- Email: [email protected]
Transplantation of the Islets of Langerhans
Transplantation of the Islets of Langerhans: New Hope for Treatment of Type 1 Diabetes Mellitus Eric H. Liu and Kevan C. Herold
For more than two decades, islet transplantation has been pursued as a curative treatment for type 1 diabetes mellitus (T1DM) with little success. It is likely that the failures of the past have involved technical difficulties in harvesting human islets, transplantation of insufficient amounts of islet tissue, the antagonistic effects of immune suppressive drugs, including calcineurin inhibitors and glucocorticoids, graft rejection and recurrent autoimmune disease. More recently, success has been reported in seven out of seven consecutive transplants using approaches that overcome the technical and therapeutic problems of the past. Although this success is noteworthy, issues remain that preclude the general application of islet transplants for treatment of the majority of patients with T1DM. These include the need for chronic immunosuppression and the requirement of large numbers of islets. Efforts are under way, using a variety of immunological, molecular and cellular strategies, to make this promising treatment available to the majority of patients with this disease. Since Banting and Best first isolated insulin (then known as the ‘internal secretion’) from purified islets of Langerhans on 30 July 1921, and showed that it could reduce the level of blood sugar in a patient with type 1 diabetes mellitus (T1DM), the replacement of pancreatic islets has remained the single best hope for a cure of the disease1. However, the task has proven to be more difficult than originally anticipated. Minkowski first proposed the idea of injecting the extract of pulverized pancreas in 1889, but found the toxic effects unacceptable. Seventy-six years later Moskalewski demonstrated that islets could be purified from a guineapig pancreas2. Since then, much work
E.H. Liu and K.C. Herold are at the Naomi Berrie Diabetes Center, Departments of Surgery and Medicine, College of Physicians and Surgeons, Columbia University, 1150 St Nicholas Ave, New York, NY 10032, USA. Tel: 11 212 304 5492, Fax: 11 212 304 5493, e-mail: [email protected]
TEM Vol. 11, No. 9, 2000
has gone into the refinement of the isolation procedure in numerous species, including humans. The rationale for islet transplantation is to provide physiological control of the metabolic state of T1DM and to prevent the long-term complications of the disease. The need for this has been emphasized by the findings of the Diabetes Control and Complications Trial: intensive control of the disease was shown to reduce and delay the incidence of diabetes-related complications3. Although some individuals can achieve near-normal control of glucose levels with use of insulin injections, this cannot be done without an increased risk of severe hypoglycemia, estimated to be three–four times more common than with therapy with more relaxed goals of glucose control. Recurrent hypoglycemia with loss of glucagon and catecholamine responses can lead to hypoglycemia unawareness, which requires conscientious monitoring of glucose levels to avoid neurological and other cognitive
impairment. Thus, while the need for normal metabolic control of diabetes has now been clearly shown, a feasible means for achieving this physiological control has not been established. Whole pancreas transplantation can provide normal metabolic control of diabetes4. In fact, this approach to treatment has already been shown to prevent recurrent renal disease in recipients of kidney grafts, reverse existing lesions of diabetic complications, and improve mortality in a group of patients with autonomic neuropathy5–7. However, transplantation of the whole organ requires major surgery, lifelong immunosuppression, and imposes other risks, so that most endocrinologists have recommended this procedure to their patients only when a kidney transplant was also required. Islet transplantation offers the advantages of being minimally invasive, and thus entailing reduced risk. The safety, potential efficacy and minimal risks of the procedure account for the persistent interest in it. In addition, islet transplantation offers a novel clinical setting for testing new strategies to induce tolerance to transplanted tissues and avoid the need for life-long immunosuppression. Even in the event of failure of the islet graft, the patient would not be significantly harmed and would only revert to the insulin-requiring state. • Previous Experience with Islet Transplantation Despite its appeal, most human islet transplantation attempts have failed. Internationally, among all islet transplants performed since 1990, the oneweek insulin independence rate was only 12.4%, and at one year, only 8.2% (Ref. 8). Most of these were performed in patients who received renal grafts either before or with the islets. Isolated reports have claimed better success rates. For example, in 1997, Hering reported that nine out of 12 patients with T1DM maintained islet allograft function one year after transplantation, following meticulous peritransplant management9. Two points emerge from these experiences. First, the procedure itself is capable of reversing diabetes. Autotransplantation (i.e. from self) of fewer than
1043-2760/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1043-2760(00)00324-6
379
300 000 islets can restore normal glucose control in a patient undergoing a pancreatectomy10. In successful cases, allogeneic (i.e. from another individual) intraportal islet grafts normalize the basal hepatic glucose production and improve total tissue glucose metabolism11. Importantly, these effects occur in the absence of hypoglycemia12. Second, transplantation of islets in patients with T1DM poses unique challenges that are unlike those seen in other transplantation settings because of the threat of recurrent autoimmune disease. The success rate of islet allotransplantation in patients with nonautoimmune diabetes is 40% (Ref. 8). Thus, both allorejection and recurrent autoimmunity must be overcome with immunotherapy. • The Procedure of Islet Transplants Isolation Method One of the most important factors in the success of islet transplantation is the number of islets that are harvested. As discussed below, one of the reasons for the recent achievements of islet transplantation in Edmonton might be that more islets were transplanted into recipients than had been used previously. The basic method of islet isolation has changed very little since Lacy first adopted the technique in animals13. Though it varies from institution to institution, the major elements include: procurement of an organ from a cadaveric donor, injection of the pancreatic duct with the enzyme mixture collagenase, mechanical digestion and separation by polysucrose (Ficoll™) gradients on low speed centrifugation. The automated technique used most commonly around the world was first described by Ricordi14. This system utilizes the enzyme preparation Liberase, an enzyme blend of proteases derived from the bacteria Clostridium histolyticum15. This mixture is injected cold into the pancreatic duct, inflating the organ and filling it with enzyme. The organ is then placed into the digestion chamber (Ricordi chamber) linked to a fluid circuit designed to continually pump enzymatic solution into the chamber, while providing agitation for mechanical digestion. Marbles placed into the chamber assist in the mechanical disso-
380
ciation. Once the circuit has been filled, the chamber is continuously shaken, with samples taken to evaluate the status of the digestion. These samples are stained with the chemical diphenylcarbothiazone (Dithizone, DTZ), which stains b-cell granules red, allowing easy differentiation from acinar tissue. As free whole islets emerge from the acinar tissue, the reaction is stopped and the circuit diluted to prevent overdigestion and islet fragmentation. These tissue pellets are subsequently collected and run on a centrifuged Ficoll™ gradient, where the islets are separated from the acinar tissue based on density. Cell counts, metabolic studies, and cell viability studies are performed on the final preparation to determine the quality of the isolation. Once these highly purified islets are collected, they are injected into the patient through a percutaneous transhepatic cannulation of the portal vein, where the islets are allowed to drain into the portal system by gravity. Limiting Factors of Islet Isolation Since the automated method first came into use and the isolation procedure became more standardized, the quality of the organ and the procurement technique has emerged as a major variable affecting the outcome of the isolation. Lakey and Rajotte16 showed that older donor age, experienced local procurement team and high donor body-mass index (DBMI) were positive predictors of islet isolation; the presence of hyperglycemia, prolonged donor cardiac arrest and increased cold ischemia time were negative predictors. Among these factors, the experience of the surgical team proved to be the most important. Great care must be given to prevent venous hypertension during perfusion, to avoid lacerating the pancreatic capsule, and most importantly, to be vigilant in the icing of the surface of the pancreas while other organs are being procured. Efficient isolation of human islets is a technical challenge. In humans, islets comprise approximately 1% of the total pancreatic mass. Therefore, from an average adult organ, one might expect 400–600 mg of islet tissue, numbering one–two million islet equivalents (IEQ), where the number of islet equivalents is
an average number of islets if they all measured 150 mm in width. However, when the automated procedure was first described, only 164 000 IEQ were isolated from an average pancreas17. Furthermore, in most islet laboratories, fewer than half of the isolations made are of a quality suitable for clinical use. With successful transplants averaging 11 118 IEG kg21, it is obvious that islet mass is an important limiting factor. The actual number of islets that are needed to reverse diabetes is not known. Fewer than half the number of islets needed for allotransplantation can reverse diabetes with autotransplantation10. Part of this confusion might arise from the fact that a large number of transplanted islets might be lost in the immediate post-transplantation period, owing to damage from circulating cytokines18. These issues highlight the importance of developing methods to accurately assess transplanted b-cell mass using provocative techniques that stimulate insulin production by the grafts, such as those described by Teuscher et al.19 Immunosuppression Because most islet transplantation was performed in patients who were recipients of other solid organs in the past, the immunesuppressive regimens selected for use were those that had shown success in maintenance of the solid organ graft. Most institutions utilized an induction agent consisting of a monoclonal or polyclonal T-cell antibody, as well as a maintenance regimen of a calcineurin inhibitor, [such as cyclosporin A or tacrolimus (FK506)], an antimetabolite (such as azathioprine or mycophenolate mofetil) and glucocorticoids. These immunosuppressive agents themselves might have contributed to the lack of success in previous trials. Calcineurin inhibitors have been shown to directly inhibit insulin gene transcription and insulin production by b cells. Glucocorticoids inhibit insulin action, and both steroids and calcineurin inhibitors have been implicated in causing direct b-cell damage20–22. Furthermore, although these agents have been found to have short-term effects on progression of new onset of T1DM, none cause lasting protection against autoimmune attack. TEM Vol. 11, No. 9, 2000
Thus, in the setting of a marginal b-cell mass and conventional immunosuppressive agents, failure might have been predicted. • Recent Modifications of Islet Transplant Techniques Based on their recognition of the shortfalls of previous techniques, Shapiro et al.23 have modified the procedure, and shown recently dramatic improvement in the success rate of islet transplantation in patients with T1DM. They have achieved long-term insulin independence in seven out of seven of patients transplanted. First, modifications were made in the islet isolation techniques. Shapiro et al. carefully maintained the integrity of the organ used for isolation, beginning at the time of harvest. They refined the isolation system to include automated enzyme injection under constant pressure and separation in a refrigerated centrifuge system utilizing a continuous Ficoll™ gradient. Second, they transplanted more islets than had been used previously. This required two, and sometimes three donor pancreases. The goal of Shapiro and colleagues was to transplant sufficient mass to produce insulin independence, and they found that, on average, over 11 000 IE kg21 were needed. The transplantation was performed by the percutaneous transhepatic approach and infusion of the cells (packed-tissue volume of ,10 ml) into the portal vein. Third, rather than conventional immunosuppression, these investigators utilized an induction and maintenance immunosuppressive regimen that targeted separate steps in T-cell activation and proliferation, and importantly, eliminated glucocorticoids. The regimen consisted of induction with daclizumab, a monoclonal antibody directed against the interleukin (IL)-2 receptor, sirolimus (rapamycin), and low-dose tacrolimus. Sirolimus and tacrolimus are synergistic, and at such low doses pose no threat to the islet graft (Fig. 1). Seven consecutive diabetic patients, aged 29–54 years, were no longer insulindependent once a sufficient islet mass was transplanted. Complications of the transplantation procedure included TEM Vol. 11, No. 9, 2000
Extracellular Tacrolimus FKBP
Tacrolimus
Calcineurin
TCR
IL-2 production
IL-2 T-cell apoptosis Allograft tolerance
IL-2 receptor Daclizumab
Proliferation Sirolimus Sirolimus
FKBP
TOR trends in Endocrinology and Metabolism
Figure 1. Schematic of the immunosuppression used by Shapiro et al.23 Induction is with daclizumab, a monoclonal antibody that blocks binding at the IL-2 receptor, preventing T-cell proliferation. Immunosuppression is maintained with sirolimus (rapamycin), which binds to FKBP and inhibits the protein target of rapamycin (TOR), inhibiting cell proliferation downstream of the IL-2 receptor signal. A low dose of tacrolimus (FK506) is also used, which binds to FKBP, inhibiting the signal for production of IL-2. This novel regimen protects the islets from the deleterious effects of glucocorticoids and high-dose calcineurin inhibitors. Abbreviations: FKBP, FK-binding protein; IL, interleukin; TCR, T-cell receptor; TOR, target of rapamycin.
moderate bleeding at the site of transhepatic puncture in two out of 15 procedures. None of the patients developed cytopenia or hyperlipidemia from sirolimus, and there was no evidence of renal toxicity or development of cytomegalovirus disease, despite the fact that four out of five patients were seronegative for the virus before transplantation and received islets from a seropositive donor. There was no evidence of other infectious complications in the follow-up period of up to one year. Most importantly, all of the patients were still no longer dependent on exogenous insulin for up to 17 months, with a mean glycosylated hemoglobin of 5.7% (range 5.5–6.2). The mean amplitude of glycemic excursions decreased significantly, and all patients had detectable C-peptide levels at six months that did not decrease over time. • Challenges for the Future The recent success of islet transplantation, while proof of the feasibility of the approach for treatment of T1DM, is not a cure of the disease. There is no reason to suspect that immunosuppression can
be discontinued in the islet recipients, and therefore the procedure can be viewed as substituting lifelong immunosuppression for injected insulin–diet therapy, which may be justified in individual cases. In addition, correction of diabetes required islets from at least two donors. The number of organs available for isolation cannot meet the demand of even the majority of patients. Newer therapies to induce tolerance, which would allow acceptance of the allograft without the need for continuous immunosuppression, are in development. One such example, which has been successful in preventing recurrent diabetes in the non-obese diabetic (NOD) mouse, has been the use of the Fc receptor non-binding anti-CD3 monoclonal antibody (mAb) (Ref. 22). Inhibition of co-stimulatory signals, such as blockade of B7 with cytotoxic T-lymphocyte antigen 4 (CTLA4) Ig or anti-CD40L, also have shown promise in preclinical studies24,25. In the case of the anti-CD40L mAb, the development of thrombotic events has been an unexpected result in early clinical studies that were forced to preclude its use. Recent studies have 381
questioned previous approaches for immunosuppression and suggest reasons why they have not induced tolerance. For example, Li et al.26 and Wells et al.27 have shown that deletion of activated T cells through activation-induced cell death or growth factor withdrawal is necessary to achieve peripheral tolerance. Agents such as cyclosporine A might inhibit allograft tolerance26,27. In addition to the direct damaging effects of conventional immunosuppression methods, these studies provide support for their avoidance in attempts to induce tolerance. If existing methodologies are used, the supply of pancreases is insufficient to provide islets for even the majority of patients with T1DM. Some investigators have advocated the use of xenogenic islets, most commonly porcine. An appealing feature of porcine donors is the option of genetically manipulating the donor tissues and, therefore, the possibility of overcoming the immunologicbarriers of xenotransplantation. Others have tried to grow islet cells or islet cell lines with special culture conditions or genetic modification of the cells. Indeed, there have been reports of up to 15-cell doublings of human islets and derivation of insulin secreting cells from mouse embryonic stem cells28,29. Most recently, Ramiya et al.30 reported generation of islet cells from pancreatic ductal epithelial cells from NOD mice that were able to reverse diabetes. MacFarlane et al.31 were able to restore physiological insulin secretion to a cell line derived from an infant with persistent hyperinsulinemia of infancy by triple transfecting the cells with the homeodomain transcription factor, PDX1 and two components of K1–ATP channels31. Unlike in the past, the development of proliferative cell lines that maintain their secretory ability is extremely encouraging. • Summary The report of recent success in islet transplantation suggests that a new era in treatment of T1DM is emerging. The recent success will need confirmation by other laboratories and such a trial is now being undertaken by the Immune Tolerance Network with the Juvenile
382
Diabetes Foundation. Several questions about islet transplantation remain, including the ability of islet transplants to restore other islet functions, such as glucose counterregulation. The important new questions that are emerging for control of diabetes now fall into the realm of immunology and cell biology. But, for the first time, physiological replacement of insulin is possible. References 1 Papaspyros, N.S. (1964) The History of Diabetes Mellitus (2nd edn), Georg Thieme Verlag 2 Moskalewski, S. (1965) Isolation and culture of the islets of Langerhans of the guinea pig. Gen. Comp. Endocrinol. 5, 342–353 3 The DCCT Research Group (1993) The effect of intensive treatment of diabetes on the development of long-term complications in insulin-dependent diabetes mellitus. New Engl. J. Med. 329, 977–986 4 Sutherland, D. and Gruessner, R. (1997) Current status of pancreas transplantation for the treatment of type 1 diabetes. Clin. Diab. 2, 152–156 5 Bilous, R.W. et al. (1989) The effects of pancreas transplantation on the glomerular structure of renal allografts in patients with insulin-dependent diabetes. New Engl. J. Med. 321, 80–85 6 Fioretto, P. et al. (1998) Reversal of lesions of diabetic nephropathy after pancreas transplantation. New Engl. J. Med. 339, 69–75 7 Navarro, X. et al. (1990) Influence of pancreas transplantation on cardiorespiratory reflexes, nerve conduction, and mortality in diabetes mellitus. Diabetes 39, 802–806 8 Brendel, M. et al. (1999) International Islet Transplant Registry Report, University of Giessen 9 Hering, B. et al. (1997) Improved survival of single donor islet allografts in IDDM recipients by refined peritransplant management. Diabetes 46 (Suppl. 1), 64A 10 Pyzdrowski, K.L. et al. (1992) Preserved insulin secretion and insulin independence in recipients of islet autografts. New Engl. J. Med. 327, 220–226 11 Luzi, L. et al. (1996) Metabolic effects of successful intraportal islet transplantation in insulin-dependent diabetes mellitus. J. Clin. Invest. 97, 2611–2618 12 Alejandro, R. et al. (1997) Long-term function (6 years) of islet allografts in type 1 diabetes. Diabetes 46, 1983–1989 13 Lacy, P.E. and Kostianovsky, M. (1967) Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes 16, 35–39 14 Ricordi, C. et al. (1989) Automated islet isolation from human pancreas. Diabetes 38 (Suppl. 1), 140–142 15 Linetsky, E. et al. (1997) Improved human islet isolation using a new enzyme blend, liberase. Diabetes 46, 1120–1123 16 Lakey, J.R. et al. (1996) Variables in organ donors that affect the recovery of human islets of Langerhans. Transplantation 61, 1047–1053
17 Ricordi, C. (1992) Pancreatic Islet Transplantation 1892–1992: One Century of Transplantation for Diabetes (1st edn), R.G. Landes Co. 18 Rabinovitch, A. et al. (1996) Human pancreatic islet beta-cell destruction by cytokines involves oxygen free radicals and aldehyde production. J. Clin. Endocrinol. Metab. 81, 3197–3202 19 Teuscher, A.U. et al. (1998) Successful islet autotransplantation in humans: functional insulin secretory reserve as an estimate of surviving islet cell mass. Diabetes 47, 324–330 20 Zeng, Y.C. et al. (1993) The effect of prednisone on pancreatic islet autografts in dogs. Surgery 113, 98–102 21 Drachenberg, C.B. et al. (1999) Islet cell damage associated with tacrolimus and cyclosporine: morphological features in pancreas allograft biopsies and clinical correlation. Transplantation 68, 396–402 22 Herold, K.C. et al. (1993) Inhibition of glucose-stimulated insulin release from beta TC3 cells and rodent islets by an analog of FK506. Transplantation 55, 186–192 23 Shapiro, A.M.J. et al. (2000) Islet transplantation in patients with diabetes using glucocorticoid-free immunosuppression. New Engl. J. Med. 343, 230–238 24 Chatenoud, L. et al. (1994) Anti-CD3 antibody induces long-term remission of overt autoimmunity in nonobese diabetic mice. Proc. Natl. Acad. Sci. U. S. A. 91, 123–127 25 Kenyon, N.S. et al. (1999) Long-term survival and function of intrahepatic islet allografts in rhesus monkeys treated with humanized anti-CD154. Proc. Natl. Acad. Sci. U. S. A. 96, 8132–8137 26 Li, Y. et al. (1999) Blocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of alloreactive T cells and induction of peripheral allograft tolerance. Nat. Med. 5, 1298–1302 27 Wells, A.D. et al. (1999) Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance. Nat. Med. 5, 1303–1307 28 Beattie, G.M. et al. (1999) Sustained proliferation of PDX-11 cells derived from human islets. Diabetes 48, 1013–1019 29 Soria, B. et al. (2000) Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin induced diabetic mice. Diabetes 49, 157–161 30 Ramiya, V.K. et al. (2000) Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat. Med. 6, 278–282 31 MacFarlane, W.M. et al. (1999) Engineering a glucose-responsive human insulinsecreting cell line from islets of Langerhans isolated from a patient with persistent hyperinsulinemic hypoglycemia of infancy. J. Biol. Chem. 274, 34059–34066
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TEM Vol. 11, No. 9, 2000
For more than two decades, islet transplantation has been pursued as a curative treatment for type 1 diabetes mellitus (T1DM) with little success. It is likely that the failures of the past have involved technical difficulties in harvesting human islets, transplantation of insufficient amounts of islet tissue, the antagonistic effects of immune suppressive drugs, including calcineurin inhibitors and glucocorticoids, graft rejection and recurrent autoimmune disease. More recently, success has been reported in seven out of seven consecutive transplants using approaches that overcome the technical and therapeutic problems of the past. Although this success is noteworthy, issues remain that preclude the general application of islet transplants for treatment of the majority of patients with T1DM. These include the need for chronic immunosuppression and the requirement of large numbers of islets. Efforts are under way, using a variety of immunological, molecular and cellular strategies, to make this promising treatment available to the majority of patients with this disease. Since Banting and Best first isolated insulin (then known as the ‘internal secretion’) from purified islets of Langerhans on 30 July 1921, and showed that it could reduce the level of blood sugar in a patient with type 1 diabetes mellitus (T1DM), the replacement of pancreatic islets has remained the single best hope for a cure of the disease1. However, the task has proven to be more difficult than originally anticipated. Minkowski first proposed the idea of injecting the extract of pulverized pancreas in 1889, but found the toxic effects unacceptable. Seventy-six years later Moskalewski demonstrated that islets could be purified from a guineapig pancreas2. Since then, much work
E.H. Liu and K.C. Herold are at the Naomi Berrie Diabetes Center, Departments of Surgery and Medicine, College of Physicians and Surgeons, Columbia University, 1150 St Nicholas Ave, New York, NY 10032, USA. Tel: 11 212 304 5492, Fax: 11 212 304 5493, e-mail: [email protected]
TEM Vol. 11, No. 9, 2000
has gone into the refinement of the isolation procedure in numerous species, including humans. The rationale for islet transplantation is to provide physiological control of the metabolic state of T1DM and to prevent the long-term complications of the disease. The need for this has been emphasized by the findings of the Diabetes Control and Complications Trial: intensive control of the disease was shown to reduce and delay the incidence of diabetes-related complications3. Although some individuals can achieve near-normal control of glucose levels with use of insulin injections, this cannot be done without an increased risk of severe hypoglycemia, estimated to be three–four times more common than with therapy with more relaxed goals of glucose control. Recurrent hypoglycemia with loss of glucagon and catecholamine responses can lead to hypoglycemia unawareness, which requires conscientious monitoring of glucose levels to avoid neurological and other cognitive
impairment. Thus, while the need for normal metabolic control of diabetes has now been clearly shown, a feasible means for achieving this physiological control has not been established. Whole pancreas transplantation can provide normal metabolic control of diabetes4. In fact, this approach to treatment has already been shown to prevent recurrent renal disease in recipients of kidney grafts, reverse existing lesions of diabetic complications, and improve mortality in a group of patients with autonomic neuropathy5–7. However, transplantation of the whole organ requires major surgery, lifelong immunosuppression, and imposes other risks, so that most endocrinologists have recommended this procedure to their patients only when a kidney transplant was also required. Islet transplantation offers the advantages of being minimally invasive, and thus entailing reduced risk. The safety, potential efficacy and minimal risks of the procedure account for the persistent interest in it. In addition, islet transplantation offers a novel clinical setting for testing new strategies to induce tolerance to transplanted tissues and avoid the need for life-long immunosuppression. Even in the event of failure of the islet graft, the patient would not be significantly harmed and would only revert to the insulin-requiring state. • Previous Experience with Islet Transplantation Despite its appeal, most human islet transplantation attempts have failed. Internationally, among all islet transplants performed since 1990, the oneweek insulin independence rate was only 12.4%, and at one year, only 8.2% (Ref. 8). Most of these were performed in patients who received renal grafts either before or with the islets. Isolated reports have claimed better success rates. For example, in 1997, Hering reported that nine out of 12 patients with T1DM maintained islet allograft function one year after transplantation, following meticulous peritransplant management9. Two points emerge from these experiences. First, the procedure itself is capable of reversing diabetes. Autotransplantation (i.e. from self) of fewer than
1043-2760/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1043-2760(00)00324-6
379
300 000 islets can restore normal glucose control in a patient undergoing a pancreatectomy10. In successful cases, allogeneic (i.e. from another individual) intraportal islet grafts normalize the basal hepatic glucose production and improve total tissue glucose metabolism11. Importantly, these effects occur in the absence of hypoglycemia12. Second, transplantation of islets in patients with T1DM poses unique challenges that are unlike those seen in other transplantation settings because of the threat of recurrent autoimmune disease. The success rate of islet allotransplantation in patients with nonautoimmune diabetes is 40% (Ref. 8). Thus, both allorejection and recurrent autoimmunity must be overcome with immunotherapy. • The Procedure of Islet Transplants Isolation Method One of the most important factors in the success of islet transplantation is the number of islets that are harvested. As discussed below, one of the reasons for the recent achievements of islet transplantation in Edmonton might be that more islets were transplanted into recipients than had been used previously. The basic method of islet isolation has changed very little since Lacy first adopted the technique in animals13. Though it varies from institution to institution, the major elements include: procurement of an organ from a cadaveric donor, injection of the pancreatic duct with the enzyme mixture collagenase, mechanical digestion and separation by polysucrose (Ficoll™) gradients on low speed centrifugation. The automated technique used most commonly around the world was first described by Ricordi14. This system utilizes the enzyme preparation Liberase, an enzyme blend of proteases derived from the bacteria Clostridium histolyticum15. This mixture is injected cold into the pancreatic duct, inflating the organ and filling it with enzyme. The organ is then placed into the digestion chamber (Ricordi chamber) linked to a fluid circuit designed to continually pump enzymatic solution into the chamber, while providing agitation for mechanical digestion. Marbles placed into the chamber assist in the mechanical disso-
380
ciation. Once the circuit has been filled, the chamber is continuously shaken, with samples taken to evaluate the status of the digestion. These samples are stained with the chemical diphenylcarbothiazone (Dithizone, DTZ), which stains b-cell granules red, allowing easy differentiation from acinar tissue. As free whole islets emerge from the acinar tissue, the reaction is stopped and the circuit diluted to prevent overdigestion and islet fragmentation. These tissue pellets are subsequently collected and run on a centrifuged Ficoll™ gradient, where the islets are separated from the acinar tissue based on density. Cell counts, metabolic studies, and cell viability studies are performed on the final preparation to determine the quality of the isolation. Once these highly purified islets are collected, they are injected into the patient through a percutaneous transhepatic cannulation of the portal vein, where the islets are allowed to drain into the portal system by gravity. Limiting Factors of Islet Isolation Since the automated method first came into use and the isolation procedure became more standardized, the quality of the organ and the procurement technique has emerged as a major variable affecting the outcome of the isolation. Lakey and Rajotte16 showed that older donor age, experienced local procurement team and high donor body-mass index (DBMI) were positive predictors of islet isolation; the presence of hyperglycemia, prolonged donor cardiac arrest and increased cold ischemia time were negative predictors. Among these factors, the experience of the surgical team proved to be the most important. Great care must be given to prevent venous hypertension during perfusion, to avoid lacerating the pancreatic capsule, and most importantly, to be vigilant in the icing of the surface of the pancreas while other organs are being procured. Efficient isolation of human islets is a technical challenge. In humans, islets comprise approximately 1% of the total pancreatic mass. Therefore, from an average adult organ, one might expect 400–600 mg of islet tissue, numbering one–two million islet equivalents (IEQ), where the number of islet equivalents is
an average number of islets if they all measured 150 mm in width. However, when the automated procedure was first described, only 164 000 IEQ were isolated from an average pancreas17. Furthermore, in most islet laboratories, fewer than half of the isolations made are of a quality suitable for clinical use. With successful transplants averaging 11 118 IEG kg21, it is obvious that islet mass is an important limiting factor. The actual number of islets that are needed to reverse diabetes is not known. Fewer than half the number of islets needed for allotransplantation can reverse diabetes with autotransplantation10. Part of this confusion might arise from the fact that a large number of transplanted islets might be lost in the immediate post-transplantation period, owing to damage from circulating cytokines18. These issues highlight the importance of developing methods to accurately assess transplanted b-cell mass using provocative techniques that stimulate insulin production by the grafts, such as those described by Teuscher et al.19 Immunosuppression Because most islet transplantation was performed in patients who were recipients of other solid organs in the past, the immunesuppressive regimens selected for use were those that had shown success in maintenance of the solid organ graft. Most institutions utilized an induction agent consisting of a monoclonal or polyclonal T-cell antibody, as well as a maintenance regimen of a calcineurin inhibitor, [such as cyclosporin A or tacrolimus (FK506)], an antimetabolite (such as azathioprine or mycophenolate mofetil) and glucocorticoids. These immunosuppressive agents themselves might have contributed to the lack of success in previous trials. Calcineurin inhibitors have been shown to directly inhibit insulin gene transcription and insulin production by b cells. Glucocorticoids inhibit insulin action, and both steroids and calcineurin inhibitors have been implicated in causing direct b-cell damage20–22. Furthermore, although these agents have been found to have short-term effects on progression of new onset of T1DM, none cause lasting protection against autoimmune attack. TEM Vol. 11, No. 9, 2000
Thus, in the setting of a marginal b-cell mass and conventional immunosuppressive agents, failure might have been predicted. • Recent Modifications of Islet Transplant Techniques Based on their recognition of the shortfalls of previous techniques, Shapiro et al.23 have modified the procedure, and shown recently dramatic improvement in the success rate of islet transplantation in patients with T1DM. They have achieved long-term insulin independence in seven out of seven of patients transplanted. First, modifications were made in the islet isolation techniques. Shapiro et al. carefully maintained the integrity of the organ used for isolation, beginning at the time of harvest. They refined the isolation system to include automated enzyme injection under constant pressure and separation in a refrigerated centrifuge system utilizing a continuous Ficoll™ gradient. Second, they transplanted more islets than had been used previously. This required two, and sometimes three donor pancreases. The goal of Shapiro and colleagues was to transplant sufficient mass to produce insulin independence, and they found that, on average, over 11 000 IE kg21 were needed. The transplantation was performed by the percutaneous transhepatic approach and infusion of the cells (packed-tissue volume of ,10 ml) into the portal vein. Third, rather than conventional immunosuppression, these investigators utilized an induction and maintenance immunosuppressive regimen that targeted separate steps in T-cell activation and proliferation, and importantly, eliminated glucocorticoids. The regimen consisted of induction with daclizumab, a monoclonal antibody directed against the interleukin (IL)-2 receptor, sirolimus (rapamycin), and low-dose tacrolimus. Sirolimus and tacrolimus are synergistic, and at such low doses pose no threat to the islet graft (Fig. 1). Seven consecutive diabetic patients, aged 29–54 years, were no longer insulindependent once a sufficient islet mass was transplanted. Complications of the transplantation procedure included TEM Vol. 11, No. 9, 2000
Extracellular Tacrolimus FKBP
Tacrolimus
Calcineurin
TCR
IL-2 production
IL-2 T-cell apoptosis Allograft tolerance
IL-2 receptor Daclizumab
Proliferation Sirolimus Sirolimus
FKBP
TOR trends in Endocrinology and Metabolism
Figure 1. Schematic of the immunosuppression used by Shapiro et al.23 Induction is with daclizumab, a monoclonal antibody that blocks binding at the IL-2 receptor, preventing T-cell proliferation. Immunosuppression is maintained with sirolimus (rapamycin), which binds to FKBP and inhibits the protein target of rapamycin (TOR), inhibiting cell proliferation downstream of the IL-2 receptor signal. A low dose of tacrolimus (FK506) is also used, which binds to FKBP, inhibiting the signal for production of IL-2. This novel regimen protects the islets from the deleterious effects of glucocorticoids and high-dose calcineurin inhibitors. Abbreviations: FKBP, FK-binding protein; IL, interleukin; TCR, T-cell receptor; TOR, target of rapamycin.
moderate bleeding at the site of transhepatic puncture in two out of 15 procedures. None of the patients developed cytopenia or hyperlipidemia from sirolimus, and there was no evidence of renal toxicity or development of cytomegalovirus disease, despite the fact that four out of five patients were seronegative for the virus before transplantation and received islets from a seropositive donor. There was no evidence of other infectious complications in the follow-up period of up to one year. Most importantly, all of the patients were still no longer dependent on exogenous insulin for up to 17 months, with a mean glycosylated hemoglobin of 5.7% (range 5.5–6.2). The mean amplitude of glycemic excursions decreased significantly, and all patients had detectable C-peptide levels at six months that did not decrease over time. • Challenges for the Future The recent success of islet transplantation, while proof of the feasibility of the approach for treatment of T1DM, is not a cure of the disease. There is no reason to suspect that immunosuppression can
be discontinued in the islet recipients, and therefore the procedure can be viewed as substituting lifelong immunosuppression for injected insulin–diet therapy, which may be justified in individual cases. In addition, correction of diabetes required islets from at least two donors. The number of organs available for isolation cannot meet the demand of even the majority of patients. Newer therapies to induce tolerance, which would allow acceptance of the allograft without the need for continuous immunosuppression, are in development. One such example, which has been successful in preventing recurrent diabetes in the non-obese diabetic (NOD) mouse, has been the use of the Fc receptor non-binding anti-CD3 monoclonal antibody (mAb) (Ref. 22). Inhibition of co-stimulatory signals, such as blockade of B7 with cytotoxic T-lymphocyte antigen 4 (CTLA4) Ig or anti-CD40L, also have shown promise in preclinical studies24,25. In the case of the anti-CD40L mAb, the development of thrombotic events has been an unexpected result in early clinical studies that were forced to preclude its use. Recent studies have 381
questioned previous approaches for immunosuppression and suggest reasons why they have not induced tolerance. For example, Li et al.26 and Wells et al.27 have shown that deletion of activated T cells through activation-induced cell death or growth factor withdrawal is necessary to achieve peripheral tolerance. Agents such as cyclosporine A might inhibit allograft tolerance26,27. In addition to the direct damaging effects of conventional immunosuppression methods, these studies provide support for their avoidance in attempts to induce tolerance. If existing methodologies are used, the supply of pancreases is insufficient to provide islets for even the majority of patients with T1DM. Some investigators have advocated the use of xenogenic islets, most commonly porcine. An appealing feature of porcine donors is the option of genetically manipulating the donor tissues and, therefore, the possibility of overcoming the immunologicbarriers of xenotransplantation. Others have tried to grow islet cells or islet cell lines with special culture conditions or genetic modification of the cells. Indeed, there have been reports of up to 15-cell doublings of human islets and derivation of insulin secreting cells from mouse embryonic stem cells28,29. Most recently, Ramiya et al.30 reported generation of islet cells from pancreatic ductal epithelial cells from NOD mice that were able to reverse diabetes. MacFarlane et al.31 were able to restore physiological insulin secretion to a cell line derived from an infant with persistent hyperinsulinemia of infancy by triple transfecting the cells with the homeodomain transcription factor, PDX1 and two components of K1–ATP channels31. Unlike in the past, the development of proliferative cell lines that maintain their secretory ability is extremely encouraging. • Summary The report of recent success in islet transplantation suggests that a new era in treatment of T1DM is emerging. The recent success will need confirmation by other laboratories and such a trial is now being undertaken by the Immune Tolerance Network with the Juvenile
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Diabetes Foundation. Several questions about islet transplantation remain, including the ability of islet transplants to restore other islet functions, such as glucose counterregulation. The important new questions that are emerging for control of diabetes now fall into the realm of immunology and cell biology. But, for the first time, physiological replacement of insulin is possible. References 1 Papaspyros, N.S. (1964) The History of Diabetes Mellitus (2nd edn), Georg Thieme Verlag 2 Moskalewski, S. (1965) Isolation and culture of the islets of Langerhans of the guinea pig. Gen. Comp. Endocrinol. 5, 342–353 3 The DCCT Research Group (1993) The effect of intensive treatment of diabetes on the development of long-term complications in insulin-dependent diabetes mellitus. New Engl. J. Med. 329, 977–986 4 Sutherland, D. and Gruessner, R. (1997) Current status of pancreas transplantation for the treatment of type 1 diabetes. Clin. Diab. 2, 152–156 5 Bilous, R.W. et al. (1989) The effects of pancreas transplantation on the glomerular structure of renal allografts in patients with insulin-dependent diabetes. New Engl. J. Med. 321, 80–85 6 Fioretto, P. et al. (1998) Reversal of lesions of diabetic nephropathy after pancreas transplantation. New Engl. J. Med. 339, 69–75 7 Navarro, X. et al. (1990) Influence of pancreas transplantation on cardiorespiratory reflexes, nerve conduction, and mortality in diabetes mellitus. Diabetes 39, 802–806 8 Brendel, M. et al. (1999) International Islet Transplant Registry Report, University of Giessen 9 Hering, B. et al. (1997) Improved survival of single donor islet allografts in IDDM recipients by refined peritransplant management. Diabetes 46 (Suppl. 1), 64A 10 Pyzdrowski, K.L. et al. (1992) Preserved insulin secretion and insulin independence in recipients of islet autografts. New Engl. J. Med. 327, 220–226 11 Luzi, L. et al. (1996) Metabolic effects of successful intraportal islet transplantation in insulin-dependent diabetes mellitus. J. Clin. Invest. 97, 2611–2618 12 Alejandro, R. et al. (1997) Long-term function (6 years) of islet allografts in type 1 diabetes. Diabetes 46, 1983–1989 13 Lacy, P.E. and Kostianovsky, M. (1967) Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes 16, 35–39 14 Ricordi, C. et al. (1989) Automated islet isolation from human pancreas. Diabetes 38 (Suppl. 1), 140–142 15 Linetsky, E. et al. (1997) Improved human islet isolation using a new enzyme blend, liberase. Diabetes 46, 1120–1123 16 Lakey, J.R. et al. (1996) Variables in organ donors that affect the recovery of human islets of Langerhans. Transplantation 61, 1047–1053
17 Ricordi, C. (1992) Pancreatic Islet Transplantation 1892–1992: One Century of Transplantation for Diabetes (1st edn), R.G. Landes Co. 18 Rabinovitch, A. et al. (1996) Human pancreatic islet beta-cell destruction by cytokines involves oxygen free radicals and aldehyde production. J. Clin. Endocrinol. Metab. 81, 3197–3202 19 Teuscher, A.U. et al. (1998) Successful islet autotransplantation in humans: functional insulin secretory reserve as an estimate of surviving islet cell mass. Diabetes 47, 324–330 20 Zeng, Y.C. et al. (1993) The effect of prednisone on pancreatic islet autografts in dogs. Surgery 113, 98–102 21 Drachenberg, C.B. et al. (1999) Islet cell damage associated with tacrolimus and cyclosporine: morphological features in pancreas allograft biopsies and clinical correlation. Transplantation 68, 396–402 22 Herold, K.C. et al. (1993) Inhibition of glucose-stimulated insulin release from beta TC3 cells and rodent islets by an analog of FK506. Transplantation 55, 186–192 23 Shapiro, A.M.J. et al. (2000) Islet transplantation in patients with diabetes using glucocorticoid-free immunosuppression. New Engl. J. Med. 343, 230–238 24 Chatenoud, L. et al. (1994) Anti-CD3 antibody induces long-term remission of overt autoimmunity in nonobese diabetic mice. Proc. Natl. Acad. Sci. U. S. A. 91, 123–127 25 Kenyon, N.S. et al. (1999) Long-term survival and function of intrahepatic islet allografts in rhesus monkeys treated with humanized anti-CD154. Proc. Natl. Acad. Sci. U. S. A. 96, 8132–8137 26 Li, Y. et al. (1999) Blocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of alloreactive T cells and induction of peripheral allograft tolerance. Nat. Med. 5, 1298–1302 27 Wells, A.D. et al. (1999) Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance. Nat. Med. 5, 1303–1307 28 Beattie, G.M. et al. (1999) Sustained proliferation of PDX-11 cells derived from human islets. Diabetes 48, 1013–1019 29 Soria, B. et al. (2000) Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin induced diabetic mice. Diabetes 49, 157–161 30 Ramiya, V.K. et al. (2000) Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat. Med. 6, 278–282 31 MacFarlane, W.M. et al. (1999) Engineering a glucose-responsive human insulinsecreting cell line from islets of Langerhans isolated from a patient with persistent hyperinsulinemic hypoglycemia of infancy. J. Biol. Chem. 274, 34059–34066
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TEM Vol. 11, No. 9, 2000