).

Data and Safety Monitoring Board Most clinical research trials use some form of data and safety monitoring. This can be obtained either by the principal investigator, an independent clinician, or by an entire DSMB. Due to the complexity and potential risk to human subjects, clinical trials in islet cell transplantation tend to lean towards using a DSMB. This DSMB should be comprised of external people without any direct link to the clinical trial. Ideally, all members should have relevant experience in the same area as the study. A statistician, endocrinologist, ethicist, solid organ transplant physician, or a physician who directly has experience with immunosuppression are examples of appropriate members for an islet cell transplantation DSMB. The National Institute of Health (NIH) has developed a policy that all NIH supported clinical trials have some form of data and safety monitoring

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in place for the appropriate oversight and monitoring of the conduct of clinical trials to ensure the safety of participants and the validity and integrity of the data. Multi-site clinical trials involving interventions that entail potential risk to human subjects should have an established DSMB. The requirements for a study review and approval by an IRB are separate from the requirements for data safety monitoring oversight (11).

CONCLUSIONS Setting up clinical trials in islet cell transplantation remains challenging. However, the trail has been previously traveled—protocols, procedures, and practices in islet cell transplantation have been established for over a decade. Efficient and resourceful collaboration with experienced centers around the world is a principal factor in establishing successful clinical trials in islet cell transplantation. Furthermore, it is essential that institutions all over the world collaborate their efforts with comprehensive standardization in mind. Successful clinical trials in islet cell transplantation will influence healthrelated polices at all levels. Keep in mind that project management and regulatory compliance are the backbone to proficient and successful clinical trials.

REFERENCES 1.

Brendel M, Hering B, Schulz A, Bretzel R. International Islet Transplant Registry report. Giessen, Germany: University of Giessen, 1999:1–20. 2. Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Eng J Med 2000; 343:230–238. 3. http://www.unos.org/whoWeAre/theOPTN.asp (accessed February 2006). 4. http://www.unos.org/whoWeAre/transplantCenters.asp (accessed February 2006). 5. Based on the OPTN database February 17, 2006. This work was supported in part by Health Resources and Services Administration contract 231-00-0115. 6. Code of Federal Regulations: Protection of human subjects, 21 CFR 50.25, Revised April 1, 2005. 7. U.S Food and Drug Administration: Letter to Transplant Centers Allogeneic Pancreatic Islets for Transplantation, September 8, 2000. 8. Code of Federal Regulations: Investigational New Drug Application, 21 CFR 312, Revised April 1, 2005. 9. http://www.fda.gov/cber/ind/ind.htm. 10. Code of Federal Regulations: Institutional Review Boards, 21 CFR 56, Revised April 1, 2002. 11. NIH Policy for Data Safety Monitoring, June 10, 1998; http://grants.nih.gov/ grants/guide/notice-files/not98-084.html.

13 Clinical Outcomes and Future Directions in Islet Transplantation Faisal Al-Saif and A. M. James Shapiro Department of Surgery, Walter C. Mackenzie Health Center, University of Alberta, Edmonton, Alberta, Canada

INTRODUCTION The first human pancreatic islet transplant was reported in 1924 when Pybus transplanted fragments of cadaveric pancreatic tissue into two patients, one of whom had a reduction in glucose excretion (1). It was not until 1990 when the first human case of insulin independence following islet transplantation was reported in a 36-year-old patient who had undergone previous kidney transplantation (2). Success was, however, short-lived, and insulin had to be restarted 25 days post-transplantation. In the following decade, although results varied between centers, outcomes of pancreatic islet cell transplant remained generally poor overall with a 1-year insulin independence of only approximately 11% (3). Ricordi, in 1992, reported 50% insulin independence in 10 patients who underwent liver and pancreatic islet allotransplantation following upper abdominal exenteration for carcinoma. These patients had primary or metastatic liver tumors, which were too extensive to be removed locally; therefore, upper abdominal exenteration, including total hepatectomy and pancreatectomy, was performed with cadaveric liver and autoislet transplantation. All patients received tacrolimus monotherapy without the use of steroids. Insulin independence was not achieved by any patients with previous diabetes (4).

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In the late 1990s, the group in Giessen, Germany, implemented a new protocol to improve islet transplant outcomes. This new protocol included the use of highly purified islet preparations, islet culture for 24–72 hours, HLA matching, and induction with T-cell antibodies and steroids starting 3 days prior to transplantation. It also included intravenous insulin infusion, total parenteral nutrition, and intravenous Cyclosporine A to avoid islettoxic substrate concentrations in the portal vein for better islet engraftment. Islet function at 3 months was documented in 75% of the islet-after-kidney group and in all patients who had undergone simultaneous islet and kidney transplantation (5). An improvement in islet allotransplant outcomes was reported by the Swiss-French GRAGIL consortium where 20% insulin independence at one year was achieved. This success was mostly attributed to principles adopted from the Giessen experience (6). Seven out of 20 patients (35%) achieved insulin independence at the University of Milan, with all patients receiving islet after, or in conjunction with, kidney transplantation. Patients received an average of 9400 islet equivalent/kg body weight and underwent strict metabolic control post-transplant with intensive insulin therapy to protect the islet cells during engraftment (7). A landmark in the history of islet transplantation came with the introduction of the Edmonton Protocol in 2000 (8). With the use of a steroid-free sirolimus-based immunosuppressive protocol and the infusion of adequate islet mass, 100% insulin independence was achieved. More than 550 islet transplants have been performed over the last five years in comparison to 237 performed in the decade preceding the Edmonton Protocol (3,9). This success, although unique, was largely a result of lessons learned from previous experiences in Edmonton and around the world in the areas of islet isolation, transplantation, and immunosuppression (Fig. 1). Although the medical community and the public place a great emphasis on insulin independence as a mark of success, good glycemic control should be the main goal of islet transplantation. It has been proven that lowering glycosylated hemoglobin (HbA1c) with intensive insulin therapy reduces incidence of long-term diabetic complications, even when HbA1c remains above the normal range (10).

EFFORTS TO IMPROVE OUTCOMES Initial success in islet transplantation was achieved following autotransplantation without the use of immunosuppression. Islet preparations were large in volume and contained a vast amount of debris and exocrine tissues, which led to serious and potentially life-threatening complications, such as portal vein thrombosis, portal hypertension, and liver failure (11). Therefore, islet allotransplants were primarily performed in combination

Clinical Outcomes and Future Directions in Islet Transplantation Europe

>50 Institutions: >600 Patients Approximately 200 islet infusions in 2005 (120 in N America, 70 in Europe) North America Edmonton (93) Miami (30) Minneapolis (20) Vancouver (12) U Penn, (12) Houston (11) Harvard, Boston (10) Birmingham AL (3) Northwestern, (8) St. Louis (8) U Illinois (5) Emory, Atlanta (7) Cincinnati (6) NIH (6) Seattle (6) City of Hope CA (5) Memphis (3) U Maryland (2) Columbia NY (2) U Mass (2) UC San Francisco (2) Carolina Med Center (1) Cornell NY (1) Denver (1)

Figure 1

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Geneva+GRAGIL (48) Milan (39) Giessen (31) Brussels/Free Univ(25) Nordic Network (25) Brussels/Louvain (20) Nantes (1)

Zurich (12) Innsbruck (11) Lille (7) Budapest/Geneva (3) King’s UK (4) Royal Free UK (3) Oxford (1) Stockholm/Giessen (2)

Asia and Australia

South America Santiago Chili (1) San Paulo (3) Buenos Aires (11)

Sydney (6) Kyoto (6) Tokyo (1) Seoul (2) Chiba (1) Harbin (1) Shanghai (1)

Islet Transplantation Activity (1999–2006).

with other organ transplants, mainly liver and kidney, which required immunosuppression. Numerous improvements in pancreas procurement and transportation, islet isolation and culture, transplantation procedure, and immunosuppression have paved the way to the success of the Edmonton Protocol and the beginning of a new era in islet transplantation.

Pancreas Procurement The pancreas itself is very sensitive to hypotension, ischemia, and mechanical injuries. Any injury can stimulate pancreatic enzymes leading to autodigestion of pancreas and destruction of islets. Care of the donor pancreas begins even before starting procurement, especially in regards to adequate fluid resuscitation and avoidance of unnecessary use of inotropes, as this may reduce isolation success (12). Minimal mobilization of the pancreas during procurement along with in situ cooling and rapid transport of the pancreas to minimize cold ischemia led to stabilization of pancreatic endogenous enzymes and improvement of islet yield and functional viability (13). Procuring team experience affects isolation outcomes in general with better yields obtained from pancreata procured by a more experienced team (12).

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Pancreas Preservation and Transportation Islets are very sensitive to ischemia, and pancreata preserved with University of Wisconsin solution (UW) must be processed within 12 hours of cold storage. The two-layer method (TLM) was developed for pancreas preservation at Kobe University in Japan in 1988 (14). It consists of a layer of perfluorochemical (PFC) continuously bubbled with oxygen and covered with a layer of UW solution. PFC has a high affinity for oxygen, which diffuses to the preserved pancreas, maintaining membrane integrity, and thereby reducing ischemic changes. In comparison to UW, TLM resulted in a significant improvement in islet yield. It also allowed extension of cold ischemia time to up to 24 hours without resulting in any negative effects on outcomes, therefore allowing utilization of more distant donor pancreata (15). It also allowed the use of marginal donors that would not be used otherwise if preserved in UW, suggesting a damage reversal property of the TLM (16).

Islet Isolation Initially, pancreas digestion and islet isolation were either performed manually or by using simple mechanical choppers. Several isolation methods were tried but usually subjected the islets to significant trauma and resulted in low islet yield and purity. Gray et al. (17) described a method for isolation that was meant to be simple, quick, and resulted in minimal trauma to the pancreas. The essential features included intraductal injection of high concentration collagenase, then incubation at 39 ˚ C for a short period of time, and finally mechanical dispersion followed by filtration and centrifugation of the digestion products. This method improved isolation outcomes but purity remained low (between 10% and 40%). Ricordi in 1988 introduced the automated method for human islet isolation, which centered on 3 principles: (1) minimal traumatic action; (2) continuous digestion and collection to minimize enzymatic action on islets; and (3) minimal human intervention (Fig. 2). This approach resulted in improved islet yield and purity of around 78% along with significant improvement in islet function (18). The use of the COBE 2991 cell separator further improved islet preparation purity with no negative effect on function (19).

Enzyme Preparation Collagenase quickly digests pancreatic stroma but preserves the endocrine tissue. It has therefore been the preparation of choice for pancreatic digestion. The major issue with collagenase was inconsistent variations

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Figure 2

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Automated method for islet isolation.

between different lots and preparations. Liberase (Roche Applied Science, Indianapolis, Indiana, U.S.A.), a combination of highly purified collagenase isoforms I and II from Clostridium histoliticum and thermolysin from Bacillus thermoproteolyticus, was introduced to overcome this problem. In comparison to collagenase, Liberase-digested pancreata gave higher numbers of islets that were larger in size with less fragmentation, while islet function remained the same. Although variability between lots was less with Liberase, it remains a significant issue (20). A new enzyme blend of collagenase and neutral protease has been evaluated and has resulted in less lot-to-lot variability (21).

Culture Long-term culture leads to loss of islets. However, short-term culture is associated with loss of exocrine tissue and increase in purity of islet preparation with increase in metabolic efficacy. In animal models, single donor islets cultured for 48 hours reversed diabetes in diabetic rats, while freshly isolated islets or islets cultured for 24 hours did not (22). The same finding was observed in humans where 4 out of 6 patients became insulinfree from single donor islet preparations cultured for 24 to 48 hours prior to transplantation (23). Culturing islets prior to transplantation has many advantages, including provision of a window for achievement of an immunosuppressed state prior to transplantation and avoidance of islet loss due to

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autoimmunity, alloimmunity, or cytokine release associated with induction therapy. This also allows for islet transplantation to be performed as an elective procedure with all transplant team members present, in addition to the listing of patients from remote areas. Islets can be isolated in a central facility then transported to distant sites for transplantation, thus reducing cost and improving transplant outcomes. This method has been tried in Europe with good results, where islets freshly isolated in Geneva were then transported to sites in France and Switzerland for transplantation (6). Eleven patients underwent islet transplantation in Houston, Texas, utilizing islets isolated and established in culture at the University of Miami, Florida. All patients became C-peptide positive and seven of them (64%) are insulin independent (24).

CURRENT OUTCOMES Insulin Independence In 1990, 66 years following the first islet transplantation, the first case of insulin independence was reported (1,2). In the following decade, 237 islet transplants were reported, with only 11% having achieving insulin independence at 1 year (3). The Edmonton Protocol, introduced in 2000, implemented two major new strategies. Firstly, a steroid-free approach was used for immunosuppression, consisting of daclizumab induction followed by sirolimus and lowdose tacrolimus. Secondly, a mean islet mass of around 11,500 islet equivalent/kg, often requiring more than one transplant, was infused. These changes resulted in 100% insulin independence with a mean follow-up of 1 year (Fig. 3) (8). The success of the Edmonton Protocol has been replicated in other transplant centers. Combined data from Edmonton, Miami, and Minnesota reveal an initial 90% insulin-independence rate, although at newly developed sites the insulin-independence rate is averaging only 23% (25). Ten transplantation centers across North America and Europe participated in a multi-center trial of islet transplantation using the Edmonton Protocol. The 1-year insulin independence was 44%, but there was a great variation between different centers, with superior outcomes in more experienced centers. The majority of patients achieved insulin independence after 2 infusions. A minority became insulin-free after single infusion, and rarely were more than 2 infusions required (8,26,27). The number of islets needed to render patients insulin-free continues to remain unknown. In the original series of seven patients within Edmonton Protocol, 11,000 islet equivalent/kg in two islet infusions were required to achieve insulin independence (8). In a follow-up report, around 9000 IE/kg was apparently required to achieve insulin independence (28). Recently,

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Daclizumab(2 mg/kg -2 hours prior, for 5 days) Sirolimus (12-15 ng/ml -3 months) (7-10 ng/ml after 3 months) Tacrolimus(4-6 ng/ml)

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Figure 3

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insulin independence from a single donor was achieved with around 7000 IE/kg (29). In a 5-year follow-up data review at the University of Alberta, it would appear that there is no predictable relationship between the number of islets and insulin independence, as other factors including islet quality, viability, engraftment, and function are equally important (Fig. 4) (26). In spite of the high percentage of insulin-independent patients at 1 year following transplant, this quickly starts to decline thereafter, with 40–50%

1.2

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Figure 5 Survival analysis for insulin independence over time post-islet transplantation in the University of Alberta program.

remaining insulin-free at 3 years and only 10% at 5 years (Fig. 5) (26). However, being insulin-dependent again after a period of insulin independence does not necessarily equate with complete loss of graft function and/or poor glycemic control, as 80% of patients were C-peptide positive at 5 years (Fig. 6) (26). Potential reasons for graft loss remain unknown but alloimmunity could be a major factor. A significant number of patients will become panel reactive antibody (PRA) positive following transplantation, and around half of those with high PRA may lose graft function (26). Autoimmunity is another potential factor whereby patients who develop autoantibodies may exhibit a decrease in graft function (27). Metabolic Control Good glycemic control should be the main goal of islet transplantation. In the Diabetes Control and Complications Trial, the risk of microvascular complications was reduced significantly through a 2% reduction in HbA1c; the risk of hypoglycemia, however, increased threefold despite the mean HbA1c value remaining abnormal with a mean of 7% (10). Although insulin independence post-islet transplantation ranges from 80–100% at 1 year, falling to 10% at 5 years, the percentage of patients who

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Figure 6 Survival analysis for C-peptide secretion over time post-islet transplantation in the University of Alberta program.

are C-peptide positive with functioning grafts may be as high as 100% at 1 year and 80% at 5 years (8,26,27,30). For patients who became insulin dependent but remained C-peptide positive, their insulin requirement decreased by half compared to pre-transplantation. In C-peptide negative patients, on the other hand, insulin dose remained the same or increased (26). Patients with functioning grafts, insulin dependent or independent, achieved significant reduction in HbA1c to near-normal levels (6.2% in insulin independent and 6.8% in insulin dependent) in comparison to both pre-transplant levels and patients who lost graft function (8.1%) (Fig. 7) (26). Hypoglycemia and glycemic lability are the main indications for islet transplantation. Both were significantly reduced in all C-peptide positive patients, insulin-free or not, and hypoglycemia was rarely encountered (Fig. 8A and B) (27,31). Fear of hypoglycemia is significantly reduced in islet transplantation recipients, resulting in an improved quality of life (32). The majority of insulin independent patients will demonstrate impaired glucose tolerance, which could be attributed to the mass of islets being transplanted, which is always less than that of a normal pancreas (8). C-peptide secretion and glucose tolerance in response to a mixed meal test was superior in insulin-independent individuals in comparison to C-peptide positive, insulindependent patients. A decline in C-peptide increment following mixed meal testing seemed to precede resumption of insulin therapy (26).

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Figure 7 Mean ± SE of HbA1c over time post-transplantation in the University of Alberta program in those whose transplant failed (—•—), those whose graft remained functional but had to resume insulin (—¡—), and those who remained insulin independent (—♦—).

Prevention of Long-Term Diabetes Complications Sirolimus is known to cause hypercholesterolemia (33), which is one of the most common complications of islet transplantation. Sixty to ninety percent of patients required introduction or increased doses of lipid lowering medications (Fig. 9) (26,27,30). A significant number of patients (25–60%) were on anti-hypertensive medications pre-transplantation. About half of those not receiving treatment needed anti-hypertensive therapy post-transplantation and the need to use multiple anti-hypertensive medications increased by sevenfold. In spite of the increased need for anti-hypertensive therapy, the mean systolic and diastolic blood pressure remained unchanged pre- and posttransplantation (26). The increased incidence of hypertension and hypercholesterolemia does not seem to increase the risk of cardiovascular complications. In isletafter-kidney transplant recipients, C-peptide positive patients had a lower rate of cardiovascular mortality and decreased signs of endothelial injury in comparison to kidney-alone transplant recipients (34). An improvement in cardiac function determined by increased ejection fraction and peak-filling rate in end-diastolic volume was also noted (35). Kidney function is a major concern in patients with diabetes. Microalbuminuria can develop post-transplantation in at least 10% of patients. Approximately half of patients with microalbuminuria pretransplant may progress to frank proteinuria (26). With discontinuation of

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Figure 8 The HYPO score (A) and Lability Index (B) pre- and post-transplant in C-peptide positive patients.

sirolimus and conversion to MMF therapy, both proteinuria and microalbuminuria may resolve (36). It has been shown previously that whole organ pancreas transplantation can reverse lesions of diabetic nephropathy in the long term (37). Islet transplantation had a similar effect in animal models but only on early lesions (38). There is a trend to increased creatinine and decreased creatinine clearance post-islet transplantation. Those individuals with abnormal creatinine pre-transplantation observed a significant increase in creatinine posttransplantation (26,30). Interestingly, it would appear that islet-after-kidney transplantation is associated with improved kidney graft survival rate in comparison to patients with Type 1 diabetes who underwent kidney transplantation alone (39). Rapid improvement in overall glycemic control has been associated with deterioration of diabetic retinopathy (40,41). In islet transplantation a

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Neurotoxicity • Mouth ulcers Hypertension Hypercholesterolemia Immunosuppression Diabetes Nephrotoxicity

• Peripheral edema • Ovarian cysts • Proteinuria • Hypertension • Hypercholesterolemia • Anti-angiogenic

4/93 ( 4%) stopped for side effects 12/93 (13%) stopped after loss of function

Figure 9

• Islet ‘unfriendly’ • Tolerance ‘unfriendly’

Edmonton Protocol drug side effects.

similar finding has been noted, with exacerbation of diabetic retinopathy in the initial few months followed by stabilization (26). Post-Transplant Complications Procedure-Related Bleeding from the liver puncture site had been a significant problem in islet transplantation with occurrences in up to 12% of procedures. Usually it was self-limited and managed by either observation or limited blood transfusion. Laparotomy to control bleeding was rarely required (26,27,30). Several methods were attempted to seal the tract in order to prevent bleeding, including laser, gelfoam, and coils, but the most effective approach was the combination use of coils and tissue fibrin glue (Tiseel; Baxter Healthcare, Ltd., Mississauga, Ontario, Canada) (42). Currently, a microfibrillar collagen (Avitene; Davol, Woburn, Massachusetts, U.S.A.) is being used without coils. It is easy to prepare and can be mixed with contrast to allow visualization and prevention of portal vein embolization. Omission of coils avoids artifacts in future radiological studies to assess patient and graft function. No cases of main portal vein thrombosis have been encountered in Edmonton patients, but thrombi in secondary branches, although rare, were seen and managed with short-term anticoagulation with no major sequelae (26).

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There is a risk of puncturing the gall bladder during the procedure. However, no surgical intervention is generally required, and only observation is warranted (26). Transient increases in liver transaminases will occur in approximately 50% of recipients and usually resolves in 3–4 weeks (26,27,30,43). Immunosuppression-Related Several side effects related to immunosuppression have been reported. Mouth ulceration occurs in approximately 90% of patients. It is usually selflimiting or responsive to topical steroid therapy and/or reduction of sirolimus dose. In one case surgical debridement was required (26). Small bowel ulceration has been reported in two cases and was attributed to elevated sirolimus levels (44). Neutropenia can affect about half of the patients but is only severe in 13%. Anemia is very common and may occasionally necessitate erythropoietin therapy (26). Nausea, vomiting and diarrhea are observed in more than half the patients and can be related to diabetic autonomic neuropathy and/or immunosuppression. Prokinetic agents can be an effective treatment (26). Sirolimus can cause significant edema and in some of the affected patients, sirolimus needed to be replaced, usually with MMF (26). Other complications include hypercholesterolemia, hypertension, anxiety, tremor, headache, acne, fatigue, ovarian cysts, menorrhagia, fever, chest pain, pericardial effusion, pyelonephritis, worsening genital herpes, and appendicular abscess (26,27). Other Complications Fatty infiltration of the liver was seen in about 20–30% of patients who underwent MRI post-islet transplantation (45). Although the significance of this remains unknown, is thought to be related to insulin secretion from intrahepatic islets, as it resolved completely in one patient who lost graft function (45). About one in five patients who are PRA negative (<15%) pretransplantation will become PRA positive (≥15%) post-transplantation, and, of this group, approximately one half will lose graft function (26). Cytomegalovirus (CMV) infection is a major cause of morbidity and mortality in solid organ transplant recipients. The prevalence of CMV seropositivity in the general population is between 30% and 97%. CMV infection usually occurs in the first 3 months post-transplantation. In the absence of prophylaxis, the risk of CMV disease in transplant patients is between 8% and 39%, highest in heart-lung recipients and lowest in kidney recipients. CMV disease is most common in donor-positive, recipient-negative, solid organ transplant recipients. The risk of CMV disease increases 3–4-fold with the use of lymphocyte-depleting therapy.

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CMV infection increases the probability of other infectious complications in addition to acute and chronic rejection (46). Very few case of seroconversion have been reported and there have been no cases of CMV reinfection, reactivation, or invasive disease in islet transplant recipients, which reflects the small amount of highly purified and lymphocyte-free tissue that is transplanted (8,26,27,47). Our current protocol is to give CMV prophylaxis for 3 months to CMV mismatch patients (D þ / R
) and to any patient receiving lymphocyte depletion therapy. Post-transplant lymphoproliferative disorders (PTLDs) are a potentially life-threatening complication after solid organ and bone marrow transplantation. They range from infectious mononucleosis and lymphoid hyperplasia to highly invasive malignant lymphoma. There is a strong association with Epstein Barr virus (EBV) that leads to uncontrolled B-cell proliferation and tumor formation. PTLD affects about 10% of transplant recipients with 50% mortality. It is most common in lung transplant recipients due to the potential for a large number of EBV infected donor lymphocytes in bronchus-associated lymphoid tissues (48). To date, no cases of PTLD have been reported post-islet transplantation, which could be related to the very small lymphocyte load in islet preparations. In Edmonton, two deaths, not directly related to islet transplantation, have occurred. The first patient was a 43-year-old man who underwent two islet transplants but remained insulin dependent despite positive C-peptide. He had been taking high-dose methadone for eye pain but treatment had been stopped for 4 years. The patient had an upper respiratory tract infection and took diphenhydramine and the same dose of methadone he had taken in the past. This resulted in respiratory depression and death 20 months post-transplantation. The second patient was a 40-year-old female who underwent two transplant procedures and became C-peptide positive with excellent glycemic control. She had significant problems with gastroparesis-associated nausea and vomiting. She was admitted to hospital with dehydration and a central line was inserted for fluid resuscitation. She developed a central line infection and died of sepsis 4 years posttransplantation.

NEW STRATEGIES TO IMPROVE OUTCOMES Single Donor Islet Transplantation Donor shortage is a major obstacle in organ transplantation; it is an even greater problem in islet transplantation considering that most patients require more than one transplant to achieve insulin independence. Only a small percentage will achieve insulin independence with a single donor (26,27). Hering et al. (23) reported insulin independence in 4 out of 6 patients after single donor islet transplantation. All patients received induction

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therapy with anti-CD3 monoclonal antibody hOKT3 1 (Ala-Ala) and a mean islet dose of 10,302 IEQ/kg. Recently, the same group reported 100% insulin independence in 8 patients following single donor transplantation. Anti-thymocyte globulin and etanercept were given pre-transplantation along with a mean islet mass of 7300 IEQ/kg. Recipient mean body weight was 60 kg. Potent induction therapy might be the main factor underlying this success, but careful patient selection was clearly another important factor.

Living-Related Donation Living-related donation (LRD) has been adopted in many organ transplant programs to compensate for organ shortages. However, initial results in living donor segmental pancreas transplantation at the University of Minnesota revealed limitations. Besides the expected potential complications from distal pancreatectomy, donors have a higher chance of developing diabetes. 35% of living related donors for pancreas transplantation developed an abnormal oral glucose tolerance test and 4 out of 8 donors developed diabetes in the long-term (49,50). Therefore, careful selection of donors is vital. In Kyoto, Japan, Matsumoto et al. (51) reported the first successful living donor islet transplant. The recipient was a 27-year-old woman with hyper-labile insulin-requiring diabetes, secondary to chronic pancreatitis, who received just over 400,000 islets isolated from the distal pancreas of her 56-year-old mother. The patient achieved insulin independence and the donor had no complications (51). LRD has the advantages of avoiding the inflammatory changes seen in deceased donors, shortening ischemia time, and elective scheduling of the procedure.

Islet-After-Kidney Transplantation Islet-after-kidney transplantation can cause sensitization of the recipient and destabilization of the transplanted kidney; in addition, most kidney transplant immunosuppression protocols are steroid-based. On the other hand, kidney transplant patients are already immunosuppressed and the islet transplant procedure therefore caries a minimal risk. The presence of a transplanted kidney also provides an easier means to diagnose rejection. Islet transplantation has been performed in selected groups of kidney transplant patients after conversion to steroid-free immunosuppression with results comparable to islet transplantation alone patients (52,53). In addition, islet-after-kidney patients experienced prolonged transplant kidney graft survival and lower cardiovascular mortality (34,39). A multi-center

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trial of islet-after-kidney transplantation is being conducted at the present time. Lehmann et al. (54) performed simultaneous islet and kidney transplantation in nine patients using a steroid-free protocol (daclizumab, sirolimus, and low dose tacrolimus) with a median follow up of 2.3 years. There was only one renal graft failure due to primary non-function. Of six patients who received adequate islet mass (>8500 IE/kg), five remained insulin-free.

FUTURE DIRECTIONS Improving Engraftment Islets comprise only 1% of the pancreas but receive up to 15% of its blood supply. Engraftment is the process whereby islets become revascularized in the recipient hepatic sinusoids, a process usually taking approximately 2 weeks. Transplanted islets express tissue factor, which activates the clotting cascade thereby causing an instant blood mediated inflammatory reaction (IBMIR). IBMIR can activate alloimmune responses or cause the formation of a fibrin capsule around the islets, preventing proper engraftment. The use of agents to prevent IBMIR, such as nicotinamide or low molecular weight dextran, improves islet engraftment (55,56).

Tolerance Induction Initially, most islet transplant immunosuppression protocols included steroids. The Edmonton Protocol presented a steroid-free, “islet-friendly” regimen that, in combination with other factors, led to superior outcomes. Prolonged immunosuppression can lead to serious and potentially lifethreatening complications, including infection and malignancy. If tolerance can be induced, a lower dose of immunosuppressive medications would be required with fewer side effects. A wide range of experimental studies have been focused on finding “islet-friendly,” tolerance-inducing medications. Anti-CD154 induced longterm insulin independence in rhesus monkeys that received intraportal islet infusions (57). LEA29Y (Belatacept; Bristol-Myers Squibb, New Brunswick, New Jersey, U.S.A.), which blocks CD28 signaling, prolonged islet allograft survival in animal models in comparison to conventional immunosuppression (58). Anti-CD40 monoclonal antibody showed a synergistic effect when used with Belatacept (59). Renal transplant patients were maintained successfully on half the dose of cyclosporine without additional maintenance therapy following induction with anti-CD52 (alemtuzumab) (60). This approach is currently being explored in islet transplantation.

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Islet and Sertoli cell co-transplantation produced a dose-dependent prolongation of islet graft survival in a rodent model, indicating a potential immunoprotective role of Sertoli cells (61). Simultaneous islet and donor bone marrow-derived cell transplant is a promising approach but would be associated with risk of graft-versus-host disease (62). Alternative Sources of Islets Ductal structures in adult pancreata appear to contain stem cells that can differentiate into insulin-producing cells. When transplanted into diabetic mice, insulin-dependent diabetes has been successfully reversed (63). Insulin-secreting cells have also been derived from mouse embryonic stem cells. They produced insulin in response to glucose stimulation in vitro, underwent rapid vascularization, and maintained a clustered, islet-like organization when injected into diabetic mice (64). Porcine insulin was the standard therapy for diabetes for many years before the introduction of recombinant insulin. Xenotransplantation of porcine islets is being explored and will potentially provide an unlimited supply of islets. The main risks of xenotransplantation, however, are hyperacute rejection mediated by alpha-Gal and other xenoantigens and zoonoses (65). REFERENCES 1. 2. 3. 4. 5.

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Shapiro AM, Lakey JR, Paty BW, Senior PA, Bigam DL, Ryan EA. Strategic opportunities in clinical islet transplantation. Transplantation 2005; 79(10): 1304–1307. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of longterm complications in insulin-dependent diabetes mellitus. N Engl J Med 1993; 329(14):977–986. Walsh TJ, Eggleston JC, Cameron JL. Portal hypertension, hepatic infarction, and liver failure complicating pancreatic islet autotransplantation. Surgery 1982; 91(4):485–487. O’Gorman D, Kin T, Murdoch T, et al. The standardization of pancreatic donors for islet isolations. Transplantation 2005; 80(6):801–806. Lakey JR, Kneteman NM, Rajotte RV, Wu DC, Bigam D, Shapiro AM. Effect of core pancreas temperature during cadaveric procurement on human islet isolation and functional viability. Transplantation 2002; 73(7):1106–1110. Kuroda Y, Kawamura T, Suzuki Y, Fujiwara H, Yamamoto K, Saitoh Y. A new, simple method for cold storage of the pancreas using perfluorochemical. Transplantation 1988; 46(3):457–460. Matsumoto S, Qualley SA, Goel S, et al. Effect of the two-layer (University of Wisconsin solution-perfluorochemical plus O2) method of pancreas preservation on human islet isolation, as assessed by the Edmonton Isolation Protocol. Transplantation 2002; 74(10):1414–1419. Ricordi C, Fraker C, Szust J, et al. Improved human islet isolation outcome from marginal donors following addition of oxygenated perfluorocarbon to the cold-storage solution. Transplantation 2003; 75(9):1524–1527. Gray DW, McShane P, Grant A, Morris PJ. A method for isolation of islets of Langerhans from the human pancreas. Diabetes 1984; 33(11):1055–1061. Ricordi C, Lacy PE, Finke EH, Olack BJ, Scharp DW. Automated method for isolation of human pancreatic islets. Diabetes 1988; 37(4):413–420. Lake SP, Bassett PD, Larkins A, et al. Large-scale purification of human islets utilizing discontinuous albumin gradient on IBM 2991 cell separator. Diabetes 1989; 38(Suppl 1):143–145. Linetsky E, Bottino R, Lehmann R, Alejandro R, Inverardi L, Ricordi C. Improved human islet isolation using a new enzyme blend, liberase. Diabetes 1997; 46(7):1120–1123. Bucher P, Mathe Z, Morel P, et al. Assessment of a novel two-component enzyme preparation for human islet isolation and transplantation. Transplantation 2005; 79(1):91–97. Jahr H, Hussmann B, Eckhardt T, Bretzel RG. Successful single donor islet allotransplantation in the streptozotocin diabetes rat model. Cell Transplant 2002; 11(6):513–518. Hering BJ, Kandaswamy R, Harmon JV, et al. Transplantation of cultured islets from two-layer preserved pancreases in type 1 diabetes with anti-CD3 antibody. Am J Transplant 2004; 4(3):390–401. Barshes NR, Lee T, Goodpasture S, et al. Achievement of insulin independence via pancreatic islet transplantation using a remote isolation center: a first-year review. Transplant Proc 2004; 36(4):1127–1129.

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25. Shapiro AMJ, Ricordi C, Hering B. Edmonton’s islet success has indeed been replicated elsewhere. Lancet 2003; 362:1242. 26. Ryan EA, Paty BW, Senior PA, et al. Five-year follow-up after clinical islet transplantation. Diabetes 2005; 54(7):2060–2069. 27. Froud T, Ricordi C, Baidal DA, et al. Islet transplantation in type 1 diabetes mellitus using cultured islets and steroid-free immunosuppression: Miami experience. Am J Transplant 2005; 5(8):2037–2046. 28. Ryan EA, Lakey JR, Rajotte RV, et al. Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol. Diabetes 2001; 50(4):710–719. 29. Hering BJ, Kandaswamy R, Ansite JD, et al. Single-donor, marginal-dose islet transplantation in patients with type 1 diabetes. Jama 2005; 293(7):830–835. 30. Ryan EA, Lakey JR, Paty BW, et al. Successful islet transplantation: continued insulin reserve provides long-term glycemic control. Diabetes 2002; 51(7):2148–2157. 31. Ryan EA, Shandro T, Green K, et al. Assessment of the severity of hypoglycemia and glycemic lability in type 1 diabetic subjects undergoing islet transplantation. Diabetes 2004; 53(4):955–962. 32. Johnson JA, Kotovych M, Ryan EA, Shapiro AM. Reduced fear of hypoglycemia in successful islet transplantation. Diabetes Care 2004; 27(2): 624–625. 33. Kahan BD. Efficacy of sirolimus compared with azathioprine for reduction of acute renal allograft rejection: a randomised multicentre study. The Rapamune US Study Group. Lancet 2000; 356(9225):194–202. 34. Fiorina P, Folli F, Maffi P, et al. Islet transplantation improves vascular diabetic complications in patients with diabetes who underwent kidney transplantation: a comparison between kidney-pancreas and kidney-alone transplantation. Transplantation 2003; 75(8):1296–1301. 35. Fiorina P, Gremizzi C, Maffi P, et al. Islet transplantation is associated with an improvement of cardiovascular function in type 1 diabetic kidney transplant patients. Diabetes Care 2005; 28(6):1358–1365. 36. Senior PA, Paty BW, Cockfield SM, Ryan EA, Shapiro AM. Proteinuria developing after clinical islet transplantation resolves with sirolimus withdrawal and increased tacrolimus dosing. Am J Transplant 2005; 5(9): 2318–2323. 37. Fioretto P, Steffes MW, Sutherland DE, Goetz FC, Mauer M. Reversal of lesions of diabetic nephropathy after pancreas transplantation. N Engl J Med 1998; 339(2):69–75. 38. Pugliese G, Pricci F, Pesce C, et al. Early, but not advanced, glomerulopathy is reversed by pancreatic islet transplants in experimental diabetic rats: correlation with glomerular extracellular matrix mRNA levels. Diabetes 1997; 46(7):1198–1206. 39. Fiorina P, Folli F, Zerbini G, et al. Islet transplantation is associated with improvement of renal function among uremic patients with type I diabetes mellitus and kidney transplants. J Am Soc Nephrol 2003; 14(8):2150–2158. 40. Early worsening of diabetic retinopathy in the Diabetes Control and Complications Trial. Arch Ophthalmol 1998; 116(7):874–886.

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57. Kenyon NS, Chatzipetrou M, Masetti M, et al. Long-term survival and function of intrahepatic islet allografts in rhesus monkeys treated with humanized anti-CD154. Proc Natl Acad Sci U S A 1999; 96(14):8132–8137. 58. Adams AB, Shirasugi N, Durham MM, et al. Calcineurin inhibitor-free CD28 blockade-based protocol protects allogeneic islets in nonhuman primates. Diabetes 2002; 51(2):265–270. 59. Adams AB, Shirasugi N, Jones TR, et al. Development of a chimeric antiCD40 monoclonal antibody that synergizes with LEA29Y to prolong islet allograft survival. J Immunol 2005; 174(1):542–550. 60. Calne R, Moffatt SD, Friend PJ, et al. Prope tolerance with induction using Campath 1H and low-dose cyclosporin monotherapy in 31 cadaveric renal allograft recipients. Nippon Geka Gakkai Zasshi 2000; 101(3):301–306. 61. Korbutt GS, Suarez-Pinzon WL, Power RF, Rajotte RV, Rabinovitch A. Testicular Sertoli cells exert both protective and destructive effects on syngeneic islet grafts in non-obese diabetic mice. Diabetologia 2000; 43(4): 474–480. 62. Ricordi C, Karatzas T, Nery J, et al. Effect of timing of donor bone marrow infusions on liver allograft survival. Transplant Proc 1997; 29(1–2):1186. 63. Ramiya VK, Maraist M, Arfors KE, Schatz DA, Peck AB, Cornelius JG. Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat Med 2000; 6(3):278–282. 64. Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, McKay R. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 2001; 292(5520):1389–1394. 65. Cooper DK, Gollackner B, Knosalla C, Teranishi K. Xenotransplantation— how far have we come? Transpl Immunol 2002; 9(2–4):251–256.

14 Culture and Transportation of Human Islets Between Centers Hirohito Ichii, Antonello Pileggi, Aisha Khan, Chris Fraker, and Camillo Ricordi DeWitt Daughtry Family Department of Surgery, University of Miami Leonard M. Miller School of Medicine, and Clinical Islet Transplant Program, Cell Transplant Center, Diabetes Research Institute, Miami, Florida, U.S.A.

INTRODUCTION Significant progress has been achieved in clinical islet transplantation in the past two decades (1–7). Islet transplantation continues to gain promise as a potential cure for patients with Type 1 diabetes mellitus (8). Numerous technological improvements have been introduced in pancreas procurement (9) and preservation (10–12), islet isolation (13–15), purification (16–18), assessment (19,20), and culture (21), as well as in the management of islet transplant recipients (1–6). The need for physical detachment of islets from the pancreatic environment through the digestion process may alter the native extracellular matrix surrounding the islet cell clusters, therefore depriving them of physiological signals required for homeostasis and survival, a phenomenon referred to as anoikis (22). In addition, reduction of oxygen availability to the islets during culture may result in loss of islet b-cell mass and functional impairment. Optimal culture conditions for isolated human islets should provide sufficient oxygen and nutrients in order to allow for islet cells to recover from isolation-induced damage, maintaining 3-dimensional structure of the clusters and reducing islet mass loss. Numerous approaches have been utilized over the years for the culture of human islets prior to transplantation. Protocols for islet culture have not completely been standardized and practice may vary between centers, although it is 251

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commonly accepted that extensive period of times should be avoided as they are associated with loss of islet mass and function. Initial clinical trials used culturing islets prior to transplantation in order to allow for assessment of sterility and for pooling islet preparations from multiple donors if needed to obtain large islet masses for implantation (23–27). The Edmonton Protocol required that freshly-isolated islets were transplanted without keeping them in culture (3). More recently, most transplant centers are culturing isolated human islet preparations pretransplantation with results comparable to those with non-cultured islets (5,6,15,28–32). Advantages of the use of cultured islet preparations over freshly isolated islets are multifold and include (i) practicality, as it allows for arranging logistics of patient admission to the hospital and implementation of pre-conditioning therapy prior to transplantation, and (ii) additional time to assess the safety of the islet preparation by performing microbiological (i.e., mycoplasma, aerobic and anaerobic cultures) and pyrogenic (endotoxin) tests. Additionally, pre-culturing may be of assistance in modifying the islet preparations reducing immunogenicity (i.e., due to the reduction of the number of leukocytes or endothelial cells in the final product for transplantation, or reducing the expression of pro-inflammatory molecules) (21,24,33–38). The development of methods of culturing islet preparations for 48–72 hours prior to transplantation has also allowed shipping of isolated islets to remote centers where islet transplantation is performed (28,39–41). This approach has been instrumental in substantially reducing the costs for infrastructure and specific training in islet isolation technology at each center, while maximizing utilization of donor organs for transplantation. Transplantation of islets isolated at remote centers has been associated with very favorable clinical outcomes (9,18,21,39,40). Indeed, although several centers have reported high rates of success in islet transplantation trials worldwide (1–7,30,31), transferring expertise and technology required for islet isolation to newly created centers still represents an obstacle toward achieving reproducible success rates in clinical trials. Optimization of shipping conditions is, however, required to ensure that sufficient oxygen and nutrients are available in order to maintain islet cell mass and potency until transplantation. This chapter provides a review of the most recent methods utilized for the culture of human islet preparations and for their shipment to remote transplantation centers.

HUMAN ISLET CULTURE Islets are obtained by a mechanically enhanced enzymatic digestion (13,14) resulting in fragmentation of pancreatic tissue while preserving islet

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integrity, followed by purification on density gradients (16–18). Removal of islets from the native pancreatic environment may result in a traumatic removal of extracellular matrix proteins essential to maintain homeostasis of the cell subsets comprising the cluster and to prevent functional impairment and/or cell death (22,42–46). In addition, islets are highly vascularized structures that are continuously exposed to sufficient oxygen tension in the native pancreas (47–49); reproducing similar conditions in vitro is difficult, mainly due to the diffusion kinetics of oxygen in the culture medium and into the islet core. Furthermore, the generation of pro-inflammatory mediators by islet cells (i.e., cytokines and chemokines) (50–56) may contribute to the demise of islet cells in vitro and amplify inflammation upon islet infusion in the recipient’s liver (50,57). Optimal culture conditions for isolated human islet preparations should aim at providing sufficient oxygen and nutrients to allow for recovery from isolation-induced damage, while maintaining 3-dimensional structure of the clusters and preventing islet mass loss (58–60). Numerous media formulations and culture protocols have been utilized for human islets since the 1970s (61,62). Current practice for islet culture varies substantially between centers, suggesting that, despite the steady success reported in the clinical setting of islet transplantation in recent years, standardization has not been achieved, and considerable scope for improvement in the current methods utilized for the culture of human islets still exists. Development of efficient culture media formulations and of protocols specific for human islet preparations may improve preservation of b-cell mass and quality, thereby increasing the success rate of islet transplantation in the clinical setting.

Human Islet Culture at the University of Miami At the end of the purification phase, islets are resuspended in culture medium and transferred into the culture flasks. The Connaugh Medical Research Laboratories (CMRL) 1066 medium is commonly utilized for islet culture (63–67). The medium formulation used at our center is a CMRLbased medium with the inclusion of supplements (Table 1) (6,21). Serum supplementation of the medium is beneficial for cell survival in culture. The use of xenogeneic proteins (namely, fetal calf serum) is generally avoided in order to possibly reduce immunogenicity of the grafted tissue (33). Therefore, human serum albumin (HSA), although poorer in growth factors than fetal calf serum, is currently preferred. Culturing islet preparations with the optimum concentration of glucose is essential for successful clinical islet transplantation. Glucose concentration of our culture media is 5.38 mM based on the previous studies (61,68), indicating that human islets cultured at 11 or 28 mM glucose had

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Table 1

Composition of the Miami-Modified Medium-1 (MM1) for Human Islet Culturea

Chemical CaCl2 (anhydride) MgSO4 (anhydride) Na acetate (anhydride) NaH2PO4.H2O ZnSO4.H2O Dextrose KCl NaCl Na glucuronate H2O Selenious acid L-alanine L-arginine HCl L-aspartic acid L-cysteine HCl.H2O L-cystine 2HCl L-glutamic acid Glycine L-histidine HCl.H2O Hydroxy-L-proline L-isoleucine L-leucine L-lysine HCl L-methionine L-phenylalanine L-proline L-serine L-threonine L-tryptophan L-valine L-tyrosine.2Na.2H2O L-alanyl-L-glutamine Biotin Folic acid Riboflavin Cocarboxylase Coenzyme A Li3 Salt 2H2O Coenzymase FAD disodium salt TPN, Na UTP, Na3 2H2O Trolox Insulin–human recombinant

Concentration (g/L) 0.19512 0.09532 0.04878 0.13659 0.00468 0.97561 0.39024 6.79024 0.00410 0.00001 0.02439 0.06829 0.02927 0.25366 0.02537 0.07317 0.04878 0.01951 0.00976 0.01951 0.05854 0.06829 0.01463 0.02439 0.03902 0.02439 0.02927 0.00976 0.02439 0.05659 0.42380 0.00001 0.00001 0.00001 0.00098 0.00244 0.00683 0.00098 0.00098 0.00098 0.00244 0.00610 (Continued)

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Table 1

Composition of the Miami-Modified Medium-1 (MM1) for Human Islet Culturea (Continued )

Chemical

Concentration (g/L)

Ascorbic acid D-Ca-pantothenate Choline chloride i-Inositol Nicotinic acid Nicotinamide PABA Pyridoxine HCl Na pyruvate Thiamine.HCl Linoleic acid Human trnsferrin (iron poor) Thymidine 2D-adenosine 2D-cytidine HCl 2D-guanosine 5-methyl-2’-dexycytidine Cholesterol Tween 80 HEPES NaHCO3 NSA-human 25% ml/L

0.04878 0.00001 0.00049 0.00005 0.00002 1.19024 0.00005 0.00005 0.53659 0.00001 0.00001 0.00610 0.00976 0.00976 0.00976 0.00976 0.00010 0.00020 0.00488 5.80860 2.14634 24.3902

a

Ciprofloxacin (0.2 mg/ml) is freshly added.

40% and 60% lower insulin content, respectively, when compared to islets cultured at 5.6 mM glucose. Insulin induces growth and proliferation of many somatic cell lines interacting with other hormones (69). Moreover, insulin supplementation of serum-free medium improves islet survival during culture (70). The addition of transferrin to serum-free medium also enhances islet cell preservation through its iron binding properties (69,71). Supplementation of islet culture media with nicotinamide has been introduced, based on its known cytoprotective properties. Nicotinamide has been shown to ameliorate the effects of islet cell injury caused by noxious stimuli such as hydrogen peroxide (72) and cytokines (73,74). The poly adenosine diphosphate (ADP)-ribose polymerase (PARP) inhibition induced by nicotinamide prevents nicotinamide adenine dinucleotide (NAD) consumption and decrease in cellular adenosine triphosphate (ATP) content, thereby preventing necrosis by inducing a shift from necrosis to apoptosis (75). As a result, cell viability is preserved (72). The process of human islet isolation induces considerable stress in the islets, including

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induction of apoptosis and necrosis and the production of pro-inflammatory cytokines and chemokines (51–53). In particular, oxidative stress seems to play a major role in triggering the death of islets during isolation (52). The activation of nuclear factor-κB (NF-κB) and PARP, two of the major pathways responsible for cellular responses to stress, are shown to be upregulated significantly in pancreatic cells during the isolation procedure (52). Furthermore, it has been recently reported that islets express and synthesize tissue factor (TF), which triggers a detrimental thrombotic reaction after hepatic portal islet infusion (21,76,77). Other reports have shown that macrophage chemoattractant protein-1 (MCP-1) is also expressed and secreted by islets (67,78–80). MCP-1 has a potent chemotactic activity for monocytes, and MCP-1 levels inversely correlate with the outcome of clinical islet transplantation (79). Nicotinamide supplementation of culture medium has been shown to reduce both TF and MCP-1 production (38). Based on these premises, we currently use nicotinamide supplementation in the culture media of human islet preparations for clinical use (6,21). Vitamin E (Trolox) is a well-known antioxidant, free radical scavenger (81,82). Islet cells are exposed to reactive oxygen species (ROS) during islet processing procedures, such as preservation, isolation, and culture. Vitamin E might be beneficial for reducing and preventing islet cell damage mediated by the isolation procedure. The Food and Drug Administration (FDA) regulates islet transplantation under the category of Investigational New Drug (83–85). For this reason, all reagents utilized in the manufacturing of human islets for transplantation should be free of adventitious substances that may harm the recipient of the islet cell product transplanted (7). The medium is prepared using clinical grade reagents that are tested for contamination from microorganisms and endotoxin. After addition of supplements to the base medium, the solution is filtered through a 0.2 µm filter for sterilization. Prophylaxis with broad-spectrum chemotherapy (Ciprofloxacin 0.2 mg/ml) is commonly part of the culture media formulation with the aim of preventing growth of microorganisms during culture. Culture Density After purification, islet preparations are separated into 2–3 groups according to purity (e.g., 90%, 60%, and 30%) assessed by dithizone staining (86,87). The culture conditions represent a compromise between the need for nutrients, optimized by a larger volume of media, and the need for good oxygen diffusion optimized by smaller volume of media to maximize gas exchange at the air/liquid interface. In our facility, islet preparations are therefore cultured at a density of approximately 20,000 IEQ in 30 ml of culture media using a 175 cm2 non-tissue culture treated flask (Sarstedt, Inc., Newton, North Carolina, U.S.A.) when islet purity is >90%. Islet fractions

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are separately cultured until the day of transplantation or shipment. Nonendocrine cells can produce noxious cytokines and chemokines that may contribute to loss of islet mass and function during culture. To minimize this, islet fractions with high and low purity are separately cultured until the day of transplantation. Less pure islet preparations are generally cultured at a density calculated using the following formula for number of islets per flask:   Purity % IEQ per flask ¼ 20;000 IEQ
100 The seeding density of islet preparations during culture plays an important role in islet survival. After isolation, most islet cells will be hypoxic and significantly damaged. Oxygen diffusion in the media will be critical for islet survival during culture. The typical flask loading is equivalent to 115–133 IEQ/cm2 of surface area. Given a uniform islet size of 150 µm diameter (87), this translates to a 1.2–2.4% flask surface utilization. When the cell density exceeds the above cutoff, cells begin to experience anoxia and cell death in the core regions. When the cell number is increased, the anoxic tissue fraction increases precipitously to approximately 65% at a seeding density of approximately 500 IEQ/cm2. Culture Temperature Human islet preparations are cultured in humidified incubators (95% air, 5% CO2). There is no consensus regarding the standardization of incubating temperature for human islet preparation culture. At our facility, islets are cultured at 37 ˚ C for the first 24 hours and at 22 ˚ C for the remaining 24–48 hours until transplantation (6,18,21). Culture at 37 ˚ C for the first 24 hours may be helpful for the recovery of islet cells from the damage during isolation. Culturing islet preparations at 22 ˚ C until transplantation might favor reduction of exocrine cells, therefore improving the purity of the final preparations for transplantation. Preparation of Human Islets Cultures The human islet preparation is cultured in a humidified incubator (95% air, 5% CO2) in supplemented Miami-Modified Media (MM1, Table 1) containing 0.5% HSA. Preparation of the islet suspension for plating begins by adding enough MM1 to the transfer container (i.e., 250 ml conical bottle or 100 ml beaker) containing the islet preparations so that the number of islets to be cultured in each flask is re-suspended in 10 ml of medium. For instance, if 5 flasks are to be cultured, the islet preparation should be re-suspended in a total volume of 50 ml. Islet cells will be gently resuspended by using wide mouth pipettes, and 2 ml of the islet cell suspension are aspirated immediately from the containers and transfered into each

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culture flask. In order to distribute the islets evenly between flasks, a total of 10 ml of re-suspended islets is transferred into a given flask in five 2 ml aliquots. The transfer container is then rinsed with an additional 10 ml of culture medium that are then distributed in 2 ml aliquots at a time to each flask before adding further medium to a final volume of 30 ml per flask. The caps are then replaced on all the flasks and closed in the vented position. The caps are gas-permeable so gas exchange will occur. Each flask is labeled in order to identify islet batch number, date, number of islets plated, and the fraction number (denoting endocrine purity). All flasks are then transferred into the 37 ˚ C CO2 incubator where they are kept for the first 24 hours of culture. Flasks are inspected after the first 24 hours before replacing existing with fresh media. After inspection, the flasks are placed in a Class II Biological Safety Cabinet (BSC) and tilted at a 45 ˚ angle in order to allow islets to settle down for approximately 5 minutes. Twenty ml of culture media are removed aseptically and replaced with fresh MM1. After completion of the media change, flasks are transferred into the 22 ˚ C CO2 incubator for another 24–48 hrs prior to transplant. SHIPPING HUMAN ISLETS BETWEEN CENTERS Transfer of knowledge and expertise required for acquiring sufficient number and quality of islets for a Clinical Islet Transplant Program (CITP) has been the most difficult challenge for a newly established Islet Cell Processing Center (ICPC). The need for specific infrastructures, dedicated personnel, and acquisition of specialized expertise in islet cell isolation and purification has substantial economical impact and may require long-term commitment for a center starting a CITP (5,7). This is intrinsic to the technology required for human islet cell isolation and culture. In addition, islet cell products are regulated by the FDA as an Investigational New Drug under the somatic cell therapy category, which mandates implementation of current Good Manufacturing Practices (cGMP) with high demand for quality control and quality assurance, and use of standardized protocols for cell processing and characterization (83–85). Recent clinical multi-center trials have highlighted the critical role of established center experience in the success rate of the CITP (88,89). This observation, together with the burden of demanding regulations for the production of clinical cell products for transplantation have favored increasing interest in the implementation of regional ICPCs that isolate high quality human islet cells for transplantation in clinical trials at a remote CITP. This approach has proven successful to maximize the success rate of islet isolation while containing the costs of setting up a fully operational cGMP facility in recent clinical trials. This strategy has enabled successful clinical trials both in United States and Europe with excellent outcomes (28,39–41,90–93). The need to ship islets between distant centers requires optimization of shipping

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conditions that allow maximal preservation of islet mass, viability, potency, and sterility. This will be of utmost importance in favorable outcomes of clinical islet transplantation trials, but also in improve distribution of human islet preparations to researchers at distant institutions without altering islet cell quality and physiology for further understanding of human islet cell biology. Very few data are currently available regarding islet shipping protocols at different centers, suggesting that optimization and standardization are still not well defined. In the Group Rhin-Rhone-Alpes-Geneve pour la Transplantation d’Ilots de Langerhans (GRAGIL) experience, islets are transported by an ambulance to the remote transplant centers where they are transplanted within a few hours loaded in a syringe or infusion bag utilized for islet implantation (40,41). The University of California Islet Transplant Consortium reported shipping islets in the infusion bag to the remote centers within hours of isolation, after culture, or cryo-preserved prior to shipment (90,91). Although the Minneapolis-Seattle collaboration have reported shipment of islet preparations for clinical transplantation, the detailed protocol for shipment was not published (93). We have recently established partnerships between our center and distant CITPs where islets are shipped for transplant by charter jet (28,39). To comply with FDA regulations, islet cell products are assessed before (at the islet cell processing facility) and after shipment (at the remote CITP) to confirm that adequate quality (i.e., viability and sterility) and mass of islets are available for transplantation (7,39). In the recently published series of islet transplant alone performed using the consortium concept, islets were isolated at our center from pancreata procured at the remote center, the Methodist Hospital at the Baylor’s College of Medicine in Houston (9). Isolated islets were shipped to the remote partner CITP where product release and quality control were repeated before intrahepatic islet transplantation into patients with Type 1 diabetes (28,39,94). The clinical results obtained in our islet consortium experience are quite promising (6,28,39): insulin independence has been achieved consistently using sequential islet infusions with data that are comparable to those obtained without shipping islets at our Center (6,28,39) and those reported by other CITPs (30). Herein we summarize the protocol developed at our center for the shipment of clinical-grade human islet shipment that has been recently utilized in the ongoing clinical trials with remote sites (28,39,94). Shipping Bag The most critical issue in islet shipment is prevention of hypoxia in transit, which could impair islet cell functional integrity and mass. In order to address this issue, at our center islets are shipped using sterile Lifecell Tissue Culture bags (PL-732 polyolefin lifecell; Nexell Therapeutics, Inc., Irvine,

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Figure 1

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Gas-permeable shipping bag and stainless steel racks.

California, U.S.A.) (Fig. 1), which are gas-permeable and made from the same material used for platelet product storage and shipment (95). Oxygen diffusion from ambient air into the gas-permeable bags utilized in our ongoing clinical trial may play a role in maintaining quality of human islet cell products during shipment. Availability of sufficient oxygen diffusion to islet preparations during shipment at 25 ˚ C using polyolefin bags may achieve a peri-islet oxygen partial pressure that never reaches a level that would create diffusion limitations to the core regions of the islets, therefore helping preserve islet cell health over time. On the day of shipment, the islet preparation for transportation is transferred into the Lifecell Tissue Culture bag under aseptic conditions in a BSC. Layers with different purities are generally kept separately, and those with comparable purity are collected from the culture flasks and pooled into a 250 ml conical bottle. After two centrifugation steps (1,000 rpm for 1 minute) aimed at replacing all medium, samples are obtained prior to loading the islet preparation in the shipping bags for product release assessment and quality assurance (including cell counts, determination of overall purity, and final volume of tissue). In a BSC, a 60 ml syringe without plunger is connected to the corresponding luer-lock found at the end of the tissue culture bag tubing. The syringe is used as a funnel to load the tissue into the shipping bag, and it is kept vertical by a metal stand. Final islet density and medium volume in the bag are calculated according to the bag size utilized and the purity of the cell product (Table 2). Loading of the bags is completed by adding culture medium without bicarbonate. The bags are not filled to capacity, so that the height of the media is kept constant in all bag sizes (0.733 cm) in order to prevent impairment of oxygen diffusion to the tissue. The same medium used during culture (Table 1) is also utilized for

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Table 2

Human Islet Density into Different Size Gas-Permeable Shipping Bags

Bag volume (L) 0.3 1.5 3.0

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Total IEQ/bag

Maximum volume (ml)

Bag surface area (cm2)

Media height (cm)

47,880
purity/100 79,800
purity/100 200,000
purity/100

132 219 550

180 300 750

0.733 0.733 0.733

shipment, with the only exception being absence of bicarbonate buffer in the shipping formulation to prevent pH alterations in the absence of controlled CO2 during shipment. After loading the cell product and culture medium into the bags, the bag is heat-sealed. Shipping Container Currently utilized shipping containers consist of large coolers for transport (Fig. 2). The bottom of each large insulated cooler is lined with absorbent material, and stainless steel racks are assembled into it. Each cooler can carry a maximum of three shipping bags (one per shelf). One shipping bag is placed on each shelf, ensuring that it is lying flat and not hanging off the

Figure 2 Shipping containers consist of large coolers for transport. The bottom of the large insulated cooler is lined with absorbent material and stainless steel racks are assembled into it. Each cooler can carry a maximum of three shipping bags (one per shelf). One shipping bag is placed on each shelf, ensuring that it is lying flat and not hanging off the edge of the shelf. This will allow for maximal exposure of the bag surfaces to favor gas exchange.

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edge of the shelf. This favors optimal gas exchange. The container is sealed and shipped to the remote CITP by airplane (28,39). Temperature Control In order to stabilize temperature between 20–24 ˚ C inside the shipping container during transport, coolant packs (Blood/Platelet CoolantTM; model 1290 SEBRA, Tucson, Arizona, U.S.A.) are included. The temperature is logged using a portable Internal/External Temperature Logger computer system (StowAway XTI Logger; ONSET Computer Corp., Bourne, Massachusetts, U.S.A.) designed to log real-time temperatures from months to years at intervals of the user’s choosing (seconds to hours). This allows for proper recording of all temperatures to which the human islet cell product has been exposed during shipment. The electronic data are subsequently uploaded on a computer using dedicated software. Islet Collection and Assessment at the Remote Site Once the shipping container has been received at the remote site, under aseptic conditions and in a BSC, the contents of the bags are transferred to 250 ml conical bottles and pooled for transplantation. Standard product release and quality assurance testing are repeated (assessment of islet numbers, purity, viability, sterility, etc.) to ascertain that adequate islets for transplantation are available and that the shipping did not affect the quality of the cell product (96).

CONCLUSIONS The results of clinical islet transplantation have dramatically improved in recent years thanks to the introduction of more efficient techniques in pancreatic processing and more effective immunosuppressive strategies. Islet transplantation is becoming an increasingly promising option for the treatment of patients with Type 1 diabetes. Improved islet culture and shipping methods have greatly contributed to the steady evolution of this therapeutic approach. The results obtained in the ongoing clinical trials with islets isolated and cultured at our centralized, regional ICPC and transplanted at distant CITPs after shipment have confirmed the feasibility of the islet consortium, which we are currently extending to other centers. Our constant efforts to optimize culture and shipping conditions may be of assistance in further improving the success rate of islet transplantation in the future. This would facilitate increases in the number of transplants and benefit larger cohorts of patients with diabetes. We strongly encourage the implementation of such an approach between centers because of the

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substantial benefits in pancreas utilization and in attainment of consistently high quality islet preparations for transplantation. ACKNOWLEDGMENTS This work has been possible thanks to: the National Institutes of Health/ National Center for Research Resources (U42 RR016603, M01RR16587), the Juvenile Diabetes Research Foundation International (#4-2000-946 and 2003), the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases (5 R01 DK55347, 5 R01 DK056953), the State of Florida, the American Diabetes Association, and continuous support by the Diabetes Research Institute Foundation (www.diabetesresearch.org). REFERENCES 1.

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79. Bertuzzi F, Marzorati S, Maffi P, et al. Tissue factor and CCL2/monocyte chemoattractant protein-1 released by human islets affect islet engraftment in type 1 diabetic recipients. J Clin Endocrinol Metab 2004; 89(11):5724–8. 80. Chen MC, Proost P, Gysemans C, Mathieu C, Eizirik DL. Monocyte chemoattractant protein-1 is expressed in pancreatic islets from prediabetic NOD mice and in interleukin-1 b-exposed human and rat islet cells. Diabetologia 2001; 44(3):325–32. 81. Luca G, Nastruzzi C, Basta G, et al. Effects of anti-oxidizing vitamins on in vitro cultured porcine neonatal pancreatic islet cells. Diabetes Nutr Metab 2000; 13(6):301–7. 82. Burkart V, Gross-Eick A, Bellmann K, Radons J, Kolb H. Suppression of nitric oxide toxicity in islet cells by alpha-tocopherol. FEBS Lett 1995; 364(3): 259–63. 83. Weber DJ. FDA regulation of allogeneic islets as a biological product. Cell Biochem Biophys 2004; 40(3 Suppl):19–22. 84. Weber DJ, McFarland RD, Irony I. Selected Food and Drug Administration review issues for regulation of allogeneic islets of langerhans as somatic cell therapy. Transplantation 2002; 74(12):1816–20. 85. Wonnacott K. Update on regulatory issues in pancreatic islet transplantation. Am J Ther 2005; 12(6):600–4. 86. Latif ZA, Noel J, Alejandro R. A simple method of staining fresh and cultured islets. Transplantation 1988; 45(4):827–30. 87. Ricordi C. Quantitative and qualitative standards for islet isolation assessment in humans and large mammals. Pancreas 1991; 6(2):242–4. 88. Shapiro AM, Ricordi C. Unraveling the secrets of single donor success in islet transplantation. Am J Transplant 2004; 4(3):295–8. 89. Shapiro AM, Ricordi C, Hering B. Edmonton’s islet success has indeed been replicated elsewhere. Lancet 2003; 362(9391):1242. 90. Brunicardi FC, Atiya A, Stock P, et al. Clinical islet transplantation experience of the University of California Islet Transplant Consortium. Surgery 1995; 118(6):967–71; discussion 971–2. 91. Brunicardi FC. Clinical islet transplantation: a consortium model. Transplant Proc 1996; 28(4):2138–40. 92. Rabkin JM, Leone JP, Sutherland DE, et al. Transcontinental shipping of pancreatic islets for autotransplantation after total pancreatectomy. Pancreas 1997; 15(4):416–9. 93. Rabkin JM, Olyaei AJ, Orloff SL, et al. Distant processing of pancreas islets for autotransplantation following total pancreatectomy. Am J Surg 1999; 177 (5):423–7. 94. Goss JA, Soltes G, Goodpastor SE, et al. Pancreatic islet transplantation: the radiographic approach. Transplantation 2003; 76(1):199–203. 95. Lemoli RM, Tafuri A, Strife A, Andreeff M, Clarkson BD, Gulati SC. Proliferation of human hematopoietic progenitors in long–term bone marrow cultures in gas-permeable plastic bags is enhanced by colony-stimulating factors. Exp Hematol 1992; 20(5):569–75. 96. Ichii H, Sakuma Y, Pileggi A, Fraker C, et al. Shipment of human islet for transplantation. Am J Transplantation 2007; 7(4):1010–1020.

15 Development and Application of Contemporary Immunosuppression in Human Islet Transplantation Dixon B. Kaufman Department of Surgery, Division of Transplantation, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, U.S.A.

INTRODUCTION An effective and safe immunosuppression protocol for successful islet transplantation will prevent a variety of cytodestructive auto- and alloimmune host responses without inhibiting the secretion or peripheral action of insulin and, of course, without imparting detrimental effects on recipient well-being. Perfection in this realm does not exist. Nevertheless, important strides have been made over the past several years that have resulted in meaningful breakthroughs, positioning the field of islet transplantation into pivotal licensing studies to establish it as a truly therapeutic treatment option for treatment of diabetes in selected patients. This chapter reviews the development of the current state-of-the-art immunosuppressive approach to islet allotransplantation. Many similarities exist between the immunosuppressive properties needed for successful solid organ transplants and islet transplantation. However, there are several considerations pertinent to islet transplantation that differentiate it from solid organ transplants. First, the effectiveness of the anti-rejection properties in preventing a variety of cytodestructive host immune responses must be nearly perfect because the marginal mass of islets that ultimately engraft has virtually no excess capacity to tolerate b-cell injury without reaching the tipping point causing metabolic dysfunction. The islets also appear to have essentially null capacity to regenerate if 269

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injured. Because surrogate serum markers of very early islet graft rejection do not exist, there is currently no timely prompt to the clinician to guide decision-making regarding application of anti-rejection therapy for the precise purpose of interrupting the immune destructive processes. These challenges have not held back the field, and a brief glance back sheds some light on what has been accomplished and why, and provides some direction on where the field is heading. THE EVOLUTION OF MODERN IMMUNOSUPPRESSION FOR CLINICAL ISLET TRANSPLANTATION Prior to 2000 the success rate of islet transplantation was relatively low. Discerning the technical from the immunological causes of failure was difficult. At that time the technical methods for manufacturing a consistently potent islet product were becoming quite sophisticated but were still in development. The use of pre-transplant culture was not in practice, and there were no validated measures of islet potency that could be quantified prior to transplant that would correlate with clinical success or failure. Despite these shortcomings, a relatively high success rate of islet autotransplantation (1) was being accomplished and suggested that the technical aspects of islet isolation were not holding the field back. Rather, the failure of islet allotransplants appeared to be largely due to inadequacies of the immunosuppression method (2). The immunosuppression approach at that time was strongly influenced by the renal solid organ perspective. Islet transplants performed in the 1990s placed greater emphasis on application to Type 1 diabetic patients with nephropathy that were to receive the islets simultaneously with, or after, a previous kidney transplant. The immunosuppression armamentarium consisted of the same agents that were used to maintain function of the kidney grafts. Unfortunately, those agents were toxic to the b-cells and insufficient in potency to minimize destructive host immune responses. For example, chronic corticosteroid maintenance therapy was an important component of the immunosuppressive approach. Also, the potency of the combination of tacrolimus and sirolimus was unknown. In fact, concerns that the properties of those two agents would mutually exclude their combination held back the field. So, the many failures of islet transplants at that time were due to the routine practice of implanting an impotent islet mass and using toxic and marginally effective immunosuppressive therapy. A few key studies in large animal pre-clinical models helped shape the things that were to emerge as true breakthroughs in human pilot studies. The benefits of induction therapy were shown to have a promising effect on outcome in functional large animal models of islet allotransplantation (3,4). With respect to maintenance immunotherapy, the diabetogenic side effects of chronic corticosteroid therapy were shown to be particularly deleterious

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in the presence of a marginal islet mass. Even short courses of low-dose prednisone therapy immediately post-transplant were shown to significantly shorten the durability of islet function in dogs that underwent total pancreatectomy and pancreatic islet autotransplantation (3,5,6). Calcineurin inhibitor therapy has become the backbone of virtually all immunosuppressive protocols since 1983. No significant deleterious effects of cyclosporine monotherapy on islet graft metabolic function were found (7). However, the combination of steroids and calcineurin inhibitors was especially detrimental to islet function, as demonstrated in dogs with stable long-term islet autograft function (8). Of particular importance was the report in 1996 that sirolimus had a beneficial metabolic impact in canine islet autograft recipients (9). Sirolimus was associated with increased glucose clearance, increased total and stimulated insulin release in response to glucose, and increased fasting plasma insulin level, as well as reduced insulin clearance. Studies in the pre-clinical pig islet allotransplant model strongly suggested efficacy of sirolimus, combined with low-dose calcineurin inhibitors, in preventing islet allograft rejection (10). In summary, the large animal models indicated that induction therapy in combination with a sirolimus-based maintenance regimen was of benefit and that corticosteroids were deleterious. In the clinical arena, McAlister, et al. (11) reported on a successful new maintenance immunosuppressive approach in 32 kidney, pancreas, and liver recipients that were transplanted from April 1998 through May 1999 with low-dose tacrolimus (trough concentration 3–7 ng/mL) and sirolimus (trough concentration 6–12 ng/mL) combination immunosuppression. THE EDMONTON IMMUNOSUPPRESSION PROTOCOL Investigators from the University of Alberta developed a steroid-free version of this protocol for human islet transplantation that had remarkable results. The Edmonton Protocol was born. It consisted of induction therapy with the IL-2 receptor antagonist, daclizumab. High-dose sirolimus combined with relatively low-dose tacrolimus was used for maintenance therapy (12). No glucocorticoids were given at any time. Daclizumab induction therapy was given intravenously at a dose of 1 mg/kg every 14 days for a total of 5 doses over a 10-week period, thus allowing an extended period for a supplemental islet transplant procedure. If the second islet transplant procedure occurred more than 10 weeks after the first, the course of daclizumab was repeated. Sirolimus was dosed to achieve and maintain trough levels of 12 to 15 ng/mL for the first 3 months and of 7 to 10 ng/mL thereafter. Tacrolimus was administered at an initial dose of 1 mg twice daily, then adjusted to maintain a trough concentration at 12 hours of 3 to 6 ng/mL. Type 1 diabetic islet allograft recipients reliably achieved and maintained freedom from the need for exogenous insulin after

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transplantation of an adequate mass of islets prepared from 2–4 donor organs, suggesting that the Shapiro et al. protocol protected against alloimmune and autoimmune reactivity. For any type of immunosuppression, efficacy and toxicity must be balanced. A recent analysis of adverse events observed in the first 31 Edmonton islet transplant recipients treated with daclizumab, sirolimus, and reduced-dose tacrolimus (follow-up, up to 3 years) revealed dyslipidemia in 62% and mouth ulceration in 81% (13). A significant increase in serum creatinine was only seen in two recipients who had elevated creatinine levels pre-transplant. Opportunistic infections and malignancies were not noted in any of the recipients followed for up to 3 years. The consistently successful outcomes reported by the University of Alberta group have been duplicated at other institutions (14–17). Immunosuppression with daclizumab, sirolimus, and reduced-dose tacrolimus has evolved as the gold standard for Type 1 diabetic islet transplant recipients. A few modifications of the induction therapy involving the use of a T-cell depleting agent, anti-thymocyte globulin (rabbit), in combination with TNF-α blockade are showing promising results.

INDUCTION AND ANTI-CYTOKINE AGENTS Rabbit anti-thymocyte globulin (Thymoglobulin; Genzyme, Cambridge, Massachusetts, U.S.A.) was approved by the FDA in 1999 for the treatment for acute renal graft rejection. It is a polyclonal IgG antibody generated by rabbits immunized with human thymocytes. Rabbit anti-thymocyte globulin contains cytotoxic antibodies directed against antigens expressed on human T lymphocytes. The rationale for anti-thymocyte globulin induction immunosuppression includes prevention of autoimmune recurrence in transplanted islets via deletion of autoreactive memory cells, prophylaxis of islet allo-rejection, avoidance of the use of calcineurin inhibitors in the immediate post-transplant period, and attenuation of non-specific inflammatory responses to transplanted islets, thereby maximizing engraftment and functional survival. Rabbit anti-thymocyte globulin has shown a consistent safety profile, with most adverse events being manageable and reversible; the most common events are fever, chills, and leukopenia. While rare, the most severe events include allergic or anaphylactoid reactions and serum sickness. As with all immunosuppression, administration of rabbit anti-thymocyte globulin may be associated with an increased risk of infection and development of malignancy (especially of the skin and lymphoid system). Published results of the use of anti-thymocyte globulin in clinical and experimental islet transplantation are limited to relatively small cohorts. Hirshberg et al. (4) described the successful role of rabbit anti-thymocyte

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globulin and sirolimus in reducing rejection of islet allografts in primates. Hering et al. (14) described a beneficial role of rabbit anti-thymocyte globulin induction (6 mg/kg) in 8 consecutive patients. They received an average of 7271 ± 1035 IE/kg from a single donor pancreas, and all achieved insulin independence and were protected against recurrence of hypoglycemia (14). Etanercept is a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human 75 kilodalton (p75) TNF receptor linked to the Fc portion of human IgG1. Etanercept inhibits binding of both TNF-α and TNF-b to cell surface TNF receptors, rendering TNF biologically inactive (18,19). It is FDA-approved for use in severe rheumatoid arthritis, juvenile arthritis, ankylosing spondylitis, and psoriatic arthritis. The basic premise behind the treatment is that peri-transplant administration of etanercept will interfere with the biological activity of TNF-α released early post-transplant as part of the activation of the innate immune response. Blockade of TNF-α in the early post-transplant period is expected to lessen early islet loss. TNF-α is known to be cytotoxic to human islet b-cells (20). In murine models, selective inhibition of TNF-α in the peritransplant period has promoted reversal of diabetes after marginal-mass islet isografts (21). Experience with anti-TNF-α therapies in clinical islet transplantation has been limited. Nineteen islet transplant recipients have received etanercept in the peri-transplant period for the purpose of enhancing engraftment and functional survival of transplanted human islets at the University of Minnesota, University of California San Francisco (UCSF), and the University of Miami. Etanercept was administered as follows: 50 mg IV at 1 hr prior to transplant, 25 mg subcutaneous on days þ3, þ7, and þ10 post-transplant. In patients transplanted at the University of Minnesota, etanercept was combined with anti-thymocyte globulin, and in two patients at UCSF, etanercept was combined with hOKT3 (Ala-Ala) for induction immunotherapy. At the University of Miami, etanercept was combined with daclizumab (n ¼ 4) or alemtuzumab (n ¼ 3) induction immunotherapy. No adverse events related to etanercept were encountered in these 19 patients. The Minnesota/UCSF pilot study was promising with all four patients each receiving islets from a single donor pancreas. One patient became insulin-independent and the other three achieved markedly improved glycemic control on substantially reduced exogenous insulin doses. At the University of Miami, one of the three subjects that received alemtuzumab (Campath; Genzyme) and etanercept became insulin-independent after receiving islets from a single donor. The follow-up on the first 8 Minnesota patients is more complete and insulin independence was achieved in all with islets prepared from a single pancreas as described above.

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MODERN IMMUNOSUPPRESSION FOR CLINICAL ISLET TRANSPLANTATION Figure 1 shows an algorithm of a contemporary immunosuppressive regimen for islet transplantation. In this scenario, the isolated islets are determined to meet preliminary release criteria for transplantation 2 days prior to infusion. They are then placed in culture. During the culture period, anti-thymocyte globulin is administered to the recipient. The 48-hour leadtime allows for resolution of possible antibody-induced cytokine release prior to islet infusion. Tacrolimus-based maintenance immunotherapy (usually in combination with sirolimus) is also initiated 48 hours prior to infusion. Chronic corticosteroids are not administered. Guidelines for contemporary immunosuppression medications are outlined below. For the initial islet infusion, rabbit anti-thymocyte globulin is administered as induction therapy two days prior to islet infusion. A total of 6 mg/kg is given. The dose is 0.5 mg/kg on day
2, 1.0 mg/kg on day
1, and 1.5 mg/kg on days 0, þ1, and þ2. Premedications include: n n n n

Acetaminophen (paracetamol) 650 mg ½ hr before and midway through ATG infusion. Diphenhydramine 50 mg p.o. ½ hr before and midway through ATG infusion. Methylprednisolone 1 mg/kg i.v. one hour prior to and midway through the first ATG infusion only (i.e. on day –2). Pentoxifylline 400 p.o three times daily to be initiated one hour prior to the first ATG infusion and to be continued through day þ7.

Tacrolimus-based maintenance immunosuppression (tacrolimus / sirolimus) Antilymphocyte globulin 6 mg/kg over 4-6 days beginning day -2 Etanercept day 0, 3, 7, 10 Daclizumab day 0 and q 2 weeks x 4 (re-transplants)

-2

-1

0

+14

+28

+42

Days post transplant Islet isolation

Islet transplant

Islet culture (~ 48 hours)

Figure 1

Islet transplant immunosuppression protocol.

+56

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For anti-inflammatory therapy, etanercept (Enbrel) may be administered at a dose of 50 mg IV on day 0 (1 hr prior to transplant), and 25 mg subcutaneous on days þ3, þ7, and þ10 post-transplant. For maintenance therapy, sirolimus is administered at an initial dose 0.2 mg/kg p.o. on day
2 relative to islet transplant, followed by 0.1 mg/kg four times daily. The daily dose will be adjusted to achieve a whole blood 24-hr trough concentration of 10–15 ng/mL for the first 3 months and 8–12 ng/mL thereafter. Tacrolimus is administered at an initial dose 0.015 mg/kg p.o. twice daily on day þ1 to achieve a whole blood 12-hour trough concentration of 3–6 ng/mL. If subsequent islet infusions are required, then the immunosuppressive regimen will be identical to the regimen for the initial islet transplant with the following exceptions. Daclizumab is used instead of rabbit antithymocyte globulin for all subsequent islet transplants. The first dose is 2 mg/kg and is given within two hours prior to islet transplant. Doses 2–5 are at 1 mg/kg and are given every 2 weeks starting on day 14 after the subsequent transplant. If a third transplant is deemed necessary and performed between 30 and 70 days after the second transplant, no additional doses of daclizumab are required. If a third islet transplant is performed more than 70 days after the second transplant, all five doses of daclizumab should be repeated.

THE NORTH AMERICAN EXPERIENCE OF IMMUNOSUPPRESSION FOR ISLET TRANSPLANTATION The Collaborative Islet Transplant Registry (CITR) has collected data on 203 islet transplantation alone (ITA) recipients. The immunosuppressive regimens used are described in Tables 1 and Table 2. The most commonly Table 1 Islet Alone Recipients—Induction Immunosuppression Regimen at Time of First Islet Infusion Total

N 194

% 100.0

Daclizumab alone Anti-thymocyte globulin and daclizumab Alemtuzumab and daclizumab Basiliximab alone Anti-thymocyte globulin alone hOKT3-1(Ala-Ala) alone Alemtuzumab alone None

150 11 9 9 8 3 3 1

77.3 5.7 4.6 4.6 4.1 1.5 1.5 0.5

Source: From the Collaborative Islet Transplant Registry, Annual Report, 2006:86.

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Table 2

Islet Alone Recipients—Maintenance Immunosuppression Regimen at Time of First Islet Infusion

Total

N 203

% 100.0

Sirolimus þ tacrolimus Sirolimus þ tacrolimus þ MMF Sirolimus þ MMF Sirolimus þ tacrolimus þ MMF þ methylprednisolone Sirolimus þ MMF þ methylprednisolone Neoral cyclosporine þ everolimus Neoral cyclosporine þ methylprednisolone Neoral cyclosporine þ methylprednisolone þ everolimus Missing information on immunosuppression

176 3 1 7 1 1 1 4 9

86.7 1.5 0.5 2.5 0.5 0.5 0.5 2.0 4.4

Source: From the Collaborative Islet Transplant Registry, Annual Report, 2006:85.

used class of induction agent was an IL-2 receptor antagonist. Daclizumab was used in virtually all of those cases. T-cell depleting agents were not commonly used. However, rabbit anti-thymocyte globulin is being more commonly used following the report by Hering et al. (14) on the success with single donor islet infusions to achieve insulin independence. The most commonly used maintenance agents are tacrolimus and sirolimus in nearly 90% of cases. Corticosteroids are infrequently used. In approximately 25% of cases, anti-inflammatory agents that suppress cytokines (usually TNF) are administered in the early post-transplant course (Table 3).

IMMUNOSUPPRESSION FOR ISLET-AFTER-KIDNEY TRANSPLANTATION Individuals with Type 1 diabetes and a successful kidney allograft are particularly appropriate candidates for an islet after kidney (IAK) Table 3 Islet Alone Recipients—Anti-Inflammatory Agents Used at Time of First Islet Infusion Total

N 203

% 100.0

Infliximab Etanercept 15-deoxyspergualin Infliximab þ etanercept None Missing information on immunosuppression

17 24 5 2 146 9

8.4 11.8 2.5 1.0 71.9 4.4

Source: From Collaborative Islet Transplant Registry, Annual Report, 2006:85.

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transplant procedure because they are already committed to life-long chronic immunosuppression. Thus, the nominal risk of the islet transplant procedure itself is minimal compared to the risk of initiating new immunosuppression in non-uremic patients with diabetes in addition to the transplant procedure (22–24). Also relevant is the preliminary evidence from uncontrolled studies suggesting that transplanted islets in patients who have already received a kidney transplant may provide beneficial vascular and renal effects (25,26). Candidates for IAK transplants are distinguished from ITA recipients in several respects. First, recipients that have a successful kidney transplant and are prescribed an immunosuppressive regimen that differs from the corticosteroid-free protocol described by the Edmonton group may be required to convert to the Edmonton immunosuppression protocol prior to islet transplantation. There are at least two situations where approaches to kidney transplant immunosuppression have implications with respect to preparation for a subsequent IAK transplant. The first scenario includes renal transplant recipients with Type 1 diabetes that were initially prescribed a “conventional” immunosuppression regimen including corticosteroids at the time of kidney transplantation prior to any consideration of an IAK procedure. A typical immunosuppression protocol might entail cyclosporine, azathioprine (or mycophenolate mofetil), and prednisone. This situation in which immunosuppression was initially prescribed in consideration of the kidney transplant only is referred to as a “casual approach.” In this circumstance the patient may undergo immunotherapy conversion to that resembling the Edmonton Protocol of tacrolimus and sirolimus without corticosteroids, prior to the IAK transplant. The issue of converting a patient on a cyclosporine-based maintenance therapy to tacrolimus is relatively straightforward, as is the situation of conversion from an anti-metabolite (azathioprine or mycophenolate mofetil) to sirolimus. A typical immunotherapy conversion protocol starts with a conversion from cyclosporine to tacrolimus. Tacrolimus 12-hr trough concentration target levels are 4–6 ng/mL. After a period of 3 months, if renal function is stable, then the anti-metabolite (azathioprine or MMF) is converted to sirolimus. Sirolimus 24-hr trough concentration target levels are 6–8 ng/mL. After another 3-month period of observation the corticosteroids are slowly weaned off. Usually, patients require only 2.5–7.5 mg per day of prednisone. If the dose is 7.5 mg, then an immediate cut to 5 mg per day is made when the tacrolimus is started. The last 5 mg is tapered off over a period of 3 months after successful conversion to tacrolimus and sirolimus. It is prudent that baseline measures of kidney transplant function (e.g., 24 hour collection for creatinine clearance and protein) are obtained prior to immunosuppression conversion to fully determine any subtle changes in renal transplant function.

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It is important that patients are informed that some corticosteroid withdrawal protocols are associated with an increased risk of acute rejection and renal allograft loss. Virtually all of the reports of corticosteroid withdrawal failure, however, are reported in recipients maintained on cyclosporine and either azathioprine or MMF (27–29). That is not to say that successful steroid withdrawal protocols have not been described in kidney transplant recipients on cyclosporine (Neoral; Novartis, East Hanoever, New Jersey, U.S.A.) and MMF (30). Recent reports with the combination of cyclosporine (Neoral) and sirolimus appear promising as well (31). There is also published experience with steroid withdrawal in patients receiving tacrolimus-based immunosuppression, but it is relatively limited (32,33). In short, the benefits of the subsequent IAK transplant must outweigh the risks of withdrawing corticosteroids and precipitating an acute renal allograft rejection episode. The other scenario includes renal transplant recipients with Type 1 diabetes that are prescribed an immunosuppression regimen in which the corticosteroids are avoided or immediately rapidly eliminated following the kidney transplantation in anticipation of the subsequent islet transplant. This approach is referred to as “expectant immunosuppression.” Rapid corticosteroid elimination (avoidance) appears to be associated with less risk of graft loss from rejection compared to the slow steroid withdrawal protocols described above. We have previously reported that the combined use of tacrolimus and MMF with an IL-2 receptor antagonist allows corticosteroids to be withdrawn within 3 days of renal transplantation (34). The risk of a renal allograft rejection episode is approximately 13% (85–90% of occurring within three weeks of transplantation), and all episodes were reversible with appropriate anti-rejection therapy. No increase in risk of graft loss was observed versus the conventional chronic corticosteroid therapy approach. Others have described success with rapid corticosteroid elimination protocols in kidney transplantation. Matas et al. (35) have described a rapid steroid elimination protocol utilizing cyclosporine (Neoral), MMF, and anti-thymocyte globulin (rabbit). Cole et al. (36) reported on the efficacy of a steroid-free protocol using cyclosporine (Neoral), MMF, and daclizumab. In both studies, no clinically significant increase in risk of graft loss or rejection ensued compared to historical control groups. One of the criticisms of steroid avoidance protocols is that the long-term results are not known. The long-term outcome of steroid avoidance has been addressed by Birkeland (37). A five-year follow-up of 100 kidney transplant recipients indicated that renal allograft survival was not compromised by omitting chronic steroid exposure. Application of these immunosuppression options for IAK recipients would be advisable in programs with established successful steroid-sparing protocols already in place in the renal transplant program. A coordinated approach to renal transplant immunotherapy with subsequent medical

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conversion including possible corticosteroid withdrawal in preparation for islet transplantation requires integration of the kidney and islet transplant programs. A functionally integrated program enables consideration of whole pancreas transplantation for some individuals with an existing kidney allograft. Transplantation of the whole pancreas after a kidney transplant does not require corticosteroid withdrawal. Maintenance agents are often converted to a tacrolimus/MMF regimen, although this is not an absolute requirement. The second distinguishing feature is that an IAK candidate already has a functioning renal transplant, and maintenance of optimal kidney transplant function becomes paramount. This difference requires consideration of treatment protocols for islet transplantation to take a backseat to those needed to maintain optimal kidney transplant function. Conflicts involving approaches to immunosuppression could arise when application of tacrolimus and sirolimus immunosuppression suited for islet transplantation unexpectedly compromises renal allograft function due to nephrotoxicity. For example, an individual who has enjoyed good and stable kidney transplant function for years while receiving cyclosporine, azathioprine, and prednisone for immunosuppression is converted to tacrolimus and sirolimus at levels appropriate for the subsequent islet transplantation begins to demonstrate deteriorating renal function. In such a case, it may not be possible to achieve the target levels of tacrolimus and sirolimus that have been established in the Edmonton Protocol. Moving ahead with the subsequent islet transplantation in the face of “sub-therapeutic” immunosuppression may result in inferior outcomes and risk of worsening renal function. Third, IAK transplant recipients would be pre-immunosuppressed prior to islet transplantation. This may result in more efficient islet engraftment such that insulin independence could be achieved with a mass of islets significantly less than the threshold of approximately 8–10,000 islet equivalents/kilogram body weight, which usually requires two or more cadaveric donors. The Edmonton group and others have observed that insulin can often be stopped almost immediately following the second islet transplant. There are at least two potential contributory factors differing from the first transplant: the greater overall mass of islets and the preimmunosuppressed state. Since both may play a role, this raises the theoretical possibility that the pre-immunosuppressed state could result in more efficient early islet survival such that insulin independence may be achieved with a mass of islets significantly less than the threshold of approximately 8–10,000 islet equivalents/kilogram body weight, increasing likelihood of single donor success. The caveat is that the patient selection criteria for IAK transplants may not necessarily be identical to those applied in ITA candidate selection. Body mass index (generally <25) and insulin requirements (generally <0.7 U/kg body weight) are objective measures that can be standardized. Insulin sensitivity, which is not always formally

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assessed, may, however, be reduced in renal transplant recipients on established immunosuppression. Frequency and severity of hypoglycemic episodes may serve both as a surrogate marker of sufficient insulin sensitivity for islet transplant benefit and as the primary indication for islet transplantation in those with and without an existing renal graft. Because IAK transplant candidates have already accepted the risk of immunosuppression, looser criteria regarding severity of hypoglycemia necessary for proceeding with IAK may pertain. This may be a reasonable and justifiable approach, but impact of potential differences in baseline status and clinical indications on outcome must be carefully considered. CONCLUSIONS Since 2000, the Edmonton immunosuppression protocol for islet transplantation has become established. This has enabled reproducible success in programs worldwide. Induction and maintenance immunosuppression has continued to evolve, enabling further progress towards the ultimate goal of reproducible long-term insulin independence in ITA and IAK transplant recipients following transplantation of islets derived from a single deceased donor.

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Shapiro AM, Hao E, Lakey JR, Finegood D, Rajotte RV, Kneteman NM. Diabetogenic synergism in canine islet autografts from cyclosporine and steroids in combination. Transplant Proc 1998; 0:527. Kneteman NM, Lakey JR, Wagner T, Finegood D. The metabolic impact of rapamycin (sirolimus) in chronic canine islet graft recipients. Transplantation 1996; 61:1206–1210. Shibata S, Matsumoto S, Sageshima J, et al. Temporary treatment with sirolimus and low-trough cyclosporine prevents acute islet allograft rejection, and combination with starch-conjugated deferoxamine promotes islet engraftment in the preclinical pig model. Transplant Proc 2001; 33:509. McAlister VC, Gao Z, Peltekian K, Domingues J, Mahalati K, MacDonald AS. Sirolimus-tacrolimus combination immunosuppression. Lancet. 2000; 355:376–7. Shapiro AMJ, Lakey JRT, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000; 343:230–238. Ryan EA, Paty BW, Senior PA, Shapiro AM. Risks and side effects of islet transplantation. Curr Diab Rep 2004; 4:304–309. Hering B, Kandaswamy R, Ansite J, et al. Single-donor, marginal-dose islet transplantation in patients with type 1 diabetes. JAMA 2005; 293:830–835. Alejandro R, Ferreira JV, Caulfield A, et al. Insulin independence in 7 patients following transplantation of cultured human islets. Transplantation 2002; 2 (Suppl. 3):227. Rother KI, Hirschberg B, Gaglia JL, et al. Islet transplantation in patients with type 1 diabetes. NIH experience in 6 patients. Transplantation 2002; 2 (Suppl. 3):228. Markmann JF, Deng S, Huang X, et al. Reversal of type 1 diabetes by transplanting isolated pancreatic islets from single donors. Transplantation 2002; 2(Suppl. 3):228. Eason J, Wee S, Kawai T, et al. Inhibition of the effects of TNF in renal allograft recipients using recombinant human dimeric tumor necrosis factor receptors. Transplantation 1995; 59:300–305. Wee S, Pascual M, Eason J, et al. Biological effects and fate of a soluble, dimeric, 80–kDa tumor necrosis factor receptor in renal transplant recipients who receive OKT3 therapy. Transplantation 1997; 61:570–577. Rabinovitch A, Sumoski W, Rajotte R, Warnock G. Cytotoxic effects of cytokines on human pancreatic islet cells in monolayer culture. J Clin Endocrinol Metab 1990; 71(1):152–6. Farney A, Xenos E, Sutherland D, et al. Inhibition of pancreatic islet b-cell function by tumor necrosis factor is blocked by a soluble tumor necrosis factor receptor. Transplant Proc 1993; 25:865–866. Davalli A, Maffi P, Socci C, et al. Insights from a successful case of intrahepatic islet transplantation into a type 1 diabetic patient. J Clin Endocrinol Metab 2000; 85:3847–3852. Bertuzzi F, Grohovaz F, Maffi P, et al. Successful transplantation of human islets in recipients bearing a kidney graft. Diabetologia 2002; 45:77–84.

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24. Kaufman D, Baker M, Chen X, Leventhal J, Stuart F. Sequential kidney/islet transplantation using prednisone-free immunosuppression. Am J Transplant 2002; 2:674–677. 25. Fiorina P, Folli F, Maffi P, et al. Islet transplantation improves vascular diabetic complications in patients with diabetes who underwent kidney transplantation: a comparison between kidney-pancreas and kidney-alone transplantation. Transplantation 2003; 75:1296–1301. 26. Fiorina P, Folli F, Bertuzzi F, et al. Long-term beneficial effect of islet transplantation on diabetic macro-/microangiopathy in type 1 diabetic kidneytransplanted patients. Diabetes Care 2003; 26:1129–1136. 27. Sinclair NR. Low-dose steroid therapy in cyclosporine-treated renal transplant recipients with well-functioning grafts. The Canadian Multicentre Transplant Study Group. Can Med Assoc J 1992; 147:645–57. 28. Schulak JA, Mayes JT, Moritz CE, Hricik DE. A prospective randomized trial of prednisone versus no prednisone maintenance therapy in cyclosporinetreated and azathioprine-treated renal transplant patients. Transplantation 1990; 49:327–32. 29. Ahsan N, Hricik D, Matas A, et al. Prednisone withdrawal in kidney transplant recipients on cyclosporine and mycophenolate mofetil—a prospective randomized study. Steroid Withdrawal Study Group. Transplantation 1999; 68:1865–74. 30. Grinyo JM, Gil-Vernet S, Seron D, et al. Steroid withdrawal in mycophenolate mofetil-treated renal allograft recipients. Transplantation 1997; 63:1688–90. 31. Mahalati K, Kahan BD. An open-labeled pilot study of steroid withdrawal from kidney transplant recipients on sirolimus-cyclosporine combination. Amer J Transplanation 2001; 1:S247. 32. Chakrabarti P, Wong HY, Toyofuku A, et al. Outcome after steroid withdrawal in adult renal transplant patients receiving tacrolimus-based immunosuppression. Transplant Proc 2001; 3:1235–6. 33. Shapiro R, Jordan ML, Scantlebury VP, et al. Outcome after steroid withdrawal in renal transplant patients receiving tacrolimus-based immunosuppression. Transplant Proc 1998; 30:1375–7. 34. Kaufman DB, Leventhal JR, Fryer JP, Abecassis MI, Stuart FP. Kidney transplantation without prednisone. Transplantation 2000; 69:S133. 35. Matas AJ, Rancharan T, Paraskevas S, Sutherland DER: Rapid discontinuation of steroids in living donor kidney transplantation: A pilot study. Amer J Transplantation 2001; 1:278–85. 36. Cole E, Landsberg D, Russell D, et al. A pilot study of steroid-free immunosuppression in the prevention of acute rejection in renal allograft recipients.Transplantation 2001; 72:845–50. 37. Birkeland SA. Steroid-free immunosuppression in renal transplantation: a long-term follow-up of 100 consecutive patients. Transplantation 2001; 71: 1089–91.

16 Pig Islet Xenotransplantation— Update and Context Daniel R. Salomon Department of Molecular and Experimental Medicine, The Scripps Research Institute, Center for Organ and Cell Transplantation, Scripps Health, La Jolla, California, U.S.A.

INTRODUCTION In a comprehensive new text on islet transplantation, the key challenge of a chapter on islet xenotransplantation is to examine how it is different than transplantation of allogeneic human islets. Thus, there is much in common in these two endeavors; for example, islet isolation technologies, current choice of implantation site, and the huge difficulty of protecting the survival and function of the islets in the early post-transplant period. But there are also things that are specific to xenotransplantation, for example, the potential of genetically engineering the donor animals, the additional barrier of the xenoimmune response, and the potential of an infectious disease risk due to cross-species transmission of animal pathogens to immunosuppressed human patients. Three recent reviews of islet xenotransplantation have been published that the reader should also find useful to reference (1–3). Finally, for reasons of focus, the primary assumption of this chapter is that the pig will be the donor source animal for islet xenotransplantation, at least for now.

ORGAN SHORTAGE AND FISCAL REALITY A key point to make at the start is that the driver for islet xenotransplantation is a combination of two factors: the human donor organ shortage (always noted) and cost (rarely mentioned). There is still more that can be 283

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done to optimize the current use of human pancreata from deceased donors. In the United States, the U.S. Health Resources and Services Administration (HRSA), in partnership with the Organ Procurement Organizations (OPOs) and local hospitals, deserves tremendous credit for their very successful advocacy efforts that have advanced the efficiency and number of organs donated nationwide. But even if completely optimized, including use of extended criteria donors and non–heart-beating donors, the total number of pancreata available yearly would be about 10,000. This is far short of what will be needed to treat over a million individuals with Type 1 insulin-dependent diabetes. The second issue is cost. Interestingly, this issue has had much less attention in the context of xenotransplantation. The established position of the Centers for Medicare and Medicaid Services (CMS) that controls the funding for clinical organ transplantation in the United States is that a pancreas procured for clinical transplantation must be priced at the full organ procurement cost of approximately $25,000–30,000. Thus, the fact that many patients require islet transplants from two or more pancreata to achieve a sufficient functional mass is at least as major a disincentive to further development of human islet transplantation in the clinic as pancreas availability. In this situation, the potential of developing alternative sources of islets for transplantation, such as the use of pigs as donors, is compelling.

PIG ISLET ISOLATION The technology to isolate pig islets is not substantially different from that currently used for human islet isolation already reviewed in detail in other chapters of this book. Technology includes the use of Ricordi chambers and infusion at the time of organ procurement of complex collagenase preparations followed by a measured physical disruption of the organ to yield the islets for further purification on density gradients by centrifugation. There are some differences that are important to recognize. First, it appears that the impact of collagenase batches on pig pancreata is sufficiently different from human pancreata to necessitate specific screening in pig isolations (unpublished observations). In fact, considering the anatomy and functional context of a pig compared to a human, it is not surprising that this would be the case. Second, pig islets tend to be significantly smaller than human islets and have less individual islet integrity even after short-term culture. This has been attributed to the less-developed capsule surrounding pig islets. A recent paper by Yonekawa et al. reviews some of these issues and proposes one strategy for effective and high quality pig islet isolations (4). Third, the spectrum of experience goes from fetal to neonatal to young pig to retired breeder sows. Indeed, the latter by size, age, and fat content have the highest consistent yields. Thus, it is also true that

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factors such as age, diet, fat content, and pig strain will contribute to both yields and functionality (2). In general, the operational notion is that fetal and neonatal islets or islet progenitors are more robust with respect to surviving early post transplant trauma and this may be linked to their potential to proliferate or differentiate in situ (3,5). While use of pig donors obviates the ethical and political issues raised by a similar approach with human fetal or neonatal tissues, the major limitation remains the small number of islets or progenitors that are presently yielded compared to the large islet mass requirements to cure an adult human of insulin-dependent diabetes. Thus, despite the theoretical advantages of fetal or neonatal cells, the technical limitation of pure numbers remains insurmountable at the present time. That will only change with the introduction of a successful strategy to proliferatively expand fetal or neonatal islets or progenitors postisolation (6). Moreover, in practical terms, there is no way to base a clinical strategy on retired breeders that would take years to accumulate in sufficient numbers. Thus, the only viable commercial targets for pig islet xenotransplantation are juvenile animals from 6 to 24 months of age that can be born and raised in specialized facilities very efficiently and relatively rapidly. The rest is still laboratory science.

RECENT EXPERIENCE WITH PIG ISLET XENOTRANSPLANTATION IN PRIMATES First, the premise that pig insulin works in human beings is well established by the clinical use of porcine insulin as exogenous therapy before the advent of recombinant human insulin preparations and its selective continued use (7). There are 4 amino acid differences between pig and human insulin in the 59-residue hormone (NCBI protein sequence database). Second, the success in transplanting pig islets to various mouse models of diabetes and islet transplantation is well established and includes successful immunosuppressive strategies (8–12), tolerance induction (13), and encapsulation methods (14,15) (manuscript in preparation). Thus, the real challenge at the present time is to demonstrate success in a non-human primate model of diabetes or humans with a strategy that is feasible for application to human patients. Recent progress in this effort has been significant. In the first of the more recent reports of successful islet xenotransplantation in non-human primates, Macaca mulatta donor islets were transplanted into two Macacca fascicularis and one Ceropithecus aethiops monkeys in a concordant xenograft model (16). Immunosuppression was a novel protocol comprised of two doses of an anti-CD3 antibody labeled with an immunotoxin to profoundly deplete host T cells in all tissue compartments and a 4-day course of cyclosporine and steroids. All three animals demonstrated significant islet function post-transplant and were

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insulin independent for several years without any additional immunosuppression, a set of conditions that reasonably defines tolerance. Even if we consider the special issues of doing concordant xenografts from non-human primate to human recipients [specifically prohibited by current Food and Drug Administration (FDA) guidelines for xenotransplantation], the implication of this work is that this immunosuppression approach should be tested for human islet allografts. Thus, it is not clear what obstacles precluded clinical translation of this extremely promising study. The second study involved xenotransplantation of pig islets into 12 human children with insulin-dependent Type 1 diabetes (17). The strategy did not involve immunosuppression but rather the use of pig Sertoli cells isolated from the testes, long thought to produce immune inhibitory substances as part of their natural role in creating a site of immune privilege in the testes where the male germ line cells (sperm) are produced (18,19). In addition, four separate stainless steel tubes with teflon inserts were transplanted in the subcutaneous tissue of the abdomen to create revascularized, collagen-rich transplantation sites. Several months later, the teflon inserts were removed and the pig islet/pig Sertoli cell mixtures placed inside these discrete spaces. The most recent report reviews the fouryear results for the 12 transplanted children revealing no complications, “significant” reductions in insulin requirements in half the patients as compared to pre-transplant levels, and measurements of pig insulin with glucose stimulation in three of the patients. In four patients, one of the implanted devices was removed. All explants demonstrated some evidence of insulin-positive cells. This study initially generated significant controversy for two reasons. First, the process directly challenged the approaches to xenotransplantation that were being developed at the time by the FDA, Health Canada, Great Britain, New Zealand, and the European Union, particularly by not using designated pathogen-free pigs or having in place accepted screening methods for potential infectious pathogens. Secondly, there were no data given for glucose-stimulated or arginine-stimulated pig insulin or C-peptide in any patients. Confusion over how pre-transplant insulin requirements and quality of diabetic management were defined and recorded was also a concern, given that they became the primary metrics for the study. However, with the follow-up results (17) it is now shown that pig insulin could be detected after 4 years in some patients with glucose stimulation, and positive histology for insulin at the transplant sites is also encouraging. Nonetheless, it does not appear that any additional transplants have been undertaken. Interestingly, another experienced group recently tried a similar approach using pig Sertoli cells and islets transplanted into seven nondiabetic non-human primates and also used no immunosuppression (20). Though porcine C-peptide was detected at “very low levels” in all animals, there was no evidence of glucose-stimulated secretion, and histology of the

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transplant sites failed to demonstrate the presence of insulin-positive islets. While non-human primates are obviously a different model than humans with autoimmune Type 1 diabetes, these results are disappointing and it remains unclear what final conclusions can be made regarding the use of pig islet/Sertoli xenografts. More recently, two separate reports demonstrate that pig islet xenotransplantation into the portal circulation of the liver, essentially as currently performed for human alloislet transplantation, can be successful with the right combination of immunosuppressive drugs. The first report documented more than 100 days of functional pig islet survival in selected animals after transplants in cynomolgus macaque monkeys with STZinduced diabetes (21). The successful immunosuppressive regime comprised anti-CD25 and anti-CD154 monoclonal antibodies, FTY720 or tacrolimus, everolimus (a rapamycin analog), and leflunomide (n=5). While these results constitute a major step forward, they are mitigated somewhat by the death of 5 of the 12 animals transplanted due to complications of immunosuppression, suggesting that this specific combination of immunosuppressive drugs is very toxic, at least in this animal model. The second study involved neonatal pig islets transplanted into the portal hepatic circulation of rhesus macaque monkeys (22). Immunosuppression involved a novel combination comprising induction with antiCD25 and anti-CD154 followed by long term administration of sirolimus (rapamycin) and belatacept, a second-generation high-affinity derivative of CTLA4-Ig. Insulin independence was achieved in all 7 immunosuppressed animals by 60 days post-transplantation, suggesting proliferation and/or differentiation following engraftment of neonatal islets. However, in the first immunosuppressed cohort, all the animals developed simian CMV and SV40 virus and faired poorly. In the next cohort of 4 animals the investigators modified the protocol to include antiviral prophylaxis and improved nutritional support with much better results. One animal rejected the islet graft on day 76, which was probably caused by an inability to achieve a therapeutic level of sirolimus. The remaining three demonstrated stable insulin independence for >140, >155, and >260 days, respectively, although the first developed simian CMV and SV40 viremia without disease. Thus, while there remain concerns that the aggressive level of immunosuppression used might limit this strategy in human patients, there is now evidence from both these studies that pig islet xenografts in these nonhuman primate diabetic models can indeed provide a “cure” of the disease. Finally, taking a very different approach, the last recent study transplanted neonatal pig islets after microencapsulation in alginatepolyornithine-alginate into the intraperitoneal spaces of eight diabetic cynomolgus macaque monkeys (23). All animals received a second transplant 3 months after the first. There was no immunosuppression given and two animals died of causes not related to the transplants. In the final analysis the

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exogenous insulin requirement of the transplanted animals compared with the control group was decreased by a mean of 43% with equally good blood glucose control. While the potential of biomaterial encapsulation of islets to prevent immune rejection and avoid a need for long term immunosuppression has been discussed for many years with evanescent success reported, this latest iteration of the technology must be considered promising. The potential of using encapsulation to reduce or eliminate the requirement for long term immunosuppression for islet xenografts is an area that merits continued work, particularly when taken in the context of the concerns raised regarding the intensity and toxicity of the two successful immunosuppressive strategies already reviewed.

POTENTIAL RISK OF PORCINE ENDOGENOUS RETROVIRUS One focus of work in my own group has been on the potential risk of crossspecies transmission of porcine endogenous retrovirus (PERV) from pig donor tissues to immunosuppressed human patients [reviewed in (24)]. While a complete review of this area is not intended here, it is important to briefly update the reader. First, a collaboration led by Dr. Clive Patience and including our group cloned the human PERV receptor and identified two new genes that appear to have arisen by gene duplication (25). Interestingly, orthologs of the human PERV receptor, now called HuPAR, are found all the way down the evolutionary tree to C. elegans, xenopus, and drosophila, consistent with the conclusion that this receptor family are critical cellular molecules. By inference from several other known retrovirus receptors, the HuPAR may be cellular nutrient or electrolyte transporters. This is further supported by the fact that we believe by structural analysis that they are multi-membrane–spanning proteins. Second, we reported that non-human primate cell lines and a number of primary cells could not be infected with PERV (26). Indeed, non-human primate cells have two different defects blocking PERV infection, one at the level of viral entry and the other at the level of viral assembly. In this situation, the potential of productive PERV infection in a non-human primate is extremely unlikely. In contrast, many human cells and cell lines studied have no such defects to productive PERV infection (24). The obvious conclusion is that all the evidence accumulating in different non-human primate models that have been cited as demonstrating no evidence of PERV infection are on one hand true but on the other hand not predictive of the situation that should be encountered in human patients. Thus, we are disappointed that all the latest papers cited above each add comments about finding no PERV infection in the non-human primate models without acknowledging at least the potential that these results are not relevant in the context of risk assessment.

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In parallel, it was recently reported that PERV could be pseudotyped by endogenous murine retrovirus (27). The model involved pig and human hematopoietic cell and tissue transplantation to create stable, long-lived pig/ human/mouse chimeras. In this case, it turned out that endogenous mouse retroviruses could package the PERV genomes and effectively carry them into the transplanted human cells. In this case, it might appear that PERV is directly infecting the human cells (and indeed this might be the case), but it is equally possible that the PERV infection of the human cells was due to the presence of the murine retrovirus, a situation obviously not present in any human patients. We followed by reporting a similar result using a simpler pig tissue transplantation model that more efficiently demonstrated the pseudotyping phenomenon (28). Thus, the previously published studies suggesting PERV infection of human cells as a risk issue for xenotransplantation based on using immunodeficient mice transplanted with human cells and pig tissues are all open to question due to this property of murine retrovirus. It is possible that the infection of the human cells was still due to PERV, but that conclusion cannot be proven and the value of this chimeric human/mouse model for these studies must be re-evaluated critically. However, it is very important to emphasize that none of these results have any impact on our original publication raising the issue of potential PERV risk to humans by demonstrating that immunodeficient mice transplanted with pig islets are infected in multiple tissue compartments by PERV (29). Pseudotyping by mouse endogenous retrovirus cannot happen to the mouse cells in the transplanted animal. Finally, we recently developed a mouse transgenic for one of the two human PERV receptors, HuPAR2, and did a series of experiments where we injected infectious PERV-containing supernatants and then evaluated the potential of infection at multiple times and in many tissue compartments (30). In this context, it is important to note that our original studies with mouse cell lines demonstrated that expression of the human PERV receptor resulted in productive PERV infection (25). Thus, we demonstrated that there are no viral entry or assembly defects in the mouse, in contrast to our findings in non-human primate cells (26). In the more recent studies, the human PERV receptor transgenic mice were productively infected with PERV in many tissue compartments including brain, kidney, liver, and bone marrow. Interestingly, we have not documented any clinical disease or tumors in these animals, though animals injected in the neonatal stage demonstrated significant growth retardation. Ongoing studies are now evaluating the immune response to PERV in these animals, including neutralizing antibodies and PERV-specific CTL as well as the impact of immunosuppression. Thus, at present, it would appear that animals, including humans, that express a functional PERV receptor will be productively infected with PERV if exposed to a sufficiently infectious dose of virus. That does not necessarily

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mean that a PERV infection will cause any disease and that the human immune system will not clear PERV infection through natural immune mechanisms. We also have no evidence to suggest that there is any risk of spreading PERV from an infected animal to a healthy littermate in a way that would support concerns of a general public health risk from PERV. It is also still possible that certain strains of pigs can be selected and bred that have a reduced level of PERV produced of the type infectious for human cells. Unfortunately, all known pigs have multiple copies of PERV in their genomes and the nature of retroviruses is to readily recombine, mutate, and adapt to enhance their infectious range and capacity. Nonetheless, we continue to believe that cautious, closely monitored clinical studies in xenotransplantation should be supported and that the possible risk of PERV simply be incorporated into the informed consent procedure. Obviously, there is also a compelling need to support ongoing research in this area. FINAL THOUGHTS The emphasis in this chapter has been on areas where the experience with pig islet xenotransplantation is different from that of human islet allograft transplantation. Nonetheless, it is important to end by reminding the reader that the major challenges of pig islet xenotransplantation are the same: effective isolation and functional islet preparations, strategies to reduce immediate post-transplant cell death, safe strategies to manage the immune response, and alternative strategies to enhance islet cell engraftment and identify the ideal site for transplantation. Moreover, the potential that stem cells (adult or embryonic) or gene-modified cells could become islet cell alternatives is of equal importance in shaping the future of islet xenotransplantation. Thus, all the elements discussed in the previous chapters on human islets are largely relevant here. Finally, it must be noted that the potential of genetically engineering the pig donors to incorporate strategies for enhanced islet isolation, survival, engraftment, resistance to rejection, proliferation, etc. create opportunities for building on the next advances in human islet transplantation and the molecular mechanisms of islet injury to a new generation of strategies for successful islet xenotransplantation. In this way, too, the futures of human islet allograft transplantation and pig islet xenotransplantation are interlinked. REFERENCES 1. 2.

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19. Dufour JM, Hamilton M, Rajotte RV, et al. Neonatal porcine Sertoli cells inhibit human natural antibody-mediated lysis. Biol Reprod 2005; 72(5): 1224–31. 20. Isaac JR, Skinner S, Elliot R, et al. Transplantation of neonatal porcine islets and sertoli cells into nonimmunosuppressed non-human primates. Transplant Proc 2005; 37(1):487–8. 21. Hering BJ, Wijkstrom M, Graham ML, et al. Prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed non-human primates. Nat Med 2006; 12(3):301–3. 22. Cardona K, Korbutt G.S., Milas Z, et al. Long-term survival of neonatal porcine islets in non-human primates by targeting costimulation pathways. Nat Med 2006; 12(3):304–6. 23. Elliott RB, Escobar L, Tan PL BB, et al. Intraperitoneal alginate-encapsulated neonatal porcine islets in a placebo-controlled study with 16 diabetic cynomolgus primates. Transplant Proc 2005; 37(8):3505–8. 24. Van der Laan L and Salomon D. Cross-species transmission of porcine endogenous retroviruses in xenotransplantation: a PERVerted reality? In: Sher L and Grinyo J, Eds. Current Opinion in Organ Transplantation. SpringerVerlag: Berlin, 2001:51–58. 25. Ericsson TA, Takeuchi Y, Templin C, et al. Identification of receptors for pig endogenous retrovirus. Proc Natl Acad Sci USA 2003; 100(11):6759–64. 26. Ritzhaupt A, Laan LJ, Salomon DR, et al. Porcine endogenous retrovirus infects but does not replicate in non-human primate primary cells and cell lines. Journal of Virology 2002; 76(22):11312–11320. 27. Yang YG, Wood JC, Lan P, et al. Mouse retrovirus mediates porcine endogenous retrovirus transmission into human cells in long-term humanporcine chimeric mice. J Clin Invest 2004; 114(5):695–700. 28. Martina Y, Kurian S, Cherqui S, et al. Pseudotyping of porcine endogenous retrovirus by xenotropic murine leukemia virus in a pig islet xenotransplantation model. Am J Transplant 2005; 5(8):1837–47. 29. Van der Laan LJ, Lockey JC, Griffeth BC, et al. Infection by porcine endogenous retrovirus after islet xenotransplantation in SCID mice. Nature 2000; 407(6800):90–4. 30. Martina Y, Marcucci KT, Cherqui S, et al. Mice transgenic for a human porcine endogenous retrovirus receptor are susceptible to productive viral infection. Journal of Virology 2006; 80(7):3135–46.

17 Approaches to b-Cell Regeneration and Neogenesis Susan C. Campbell Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle, U.K.

Wendy Macfarlane School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, U.K.

INTRODUCTION Loss of b-cell mass is the critical convergence point in the development of both Type 1 and Type 2 diabetes. Consequently, restoration of b-cell mass is the fundamental underlying premise of many current therapeutic approaches to the treatment of both forms of the disease. Improvement in the success of islet transplantation procedures has provided critical proof of the principal that the restoration of b-cell mass can reverse diabetes and restore normal insulin secretion. Hence, many research laboratories worldwide are engaged in the development of new sources of insulin-producing cells, utilizing a broad range of molecular approaches. It is clear that in a healthy individual, a fine balance is constantly maintained between b-cell apoptosis and b-cell neogenesis. In patients with diabetes, this critical balance is lost, with the resultant decrease in b-cell mass contributing to the eventual loss of normoglycemic control. This chapter reviews our current understanding of the events regulating b-cell regeneration and neogenesis, with a view to potential therapeutic intervention for the improvement of b-cell mass and function in patients with diabetes.

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REGULATION OF PANCREATIC b-CELL MASS Healthy b-cell mass is maintained and regulated both during development and in adults in response to a number of nutritional, hormonal, and environmental changes. The adaptive nature of b-cell mass allows the body to respond to changes in the cellular environment, such as prolonged high glucose concentrations (1) or peripheral insulin resistance (2), as well as fundamental changes in whole-body physiology, such as pregnancy (3), obesity (4), and pancreatic tissue damage (5). The initial events determining b-cell mass occur during the earliest development stages, when pancreatic development begins in the endodermal region of the primitive foregut. b-cell development is regulated by the hierarchical expression of a specific complement of transcription factor proteins, reviewed in (6). Beginning in cells expressing the homeodomain transcription factor PDX1 (pancreatic/duodenal homeobox 1) and repressing sonic hedgehog, both endocrine and exocrine cells of the adult pancreas arise from endodermal cells expressing PDX1 (7) and Ptf1a (8). Proliferation of progenitor cells is stimulated by the fibroblast growth factor family of proteins (9) and greatly influenced by the production of specific inductive stimuli from the surrounding developing mesenchyme (10). Transient expression of the paired domain transcription factor Pax4 in ngn3expressing cells drives the formation of b-cells, with the final b-cell transcription factor complement being driven by PDX1, Pax6, Nkx2.2, and Nkx6.1 (6). This is the point at which b-cell expansion begins. In rat models, the most rapid expansion of b-cells occurs in late gestation, with a doubling of b-cell mass occurring every day from day 16 onwards (11). In humans, a similar expansion of b-cell mass is observed in week 20 of gestation (12). The source of b-cell expansion has been the subject of much study, but it would appear that both in rats and in humans, the percentage of b-cells that are replicating is extremely low (<10%) (13). Observation of the co-expression of transcription factors, such as PDX1 in cells with pancreatic ductal markers, such as cytokeratin 19 (CK19), has led to the hypothesis that the increases in b-cell mass occur through the expansion of the ductal cells within the pancreas (13,14). Whatever the origin of the proliferating cells, it seems clear that the massive expansion of b-cell mass at this critical phase of fetal development is the result of the proliferation and differentiation of non-endocrine progenitor cells already present within the developing pancreas. A key question remains whether these progenitor cells persist in the adult pancreas and whether they are involved in normal b-cell turnover and the expansion of islet mass in response to external and environmental cues. It is clear that the cells of the adult pancreas retain the ability to adapt to changes in external environment and to expand functional b-cell mass in response to these cues (Fig. 1). A number of pharmacological and

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Figure 1 Regulation of pancreatic b-cell mass. b-cell mass is maintained and regulated in the adult in response to a number of pharmacological and nutritional stimuli, such as high glucose concentrations, insulin, EGF, gastrin, and GLP-1. It is also regulated in response to changes in whole-body physiology, such as pregnancy, obesity, and pancreatic tissue damage. A number of key animal models have been used to demonstrate this: partial pancreatectomy, destruction of the b-cells with streptozocin, cellophane wrapping of the pancreas, and by briefly occluding the pancreatic duct by a method known as “squeezing.” Potential therapeutic agents are also under investigation in a bid to enhance b-cell mass and function, including GLP-1, exendin-4, and thiazolidinediones such as rosiglitazone. Abbreviations: EGF, epidermal growth factor; GLP-1, glucagon-like peptide 1.

nutritional stimuli, such as glucose, insulin, gastrin, epidermal growth factor (EGF), and glucagon-like peptide 1 (GLP-1) have been shown to stimulate changes in functional b-cell mass. On a physiological level, during pregnancy the mass of the pancreatic b-cells can increase up to 2.5-fold (15). The role of differentiation of pancreatic progenitor cells in this process remains unclear; however, b-cell enlargement has been observed in the human pancreas during pregnancy (16), and increased b-cell number and increased b-cell mass have both been observed in rat models (15). In humans, failure to induce increases in b-cell mass during pregnancy is linked to the development of gestational diabetes, which affects 3–5% of pregnant women (17). However, pregnancy is not the only stimulus known to increase b-cell mass. In adults, increases in body mass (such as in the development of obesity) can also provoke adaptive changes in the b-cell mass of the pancreas. Obesity is defined as an increase in body fat content relative to total body mass, usually indicated by a body mass index (BMI) greater than 30 (18). Obesity also predisposes to the development of insulin resistance, a common feature in Type 2 diabetes. In response to both of these stimuli, adaptive changes can occur in the adult pancreas to compensate through

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increased b-cell mass (19). In animal models of obesity, up to a fourfold increase in b-cell mass is observed compared with lean littermates (4). Similar observations have been observed in obese human subjects. These changes are thought to occur through b-cell hyperplasia. However, in the healthy adult pancreas the rates of b-cell replication are reportedly extremely low. Given the extremely low replication rate of adult b-cells, there is great debate as to the origins of neogenic b-cells in the adult pancreas. Do adult stem cells exist in the pancreas? Or do neogenic b-cells arise from the replication of existing b-cells? Or does transdifferentiation occur from the ductal or acinar cells of the pancreas, generating neogenic b-cells from adult pancreatic precursor cells? Some recent studies have implicated b-cell replication as the key to the emergence of neogenic b-cells (20). However, this is not consistent with the known limitations of b-cell replication rates. In addition, such observations appear to contradict the increasingly large body of literature suggesting that neogenic b-cells arise predominantly through the transdifferentiation of ductal cells within the pancreas. It is worth noting, however, that several recent reports describe the process of b-cell neogenesis as a combination of firstly the de-differentiation of the adult pancreatic cells towards a ductal phenotype, with subsequent redifferentiation to a b-cell phenotype (21). Clearly the plasticity of adult pancreatic cells plays a fundamental role in the neogenesis of b-cells in the adult pancreas. Over recent years, study of the regulation of adult b-cell mass and b-cell neogenesis has been greatly enhanced by the development of a number of important animal model systems that have shed new light on the fundamental process of creating new b-cells.

ANIMAL MODELS OF NEOGENESIS In addition to pregnancy and obesity, a third set of physiological stimuli have been well characterized in terms of provoking an increase in functional b-cell mass. These are the stimuli generated upon physical damaging of the pancreas, and a number of key animal models have been generated that shed new light on these events. Partial pancreatectomy, that is, the surgical removal of a proportion of the pancreas, is known to provoke a compensatory increase in b-cell mass (5). Although the response is modest, and does not result in the total replacement of the removed tissue, nonetheless this procedure suggests that cells within the adult pancreas retain a capacity to replenish lost insulin-producing cells. During pancreatectomy, the bulk of evidence suggests that the growth response of the pancreas is proportional to the percentage of the organ removed. Hence, 70% removal of the pancreas may provoke an increase in b-cell replication, whereas a near total pancreatectomy (>90%) may produce a more dramatic

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growth pattern, with both b-cell replication and neogenesis being observed (22,23). When 90% pancreatectomy in rats was combined with the destruction of residual b-cells by streptozocin, this did not affect the capacity of the pancreas to regenerate, supporting the hypothesis that, under severe pancreatectomy conditions, neogenesis of b-cells is the dominant compensatory growth mechanism, not b-cell replication (24). Treatment of animal models with streptozocin alone has also been used to study the process of islet and b-cell neogenesis. Treatment of rodent models with the DNA-alkylating compound streptozocin results in the destruction of the b-cells of the pancreas (25). The regenerative capacity of the pancreas, as discussed above, is at its peak during the early stages of neonatal development. After 5 days, the compensatory response of the pancreas to streptozocin is muted, indicating that the early pancreas potentially contains more precursor cells that can be utilized to expand b-cell mass (26). In adult rodent models, little compensation is seen and the proliferative and differentiation capacity of the islets appears low (27). However, in studies using both streptozocin and the free radical generator alloxan, treatment with a low dose of the toxic effector, or treatment of only part of the pancreas with the toxic effector, does produce a neogenic response (28). When part of the pancreas is treated with alloxan in isolation, this part does not regenerate. However, in a response similar to that seen during pregnancy or in response to obesity, neogenesis of b-cells from pre-existing duct cells is observed in the remaining portion of the pancreas, attempting to compensate for the loss of functional b-cell mass. Combination of these toxic effector molecules with stimuli such as GLP-1, EGF, or gastrin, provokes a much more robust neogenic response. For example, combination of gastrin and EGF in response to alloxan administration in a recent study provoked a 30% increase in b-cell growth rate per day, leading to the doubling of b-cell number in only 3 days (29). This increase in b-cell mass has been attributed to neogenesis from precursor cells within the pancreas, as no increase in b-cell replication was observed. Indeed, transient co-expression of insulin and CK19 in some cells in the regenerating pancreas would support the transdifferentiation of ductal cells as the major source of neogenic b-cells in this model. In combination with the chemical induction of islet and b-cell neogenesis, several animal models utilize the ability of the pancreas to recover from physical assault. Islet neogenesis has been shown to occur following cellophane wrapping of the pancreas in rodent models (30). This model stimulates the proliferation of cells in the pancreatic ducts, eventually resulting in an increase in islet number and b-cell mass (31). Partial duct obstruction using this method was shown in streptozocin-treated animals to result in islet cell regeneration, again suggesting that neogenic b-cells were arising not from existing b-cells by replication, but by transdifferentiation of ductal cells in the adult pancreas. Brief occlusion of the main pancreatic duct by a simple

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“squeezing” technique has been shown to produce an increase in duct cell proliferation and islet mass in a similar manner to cellophane wrapping (32), again implicating ductal cells as the key precursors in the neogenesis of new b-cells (33). The production of new b-cells from ductal cells is also observed in an elegant transgenic mouse model utilizing the expression of interferon γ (IFN-γ). Transgenic mice expressing the IFN-γ gene under the control of the b-cell specific insulin gene promoter develop diabetes through b-cell destruction within 6–8 weeks after birth (34). As in the chemically induced diabetes described above, new b-cells in this model of diabetes were derived from the expansion and differentiation of ductal cells in the pancreas. This model of islet regeneration is particularly elegant, as the compensatory growth processes generating the replacement b-cells seem to mirror the normal development processes driving embryonic b-cell formation. The many animal models of islet neogenesis and b-cell regeneration currently being studied point to the ductal cells of the pancreas as the main source of precursor cells able to transdifferentiate to b-cells and hence provide compensatory b-cell expansion under conditions of b-cell stress. However, while it is clear that ductal cells may play this role in neogenic islets, several other cell types are currently under investigation for their ability to generate functional b-cells. These include the pancreatic acinar cells, liver cells, and the intestinal K cells, as discussed below.

NEW β-CELLS THROUGH TRANSDIFFERENTIATION Transdifferentiation is a coordinated alteration in gene expression and cellular phenotype that allows the conversion of one adult cell type into another adult cell type. The plasticity of adult cells is only now becoming clear. As discussed above, it is the plasticity of the ductal cells of the pancreas that many now believe drives the formation of new b-cells. However, significant literature now suggests that the generation of functional b-cells from adult precursor cells is not limited to the ductal cells, or merely to cells of the pancreas. In this section we review the use of alternative adult cell types in the derivation of new insulin-producing cells, including pancreatic acinar cells, and the cells of the liver and gut (Fig.2). The acinar cells of the pancreas are certainly a potential source of neogenic b-cells (35). In several of the animal models described above, the expansion of ductal cells and the transdifferentiation to new b-cells is preceded by an initial acino-ductal transdifferentiation (36). That is, the starting population of cells undergoing transdifferentiation from duct to b cell is thought to be comprised of mature ductal cells augmented with newly formed ductal cells generated from the acinar tissue. In IFN-γ mice (37) and in the duct ligation model described above (38), there is also evidence of direct transdifferentiation of acinar cells to b-cells, since a

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Figure 2 New sources of b-cells. A number of potential sources of new b-cells have been proposed, including replication of pre-existing b-cells; differentiation of progenitor cells within the ductal cells of the pancreas; and transdifferentiation of adult pancreatic acinar cells, either directly to b-cells or by initial acino-ductal transdifferentiation. New sources of b-cells are not limited to cells of the pancreas, and the capability of a number of alternative non-pancreatic cells is being investigated: transdifferentiation of liver cells, through the transgenic expression of key transcription factors such as pancreatic/duodenal homeobox 1 (PDX-1); transdifferentiation of intestinal cells (K cells) and bone marrow cells; and finally through differentiation of embryonic stem cells (ES) cells.

transient subpopulation of cells exists that co-express insulin with the acinar marker amylase. The transdifferentiation potential of the acinar cells can also be enhanced by expression of key developmental transcription factors such as PDX1, again demonstrating the extraordinary plasticity of the cells of the pancreas, and suggesting a further possible source of neogenic b-cells in the pancreas (36). However, this plasticity is not limited to the cells of the pancreas, as liver cells have also been shown to be capable of transdifferentiation to insulin-producing cells. In general, cells that are capable of interconversion/transdifferentiation arise from adjacent regions of the developing embryo. Hence some of the early regulatory events and stimuli driving the formation of these cells are common to both adult cell types. Such is the case for the liver and pancreas, which are both derived from the endoderm of the developing embryo, and which share some adult characteristics, such as glucose sensitivity and pathways of protein processing and secretion. Transdifferentiation of liver to pancreas (and of pancreas to liver) is a phenomenon naturally observed in rare human disease states, as well as in well-characterized animal models (39). Hepatic foci have been observed in the pancreas of humans, rodents, and simian models. Indeed, the appearance of hepatic cells in the adult pancreas has been studied for over twenty years. Whether the hepatic cells observed in the pancreas in vivo arise directly from transdifferentiation of adult pancreatic cells remains

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controversial; however, in vitro studies with transformed cell lines have shown that the transdifferentiation from liver to b-cell, and from b-cell to liver, is possible, given the right experimental conditions, or the transgenic expression of key transcription factors such as PDX1 (40). Several of the hepatic nuclear factor family of transcription factors have also been shown to be critically important to the transdifferentiation of liver to pancreas and vice versa. These include HNF1α, FoxA2, HNF4α, and HNF6 (41,42). The C/EBPα, b and, γ mRNAs have also been shown to increase during the regeneration of the pancreas in copper-deprived animal models of the transdifferentiation process (42). Although transdifferentiation of liver to pancreas occurs in disease states such as hepatic cirrhosis (43), and in several animal models and in vitro systems (39), the role of these transdifferentiation events in normal b-cell turnover and adult pancreatic b-cell function has not been established, and it seems unlikely that these events have a major role to play in the normal processes of b-cell regeneration and neogenesis. Just as liver cells emerge from the embryonic endoderm closely associated with pancreas development, so the gut cells are subjected to similar developmental stimuli. Recent studies have investigated the potential of gut K cells to produce functional insulin-secreting cells (44). Intestinal K cells have been successfully engineered in vitro to express insulin and to secrete the protein in a glucose-responsive manner (45). Gut cells, like liver cells, have the functional advantage in that they contain the appropriate secretory pathway components to regulate protein secretion. Hence, studies with liver and gut cells have proven the most successful in terms of generating glucose-responsive insulin-secreting cells by transdifferentiation. However, while these approaches have proven extraordinarily valuable in increasing our understanding of the transdifferentiation process, it seems unlikely that either cell type contributes to b-cell neogenesis in vivo. Hence, the value of these cells in terms of novel therapeutic approaches to the treatment of diabetes remains limited. Such approaches would require extensive in vitro manipulation of isolated adult cells, followed by transplantation of modified cells. While this is certainly a possibility in the long term, it seems more likely at present that successful generation of insulin-producing cells from pancreas-derived material (ductal and islet cells) will provide a more practical approach in the short-term. Current approaches to islet transplantation could certainly be enhanced by the augmentation of purified adult islets with islet-derived cells capable of transdifferentiation into b-cells. In terms of the augmentation of islet function through transdifferentiation of ductal precursor cells, or in terms of the improvement of b-cell mass and neogenesis rates in a patient with Type 2 diabetes, one candidate approach appears the most attractive at present. This approach is the augmentation of islet function by the intestinal polypeptide GLP-1. Current progress in GLP-1 research is reviewed below.

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GLP-1 GLP-1 is an incretin hormone produced and released from the L cells of the intestine (46). The insulinotropic actions of GLP-1 include the augmentation of insulin secretion and the effective lowering of circulating blood glucose concentrations (47). In b-cells, duct cells, and acinar cells, GLP-1 binds to and activates a specific GLP-1 receptor present on the cell membranes (48). Knock-out of GLP-1 receptors in transgenic mice produce defective islet function, resulting in defective regulation of blood glucose concentrations in these animals (49). In pancreatic b-cells and acinar cells, activation of the GLP-1 receptor is known to result in intracellular signaling events involving protein kinase A and changes in intracellular c-AMP levels. Characterization of these events in acinar and b-cells, and the recent finding that duct cells also express abundant functional GLP-1 receptors, indicate a key role for GLP-1 in the regulation of normal islet function (48). In addition, GLP-1 is known to regulate the expression of several key transcription factor proteins in adult b-cells, including the critical homeodomain protein PDX1 (50). However, of greatest interest in the present context is the potential role of GLP-1 in the neogenesis and regeneration of b-cells. The potential of GLP-1 to stimulate b-cell neogenesis is the subject of intense study at the present time. In early studies, investigation of the function of GLP-1 was hampered by the extremely short half-life of the protein (51). GLP-1 released from the intestinal L cells is very rapidly degraded by the dipeptidase enzyme DPPIV. The undesirably short half-life of GLP-1 has resulted in investigation of DPPIV inhibitors (52), GLP-1 agonists with longer half-lives such as Exendin 4 (Ex4) (53), and shorter fragments of active GLP-1, such as the functional 7-36 Amide (54) form of the peptide. Ex4 has proven of particular interest, since this has been extensively studied in the transdifferentiation of ductal cells to b-cells. Using the in vitro transformed ductal cell line Capan-1, Ex4 was shown to promote transdifferentiation to an endocrine phenotype (55). This process occurred through stimulation of the expression of the key transcription factors PDX1 and FoxA2, both of which are known to be critical in the normal development of b-cells in the embryo (7), and in the transdifferentiation process from alternative adult cell types. FoxA2 binds to and regulates the PDX1 gene promoter, events which are critical in the initiation of PDX1 gene expression and differentiation towards a b-cell phenotype. These early studies indicated that administration of GLP-1 activated both adenylate cyclase and MAP kinase signaling pathways, ultimately producing increased PDX1 gene expression and the resultant emergence of an insulin-producing b-cell phenotype (55). These data complement extensive findings suggesting that GLP-1 and Ex4 both stimulate PDX1 gene expression in ductal cells (56), with PDX1 gene expression being upregulated during the regeneration of the pancreas (57). However, even more compelling evidence comes from

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recent animal studies that firmly implicate GLP-1 in the process of b-cell neogenesis. The potential role of GLP-1 in stimulating the process of b-cell neogenesis was originally demonstrated in vivo in lean 20-day-old normoglycemic mice (58). Since these initial studies, a substantial number of studies have confirmed that GLP-1 and Ex4 exert major trophic effects on the cells of the pancreas, increasing b-cell neogenesis and boosting b-cell mass. Using the longer half-life of Ex4, recent studies have shown that administration of this GLP-1 receptor agonist to streptozotocin-treated rats results in the increased appearance of small clusters of insulin-producing cells in the ducts of these animals, and contributes to the restoration of normal blood glucose control in this model of Type 1 diabetes (48). Treatment of the animals with Ex4 resulted in a prolonged and sustained improvement in glucose tolerance, and this was shown to occur not only through the generation of neogenic b-cells from duct cells, but additionally to result in part through an increase in b-cell mass. No change was observed in the levels of b-cell apoptosis, suggesting that the effects of Ex4 were not a result of a shift in the balance between apoptosis and b-cell neogenesis, but rather a simple positive effect of increasing b-cell number and mass (48). These interesting findings indicate that Ex4 can increase b-cell neogenesis and b-cell mass in diabetic animals, restoring islet function and allowing the control of circulating blood glucose concentrations. Of most clinical relevance, these results were also observed in isolated human pancreatic ducts, where treatment with Ex4 again resulted in the increased appearance of insulin-positive clusters of cells (48). It has been reported that the insulin secretory response to glucose in islets following transplantation, a measure of functional b-cell mass, correlates significantly with the number of ductal cells originally present in the transplanted islets (59). Hence the finding of abundant GLP-1 receptors on human ductal cells is consistent with a key role for GLP-1 in the b-cell neogenesis process. These findings complement many recent animal studies. GLP-1 was found to be a potent stimulator of b-cell neogenesis following partial pancreatectomy (60). Interestingly, GLP-1 receptor knock-out mice showed much poorer glucose tolerance following partial pancreatectomy, identifying a significant defect in b-cell mass regeneration in these animals compared to wild-type animals with an intact GLP-1 response (60). In obese hyperglycemic ob/ob mice, Ex4 increases b-cell mass and improves b-cell function, restoring normal glycemic control (58). In other mouse models of diabetes, GLP-1 has been shown to increase b-cell mass through neogenesis of b-cell from ductal cells expressing PDX1 (61), and GLP-1 administration even delays the onset of diabetes in the db/db mouse model (62). These recent studies add to a growing weight of evidence suggesting that GLP-1 and its analogues may be key regulators of b-cell regeneration and neogenesis. From the original studies in normoglycemic mice, through

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study of pancreatectomized animals, GLP-1 receptor knock-out mice, aging mice (63), neonatal streptozotocin-treated animals (64), the Goto-Kakizaki (GK) rat model (65), in vitro analysis of isolated cell lines, right through to the recent identification of abundant GLP-1 receptors on rat and human ductal cells, the weight of data now suggests a potentially exciting therapeutic role for GLP-1 administration in the promotion of b-cell neogenesis, and the restoration of b-cell mass and GLP-1 may represent one of the best therapeutic candidates for the improvement of b-cell mass and function in patients with diabetes. ENHANCING β-CELL MASS AND NEOGENESIS GLP-1 represents an exciting potential therapeutic agent to increase b-cell neogenesis and b-cell mass in the treatment of diabetes. However, several other approaches to the enhancement of b-cell neogenesis and mass are currently under investigation. Two of the major alternative approaches under current investigation are combination therapy through treatment of islets with gastrin and EGF, and the enhancement and protection of b-cell mass through administration of thiazolidinediones. It remains possible that a combination of these approaches may prove beneficial in enhancing the function of b-cells in patients with diabetes, or following islet transplantation. Combined treatment of streptozocin-treated rats with gastrin and EGF has been reported to not only increase b-cell mass, but to reduce hyperglycemia (66). In alloxan-treated animals, the same combination also resulted in restored normoglycemia through induction of b-cell neogenesis from ductal cells (67). Hence, further studies have begun to investigate the combined effects of these stimuli on b-cell regeneration and islet function. Culture of isolated human islets (containing ductal cells) with gastrin and EGF resulted in a significant increase in functional b-cell mass (68). This increase was related to increased expression of the transcription factor PDX1 and resulted in increased production of both insulin and C-peptide from the expanded b-cell mass in vitro. When human islets were transplanted into NOD/SCID mice, an immunocompromised mouse model of type 1 diabetes, administration of gastrin and EGF in combination increased functional b-cell mass and insulin secretion, as well as increasing the insulin secretory response of the transplanted islets to oral glucose challenge (68). Hence, recent studies indicate that combination of gastrin and EGF increases b-cell mass and function in isolated human islets in vitro and in vivo, through induction of b-cell neogenesis from ductal cells. Combination of gastrin and EGF, or the use of GLP-1 analogues, has the potential to generate an increased b-cell mass in vivo and in vitro in isolated and subsequently transplanted islets. A third therapeutic approach currently under intense scrutiny is the potential protection and enhancement of neogenic b-cell function through administration of thiazolidinediones

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such as rosiglitazone. Rosiglitazone is a commonly prescribed antihyperglycemic drug prescribed to those with Type 2 diabetes (69). Initially characterized as a modulator of adipocyte cell function, the agonistic actions of rosiglitazone on the nuclear hormones receptor peroxisome proliferator activator gamma (PPAR) are known to result in decreased hepatic glucose output, increased peripheral glucose uptake, and alterations in adipocyte metabolism. However, recent studies indicate that in addition to these wellcharacterized effects, rosiglitazone acts directly on the pancreatic b-cells (70). Rosiglitazone administration has been shown to stimulate PDX1 gene expression in b-cells in culture, as well as increasing the expression and nuclear localization of the PDX1 and FoxA2 proteins, both of which are well-characterized regulators of b-cell growth and development. These key development transcription factors have also been repeatedly implicated in the process of b-cell neogenesis and increases in b-cell mass. Consistent with these observations of the direct effects of rosiglitazone on b-cell function, and the success of rosiglitazone in improving (71) and protecting b-cell function in vivo (72), administration of rosiglitazone has also been shown to enhance the function of transplanted islets (73). This raises the interesting possibility that administration of rosiglitazone may perform three roles in patients undergoing islet transplantation. First, rosiglitazone may enhance b-cell function and increase b-cell mass; second, systemic effects of rosiglitazone are known to improve insulin sensitivity and decrease hyperglycemia through targeting liver, muscle, and adipocyte cell function; and last, rosiglitazone may protect not only the transplanted islets, but the neogenic b-cells emerging from transplanted ductal material. Hence, mounting evidence supports the idea that rosiglitazone administration may have an overall beneficial effect on b-cell neogenesis and function, both in vivo and following transplantation of isolated islets. In summary, the augmentation of transplanted islets with increased neogenic potential may well be achieved through stimulation with GLP-1 (Ex4) in combination with rosiglitazone. This would promote b-cell neogenesis and an increase in b-cell mass while endowing the neogenic b-cells with an element of protection and promoting increased function. Only time will tell whether this type of combined therapy can improve islet graft function, and in the long term, potentially also improve islet function of patients with Type 2 diabetes. As described in the sections above, an increasingly large volume of work indicates that the plasticity of the adult cells of the pancreas can be utilized to generate new sources of insulin-producing cells. The optimal regeneration of b-cell function, and the promotion of b-cell neogenesis, may require a combination of one or more of these approaches. In the longer term, limitations on the amount of available donor islet tissue may result in a drive towards the successful generation of functional b-cells through transdifferentiation of other adult cell types such as liver cells. Finally, a worldwide effort is underway to generate functional b-cells

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from embryonic stem cells in culture (74) and from bone-marrow derived stem cells (75). Successful generation of functional b-cells from embryonic stem cells would generate a tissue bank of insulin-producing cells for the treatment of large numbers of patients with diabetes. However, as reviewed here, the events controlling the emergence of a mature b-cell phenotype, and the balance between proliferation and apoptosis of the b-cells, are extremely complex. Hence, differentiation of embryonic stem cells to a fully functional and self-renewing b-cell phenotype is certainly a challenging prospect. Current stem-cell approaches for the generation of new b-cells are the subject of the chapter that follows. REFERENCES 1.

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14. Bouwens L, De Blay E. Islet morphogenesis and stem cell markers in rat pancreas. J Histochem Cytochem 1996; 44:947–951. 15. Van Assche FA, Gepts W, Aerts L. Immunocytochemical study of the endocrine pancreas in the rat during normal pregnancy and during experimental diabetic pregnancy. Diabetologia 1980; 18:487–491. 16. Van Assche FA, Aerts L, De Prins F. A morphological study of the endocrine pancreas in human pregnancy. Br J Obstet Gynaecol 1978; 85:818–820. 17. Allen SR. Gestational diabetes: a review of the treatment options. Treat Endocrinol 2003; 2(5):357–65. 18. Greenberg AS, Obin MS. Obesity and the role of adipose tissue in inflammation and metabolism. Am J Clin Nutr 2006; 83(2):461S–465S. 19. Kloppel G, Lohr M, Habich K, Oberholzer M, Heitz PU. Islet pathology and the pathogenesis of type 1 and type 2 diabetes mellitus revisited. Surv Synth Pathol Res 1985; 4:110–125. 20. Dor Y, Brown J, Martinez OI, Melton DA. Adult pancreatic b-cells are formed by self-duplication rather than stem-cell differentiation. Nature 2004; 429:41–46. 21. Gao R, Ustinov J, Korsgren O, Otonkoski T. In vitro neogenesis of human islets reflects the plasticity of differentiated human pancreatic cells. Diabetologia 2005; 48(11):2296–304. 22. Bonner-Weir S, Baxter LA, Schuppin GT, Smith FE. A second pathway for regeneration of adult exocrine and endocrine pancreas. A possible recapitulation of embryonic development. Diabetes 1993; 42:1715–1720. 23. Dor Y, Brown J, Martinez OI, Melton DA. Adult pancreatic b-cells are formed by self-duplication rather than stem-cell differentiation. Nature 2004; 429:41–46. 24. Finegood DT, Weir GC, Bonner-Weir S. Prior streptozotocin treatment does not inhibit pancreas regeneration after 90% pancreatectomy in rats. Am J Physiol Endocrinol Metab 1999; 276:E822–E827. 25. Szkudelski T. The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas. Physiol Res 2001; 50(6):537–46. 26. Wang RN, Bouwens L, Klo¨ppel G. Beta cell growth in adolescent and adult rats treated with streptozotocin during the neonatal period. Diabetologia 1996; 39:548–557. 27. Ahren B, Sundkvist G. Long-term effects of alloxan in mice. Int J Pancreatol 1995; 17:197–201. 28. Waguri M, Yamamoto K, Miyagawa JI, et al. Demonstration of two different processes of b-cell regeneration in a new diabetic mouse model induced by selective perfusion of alloxan. Diabetes 1997; 46:1281–1290. 29. Rooman I, Bouwens L. Combined gastrin and epidermal growth factor treatment induces islet regeneration and restores normoglycaemia in C57Bl6/J mice treated with alloxan. Diabetologia 2004; 47:259–265. 30. Rafaeloff R, Pittenger GL, Barlow SW, et al. Cloning and sequencing of the pancreatic islet neogenesis associated protein (INGAP) gene and its expression in islet neogenesis in hamsters. J Clin Invest 1999; 99:2100–2109. 31. Rosenberg L, Clas D, Duguid. Trophic stimulation of the ductal/islet cell axis: a new approach to the treatment of diabetes. Surgery 1990; 108:191–197.

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32. Woodroof CW, de Villiers C, Page BJ, van der Merwe L, Ferris WF. Islet neogenesis is stimulated by brief occlusion of the main pancreatic duct. S Afr Med J 2004; 94(1):54–7. 33. Ferris WF, van der Merwe L, Campbell SC, Macfarlane WM. Glucocorticoid administration and brief occlusion of the main pancreatic duct are likely to increase islet mass by a similar mechanism. Pancreas 2005; (2):132–7. 34. Sarvetnick NE, Gu D. Regeneration of pancreatic endocrine cells in interferon-γ transgenic mice. Adv Exp Med Biol 1992; 321:85–9. 35. Bouwens L. Transdifferentiation versus stem cell hypothesis for the regeneration of islet b-cells in the pancreas. Microsc Res Tech 1998; 43(4):332–6. 36. Rooman I, Heremans Y, Heimberg H, Bouwens L. Modulation of rat pancreatic acinoductal transdifferentiation and expression of PDX-1 in vitro. Diabetologia 2000; 43(7):907–14. 37. Wang RN, Kloppel G, Bouwens L. Duct- to islet-cell differentiation and islet growth in the pancreas of duct-ligated adult rats. Diabetologia 1995; 38(12): 1405–11. 38. Gu D, Arnush M, Sarvetnick N. Endocrine/exocrine intermediate cells in streptozotocin-treated Ins-IFN-gamma transgenic mice. Pancreas 1997; 15(3): 246–50. 39. Shen CN, Horb ME, Slack JM, Tosh D. Transdifferentiation of pancreas to liver. Mech Dev 2003; 120(1):107–16. 40. Koizumi M, Doi R, Toyoda E, et al. Hepatic regeneration and enforced PDX-1 expression accelerate transdifferentiation in liver. Surgery 2004; 136(2):449–57. 41. Rao MS, Yukawa M, Omori M, Thorgeirsson SS, Reddy JK. Expression of transcription factors and stem cell factor precedes hepatocyte differentiation in rat pancreas. Gene Expr 1996; 6:15–22. 42. Dabeva MD, Hurston E, Sharitz, DA. Transcription factor and liver-specific mRNA expression in facultative epithelial progenitor cells of liver and pancreas. Am J Pathol 1995; 147:1633–1648. 43. Wolf HK, Burchette JL, Garcia, JA, Michalopoulos G. Exocrine pancreatic tissue in human liver: a metaplastic process? Am J Surg Pathol 1990; 14: 590–595. 44. Fujita NY, Cheung AT, and Kieffer TJ. Harnessing the gut to treat diabetes, Pediatr Diabetes 2004; 5(Suppl 2):57–69. 45. Cheung AT, Dayanandan B, Lewis JT, et al. Glucose-dependent insulin release from genetically engineered K cells. Science 2000; 290:1959–1962. 46. Kieffer TJ, Habener JF. The glucagon-like peptide. Endocr Rev 1999; 20: 876–913. 47. Gutniak M, Orskov C, Holst JJ, Ahren B, Efendis S. Antidiabetogenic effect of glucagon-like peptide-1 (7-36) amide in normal subjects and patients with diabetes mellitus. N Engl J Med 1992; 326:1316–1322. 48. Xu G, Kaneto H, Lopez-Avalos MD, Weir GC, Bonner-Weir S. GLP-1/ exendin-4 facilitates b-cell neogenesis in rat and human pancreatic ducts. Diabetes Res Clin Pract 2006; 73(1):107–10. 49. Flamez D, Gilon P, Moens K, et al. Altered cAMP and Ca2þ signaling in mouse pancreatic islets with glucagon-like peptide-1 receptor null phenotype. Diabetes 1999; 48(10):1979–86.

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50. Campbell SC, Macfarlane WM. Regulation of the pdx1 gene promoter in pancreatic b-cells. Biochem Biophys Res Commun 2002; 299(2):277–84. 51. Deacon CF, Johnsen AH, Holst JJ. Degradation of glucagon-like peptide-1 by human plasma in vitro yields an N-terminally truncated peptide that is a major endogenous metabolite in vivo. J Clin Endocrinol Metab 1995; 80:952–957. 52. Holst JJ. Treatment of type 2 diabetes mellitus with agonists of the GLP-1 receptor or DPP-IV inhibitors. Expert Opin Emerg Drugs 2004; 9(1):155–66. 53. Nielsen LL, Young AA, Parkes DG. Pharmacology of exenatide (synthetic exendin-4): a potential therapeutic for improved glycemic control of type 2 diabetes. Regul Pept 2004; 117(2):77–88. 54. Nauck MA, Kleine N, Ørskov C, Holst JJ, B. Willms, Creutzfeldt W. Normalization of fasting hyperglycaemia by exogenous glucagon-like peptide 1 (7–36 amide) in type 2 (non-insulin-dependent) diabetic patients, Diabetologia 1993; 36:741–744. 55. Zhou J, Pineyro MA, Wang X, Doyle ME, Egan JM. Exendin-4 differentiation of a human pancreatic duct cell line into endocrine cells: involvement of PDX-1 and HNF3beta transcription factors. J Cell Physiol 2002; 192(3): 304–14. 56. Stoffers DA, Kieffer TJ, Hussain MA, et al. Insulinotropic glucagon-like peptide 1 agonists stimulate expression of homeodomain protein PDX-1 and increase islet size in mouse pancreas. Diabetes 2000; 49:741–748. 57. Sharma A, Zangen DH, Reitz P, et al. The homeodomain protein IDX-1 increases after an early burst of proliferation during pancreatic regeneration. Diabetes 1999; 48:507–513. 58. Edvell A, Lindstrom P. Initiation of increased pancreatic islet growth in young normoglycemic mice (Umea þ/γ).Endocrinology 1999; 140(2):778–83. 59. Street CN, Lakey JR, Shapiro AM, et al. Islet graft assessment in the Edmonton Protocol: implications for predicting long-term clinical outcome. Diabetes 2004; 53(12):3107–14. 60. De Leon DD, Deng S, Madani R, Ahima RS, Drucker DJ, Stoffers DA. Role of endogenous glucagon-like peptide-1 in islet regeneration after partial pancreatectomy. Diabetes 2003; 52:365–371. 61. Stoffers DA, Kieffer TJ, Hussain MA, et al. Insulinotropic glucagon-like peptide 1 agonists stimulate expression of homeodomain protein IDX-1 and increases islet size in mouse pancreas. Diabetes 2000; 49:741–748. 62. Wang Q and Brubaker PL. Glucagon-like peptide-1 treatment delays the onset of diabetes in 8 week-old db/db mice. Diabetologia 2002; 45:1263–1273. 63. Perfetti R, Zhou J, Doyle ME, Egan JM. Glucagon-like peptide-1 induces cell proliferation and pancreatic-duodenum homeobox-1 expression and increases endocrine cell mass in the pancreas of old, glucose-intolerant rats. Endocrinology 2000; 141(12):4600–5. 64. Tourrel C, Bailbe D, Meile MJ, Kergoat M, Portha B. Glucagon-like peptide1 and exendin-4 stimulate b-cell neogenesis in streptozotocin-treated newborn rats resulting in persistently improved glucose homeostasis at adult age. Diabetes 2001; 50(7):1562–70. 65. Tourrel C, Bailbe D, Lacorne M, Meile MJ, Kergoat M, Portha B. Persistent improvement of type 2 diabetes in the Goto-Kakizaki rat model by expansion

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18 Stem Cell Approaches for b-Cell Replacement Enrique Roche Instituto de Bioingenieria, Universidad Miguel Hernandez, Elche, Alicante, Spain

Bernat Soria CABIMER, Andalusian Center for Molecular Biology and Regenerative Medicine, Seville, Andalucia, Spain

INTRODUCTION Type 1 diabetes mellitus is a degenerative pathology caused by autoimmune destruction of pancreatic b-cells, the only cell type that in the adult organism is responsible for bioactive insulin production and secretion (1). Insulin is a key hormone in glucose and fatty acid homeostasis, enhancing uptake of these nutrients by peripheral tissues and thereby controlling their circulating levels. The function of insulin cannot be mimicked by other hormones, and, without insulin replacement, Type 1 diabetes inevitably leads to ketoacidosis and death. Daily injections have changed the fate of the disease, but the maintenance of correct glucose levels requires very well trained and motivated patients in order to diminish secondary complications, such as neuropathy, nephropathy, retinopathy, and cardiovascular disease (1). Up to 0.5% of the population in developed countries is affected by Type 1 diabetes and the incidence is increasing. This disorder appears mainly, but not exclusively, in children and young people. Indeed, recent evidence suggests that 5–15% of patients initially diagnosed with Type 2 diabetes in fact have a more slowly progressive form of diabetes, termed latent autoimmune diabetes in adults, or LADA (2). The underlying pathogenesis of Type 1 diabetes mellitus remains unclear, although it is believed that several environmental factors are capable of activating autoimmune mechanisms in genetically 311

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predisposed individuals. The HLA-DR3, in addition to HLA-DR4 in Caucasian people, is predictive of disease (3,4). Markers of islet autoimmunity include antibodies against b-cell proteins, such as surface antigens (anti-islet), glutamate decarboxylase, and tyrosine phosphatase (3,4). The pathology displays two phases: insulinitis, corresponding to islet leukocyte infiltration, and overt diabetes, corresponding to b-cell destruction. Unfortunately, in most cases Type 1 diabetes mellitus is diagnosed once more than 90% of the b-cell mass has been destroyed. Recent successes in islet transplantation have confirmed the huge potential advantages of cell therapy for diabetes in comparison to exogenous insulin injection and whole organ transplantation (5). Two major hurdles remain: the need to refine immunosuppression or immunoisolation approaches (6) and the limited number of cadaveric pancreata (7). Even in Spain, with the highest rate of organ donation in the world, only a maximum of 1500 pancreata would be available each year. This will never be sufficient for the needs of more than 100,000 Spanish people with Type 1 diabetes. This strongly suggests that new sources of islets for replacement protocols have to be found. In this context, embryonic and adult stem cells offer interesting possibilities meriting further exploration. Stem cells are defined by the potential to undergo symmetric cell divisions for self-renewal and asymmetric cell divisions for lineage commitment, for example, to form differentiated insulin-producing cells (8). Stem cells may thus offer an important and unlimited source for b-cell replacement protocols (9,10). In addition, emerging technologies, such as nuclear transfer (11–15), oocyte parthenogenesis (16,17), and cell reprogramming (18,19) by employing host-derived cells may circumvent immune rejection.

STEM CELLS Stem cells can be classified as embryonic (ESCs) and adult (ASCs). ESCs are obtained from the inner cell mass of the blastocyst, a structure formed during embryonic development at day 6 in humans and day 3.5 in mice (20). ASCs are present within tissues of adult organisms with a key role in turnover and/or repopulation (21). Although both cell types are capable of self-renewal commitment to specific cell fates, ESCs demonstrate greater plasticity (pluripotency) in terms of differentiation with potential to generate almost any cell type present in the adult organism, including the germinal lineage. ASCs appear to be committed to a more limited repertoire of cell fates (multipotency). However, the possibility that ASCs may be able to overcome cell commitment restrictions by transdifferentiation may widen possibilities for autologous transplantation (22). In this chapter, the

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potential for generation of insulin-producing cells from both ESCs and ASCs is considered further.

ADULT STEM CELLS ASCs are an attractive option representing a good alternative for cell replacement protocols in terms of immune compatibility. However, limited capacity for proliferation out from their in vivo niches with inherent commitment to specific cell fates may constrain their clinical applications (23). To achieve b-cell neogenesis, two sources of ASCs have to be considered: pancreatic and extrapancreatic stem cells.

Pancreatic Stem Cells In vitro experiments support the hypothesis that ASCs reside within the pancreatic duct with the potential to differentiate in vitro into islet-like structures, and that some ductal cells are thus islet progenitors (24,25). Culture of isolated ductal tissue from donor human pancreas under specific conditions enables formation of cell aggregates, termed cultivated human islet buds (CHIBs), that expressed (at the protein level) islet-specific hormones, including insulin and glucagon, in addition to transcription factors, such as Pdx1. A 2.5-fold increase in insulin secretion was attained in the presence of 20 mM glucose in comparison to 5 mM glucose, although this required relatively long 24-hour incubation of CHIBs (24). Another group reported isolated islet pluripotent stem cells (IPSCs) budding from pancreatic ducts isolated from pre-diabetic non-obese diabetic (NOD) mice. These IPSCs displayed increased insulin release in response to high glucose concentration (17.5 mM), and partially reversed hyperglycemia on transplantation under the kidney capsule in NOD mice (25). However, the low amount of insulin produced by IPSCs in vitro does not equate with the improvement in hyperglycemia in transplanted animals, suggesting that an as-yet uncharacterized maturation processes occurred in vivo. These reports indicate that duct epithelium obtained from cadaveric pancreata may have true potential for cell replacement protocols in Type 1 diabetes. Both CHIBs and IPSCs seem to arise from specific pluripotent cells residing within duct epithelium. Some groups believe nestin, a protein present in neurofilaments, to be a specific marker for these cells. In addition, the expression of nestin has also been reported in cells derived from islets called nestin-positive islet-derived progenitors (NIPs). NIPs isolated from rat and human islets proliferate in vitro and express endo- and exocrine pancreatic markers as well as hepatic genes (26). However, data obtained from transgenic mice indicate that nestin-positive cells seem to participate only in islet microvasculature formation (27). Therefore, whether nestin is a

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marker for islet precursors remains a matter of considerable debate. Nevertheless, the presence of islet stem cells outside ductal tissue and possibly within islets themselves remains. The Melton group have proposed that new b-cells in the adult are derived solely from replication of pre-existing b-cells and not from stem cell precursors (28). However, the experimental design that supports this conclusion does not exclude the existence of a pancreatic stem cell population. Although adult b-cells present a low rate of BrdU incorporation, it seems clear that we are not born with a mass of b-cells for the rest of our life. This suggests that these cells display a low turnover or neogenesis that could be significantly activated under specific circumstances, such as pancreatectomy or chemical insult (e.g.. streptozocin injection). However, the key molecular mechanisms underlying this process remain unclear. All these studies continue to suggest that meaningful generation of new b-cells for transplantation can be achieved from adult pancreata ex vivo or possibly even in situ in the individual with diabetes. At present, however, biosynthesized insulin remains too low for successful clinical transplantation. Moreover, it will be important to demonstrate physiological minute-tominute glucose-responsive insulin secretion. Identification of true islet progenitors and the development of small molecules to stimulate in vivo differentiation are key ongoing challenges in this field.

Extrapancreatic Stem Cells The constraints imposed by limited pancreatic tissue turnover and differentiation could be overcome by using alternative sources of ASCs. In this context, bone marrow contains a stem cell population that displays a broad plasticity, extending beyond blood and bone renewal. Dr Verfaillie’s group have isolated a multipotent cell side population from bone marrow, called Multipotent Adult Progenitor Cells (MAPCs). MAPCs have been isolated, cultured in vitro and differentiated to ectoderm, mesoderm, and endoderm lineages (29). The possibility of obtaining insulin-positive cells from bone marrow stem cells is still a matter of debate. One report claimed pancreas repopulation using highly purified bone marrow stem cells (30). Furthermore, isolated human bone marrow mesenchymal cells transfected with DNA constructs encoding for key transcription factors involved in endocrine pancreas development and cultured in islet-conditioned medium were capable of insulin gene expression. On the other hand, other laboratories failed to reproduce the results of the first report (31,32). In recent experiments progenitors circulating in peripheral blood have been coaxed to acquire an islet cell phenotype (33) and restored blood glucose control when implanted in streptozocin-diabetic immunocompromised mice. To this end, isolated circulating monocytes were dedifferentiated by adding

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interleukin-3 and macrophage colony stimulating factor to the culture medium. Subsequent exposure to epidermal and hepatic growth factors and nicotinamide resulted in insulin-containing cells (34). Liver, like the pancreas, is derived from definitive endoderm and could provide ASCs as an alternative source for bioengineering insulin-producing cells. In this context, it has been described that oval hepatic stem cells are capable of differentiation into cells positive for insulin I and II and other islet-specific markers, such as Pdx1, Pax-4, Pax-6, Nkx2.2, Nkx6.1, glucagon, pancreatic polypeptide, and the glucose transporter GLUT-2. Transplantation of these cells under the renal capsule of streptozocindiabetic NOD-SCID mice resulted in recovery of euglycemia, although graft removal for subsequent analysis and proof of restoration of the diabetic phenotype has not been reported (35). Hepatocytes share many phenotypic characteristics with b-cells, expressing key metabolic enzymes that are common to both cell types. This suggests that these cells could be interesting candidates for ectopic insulin production. This can be achieved by expressing key transcription factors essential for b-cell function such as Pdx1 or NeuroD combined with betacellulin (36,37). Hepatic expression of such proteins induces activation of the insulin gene within liver cells. Taken together, these studies offer interesting possibilities for the use of liver biopsies to generate functional and autotransplantable islet-like structures for cell replacement protocols in Type 1 diabetes. Alternatively, in situ transdifferentiation of liver cells to an insulin-secreting phenotype or b-cell neogenesis within the liver may ultimately provide a novel therapeutic strategy.

EMBRYONIC STEM CELLS ESCs provide another potential source of new b-cells. Mouse ESCs are maintained in the undifferentiated state by addition of Leukemia Inhibitory Factor (LIF), a cytokine of the interleukin-6 family, to the culture medium or by culturing on feeder layers of mitotically inactivated fibroblasts. In contrast, human ESCs do not respond to human LIF and even when cultured on feeder layers spontaneously initiate differentiation processes (38). Compared to mouse ESCs, it is thus more difficult to maintain human ESCs in an undifferentiated state in vitro. Differentiation towards specific lineages requires transfer from growth as an adherent monolayer to cell culture in suspension in the absence of LIF. Under these conditions, cells form aggregates called embryoid bodies (EBs) (38) in which markers from the three embryonic layers (ectoderm, mesoderm, and endoderm) as well as primitive endoderm are detected at different times in culture. The molecular and biophysical determinants that activate differentiation programs in EBs are still unknown, but it has been

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proposed that gradients in nutrient, oxygen, and growth factor concentrations across the EB, as well as intercellular connections, are instrumental in this process (39). Insulin expression together with detection of other pancreatic hormones has been observed during spontaneous differentiation in EBs at 10–23 d (40). However, the fraction of insulin positive cells is less than 2% of the total number of cells (41), and the total amount of insulin produced is too low to contemplate clinical transplantation trials. As stated above, the b-cell is derived from endoderm and acquires its phenotype through the expression of specific proteins, most critically insulin. In this sense, it has been accepted that insulin is the distinctive protein of pancreatic b-cells, and bioengineering strategies are targeted towards expression of this hormone. This has been the rationale behind the vast majority of culture condition manipulation protocols designed to obtain insulin-producing cells from ESCs. However, the intracellular amount of insulin obtained with these strategies is 1000 times lower than the insulin content detected in mature b-cells (42–44). The explanation may be related to the origin of the insulinpositive cells detected in the culture plates after differentiation protocols. In addition to the islet b-cell, the insulin gene is expressed in neuroectoderm-derived tissues and in primitive endoderm (45,46). The role of the hormone in these tissues has not yet been fully clarified. Recent work suggested that insulin is working as a remodeling factor in the initial stages of nervous system development when insulin-like growth factors (IGFs) are absent (47). Rodents have two insulin genes (insulin I and II). Insulin I gene is exclusively expressed in pancreatic b-cells, while insulin II is mainly expressed in yolk sack and specific neurons (46). In this context, the expression pattern of both genes may reveal important differences that should be considered in bioengineering protocols. For instance, insulin II gene is not regulated by glucose; its protein product is not fully processed, yielding thereby proinsulin from neuroectodermal tissues, and the amount of peptide produced is 1000 times lower than the amount of insulin detected in islets (48). Therefore, the presence of ectodermal-derived insulin-positive cells obtained in bioengineering protocols is a possibility that should be taken into account. This can be tested by determining the expression of insulin II gene in mouse-derived cell lines. However, humans only express one insulin gene, and complementary markers would be necessary in order to determine the exact origin of insulin-positive cells obtained from human ESCs (Fig. 1). It might thus be proposed that commitment to definitive endoderm should be integral to new protocols designed to obtain clinically useful insulin-producing cells from ESCs. Expression of insulin I and II should be used as markers at least in rodent cell lines, but appropriate markers for human cell lines must be further defined. Alternatively, based on the relative ease of generating neuroectoderm in vitro, strategies to increase the production of correctly processed insulin in cells of this origin should be considered (Fig. 2).

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Neuroectoderm ESCs -Oct3/4 -Nanog -Esg1

-Insulin II -NF-200 -GFAP -Nestin -Otx2

Primitive -Insulin II -Amnionless endoderm -Pthr1 Definitive -Insulin I -Cxcr4 endoderm -Glucagon

317 -Pdx1 -Isl1 -Pax6

-AFP -Foxa2 -Foxa3

Figure 1 Tentative scheme listing some markers detected by reverse transcriptase polymerase chain reaction (RT-PCR) that could allow distinguishing insulin-positive cells obtained in bioengineering protocols from embryonic stem cells (ESCs). Note that some markers are shared by different cell lineages. Markers for pluripotentiality of ESCs are listed as well. Source: From Refs. 39,70.

Figure 2 Strategies proposed to obtain insulin-producing cells from embryonic stem cells (ESCs). Late passages of ESCs (A) give rise to neuroectodermal insulin-positive cells (B) (39). However, these cells need to be manipulated in order to improve insulin production and correct hormone processing. (A) Transmission image of R1-ESCs passage 18. (A) and (B) images were captured at magnification X20 (Bars: 100 µm). Alternative strategies have to consider early passages of ESCs and embryoid bodies (EBs) commitment to definitive endoderm. Under these conditions different strategies could be adopted (see text for more details).

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IN VITRO DIFFERENTIATION PROTOCOLS A definitive protocol for in vitro differentiation to obtain insulin-secreting cells from ESCs or ASCs is still to be perfected. The various approaches tested so far by several laboratories can be classified into two categories: spontaneous differentiation combined with selection strategies and directed differentiation employing coaxial and reprogramming technology.

Spontaneous Differentiation Spontaneous differentiation combined with gating selection technology allows isolation of cell populations that specifically express the insulin gene (Fig. 2). To this end, particular DNA constructs are used in order to confer resistance to antibiotics under the control of b-cell specific promoters (49,50). This simple selection method has been applied to obtain other cell types, such as cardiomyocytes and neuronal-like cells (51–53). The strategy to obtain insulin-secreting cells from murine ESCs (49) consisted of a double-selection protocol based on the resistance to two antibiotics: hygromycin and neomycin. Expression of the hygromycin resistance gene under the control of the constitutive promoter of the phosphoglycerate kinase gene enabled selection of transfected single clonal cell lines. Expression of the neomycin resistance gene under the control of the regulatory regions of the insulin gene allowed selection of insulin-producing cells. The main disadvantage of this strategy is the inability to discriminate between ectodermal- and endodermal-derived insulin producing cells. One alternative would be the derivation of progenitor cells that could subsequently be differentiated to insulin-producing cells. Gating technology employing promoter elements of genes that are functionally active during intermediate stages of embryonic development provides an approach to specific derivation of islet precursor cells. In this sense, an alternative gating protocol has been developed (50) using the Nkx6.1 gene promoter controlling resistance to neomycin. Nkx6.1 is a transcription factor involved in the expansion of b-cell precursors during islet development (54). Selection with neomycin, together with specific culture conditions (presence of nicotinamide, anti-sonic hedgehog, and conditioned media from pancreatic buds), resulted in a pure population of murine cells coexpressing insulin, b-cell specific transcription factors, glucokinase, GLUT-2, and Sur-1. The obtained cells secreted insulin appropriately in response to increasing concentrations of extracellular glucose. Subsequent transplantation under the kidney capsule restored normoglycemia for 3 weeks in streptozocin-diabetic mice with reversion to hyperglycemia once the graft was removed. This new strategy offers still low but reproducible amounts of intracellular insulin content which will require further augmentation prior to any consideration of clinical transplantation trials.

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Directed Differentiation The directed differentiation approach includes both coaxial and cell reprogramming strategies (Fig. 2). Coaxial approaches are based on the design of specific culture media to drive ESCs or ASCs to insulin-positive cells. Cell reprogramming is dependent on introduction of specific proteins into the target cell either directly (cell extracts or purified proteins) or by using specific gene constructs. Coaxial Protocols A number of protocols have been developed based on the idea that nestinpositive cells could be precursors of neuronal and endocrine pancreatic cells (42). These in vitro differentiation protocols included incubation in ITSFn medium (DMEM/F12 containing insulin, transferrin, selenium, and fibronectin) and N2 medium [DMEM/F12 containing B27 supplement, insulin, transferrin, selenium, progesterone and putrescine supplemented with basic fibroblast growth factor (bFGF)]. N2 medium allowed expansion of nestin-positive cells in serum-free conditions. Withdrawal of bFGF from this medium and addition of nicotinamide in the last stage favored expression of the insulin gene (49). Insulin biosynthesis, however, was very low, and subsequent studies were performed in order to increase the number of insulin-positive cells and hormone content. Insulin content was modestly increased by addition of phosphoinositide-3-kinase inhibitors (LY294002 or wortmannin) or glucagon-like peptide-1 (GLP-1) in the last stage of the protocol (55,56). Addition of keratinocyte and epidermal growth factors (KGF and EGF, respectively) during the expansion phase of nestin-positive cells and inclusion of gating selection strategies also resulted in modest improvements (57,58). Interestingly, this nestin-selection strategy has been used for obtaining insulin-positive cells from human ESCs (59). The protocol employs culture medium containing low glucose concentration in the final incubation step. However, the co-expression of insulin with other pancreatic endocrine genes, such as glucagon and somatostatin, suggested that the final cells were still not fully differentiated (60). Although improvements to this protocol are necessary, the feasibility of using human ESCs to obtain insulin-producing cells has been confirmed, raising new hopes for a future diabetes therapy. Coaxial methodology has been applied to putative ASCs, including pancreatic duct derived insulin positive cells (24). Expansion, differentiation, and maturation of ductal cells was performed in DMEM/F12 serum-free medium containing ITS (Insulin þ Transferrin þ Selenium), KGF (to favor ductal tissue expansion), nicotinamide, 8 mM glucose, and MatrigelTM (a commercial, mouse-derived, growth-factor-rich, extracellular cell matrix that allows formation of CHIB structures; BD Biosciences, San Jose, California, U.S.A.) (25).

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ASCs from other organs, such as bone marrow or liver, have also been used in coaxial protocols to obtain insulin-producing cells (30,35,61–63). For instance, rat oval cells can be isolated and maintained undifferentiated in serum-free medium containing low glucose (5 mM) and LIF. Differentiation to insulin-containing structures can be performed by adding 10% serum, increasing glucose concentration (23 mM), and removing LIF from the culture medium. Coaxial approaches from mouse or human ESCs require rigorous application of optimized characterization criteria. In this context, insulin immunodetection is not a sufficient demonstration of endogenous hormone production. Notably, it has been reported that insulin can be sequestered from the culture medium into the cells (64), inhibiting at the same time de novo synthesis of the hormone (65). Exogenous insulin is present in serum and as a specific additive in the culture media used to select nestin-positive cells and to obtain CHIBs. This observation suggests that C-peptide matching the concentration of secreted insulin, together with the presence of insulin mRNA, should be used as more robust criteria to confirm intracellular insulin biosynthesis. Cell Reprogramming Certain proteins include membrane transduction domains that allow translocation across the plasma membrane and access to the intracellular compartment. Pdx1 posses an antennapedia-like domain that could be instrumental in reprogramming protocols. This domain was responsible for permeation of this transcription factor from the extracellular medium into pancreatic islets and ductal cells, inducing insulin gene expression (66). However, this domain is not present in all proteins and alternative permeabilization strategies would thus be required to transduce cells. Streptolysin O has been used to transiently permeabilize cellular membranes and thereby introduce protein extracts into target cells. The presence of key transcription factors and chromatin remodeling proteins in the extract may enable reprogramming by modulating the expression of a specific set of genes (67). Using this methodology, fetal rat fibroblasts have been reprogrammed to express the insulin gene following transduction with whole-cell extracts obtained from an insulinoma cell line (INS-1E) (68). These strategies therefore offer promise for induction of insulin gene expression in target cells without requiring DNA constructs overcoming the ethical issues posed by genetic manipulation. On the other hand, the use of specific gene constructs does enable constitutive or regulated expression of key proteins, such as transcription factors that are instrumental in b-cell function. Expression of Pax4 under the control of the constitutive CMV-promoter in transfected ESCs cultured following the nestin-selection strategy resulted in insulin expression (43).

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These DNA-based strategies have been used to transdifferentiate adult cells including hepatocytes. Expression of Pdx1 or NeuroD transgenes in hepatic cells resulted in induction of the insulin gene (36,37). In a similar way, human bone marrow mesenchymal stem cells were able to express insulin gene after infection with adenoviral vectors encoding key transcription factors in islet development (Pdx1, Hlxb9, and FoxA2) with subsequent incubation in islet-conditioned medium (69). CONCLUSIONS Although major further refinements are required in all protocols, a reasonable body of evidence confirms that stem cells could provide a potential source of transplantable tissue for replacement protocols in the treatment of Type 1 diabetes. The goal now has to be sharply focused on obtaining a final cell product that mimics as closely as possible the phenotype and function of mature b-cells in order to assure appropriate restoration of lost function in the recipient. Aside from insulin expression, this bioengineered cell will have to fulfill several criteria in order to accomplish a correct function in the transplanted organism. These can be classified according to three groups of proteins that work in coordination in mature pancreatic b-cells: 1. 2. 3.

The glucose-sensing machinery The regulated secretory pathway The insulin biosynthetic and processing apparatus

However, this work represents just the first part. The second step will be transplantation and this is likely to pose new challenges to scientists (7). Some of these are to: n n n n n n

select appropriate animal models to test the function of bioengineered cells before human trials; establish the best site for implantation in order to assure implant survival, and nutrient/oxygen supply; solve the problem of immune rejection by making customized tissues through gene manipulation, nuclear transfer, and oocyte parthenogenesis; enhance implant survival by placing it in well irrigated sites and considering anti-apoptotic and anti-necrotic approaches; design biosafety strategies that allow elimination of the graft in case of non-function or tumor formation; and harness in vivo differentiation processes controlling phenotypic changes in situ.

In conclusion, we are beginning to decipher the potential of stem cells in diabetes therapy. There is still much to do before new bioengineered b-cells

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from either ASCs or ESCs will be created suitable for clinical trials. The emerging technology will help to achieve this goal, but we need to further investigate the basic biology of stem cells and the determinants that control self-renewal and differentiation in order to establish reliable protocols.

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18. Western PS, Surani MA. Nuclear reprogramming—alchemy or analysis? Nat Biotechnol 2002; 20:445–446. 19. Pomerantz J, Blau HM. Nuclear reprogramming:A key to stem cell function in regenerative medicine. Nat Cell Biol 2004; 9:810–816. 20. Smith AG. Embryo-derived stem cells: Of mice and men. Annu Rev Cell Develop Biol 2001; 17:435–462. 21. Fuchs E, Tumbar T, Guasch G. Socializing with the neighbors: Stem cells and their niche. Cell 2004; 116:769–778. 22. Wagers AJ, Weissman IL. Plasticity of adult stem cells. Cell 2004; 116: 639–648. 23. Potten CS, Loeffler M. Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt. Development 1990; 110: 1001–1020. 24. Bonner-Weir S, Taneja M, Weir GC, et al. In vitro cultivation of human islets from expanded ductal tissue. Proc Natl Acad Sci USA 2000; 97:7999–8004. 25. Ramiya VK, Maraist M, Arfors KE, Schatz DA, Peck AB, Cornelius JG. Reversal of insulin-dependent diabtes using islets generated in vitro from pancreatic stem cells. Nat Med 2000; 6:278–282. 26. Zulewski H, Abraham EJ, Gerlach MJ, et al. Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine and hepatic phenotypes. Diabetes 2001; 50:521–533. 27. Treutelaar MK, Skidmore JM, Dias-Leme CL, et al. Nestin-lineage cells contribute to the microvasculature but not endocrine cells of the islet. Diabetes 2003; 52:2503–2512. 28. Dor Y, Brown J, Martı´ nez OI, Melton DA. Adult pancreatic b-cells are formed by self-duplication rather than stem-cell differentiation. Nature 2004; 429:41–46. 29. Jiang Y, Jahagirdar SN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002; 418:41–49. 30. Ianus A, Holz GG, Theise ND, Hussain MA. In vivo derivation of glucosecompetent pancreatic endocrine cells from bone marrow without evidence of cell fusion. J Clin Invest 2003; 111:843–850. 31. Choi JB, Uchino H, Azuma K, et al. Little evidence of transdifferentiation of bone marrow-derived cells into pancreatic b-cells. Diabetologia 2003; 46: 1366–1374. 32. Lechner A, Yang Y-G, Blacken RA, Wang L, Nolan AL, Habener JF. No evidence for significant transdifferentiation of bone marrow into pancreatic b-cells in vivo. Diabetes 2004; 53:616–623. 33. Runhke M, Ungefroren H. Nussler A, et al. Reprogramming human peripheral blood monocytes into functional hepatocyte and pancreatic isletlike cells. Gastroenterology 2005; 128:1774–1786 34. Runhke M, Ungefroren H, Nussler A, et al. Reprogramming human peripheral blood monocytes into functional hepatocyte and pancreatic isletlike cells. Gastroenterology 2005; 128(7):1774–1786. 35. Yang L, Li S, Hatch H, Ahrens K, Cornelius JG, Petersen BE, Peck AB. In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. Proc Natl Acad Sci USA 2002; 99:8078–8083.

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36. Ferber S, Halkin A, Cohen H, et al. Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocininduced hyperglycemia. Nat Med 2000; 6:568–572. 37. Kojima H, Fujimiya M, Matsumura K, et al. NeuroD-betacellulin gene therapy induces islet neogenesis in the liver and reverses diabetes in mice. Nat Med 2003; 9:596–603. 38. Smith AG. Culture and differentiation of embryonic stem cells. J Tissue Cult Meth 1991; 13:89–94. 39. Roche E, Sepulcre P, Reig JA, Santana A, Soria B. Ectodermal commitment of insulin-producing cells derived from mouse embryonic stem cells. FASEB J 2005; 19:1341–1343. 40. Soria B, Skoudy A, Martı´ n F. From stem cells to b-cells: new strategies in cell therapy of diabetes mellitus. Diabetologia 2001; 44:407–415. 41. Shiroi A, Yoshikawa M, Yokota H, et al. Identification of insulin-producing cells derived from embryonic stem cells by zinc-chelating dithizone. Stem Cells 2002; 20:284–292. 42. Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, McKay R. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 2001; 292:1389–1394. 43. Blyszczuk P, Czyz J, Kania G, et al. Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulinproducing cells. Proc Natl Acad Sci USA 2003; 100:998–1003. 44. Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, Tzukerman M. Insulin production by human embryonic stem cells. Diabetes 2001; 50:1691–1697. 45. Alpert S, Hanahan D, Teitelman G. Hybrid insulin genes reveal a developmental lineage for pancreatic endocrine cells and imply a relationship with neurons. Cell 1988; 53:295–308. 46. Melloul D, Marshak S, Cerasi E. Regulation of insulin gene transcription. Diabetologia 2002; 45:309–326. 47. Vicario-Abejo´n C, Yusta-Boyo MJ, Ferna´ndez-Moreno C, de Pablo F. Locally born olfactory bulb stem cells proliferate in response to insulin-related factors and require endogenous insulin-like growth factor-I for differentiation into neurons and glia. J Neurosci 2003; 23:895–906. 48. Herna´ndez-Sa´nchez C, Mansilla A, de la Rosa EJ, Pollerberg GE, Martı´ nezSalas E, de Pablo F. Upstream AUGs in embryonic proinsulin mRNA control its low translation level. EMBO J 2003; 22:5582–5592. 49. Soria B, Roche E, Berna G, Leo´n-Quinto T, Reig JA, Martı´ n, F. Insulinsecreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 2000; 49:157–162. 50. Leo´n-Quinto T, Jones J, Skoudy A, Burcin M, Soria B. In vitro directed differentiation of mouse embryonic stem cells into insulin-producing cells. Diabetologia 2004; 47:1442–1451. 51. Klug MG, Soonpa MH, Koh GY, Field LJ. Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts. J Clin Invest 1996; 98:216–224. 52. Li M, Pevny L, Lovell-Badge R, Smith A. Generation of purified neural precursors from embryonic stem cells by lineage selection. Curr Biol 1998; 8:971–974.

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53. Mu¨ller M, Fleischmann BK, Selbert S, et al. Selection of ventricular-like cardiomyocytes from ES cells in vitro. FASEB J 2000; 14:2540–2548. 54. Chakrabarti SK, Mirmira RG. Transcription factors direct the development and function of pancreatic b-cells. Trends in Endocrinol Metab 2003; 14:78–84. 55. Hori Y, Rulifson IC, Tsai BC, Heit JJ, Cahoy JD, Kim SK. Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells. Proc Natl Acad Sci USA 2002; 99:16105–16110. 56. Bai I, Meredith G, Tuch BE. Glucagon-like peptide enhances production of insulin in insulin-producing cells derived from mouse embryonic stem cells. J Endocrinol 2005; 186:343–352. 57. Moritoh Y, Yamato E, Yasui Y, Miyazaki S, Miyazaki J. Analysis of insulinproducing cells during in vitro differentiation from feeder-free embryonic stem cells. Diabetes 2003; 52:1163–1168. 58. Miyazaki S, Yamato E, Miyazaki J. Regulated expression of Pdx1 promotes in vitro differentiation of insulin-producing cells from embryonic stem cells. Diabetes 2004; 53:1030–1037. 59. Segev H, Fishman B, Ziskind A, Shulman M, Itskovitz-Eldor J. Differentiation of human embryonic stem cells into insulin-producing clusters. Stem Cells 2004; 22:265–274. 60. Polak M, Bouchareb-Banaei L, Scharfmann R, Czernichow P. Early pattern of differentiation in the human pancreas. Diabetes 2000; 49:225–232. 61. Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002; 418:41–49. 62. Hess D, Li L, Martin M, et al. Bone marrow-derived stem cells initiate pancreatic regeneration. Nat Biotechnol 2003; 21:763–770. 63. Kim SK, Hebrok M. Intercellular signals regulating pancreas development and function. Gen Develop 2001; 15:11–127. 64. Rajagopal J, Anderson WJ, Kume S, Martinez OI, Melton DA. Insulin staining of ES cell progeny from insulin uptake. Science 2003; 299:363. 65. Paek H-J, Morgan J-R, Lysaght MJ. Sequestration and synthesis: The source of insulin in cell clusters differentiated from murine embryonic stem cells. Stem Cells 2005; 23:862–867. 66. Noguchi H, Kaneto H, Weir GC, Bonner-Weir S. Pdx1 protein containing its own antennapedia-like protein transduction domain can transducer pancreatic duct and islet cells. Diabetes 2003; 52:1732–1737. 67. Collas P, Ha˚kelien A-M. Teaching cells new tricks. Trends Biotechnol 2003; 21:354–361. 68. Ha˚kelien A-M, Gaustad KG, Collas P. Transient alteration of cell fate using a nuclear and cytoplasmic extract of an insulinoma cell line. Biochem Biophys Res Commun 2004; 316:834–841. 69. Moriscot C, De Fraipont F, Richard M-J, et al. Human bone marrow mesenchymal stem cells can express insulin and key transcription factors of the endocrine pancreas developmental pathway upon genetic and/or microenvironmental manipulation in vitro. Stem Cells 2005; 23:594–604. 70. Yasunaga M, Tada S, Torikai-Nishikawa S, et al. Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells. Nat Biotech 2005; 23:1542–1550.

19 Diabetes Gene Therapy Peter S. Chapman Matthew J. Ryan Veterinary Hospital, University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

James A. M. Shaw Diabetes Research Group, Institute of Cellular Medicine, University of Newcastle, Newcastle upon Tyne, U.K.

UNDERLYING CONCEPTS AND POSSIBILITIES Gene therapy encompasses transfer of any prophylactic or therapeutic gene to human or animal cells resulting in subsequent expression in patients. Transfer of DNA can be achieved in situ by direct delivery to the host cells of the individual to be treated or performed ex vivo in cells that are subsequently transplanted. Cells or organs to be genetically manipulated prior to transplantation may be harvested and returned to the patient. Alternative sources include living or deceased human donors, animal donors, and proliferative transformed cell lines. Cell lines may be of embryonic or adult human or animal origin. The DNA sequence encoding the transgene can be incorporated into plasmid or viral vectors. In addition to employing naked plasmid DNA or a viral vector alone, approaches to achieve and enhance successful gene transfer in situ and ex vivo include plasmid complexation with a liposomal transfection agent or a wide range of other adjuvants in addition to electroporation. A wide range of targeting strategies in addition to natural tropisms associated with various viral vectors have been harnessed to attain a degree of selectivity in targeting delivery to specific organs following intravenous injection. Expression restricted to specific organs or cell types can be achieved by employing tissue-specific promoters to initiate and regulate transgene expression. These promoters may enable physiological regulation at the level of transcription. Alternatively, strong viral promoters may enable efficient constitutive expression regardless of cell type. Incompletely understood mechanisms that 327

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may have evolved as a defense against pathogenic viral expression in eukaryotic cells may, however, lead to downregulation of viral promoters [reviewed in (1)]. Promoters have been further engineered to enable pharmacological regulation of transcription following administration or withdrawal of a specific small molecule ligand (2). Regulators include tetracycline or related antibiotics, FK506 analogues, and progestogens. Each system has potential advantages and disadvantages but there has been little experience of any regulated gene therapy approach in humans to date. Viral Vectors The vast majority of clinical gene therapy trials to date (3) have been undertaken in individuals with malignant disease (67% of ongoing trials) and have employed viral vectors (70% of ongoing trials). Efficient gene delivery to a wide range of mammalian cells has been confirmed with viral vectors, but their inherent survival properties, including chromosomal insertion potentially at a site leading to oncogenic transformation, remain concerning. In addition, the mammalian immune system has evolved specific strategies to eliminate viral infection with associated risk of immune rejection and inflammatory response. The ubiquity of previous viral exposure in clinical subjects in contrast to experimental animals maintained in a specific pathogen-free environment may in particular lead to unpredicted responses. A further potential danger is re-establishment of replication competency in vivo and symptomatic viral infection. Successful gene transfer with first-generation retroviral vectors requires actively dividing cells and results in long-term expression due to insertion of the transgene into chromosomal DNA. Clinical success has been realized employing retroviral vectors to transduce host T cells in individuals with severe combined immune deficiency. Specific targeting of the vector to the promoter of an oncogene, LMO2, has, however, led to two cases of leukemic transformation in the first 10 individuals treated (4). More recently, HIV-derived lentiviral vectors have been developed. These retroviruses are able to target non-dividing cells due to an ability to traverse the intact nuclear membrane. There remain concerns regarding decreased expression over time and generation of infective wild-type HIV. Liver tumors have also been reported in pre-clinical trials (5). Adenoviral vectors can be used to transduce non-dividing, enddifferentiated cells. Risk of oncogenic transformation is low, as the vector is not incorporated into the host chromosome. A profound acute inflammatory response may be induced, however, particularly with first generation vectors. This can lead to particularly short-lived expression and has resulted in at least one death in a Phase 1 clinical study (6). Recombinant adenoassociated viral vectors have shown particular promise for successful clinical translation over the last 10 years. These are

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non-pathogenic and can mediate long-term transgene expression particularly following in vivo gene transfer. At least 20 clinical trials are underway or completed using adenoassociated viral vectors derived from AAV-2, the most common human serotype (7). These have confirmed favorable safety profile but level of expression and tissue selectivity may remain suboptimal. Neutralizing antibodies may reduce efficiency in individuals previously exposed to AAV Serotype 2. More recently, adenoassociated viral vectors have been derived from a range of other naturally occurring serotypes. AAV-1 and AAV-7 transduce skeletal muscle efficiently and AAV-8 has been employed to successfully transfer genes to liver (7). Efficient ex vivo transduction of human pancreatic islets prior to transplantation in rodent models has been confirmed using a double stranded AAV-2 vector without eliciting an immune response (8). Longterm expression in mice following in vivo administration of adenoassociated viral vectors has been confirmed following intraperitoneal and intravenous delivery. Direct cannulation and injection of the pancreatic exocrine duct with AAV-6 serotype-derived vectors appears particularly promising enabling transduction of virtually all islets (although not all cells within the core zone of each islet) minimizing viral load and extrapancreatic dissemination (9). Engineering of adenoassociated viral vectors is ongoing to further enhance tissue specificity and efficiency of gene transfer. Plasmid Vectors The excellent safety profile of plasmid DNA has led to its use in 17% of all clinical trials (3). Low efficiency of gene transfer has, however, been a major limitation. Long-term transgene expression can be achieved in skeletal muscle following simple percutaneous intramuscular plasmid injection. This approach is particularly appropriate for constitutive delivery of therapeutic proteins directly into the systemic circulation (2). Efficiency has been considerably enhanced by in situ electroporation or other adjuvant approaches so that therapeutically active circulating levels can now be achieved in large animal models of comparable weight to human patients (10). Therapeutically meaningful transfection efficiency has also been attained in liver following systemic or hepatic vein delivery of plasmid vector accompanied by hydrodynamic pressure to enhance uptake (11) or complexation with an adjuvant agent such as polyethyleneimine (PEI) (12). Plasmid-mediated gene transfer to pancreas has been attained following retrograde injection of the exocrine duct (13). Gene Therapy for Diabetes The possibilities for employing gene therapy approaches in the treatment of diabetes are seemingly limitless. Careful consideration must, however, be

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given to the potential risks and benefits in comparison to alternative conventional or novel interventions. Approaches considered include: 1. 2.

3. 4.

Prevention of disease progression and maintenance of b-cell function in Type 1 diabetes and islet transplant recipients Gene therapy approaches to insulin replacement, including derivation of conditionally transformed b-cell lines, b-cell transdifferentiation/ neogenesis, and insulin gene transfer to non-b-cells Gene transfer with non-insulin glucose-lowering genes, including insulin sensitizers and anti-obesity gene therapy Gene therapy targeted at diabetic complications

PREVENTION OF DISEASE PROGRESSION AND MAINTENANCE OF b-CELL FUNCTION IN TYPE 1 DIABETES AND ISLET TRANSPLANT RECIPIENTS Strategies for the prevention of Type 1 diabetes can be classified as: Primary prevention targeted at genetically predisposed high risk populations prior to serological evidence of established islet autoimmunity; Secondary prevention in those with islet specific antibodies evidencing established autoimmune b-cell destruction prior to clinical hyperglycemia; Tertiary prevention following presentation with diabetes aimed at attenuating or reversing further b-cell loss. Previous and current clinical Type 1 diabetes prevention trials have recently been reviewed (14). It is unlikely that gene therapy will have a role in primary prevention, as genetic screening in the absence of established disease will remain insufficient for absolute prediction of progression to diabetes given the need for an additional environmental trigger. Prior to the onset of overt clinical Type 1 diabetes, there is a long period of subclinical disease characterized by the presence of an autoimmune process specifically targeted at islet b-cells (15). Pre-clinical diagnosis of individuals with a very high likelihood of progression to overt disease is possible through presence of circulating antibodies to specific b-cell antigens, including insulin, glutamic acid decarboxylase, and the IA-2 phosphoprotein, in genetically predisposed individuals with or without evidence of subtle abnormalities in first phase insulin secretion in an intravenous glucose tolerance test. This provides a window for secondary prevention. Immunomodulation may also have a role following clinical disease onset when at least 10% of b-cell function remains, although studies in the NOD mouse model of autoimmune Type 1 diabetes have demonstrated the increased difficulty in reversing the autoimmune process once diabetes is established (16).

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The potential for recurrent b-cell autoimmunity in addition to alloimmune response following islet transplantation has been demonstrated in animal models and may play a role in loss of long-term graft function in clinical patients (17). Any successful immunoprophylactic strategy may have a further application in islet transplant recipients, and there are clearly specific opportunities for ex vivo gene transfer to islets pre-transplantation.

Antigen-Specific Tolerization Approaches There is increasing evidence for incomplete thymic deletion of islet b-cell specific T-cell precursors and defective peripheral tolerance leading to T-cell expansion, differentiation and initiation of a Type 1 immune process (18). Gene therapy approaches mediating autoantigen expression in the thymus; by circulating antigen presenting cells; or systemically may restore immune tolerance. Transgenic expression of a proinsulin cDNA under the control of the MHC Class II promoter resulted in intrathymic expression and prevention of diabetes onset in NOD mice (19). Although gene transfer to thymus following direct injection has been attained in animal models with the potential for tolerance induction (20), further studies are required to explore feasibility, safety, and efficacy of this approach for clinical translation. Injection of hematopoietic stem cells derived from the transgenic MHC promoter-driven proinsulin expressing cells resulted in prevention of spontaneous autoimmune diabetes in NOD mice (21). This provides initial proof of principle for ex vivo gene transfer to autologous cells derived from peripheral blood with subsequent reinjection. However, methods for lowrisk, efficient, and stable gene transfer to hematopoietic cells require further refinement. Moreover, the published studies required recipient bone marrow ablation, clearly unacceptable for individuals with early Type 1 diabetes or following islet transplantation. Islet autoantigens can also be expressed in muscle following direct intramuscular injection of naked plasmid DNA with uptake enhanced by in situ electroporation. Prevention of diabetes in NOD mice has been reported employing plasmids encoding the immunodominant epitope of insulin (a portion of the B chain) (22) or glutamic acid decarboxylase downstream of a signal peptide sequence mediating systemic secretion (23).

Antigen-Independent Approaches Antigen-independent gene therapy approaches to attenuating the initial autoimmune diabetogenic process or preventing recurrence in transplanted islets include expression of immunoregulatory cytokines including IL-10, IL-4, and TGF-b following in situ gene transfer to muscle or directly to

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pancreas [reviewed by Giannoukakis et al. (18)]. Alternatively autologous dendritic cells or donor islets can be engineered to express these factors ex vivo prior to transplantation (24). Direct transduction of islets to prevent antigen-presenting cell activation is an alternative approach. Examples include expression of IL-1 receptor antagonist gene to prevent IL1b b-cell toxicity and APC activation (25) and expression of a cytotoxic T-lymphocyte-associated protein 4 gene to block the second signal necessary for T-cell activation by APCs (26). Downregulation of CD40, CD80, and CD86 co-stimulatory molecule expression in harvested dendritic cells by antisense oligodeoxyribonucleotides with reimplantation by intradermal injection has recently been approved by the FDA as a Phase 1 clinical trial in individuals with Type 1 diabetes (27). An approach particularly being explored for xenograft transplantation is modification of islet cellular antigens to prevent recognition by the host immune system. Transgenic pigs have been engineered in which the alphagalactosidase transferase gene has been knocked out (28). This prevents expression of α-galactosidase, a trigger of hyperacute rejection, on the endothelial cell surface of transplanted islets. Transgenic pigs (29) and porcine islets in vitro (30) have also been engineered to express the human complementary regulatory protein to attenuate complement activation. Successful transplantation in non-human primate models of diabetes without immune rejection has been reported.

Attenuation of Apoptosis The continued presence of b-cells undergoing apoptosis even after 50 years of Type 1 diabetes (31) has highlighted the potential importance of novel anti-apoptotic therapies in preventing or reversing Type 1 diabetes and preventing loss of islet graft function over time. There is huge scope for gene therapy to have a role through in situ gene transfer to pancreas or ex vivo gene transfer to transplanted islets. Potential anti-apoptotic genes include Bcl-2, heat shock proteins, and an inhibitor of Caspase-8 activation (I-FLICE). Gene transfer strategies for cytoprotection of b-cells have recently been reviewed (32).

Enhanced Islet Engraftment It appears that less than a third of potential b-cell function is achieved following clinical islet transplantation due to poor engraftment and specifically inadequate early revascularization (33). Enhanced graft angiogenesis, insulin staining intensity, and function following transplantation under the renal capsule in immunodeficient mice with streptozocin-induced diabetes

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has been demonstrated following ex vivo vascular endothelial growth factor gene transfer to murine islets employing an adenoviral vector (34). Augmentation of b-Cell Function Within Intact Islets Gene therapy may also be employed to directly enhance b-cell mass and function in transplanted islets. Hepatocyte growth factor has been shown to induce b-cell proliferation in culture, to increase insulin secretion, and to prolong survival in transplant models. Adenoviral hepatocyte growth factor gene transfer to rat islets improved transplant outcomes following transplantation into the portal vein of an allogeneic rat strain with streptozocin-induced diabetes using daclizumab, tacrolimus, and sirolimus immunosuppression to mimic the Edmonton Protocol (35). There are a wide range of other factors with proven potential for enhancing b-cell mass and/ or function that could be delivered to isolated islets prior to transplantation employing viral vectors or non-viral transfection. These include growth factors such as epidermal growth factor, gastrin, and IGF-1 (36); key b-cell transcription factors such as PDX1 (37) and PAX4 (38); and the incretin hormone GLP-1 (39) or one of its analogues/homologues. In situ gene transfer to pancreas following endoscopic retrograde cannulation of the pancreatic duct with one or more of these factors may ultimately have a role in restoration of b-cell function in non-transplant recipients with Type 1 diabetes (40).

GENE THERAPY APPROACHES TO INSULIN REPLACEMENT It has been made clear throughout this book that severe limitations in the number of suitable donor organs will restrict availability of islet transplantation to a small percentage of those with Type 1 diabetes. It is hoped that approaches to prevent b-cell loss or restore endogenous insulin secretion will ultimately preclude the need for transplantation altogether. Augmentation of islet function, enhancement of engraftment, and prevention of immune rejection employing one or more of the approaches described in this and other chapters may enable consistent long-term insulin independence from a single donor organ. Further approaches to generate tissue banks of insulin-secreting cells sufficient for all suitable recipients with diabetes are, however, required. Detailed phenotyping of newly derived cells beyond expression of the insulin gene and positive insulin immunocytochemical staining will be imperative before any consideration of clinical trials (41). Physiological insulin expression, biosynthesis, processing, and secretion by the normal b-cell is briefly outlined below as a benchmark for comparison with genetic engineering approaches to insulin replacement. Some key components of a normal functional b-cell are illustrated in Figure 1.

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Glucose

GK

Metabolism

Glucose

G-6-P

SAPK2

K+ ATP:ADP + + + +

P Cytoplasm

PDX-1

Membrane depolarisation

Nucleus Insulin granules

2+

Ca

Ca-gated insulin secretion

P insulin mRNA

Constitutive secretory pathway

Glucose-responsive insulin gene expression Endoplasmic reticulum

Golgi apparatus

Secretory vesicles

Figure 1 The physiological b-cell. Key components of a normally functional pancreatic b-cell are highlighted, including GLUT2 and GK expression constituting the “glucose sensor,” metabolism-induced PDX1 translocation to the nucleus and binding to the insulin gene promoter to mediate “glucose-responsive insulin gene expression,” and closure of the ATP-dependent potassium channel leading to membrane depolarization and calcium influx mediating “Ca-gated insulin secretion” from storage granules within the regulated secretory pathway. Abbreviations: ADP, adenosine diphosphate; ATP, adenosine triphosphate; GK, glucokinase; GLUT2, glucose transporter 2.

Physiological Glucose-Responsive Insulin Gene Expression Insulin synthesis in the b-cell is dependent on transcription and translation of the human insulin gene located on the short arm of chromosome 11. Regulation of insulin gene transcription is dependent on promoter sequences located upstream of the transcriptional start site and the interaction of these discrete elements with a number of b-cell transcription factors. Key cis-acting elements include conserved C, E, and A elements in addition to the Z region in the human gene (42). Glucose uptake into the b-cell is facilitated by the high Km glucose transporter (GLUT2). This allows rapid equilibration of intracellular with circulating glucose levels. Glucose is then phosphorylated by glucokinase, which also has a high Km (11mmol/L) and is the key enzyme regulating flux through the glycolytic pathway (43). Glucose phosphorylation in response to raised glucose within the b-cell mediates PI3K and SAPK2 phosphorylation. This results in PDX1 modification including phosphorylation by activated PDX-1-kinase (44), enabling translocation of PDX-1 to the nucleus where it binds the four ‘A-boxes’ of the insulin gene promoter directly upregulating gene

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transcription (45). Insulin biosynthesis is further regulated at the level of translation, which is undoubtedly glucose-responsive in the physiological b-cell, although the underlying mechanisms remain incompletely understood (46). Physiological Proinsulin Biosynthesis and Processing Insulin is synthesized as a larger precursor, preproinsulin. The N-terminal signal peptide sequence targets newly synthesized proinsulin to the endoplasmic reticulum through a ubiquitous mechanism involving prepeptide cleavage by signal peptidase (47). Proinsulin is transferred through the trans-Golgi network where it is packaged into immature clathrincoated secretory granules that develop into insulin storage granules (48). The b-cell in common with other neuro-endocrine cells contains a regulated secretory pathway in addition to the ubiquitous constitutive pathway for protein secretion (49). Greater than 99% of newly synthesized proinsulin enters the regulated secretory pathway where two specific endoproteases PC2 and PC3 are uniquely expressed (50). These are responsible for posttranslational processing to mature insulin, which is completed by removal of two basic arginine residues by ubiquitously expressed carboxypeptidase E (51). Physiological Insulin Secretion Physiological glucose homeostasis is tightly controlled predominantly by minute-to-minute variation in b-cell secretion of pre-formed insulin via the regulated secretory pathway. GLUT2-mediated glucose uptake and glucokinase-mediated phosphorylation regulates flux through the glycolytic pathway. Glucose metabolism increases the adenosine triphosphate (ATP)/ adenosine diphosphate (ADP) ratio, leading to closure of ATP-dependent potassium channels and membrane depolarization, opening voltage-gated calcium channels, which, in turn, stimulates exocytosis of pre-formed insulin storage granules (52). Derivation of Proliferative b-Cells The limited capacity for b-cell proliferation within islets or as a single phenotype in cell culture has led to generation of a number of transformed b-cell lines. Sources include a radiation-induced rat insulinoma (RinM5F) (53), isolated hamster islets transformed with SV40 virus, and b-cell tumors induced in transgenic mice by SV40 large T-antigen expression (bTC and MIN6). Formation of proliferative cell lines is, however, associated with dedifferentiation altering the underlying phenotype. Insulin content may be

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decreased. Expression of the specific glucose transporter (GLUT2) and glucose phosphorylating enzyme (glucokinase), which together constitute the glucose sensor, tend to decrease with concomitant increase in more ubiquitous GLUT1 and hexokinase expression (54). This results in induction of insulin secretion at sub-physiological glucose concentrations with potential for dangerous hypoglycemia. There have been extensive efforts to iteratively engineer these cell lines to stably express human insulin, GLUT2 and glucokinase (55). Further manipulation would, however, be required to ablate expression of endogenous animal insulin, hexokinase, and GLUT1, and this approach has been largely superseded in recent years. Generation of human b-cell lines from insulinoma tissue or by oncogenic transformation of human islets has proved much more challenging. Cells with at least limited capacity to replicate in culture have been derived from patients with persistent hyperinsulinemic hypoglycemia of infancy, which is characterized by inappropriate constitutive insulin secretion and often treated by pancreatic resection. Stable transfection of one PHHI-derived cell line with the two components of the dysfunctional KATP channel (SUR-1 and Kir6.2) in addition to the key b-cell transcription factor PDX1 resulted in restoration of insulin secretion in response to changes in glucose concentration within the physiological range (56). Unregulated tumorigenic proliferation is a further hurdle to clinical transplantation of b-cell lines. Efrat and colleagues, however, pioneered a conditional b-cell immortalization approach in which transgenic mice expressing SV40 large T antigen under the control of a tetracyclineresponsive promoter were crossed with transgenic mice expressing a tetracycline-repressible transcriptional repressor downstream of the rat insulin promoter (57). Derived b-cells (designated bTCtet cells) proliferated well in vitro and led to normalization of hyperglycemia following intraperitoneal injection in mice with streptozocin-induced diabetes. Continued proliferation in vivo led to fatal hypoglycemia. Implantation together with a slow release tetracycline pellet attenuated further proliferation, with maintenance of euglycemia for up to 4 months. Proliferative b-cells have been successfully derived from human islets by over-expression of dominant oncogenes but this has been associated with dedifferentiation (58). Reversible immortalization of human islet-derived b-cells has been reported following transfection with lox-P flanked SV40 large T-antigen and human telomerase reverse transcriptase cDNAs (59). Non-tumorigenic clones were selected and infected with an adenoviral crerecombinase vector enabling removal of immortalization genes. Characterization following formation of three-dimensional cell aggregates in vitro confirmed b-cell specific gene expression, insulin storage within secretory granules with insulin content 40% of control islets, and glucose/ alternative secretagogue-stimulated insulin secretion. Sub-renal capsule

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transplantation in mice with steptozocin-induced diabetes normalized glucose levels for 30 weeks with no evidence of oncogene expression or tumor formation. Transdifferentiation/Neogenesis Approaches The potential for generating new end-differentiated non-tumorigenic b-cells following proliferation, differentiation, and selection of embryonic or adult stem cells is considered in detail in Chapter 18. Transdifferentiation approaches may ultimately enable derivation of fully functional b-cells from hepatocytes or non-endocrine pancreatic cells in situ or ex vivo as explored in Chapter 17. Neogenesis and transdifferentiation gene therapy strategies have included transduction with fluorescent marker or antibiotic resistance genes under the control of b-cell specific promoters to enable cell selection and sorting, in addition to transfection with key b-cell transcription factors (PDX1) (60), NGN-3 (61), MafA (62), and differentiation factors (GLP1). Insulin Gene Transfer to Non-b-Cells The ultimate goal of physiological glucose responsive insulin secretion may not be attainable by genetic manipulation of non-b-cells, given the highly specialized mechanism by which insulin gene transcription and secretion of fully-processed insulin is regulated by glucose uptake and metabolism in the normal pancreatic b-cell. However, the potential for long-term insulin replacement following in situ gene delivery without the need for transplantation and avoiding both alloimmune and autoimmune rejection merits further study. This may provide a cost-effective therapy with sufficient utility for the growing millions of individuals currently reliant on insulin injections, many of whom have sub-optimal control with associated risk of long-term complications and severe hypoglycemia. Ex Vivo Insulin Gene Transfer to Neuro-Endocrine Cells Neuro-endocrine cells express the regulated secretory pathway, together with the specific endoproteases, PC2 and PC3, necessary for cleavage of proinsulin to bioactive insulin. Transfection of the mouse pituitary corticotrophin cell line (AtT20) with an insulin gene construct resulted in secretion of fully processed insulin via the regulated secretory pathway, induced by membrane depolarization in the presence of calcium (63). Secretion was not glucose-responsive despite endogenous glucokinase expression. Cotransfection with GLUT2 resulted in insulin secretion in response to glucose, but in the sub-physiological range with maximum stimulation

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occurring at glucose concentration of 10 µmol/L (64). Intraperitoneal injection of insulin over-expressing AtT20 cells delayed onset of hyperglycemia induced by streptozocin by 2 weeks (65,66). Subsequent hyperglycemia may have been due to insulin resistance caused by co-secretion of ACTH. Downregulation of diabetogenic hormone expression would thus be necessary before clinical use of ex vivo engineered neuro-endocrine cells could be considered.

In Situ Insulin Gene Transfer to Neuro-Endocrine Cells K cells are endocrine cells located in the stomach, duodenum, and jejunum of mammals. They synthesize the hormone glucose-dependent insulinotropic polypeptide (GIP), which is produced and secreted in response to food ingestion in a similar way to regulation of b-cell insulin secretion according to changes in blood glucose. GIP is a potent stimulator of insulin secretion (67). K cells express PC2 and PC3 within the regulated secretory pathway in addition to glucokinase but not GLUT2 (68). The GIP promoter has been sub-cloned upstream of a human proinsulin construct and employed to stably transfect GTC-1 cells, a GIP expressing K-cell line. These cells synthesized, stored, and secreted mature insulin in response to nutrient stimulation (68,69). Transgenic expression of this construct in mice resulted in K-cell–specific insulin expression and protection from hyperglycemia induced by treatment with streptozocin in comparison to control animals (69). Meal-stimulated secretion of mature processed insulin has also been achieved in transgenic mice in which the human insulin gene has been expressed in gastric G cells under the control of the gastrin promoter (70). Specific and safe targeted gene transfer to K or G cells in vivo, possibly following oral or endoscopic delivery would, however, be required for successful clinical translation (71).

Insulin Gene Transfer to Non-Endocrine Cells Non-endocrine cells lack the regulated pathway necessary for minuteto-minute insulin storage and secretion in addition to proinsulin processing. Insulin gene transfer would thus be expected to mediate constitutive secretion of unmodified proinsulin with less than 20% of the biological activity of insulin. Several groups have engineered mutant preproinsulin constructs in which PC2 and PC3 cleavage sites at A-chain/C-peptide and B-chain/ C-peptide junctions have been altered to form tetrabasic consensus sites enabling recognition and cleavage by furin, a protease expressed ubiquitously in the trans-Golgi network of eukaryotic cells (72).

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An alternative approach has been explored in which insulin A- and B-chains were expressed linked by a peptide much shorter than the 35 residue C-peptide, a short-turn–forming heptapeptide (Gly-Gly-Gly-ProGly-Lys-Arg). The recombinant modified insulin showed much higher biological activity than proinsulin, but lower activity than human insulin, both with regard to receptor binding and glucose uptake activity, without requiring enzymatic processing (73). Transfection of the human insulin gene into a monkey kidney fibroblast cell line resulted in the expression of proinsulin but not mature, active insulin, due to the lack of PC2 and PC3 protease (74). Transfection of murine NIH 3T3 fibroblasts with a retrovirus expressing a furin-cleavable human preproinsulin gene enabled secretion of fully-processed biologically active insulin (75). In vivo implantation studies have, however, been complicated by hypoglycemia due to unregulated cell proliferation with increasing insulin secretion.

Hepatic Insulin Gene Therapy Insulin gene therapy directed to liver has several potential advantages. Hepatocytes share a glucose-sensor comparable to that in pancreatic b-cells expressing both GLUT2 and glucokinase (76). Transfection of host hepatocytes in situ can be achieved following simple intravenous injection of viral vectors or plasmid DNA pre-complexed with PEI or alternative adjuvant agent (76). Host cell transduction following injection of pharmaceutical grade vector has particular utility and cost-effectiveness for widespread clinical utilization. It avoids the need for cell culture and transplantation in addition to circumventing alloimmune rejection. Uptake and incorporation into a stable end-differentiated tissue also prevents tumorigenic proliferation with associated risk of hypoglycemia. Hepatocytes lack the regulated secretory pathway for regulated secretion of pre-formed and fully processed insulin but do benefit from endogenous genes, which are physiologically upregulated by glucose and downregulated by insulin. This offers the potential for glucose-responsive transcriptional regulation by cloning the insulin transgene downstream of regulatable promoters, including those derived from L-type pyruvate kinase, phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and GLUT2. A number of groups have explored this approach in vitro and in vivo employing furin-cleavable or single chain insulin (73) transgenes (77). The intrinsic time delay required for insulin gene transcription and translation in response to elevated glucose leads to potential for persistent hyperglycemia early after a meal with later risk of hypoglycemia. The most physiological results have been attained employing three copies of the L-PK promoter glucose-responsive element in combination with an inhibitory element

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from the insulin-like growth factor–binding protein gene in an adenoviral vector delivered to the portal vein of rats with streptozocin-induced diabetes (78).

Muscle-Targeted Insulin Gene Therapy Constitutive secretion of fully processed human insulin for up to 5 weeks has been obtained following simple injection of naked plasmid DNA encoding a furin-cleavable insulin construct into accessible skeletal muscle in rodents (79). Efficiency has been enhanced by use of in situ electroporation (80). Glucose lowering without dangerous hypoglycemia has been confirmed in mice with streptozocin-induced diabetes (81,82). Transcriptional regulation of proinsulin secretion has been confirmed following oral tetracycline administration, offering the potential for regulating basal constitutive insulin replacement (83). The promoter of the gene encoding rat transforming growth factor-b expressed upstream of a furin gene construct has been used to achieve glucose-regulated processing of transgenically expressed furin-cleavable human proinsulin in smooth muscle cells transplanted into diabetic BB rats (84). An elegant approach to storage and rapid ligand-mediated secretion of pre-formed insulin in non-endocrine cells has been developed (85). This is dependent on conditional aggregation within endoplasmic reticulum mediated by mutated FK506 binding protein domains expressed upstream of a furin cleavage site and the insulin transgene. Administration of a nonimmunosuppressive rapamycin analogue results in disaggregation and protein delivery to the trans-Golgi network, where furin cleaves off the FK506 domains and processes proinsulin secreted by the constitutive secretory pathway. Pharmacokinetics closely mimicking physiological postprandial insulin secretion have been demonstrated in transfected fused human muscle fibers in vitro (2).

GENE TRANSFER WITH NON-INSULIN GLUCOSE-LOWERING GENES Insulin Sensitizers Enhancement of insulin action by over-expression of genes that increase glucose uptake and storage by liver and muscle may provide new therapeutic options for insulin-resistant Type 2 diabetes. Over-expression of hepatic glucokinase has been shown to normalize fasting blood glucose in rodent models of insulin-deficient (86) and insulin-resistant (87) diabetes. The potential for excessive glycogen and fat accumulation in liver in addition to an increase in circulating free fatty acids and triglycerides following unregulated glucokinase over-expression is, however, a concern (88).

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A second potential target in liver is the glycogen-targeting subunits of protein phosphatase-1. These proteins target protein phosphatase-1 to the glycogen particle and bind differentially to glycogen synthase, glycogen phosphorylase, and phosphorylase kinase, serving as molecular scaffolds (89). A prominent member of this family is “protein targeting to glycogen” (PTG), which is expressed in a wide range of tissues (90). It has been demonstrated that glucose can be diverted to glycogen in normal rodents by the over-expression of PTG (91). A novel subunit, GM∆C, has been developed with a unique combination of glycogenic potency and responsiveness to glycogenolytic signals (92). Adenoviral hepatic over-expression of GM∆C prevented glucose intolerance in rats fed a high fat diet. Transgenic mice engineered to express liver glucokinase in the muscle demonstrated increased insulin sensitivity (93) and did not develop obesity, insulin resistance, or hyperglycemia on being fed a high fat diet (94). Transplantation of glucokinase-expressing myotubes attenuated hyperglycemia in mice with streptozocin-induced diabetes (95). In situ adenoviral glucokinase vector delivery to muscle also increased glucose uptake (96). Although this improved insulin sensitivity in obese rats, it did not prevent or delay the appearance of hyperglycemia and hyperinsulinemia (97). Transgenic mice over-expressing the muscle glucose transporter GLUT4 demonstrated improved glucose tolerance, which was not further enhanced by concomitant over-expression of hexokinase II (98). Most recently, intramuscular injection of streptozotocin into diabetic mice with adenoassociated viral vectors constitutively expressing insulin and hepatic glucokinase resulted in restoration of fasting and fed normoglycemia for more than four months (99). It is envisaged, however, that successful clinical translation would necessitate glucokinase gene transfer to a major proportion of total skeletal muscle to sufficiently enhance whole body glucose uptake.

Anti-Obesity Gene Therapy Increasing understanding of the neuro-endocrine modulators of appetite and weight has uncovered a wide range of novel targets for gene therapy, offering the potential for weight loss in obesity with enhanced insulin sensitivity and improved glucose tolerance in Type 2 diabetes. Decreased food intake and a reduction in body weight with lower insulin levels and maintained normoglycemia has been attained in non-diabetic mice following electroporation-enhanced plasmid-mediated leptin gene transfer to muscle (100). The leptin gene has also been transferred directly to brain by intracerebroventricular injection of an adenoassociated viral vector in an attempt to circumvent systemic pleiotropic effects of leptin [reviewed in (101)]. Normalization of insulin resistance with improved glucose tolerance

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and decreased levels of circulating fatty acids has also been demonstrated in mice with diet-induced obesity after muscle-targeted gene transfer of an adiponectin gene (102).

GENE THERAPY TARGETED AT DIABETIC COMPLICATIONS Finally, there are many potential gene therapy applications targeted at the long-term complications of diabetes that are beyond the scope of this chapter. Examples include pro-angiogenic therapy for atherosclerotic vascular disease (103). Clinical trials have recently been undertaken in which VEGF expression vectors were delivered intramuscularly to individuals with diabetes complicated by critical limb ischemia (104). In another trial, an adenoviral VEGF construct was injected directly into the myocardium of patients with severe angina (105). Results of these trials remain preliminary but safety and initial outcome data are encouraging. Gene therapy approaches for diabetic retinopathy directly targeted to the eye within the blood-retinal barrier have recently been reviewed (106). As a circulating therapeutic protein, erythropoietin is an attractive candidate for replacement following muscle-targeted gene delivery (107). This may ultimately have a role in treatment of anemia secondary to diabetic nephropathy. Similarly, muscle-targeted gene transfer of neurotrophic or neuroprotective factors in addition to delivery to the dorsal root ganglion employing herpes simplex virus-derived vectors is being explored (108).

CONCLUSION In recent years, it has often been stated that gene therapy has failed to realize its initial promise and is in many ways being superseded by alternative approaches including stem cell–derived transplantation. On the contrary, clinical success with a wide range of gene therapy approaches is accruing, and there have been dramatic recent strides in gene delivery avoiding the toxicity of early generation vectors. There is huge potential for gene transfer approaches to complement islet transplantation and b-cell replacement therapy following ex vivo transduction prior to implantation. There is also considerable as yet untapped future potential for in situ delivery of genes to individuals with diabetes without the need for transplantation or immunosuppression. Therapeutic genes may prevent diabetes progression, maintain b-cell function, enable replacement of circulating insulin and noninsulin glucose lowering peptides, or directly target long-term complications. Moreover, these approaches offer sufficient utility for cost-effective implementation in the huge number who may benefit globally with Type 1 and potentially also Type 2 diabetes.

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97. Jimenez-Chillaron JC, Telemaque-Potts S, Gomez-Valades AicG, Anderson P, Newgard CB, Gomez-Foix AM. Glucokinase gene transfer to skeletal muscle of diabetic Zucker fatty rats improves insulin-sensitive glucose uptake. Metabolism 2002; 51(1):121–6. 98. Lombardi A, Moller D, Loizeau M, Girard J, Leturque A. Phenotype of transgenic mice overexpressing GLUT4 and hexokinase II in muscle. FASEB J 1997; 11(13):1137–44. 99. Mas A, Montane J, Anguela XM, et al. Reversal of Type 1 Diabetes by Engineering a Glucose Sensor in Skeletal Muscle 10.2337/db05-1615. Diabetes 2006; 55(6):1546–53. 100. Wang XD, Liu J, Yang JC, Chen WQ, Tang JG. Mice body weight gain is prevented after naked human leptin cDNA transfer into skeletal muscle by electroporation. J Gene Med 2003; 5(11):966–76. 101. Kalra SP, Kalra PS. Gene-transfer technology: a preventive neurotherapy to curb obesity, ameliorate metabolic syndrome and extend life expectancy. Trends in Pharmacological Sciences 2005; 26(10):488–95. 102. Park JH, Lee M, Kim SW. Non-viral adiponectin gene therapy into obese type 2 diabetic mice ameliorates insulin resistance. Journal of Controlled Release 2006; 114(1):118–25. 103. Yla-Herttuala S. An update on angiogenic gene therapy: vascular endothelial growth factor and other directions. Curr Opin Mol Ther 2006; 8(4):295–300. 104. Kusumanto YH, van Weel V, Mulder NH, et al. Treatment with intramuscular vascular endothelial growth factor gene compared with placebo for patients with diabetes mellitus and critical limb ischemia: a double-blind randomized trial. Hum Gene Ther 2006; 17(6):683–91. 105. Stewart DJ, Hilton JD, Arnold JM, et al. Angiogenic gene therapy in patients with nonrevascularizable ischemic heart disease: a phase 2 randomized, controlled trial of AdVEGF(121) (AdVEGF121) versus maximum medical treatment. Gene Ther 2006. 106. Ting JHY, Martin DK. Basic and Clinical Aspects of Gene Therapy for Retinopathy Induced by Diabetes. In: Current Gene Therapy:193–214. 107. Prudhomme GJ, Glinka Y, Khan AS, Draghia-Akli R. ElectroporationEnhanced Nonviral Gene Transfer for the Prevention or Treatment of Immunological, Endocrine and Neoplastic Diseases. In: Current Gene Therapy:243–73. 108. Mata M, Chattopadhyay M, Fink DJ. Gene therapy for the treatment of sensory neuropathy. Expert Opin Biol Ther 2006; 6(5):499–507.

Index

Absolute neutrophil count (ANC), 165 Acetaminophen, 274 Addison’s disease, 67 Adenosine diphosphate (ADP), 255 Adenosine triphosphate (ATP), 32, 83, 100–102, 255 Adjuvant therapies, 160 Adult stem cells (ASCs), 312, 313–317 extrapancreatic stem cells, 314–317 pancreatic stem cells, 313–314 Alanine-amino transferase (ALT), 163 Alemtuzumab (Campath 1H), 153–154 dose, 154 mechanism of action, 153–154 side effects, 154 Allografts, islet after pancreatectomy, 11 fetal islet allografts in, 14 rejection, 2 in Type 1 diabetes, 11–13 Alloislet transplantation, 197–199 Alpha cell function, 198 Anemia, 163 Angiogenesis, 5 Angiotensin converting enzyme inhibitors (ACEIs), 170 Anticoagulation, 159 Antigen-independent approaches, for diabetes, 331–332 Antigen-specific tolerization approaches, for diabetes, 331 Anti-insulins, 33 Anti-obesity gene therapy, 341–342 Antiplatelet agents, 66 Aphthous ulcers, 166

Apoptosis, attenuation of, 332 Arginine stimulation test, 182–183 Aspartate-amino transferase (AST), 163 Aspects and challenges of islet isolation, 115–129 challenges in islet assessment, 124–126, See also under Islet assessment islet culture, 126–128: vitamins on islets, 127–128 islet purification, 122–124 pancreas digestion, 120–122: low enzymatic activity, 121 donor selection, 118–120 donor variables assessment form, 119 pancreas physical properties assessment form, 119 Assessment, islet challenges in, 124–126 islet viability, 126 islet yield, 124–126 ATG (Thymoglobulin), 152–153 Atovaquone (Mepron), 160 Autografts, islet, 10–11 Autoimmune disease, 67 Autoimmune recurrence, 12 Autoislet recipients, metabolic studies, 195–197 Avitene, 240 Azathioprine, 277–278

Beta (b) cells Beta score in islet graft function assessment, 181

351

352 [Beta (b) cells] function in Type 1 diabetes, maintenance, 330–333 regeneration and neogenesis, 293–305, See also separate entry: enhancing, 303–305 GLP-1, 301–303 pancreatic b-cell mass, regulation of, 294–296 regulatory mechanism of, 32–33 replacement, stem cell approaches for, 311–321, See also Stem cell approaches Blood glucose concentration and insulin secretion, link between, 32 Body mass index (BMI), 84, 168, 295 islet isolation and, 118–119 Bromocriptine, 162

Calcineurin inhibitor therapy, 271 California Transplant Donor Network (CADN), 222 Carbamazepine, 162 Cardiovascular disease and risk factors, 66 antiplatelet agents, 66 Care of islet transplant recipient, 147–174, See also Immunosuppressive management and Complications Carrel patch, 91 Catheter placement, 136 Cell reprogramming, 320–321 Celsior solution, 82, 99, 109 Centers for Medicare and Medicaid Services (CMS), 284 Ceropithecus aethiops, 285 Challenges in new islet transplant program setting, 203–213 Associated Research Program, 212–213 clinical islet transplantation, 209–212 ethical approval, 210 islet transplant procedure, 211–212 multidisciplinary islet transplant team, establishing, 209–210 patient recruitment, 210–211 experimental context, 203–204 islet isolation, 206–209 clean room facilities requirement, 207 personnel requirement, 209

Index [Challenges in new islet transplant program setting islet isolation] technical challenges, 207 pancreas procurement, 204–206 Cimetidine, 162 Cisapride, 162 Clarithromycin, 162 Clinical islet transplantation (CIT), 57–64, 258 challenges in, 209–212, See also under Challenges clinical assessment of individuals seeking, 73–74 contraindications to, 61–62 age, 61 alcohol, 62 drugs, 62 infection and neoplasia, 62 obesity and insulin resistance, 61 psychiatric disease, cognitive impairment, & non-compliance, 62 smoking, 62 current outcomes of, 229–244, See also individual entry goals of, 58 indications for, 58–61 glycemic lability, 60 hypoglycemia and hypoglycemia unawareness, 59–60 Type 1 diabetes, 59 modern immunosuppression for, 274–275, See also under Contemporary immunosuppression unrealistic expectations and misconceptions, 69–70 Clinical trials setting in islet cell transplantation, factors to consider, 215–227 center infrastructure, 216–218 budget, 217–218 personnel, 216–217: clinical personnel, 217 islet isolation personnel, 217 consenting, 223 data and safety monitoring board, 226–227 data management, 223–224 informed consent, key elements, 224 key elements to be included, 220–221 project management, 216, 218–219 protocol development, 219

Index [Clinical trials setting in islet cell transplantation, factors to consider project management] training by experienced islet cell transplantation centers, 219 recruitment, 221–223 regulatory issues, 225–227 institutional review board (independent ethics committee), 225–226 Investigational New Drug (IND) application, 225 regulatory, 216 space, 218 Standard Operating Procedures (SOPs), 224–225 UNOS and local OPO, 219–225 Clotrimazole, 162 Coaxial protocols, 319–320 Cold swelling, 101 Collaborative Islet Transplant Registry (CITR), 275 Concomitant medications anti-inflammatory therapy, 158–159 Etanercept, 158–159, See also separate entry Connaugh Medical Research Laboratories (CMRL), 253 Contemporary immunosuppression in human islet transplantation animal pre-clinical models, 270 development and application of, 269–280 Edmonton immunosuppression protocol, 271–272 guidelines for, 274 induction and anti-cytokine agents, 272–273 for islet-after-kidney transplantation, 276–280 modern immunosuppression for, 274–275 clinical islet transplantation, 270–271 north American experience, 275–276 Continuous glucose monitoring systems (CGMS), 60 Core-to-mantle perfusion, 5 Cultivated human islet buds (CHIBs), 313 Culture and transportation of human islets between centers, 251–263 cultured islet preparations advantages, 252 development methods, 252

353 [Culture and transportation of human islets between centers] human islet culture, 252–258, See also separate entry practicality, 252 safety, 252 shipping human islets between centers, 258–262 islet collection and assessment at the remote site, 262 shipping bag, 259–261 shipping container, 261–262 temperature control, 262 Current Good Manufacturing Practices (cGMP), 258 Current outcomes of clinical islet transplantation, 229–244 culture, 233–234 efforts to improve outcomes, 230–234 enzyme preparation, 232–233 future directions, 244 improving engraftment, 244 tolerance induction, 244 insulin independence, 234–236 islet isolation, 232 long-term diabetes complications, prevention of, 238–240 metabolic control, 236–238 new strategies to improve, 242–244 islet-after-kidney transplantation, 243–244 living-related donation (LRD), 243 single donor islet transplantation, 242–243 pancreas preservation and transportation, 232 pancreas procurement, 231 post-transplant complications, 240–242 immunosuppression-related, 241 procedure-related, 240–241 Custodial, 82, 99 Cyclosporine (CyA) (Neoral), 157, 162, 278 dose, 157 mechanism of action, 157 side effects, 157 Cytochrome P-450 enzymes, 160–161 CYP3A, 161 CYP3A4 iso-enzyme, 161 Cytomegalovirus (CMV), 160, 171–172, 241 cytomegalovirus prophylaxis, 65

354 Daclizumab (Zenapax), 148–151, 152, 275 dose, 151 mechanism of action, 148–151 side effects, 151–153 Danazol, 162 Dapsone, 160 Delavirdine, 162 Density gradient centrifugation, 123 Department of Health and Human Services (DHHS), 226 Diabetes Diabetes Control and Complications Trial (DCCT), 29–30 diabetes gene therapy, 327–342, See: Gene therapy for diabetes diabetic rodents, transplantation studies in, 4 and pancreas, link between, 3 treatment by transplantation, 45–46 Digital image analysis (DIA), for islet volume determination, 125 Diltiazem, 162 Diphenhydramine 50, 274 Disseminated intravascular coagulation (DIC), 122 Donor selection for islet isolation, 118–120 Dyslipidemia, 168

Edema, 169 Edmonton Protocol, 148, 163, 234–235, 240, 252 Efavirenz, 162 Electrolyte disorders, 169 hypomagnesemia, 169 hypophosphatemia, 169 Embryonic stem cells (ESCs), 312, 315–317 insulin-producing cells from, 317 Endocrine replacement therapy, 3 Enhanced islet engraftment, 332–333 Enzymatic activity and pancreas digestion, 121 Edmonton immunosuppression protocol, 271–272 Epstein Barr virus (EBV), 242 Erythromycin, 162 Etanercept (Enbrel), 152–153, 158–159, 273, 275 dose, 159

Index [Etanercept (Enbrel)] mechanism of action, 158 side effects, 159 Ethidium bromide (EB), 126 Euro-Collins solution, 7, 123 Euro-FicollTM, 7, 123 Ex vivo insulin gene transfer to neuroendocrine cells, 337–338

Female reproductive system, alterations of, 172 Fetal calf serum (FCS), 127 FicollTM, 4, 7, 11 FK binding protein (FKBP), 155 Fluconazole, 162 Fluorescein diacetate (FDA), 126 Food and Drug Administration (FDA), 256, 286 Function after transplantation, of islet, 194–199 alloislet transplantation, 197–199 metabolic studies of autoislet recipients, 195–197

Ganciclovir, 65 Gastrointestinal disorders, 167 Gastroparesis, 65 Gatifloxacin, 162 Gene therapy for diabetes, 327–342 anti-obesity gene therapy, 341–342 b-cell function in Type 1 diabetes, maintenance, 330–333 antigen-independent approaches, 331–332 antigen-specific tolerization approaches, 331 attenuation of apoptosis, 332 augmentation within intact islets, 333 enhanced islet engraftment, 332–333 concepts and possibilities, 327–330 viral vectors, 328–329, See also separate entry disease progression prevention, 330–333 strategies for, 330: primary prevention, 330 secondary prevention, 330 tertiary prevention, 330

Index [Gene therapy for diabetes] gene transfer with non-insulin glucoselowering genes, 340–342, See also Non-insulin glucose-lowering genes to insulin replacement, 333–340, See also separate entry plasmid vectors, 329 targeted at diabetic complications, 342 Gift of Hope Organ & Tissue Donor Network (ILIP), 222 Gift of Life Donor Program (PADV), 222 Glucagon-like peptide-1 (GLP-1), 301–303 Glucagons glucagon secretion and blood glucose, link between, 33 Glucose phosphorylation, 32 Glucose potentiation of arginine-induced insulin secretion (GPAIS), 195–196, 198 Glucose tolerance tests, 198 Glycemic lability, 60 Glycemic variability, assessment, 181–182 Glycosuria, 2 Good Manufacturing Practice (GMP), 128 Graft, islet, monitoring and imaging, 179–188 Beta Score in, 181 islet function, monitoring, 180–184 glucose stability, 180 glycemic variability, assessment, 181–182 hypoglycemia, burden of, 181–182 hypoglycemic events, 180 overall function, 180 stimulation tests, 180 islet graft biopsy, 184 islet graft imaging, 185–187 molecular monitoring, 184–185 stimulated insulin/C-peptide secretion assessment of, 182–184: arginine stimulation test, 182–183 mixed meal stimulation test, 183 Grapefruit, 162

Health Resources and Services Administration (HRSA), 284 Hematological abnormalities, 163–166 adverse events by frequency of appearance, 164–165 Anemia, 163

355 [Hematological abnormalities] Leukopenia, 163 oral ulcers, 166–167 Thrombocytopenia, 165 Hepatic disorders, 67 Hepatic insulin gene therapy, 339–340 Herpes Simplex Virus (HSV), 167 Histidine-tryptophan-ketoglutarate (HTK) solution (Bretschneider), 82, 103, 105 Human islet culture, 252–258 culture density, 256–257 culture temperature, 257 human islet culture at the University of Miami, 253–258 optimal culture conditions, 253 preparation, 257–258 at the University of Miami, 253–258 Hypercholesterolemia, 238 Hypertension, 168–169 HYPO score, 59, 239 Hypoglycemia burden of, 181–182 counter-regulatory neuro-endocrine response to, 33 definitions of, 30–31 hypoglycemia unawareness, mechanisms of, 35–36 coping strategies, 44 insulin secretion and absence of hypoglycemia, 31–33, See also Insulin new tools and devices, 44–45 treatment by transplantation, 45–46 individual risk, assessing, 37–39 islet transplantation and, 46–47 occurrence, reason for, 34–35 patient selection and assessment, 59–60 predisposing factors for, 39 problems of reversibility, 41–44 protection against hypoglycemia, strategies for, 39–41 risk of, biochemical associations, 40 diabetes related, 40 disorders of food absorption, 40 drugs and exercise, 40 endocrine diseases, 40 reduced insulin clearance, 40 symptoms of, 34 in Type 1 diabetes, 29–48

356 Hypomagnesemia, 169 Hypophosphatemia, 169 Hypothermia metabolic changes during, 100–101 Hypoxia metabolic changes in, 101–103

Immune thrombocytopenic purpura (ITP), 166 Immunosuppression, See also Contemporary immunosuppression; individual entries Immunosuppression, maintenance, 155–158 Cyclosporine (CyA) (Neoral), 157, See also separate entry Mycophenolate Mofetil, 157, See also separate entry Mycophenolate sodium (Myfortic), 158, See also separate entry Sirolimus, 155, See also separate entry Tacrolimus, 156, See also separate entry Immunosuppressive management and complications, 147–174 adjuvant therapies, 160 alternative induction therapies, 153–155 Alemtuzumab, 153, See also separate entry rabbit anti-thymocyte globulin, 154–155, See also separate entry anticoagulation, 159 concomitant medications, 158–159, See also separate entry drug metabolism and interactions, 160–161 female reproductive system, alterations of, 172 frequency of adverse events related to, 152–153 high frequency (61% or greater), 152 low frequency (10% or less), 153 medium frequency (11% to 60%), 152 gastrointestinal disorders, 167 immunosuppressive therapy-related complications, 161–173 elevation in liver function tests, 163 hematological abnormalities, 163–166, See also separate entry maintenance immunosuppression, 155–158, See also Immunosuppression

Index [Immunosuppressive management and complications] malignancy, 173 retinopathy, 173 neurological disorders, 168 prophylactic therapies, 159–160, See also separate entry protocols for, 148 current immunosuppressive protocols, 149–150: flow chart, 151 renal disorders, 168–172, See also separate entry standard induction therapy, 148–153, See also separate entry weight loss, 172 In situ insulin gene transfer to neuroendocrine cells, 338 In vitro differentiation protocols, 318–321 directed differentiation, 319–321 cell reprogramming, 320–321 coaxial protocols, 319–320 spontaneous differentiation, 318 Independent Ethics Committee (IEC), 222 Indiana Organ Procurement Organization (INOP), 222 Infectious diseases, 170–171 Instant blood mediated inflammatory reaction (IBMIR), 244 Institutional or Independent Ethics Committee (IEC), 226 Institutional Review Board (IRB), 222, 225–226 Insulin anti-insulins, 33 independence, 9, 234–236 after islet allograft transplantation, 10 insulin gene transfer to non-b-cells, 337 to non-endocrine cells, 338–339 insulin-like growth factors (IGFs), 316 as normoglycemia controller, 31–33 replacement, gene therapy approaches to, 333–340 ex vivo insulin gene transfer to neuroendocrine cells, 337–338 hepatic insulin gene therapy, 339–340 in situ insulin gene transfer to neuroendocrine cells, 338 insulin gene transfer to non- b-cells, 337

Index [Insulin replacement, gene therapy approaches to] insulin gene transfer to non-endocrine cells, 338–339 muscle-targeted insulin gene therapy, 340 neogenesis approaches, 337 physiological glucose-responsive insulin gene expression, 334–335 physiological insulin secretion, 335 physiological proinsulin biosynthesis and processing, 335 proliferative b-cells, derivation, 335–337 transdifferentiation, 337 secretion and absence of hypoglycemia, 31–33 sensitizers, 340–341 Insulinoma cell line (INS-1E), 320 Intraportal embolization, 5 Intrasplenic autotransplantation, 5 Intravenous glucose tolerance test (IVGTT), 183 Investigational New Drug (IND) application, 225–226 Ischemia reperfusion injury, 102 Islet cell infusion, 138–140 Islet Cell Processing Center (ICPC), 258 Islet cell transplant (ICT), 135 Islet culture, challenges in, 126–128 Islet equivalents (IEQ), 124 Islet graft, See: Graft, islet Islet pluripotent stem cells (IPSCs), 313 Islet purification, challenges in, 122–124 Islet quantification sheet, 125 Islet-after-kidney (IAK) transplantation, 243–244 immunosuppression for, 276–280 patients, 210 Islets of Langerhans, 194 Isolation, islet, 232 aspects and challenges of, 115–129, See also Aspects and challenges automated method for, 233 donor selection for, 118–120 history of, 116–118 pancreas preservation for, 99–110, See also Pancreas preservation with magnetic microspheres, 124 Itraconazole, 162

357 Ketoconazole, 162

Lability Index (LI), 181 Lacy, Paul E., 17–21 Latent autoimmune diabetes in adults (LADA), 311 Leukemia Inhibitory Factor (LIF), 315 Leukopenia, 163 LiberaseTM, 7, 9, 120 Life Alliance Organ Recovery Agency (FLMP), 222 Life Center Northwest Donor Network (WALC), 222 LifeGift Organ Donation Center (TXGC), 222 LifeLink of Georgia (GALL), 222 LifeNet (VATB), 222 Lifeshare of the Carolinas (NCCM), 222 LifeSource Upper Midwest Organ Procurement Organization (MNOP), 222 Liver function tests, 163 Living donor islet transplantation, 13 Living-related donation (LRD), 243 Los Angeles Preservation Solution 1 (LAP-1), 108–109

Macaca mulatta, 285 Macacca fascicularis, 285 Macrophage chemoattractant protein-1 (MCP-1), 256 MAGE (Mean Amplitude of Glycemic Excursions), 60, 181 Magnetic resonance imaging (MRI), 185 Malignancy, 173 Mammalian target of rapamycin (mTOR), 155 Mass after islet transplantation, 193–200, See also Metabolic measures Mean amplitude of glycemic excursions (MAGE), See: MAGE Metabolic measures of islet function function after transplantation, 194–199 and mass after islet transplantation, 193–200 recommended clinical monitoring

358 [Metabolic measures of islet function recommended clinical monitoring] for islet transplant recipients, 199–200 Methylprednisolone, 162, 274 Metoclopramide, 162 Miami-Modified Medium-1 (MM1) for human islet culture, 254–255 Mid-South Transplant Foundation (TNMS), 222 Minnesota/UCSF pilot study, 273 Mixed meal stimulation test, 183, 198 M-Kyoto solution, 122 Molecular monitoring, 184–185 Moxifloxacin, 162 Multi-organ harvesting, 87–88 Multipotent Adult Progenitor Cells (MAPCs), 314 Muscle-targeted insulin gene therapy, 340 Mycophenolate Mofetil (MMF) (Cellcept), 152–153, 157 dose, 158 mechanism of action, 157 side effects, 158 Mycophenolate sodium (Myfortic), 158 Myocardial perfusion imaging (MPI), 66

Nafcillin, 162 Nalgene jar, 86, 88, 93 National Institute of Health (NIH), 226 National Organ Transplant Act (NOTA), 219 Nefazadone, 162 Nelfinavir, 162 Neogenesis, 293–305, 337 animal models of, 296–298 new b-cells through transdifferentiation, 298–300 sources, 299 Neoplasia, 62 Neoral, 155, 278 Neovascularization, 5 Nephropathy, 63–64 Nestin-positive islet-derived progenitors (NIPs), 313 Neurological disorders, 168 Neuropathy, 65 autonomic neuropathy, 65 diabetic neuropathy, 65 Nevirapine, 162

Index New England Organ Bank (MAOB), 222 New Jersey Organ and Tissue Sharing Network (NJTO), 222 New York Organ Donor Network (NYRT), 222 Nicardipine, 162 Nicotinamide adenine dinucleotide (NAD), 255 Non-insulin glucose-lowering genes, gene transfer with, 340–342 insulin sensitizers, 340–341 North American experience of immunosuppression for islet transplantation, 275–276 Nursing coordinator’s perspective in clinical trials setting for islet cell transplantation 215–227, See also Clinical trials

OneLegacy (CAOP), 222 Oral glucose tolerance test (OGTT), 183 Organ Procurement Organizations (OPOs), 218, 284 Organ Procurement and Transplantation Network (OPTN), 219 at the University of Wisconsin (WIUW), 222

Packed cell volume (PCV), 138 Pancreas and diabetes, link between, 3 digestion, 117 challenges in, 120–122 pancreas preservation for islet isolation, 99–110 current preservation solutions, 103–109: EuroCollins solution (EC), 103 membrane stabilizers, 104 buffers, 104 energy substrates, 104 FRIs & ORSSs, 104 electerolytes, 104 additives, 104 Histidine-tryptophanketoglutarate solution (Bretschneider), 105 University of Wisconsin solution, 105–106 twolayer method (TLM), 106–108 Los Angeles Preservation Solution 1 (LAP-1), 108–109 superoxide dismutase (SOD), 108

Index [Pancreas pancreas preservation for islet isolation] metabolic changes: during hypothermia, 100–101 in hypoxia, 101–103 pancreas procurement, 204–206, 231 donor organs allocation for research, 206 models, 204 whole pancreas vs. islet transplant allocation, 205–206 pancreatic b-cell mass regulation of, 294–296 pancreatic stem cells, 313–314 extrapancreatic stem cells, 314–317 pancreatic tissue fragments, transplant, 2 transplantation, 14–16, 115 pancreatic tissue dissociating methods, 117 Pancreatic islet transplantation pancreas procurement for, surgical aspects of, 81–94 back-table preparation and cold storage, 92–93 dissection of pancreas prior to crossclamping, 88–91 donors selection, 83–86: criteria, 84–85 exposure for multi-organ harvesting, 87–88 general surgical principles in, 93 perfusion and cross-clamping and retrieval, 91–92 preparation prior to procurement, 86–87 technique, 83–93 Panel reactive antibody (PRA), 236 Parenchymal track embolization, 140–141 Avitene paste, 140–141, 143 gelatin-sponge, 140 stainless steel or platinum embolization coils, 140 thrombin saturated Gelfoam collagen paste, 140 Tisseal, 140 Patient selection and assessment, 57–77, See also Clinical islet transplantation diabetes complications and other comorbidities, 62–67 autoimmune disease, 67 autoimmune thyroid disease, 67 cardiovascular disease and risk factors, 66 hepatic disorders, 67

359 [Patient selection and assessment diabetes complications and other comorbidities] nephropathy, 63–64 neuropathy, 65, See also individual entry retinopathy, 63 endocrinologist’s perspective, 57–77 islet transplantation assessment, approach to, 67–76, See also under Islet transplantation overall purpose of, 57 Pentamidine Isethionate (Pentam), 160 Pentoxifylline, 274 Percutaneous portal vein access choice of access, 135–136 equipment, 136 islet cell infusion, 138–140 set-up, 140 parenchymal track embolization, 140–141, See also separate entry post-procedural management and followup, 144 pre-procedure requirements and contraindications, 136–137 procedure, 137–138 aseptic technique, 137 sedation, 137 stiffened micropuncture set, 138 procedure-related complications, 141–144 radiological aspects, 135–144 subsequent transplants and secondary interventions, 144 ultrasound, 136 Perfluorocarbon (PFC), 83, 93 Peroxisome proliferator activator gamma (PPAR), 304 Phenobarbital, 162 Phenytoin, 162 Physiology, islet, 194 alpha cell, 194 delta cell, 194 insulin secretion, 194 Islets of Langerhans, 194 Pig islet xenotransplantation, 283–290 in primates, 285–288 Ceropithecus aethiops, 285 Macaca mulatta, 285 Macacca fascicularis, 285 organ shortage and fiscal reality, 283–284

360 [Pig islet xenotransplantation] porcine endogenous retrovirus, potential risk of, 288–290 Plasmid vectors, 329 Pneumocystis carinii prophylaxis, 65, 159–160 Atovaquone (Mepron), 160 Dapsone, 160 Pentamidine Isethionate (Pentam), 160 Trimethoprim-Sulfamethoxazole (Bactrim), 159 Poly adenosine diphosphate ribose polymerase (PARP) inhibition, 255 Polyethyleneimine (PEI), 329 Polyuria, 2 Porcine endogenous retrovirus (PERV), 288–290 Portal embolization, 5 Portal vein thrombosis (PVT), 141–142 Positron-emission tomography (PET), 185–187 Post-transplant amnesia and moving goal posts, 70–71 Post-transplant lymphoproliferative disorders (PTLDs), 242 Prograf, 148 Proliferative b-cells, derivation, 335–337 derived b-cells, 336 proliferative b-cells, 336 Prophylactic therapies, 159–160 Pneumocystis carinii prophylaxis, 159–160, See also separate entry Proteinuria, 169–170 Public Health Service Act (PHS Act), 225

Rabbit anti-thymocyte globulin (ATG, Thymoglobulin), 154–155 dose, 155 mechanism of action, 154 side effects, 155 Rapamune, 148 Renal disorders, 168–172 cytomegalovirus, 171–172 edema, 169 electrolyte disorders, 169 hypertension, 168–169 infectious diseases, 170–171 kidney function alterations, 169 proteinuria, 169–170 skin manifestations, 170

Index Retinopathy, 63, 173 Reverse transcription polymerase chain reaction (RT-PCR), 184 Reversibility, problems of, 41–44 Ricordi continuous digestion chamber, 7, 121 Rifabutin, 162 Rifampin, 162 Rifapentine, 162 Ritonavir, 162

Secretory Unit of Islet Transplant Objects (SUITO), 181 Semi-automated dissociation chamber, 6 Simultaneous islet and kidney (SIK) patients, 210 Simultaneous pancreas-kidney (SPK) grafts, 15, 64 Single donor islet transplantation, 242–243 Sirolimus (Rapamune), 65, 152, 155, 238, 275, 277 dose, 156 mechanism of action, 155 side effects, 156 Skin manifestations, 170 Southwest Transplant Alliance (TXSB), 222 SPIO labeling, 185 Standard induction therapy, 148–153 Daclizumab, 148–151, See also separate entry Standard Operating Procedures (SOPs), 221, 224–225 Stem cell approaches for b-cell replacement, 311–321 adult stem cells (ASCs), 312–317, See also separate entry embryonic stem cells (ESCs), 312, 315–317, See also separate entry in vitro differentiation protocols, 318–321, See also separate entry Superoxide dismutase (SOD), 108 Super-paramagnetic iron oxide (SPIO) labeling, 185

Tacrolimus (Prograf) (FK506), 152–153, 156, 277 dose, 156

Index [Tacrolimus (Prograf) (FK506)] mechanism of action, 156 side effects, 156–157 Thrombocytopenia, 165 Thymoglobulin, 272 Tiseel, 240 Transdifferentiation, 337 new b-cells through, 298–300 Transhepatic catheterization, 136 Transplantation, diabetes treatment by, 45–46 Transplantation, islet, See also Pancreatic islet transplantation assessment, approach to, 67–76 clinical assessment, 72 patient expectations, goals, and aspirations, 68–69 post-transplant amnesia and moving goal posts, 70–71 social, financial, and psychological considerations, 71–72 suggested investigations and additional assessments, 75–76 transplant endocrinologist’s role, 67–68 unrealistic expectations and misconceptions, 69 waiting list and reassessment, 72–76 categories of, 10 components of, 204–213 pancreas procurement, 204–206, See also separate entry early clinical trials of, 9–10 experimental and clinical, 1–21 British Medical Journal article, 1 historical perspective, 1–21 The Lancet article, 3 fetal islet allografts in type 1 diabetes, 14 hypoglycemia AND, 46–47 islet allografts after pancreatectomy, 11, See also Allografts islet autografts, 10–11 Islet Transplantation Activity (1999–2006), 231 islet transplantation alone (ITA), 275 living donor islet transplantation, 13 pancreas transplantation, 14–16 Paul E. Lacy’s lecture on, 17–21 setting, challenges in, 203–213, See also under Challenges

361 [Transplantation, islet] transplant recipient, care of, 147–174, See also Immunosuppressive management and complications Trimethoprim-Sulfamethoxazole (Bactrim), 65, 159 Tumor necrosis factor α (TNF-α), 158 Two-layer method (TLM), 106–108, 232 Type 1 diabetes (T1DM), 57–77 hypoglycemia in, 29–48, See also Hypoglycemia

United Network of Organ Sharing (UNOS), 218 University of Wisconsin (UW) solution, 105–106

Verapamil, 162 Vesicular monoamine transporter 2 (VMAT2), 187 Viral vectors, 328–329 adenoviral vectors, 328 recombinant adenoassociated viral vectors, 328 retroviral vectors, 328 Vitamin E (Trolox), 256 Vitamins on islets, 127–128 Voriconazole, 162

Washington Regional Transplant Consortium (DCTC), 222 Weight loss, 172 World Health Organization (WHO), 128

Xenograft pancreas tissue, 2

Zenapax, 148 Zetia, 168