Online Issues

ILAR Journal Vol 45(3)

Nonhuman Primate Models in Type 1 Diabetes Research

Juan L. Contreras, Cheryl A. Smyth, David T. Curiel, and Devin E. Eckhoff

Juan L. Contreras, M.D., Cheryl A. Smyth, M.S., and Devin E. Eckhoff, M.D., are in the Department of Surgery, Division of Transplantation and Division of Human Gene Therapy and Gene Therapy Center, University of Alabama at Birmingham. David T. Curiel, M.D., Ph.D., is in the Division of Human Gene Therapy and Gene Therapy Center, University of Alabama at Birmingham.

Abstract

The recent success of "steroid-free" immunosuppressive protocols and improvements in islet preparation techniques have proven that pancreatic islet transplantation (PIT) is a valid therapeutic approach for patients with type 1 diabetes. However, there are major obstacles to overcome before PIT can become a routine therapeutic procedure, such as the need for chronic immunosuppression, the loss of functional islet mass after transplantation requiring multiple islet infusion to achieve euglycemia without exogenous administration of insulin, and the shortage of human tissue for transplantation. With reference to the first obstacle, stable islet allograft function without immunosuppressive therapy has been achieved after tolerance was induced in diabetic primates. With reference to the second obstacle, different strategies, including gene transfer of antiapoptotic genes, have been used to protect isolated islets before and after transplantation. With reference to the third obstacle, pigs are an attractive islet source because they breed rapidly, there is a long history of porcine insulin use in humans, and there is the potential for genetic engineering. To accomplish islet transplantation, experimental opportunities must be balanced by complementary characteristics of basic mouse and rat models and preclinical large animal models. Well-designed preclinical studies in primates can provide the quality of information required to translate islet transplant research safely into clinical transplantation.

Key Words: apoptosis; diabetes; gene therapy; islet transplantation; nonhuman primates; tolerance; xenotransplantation

Introduction

Diabetes mellitus is a disease of metabolic dysregulation that is characterized by inappropriate hyperglycemia resulting from progressive loss of insulin secretion or action. Diabetes affects people of every age, race, and nationality, including approximately 17 million Americans. It contributes $92 to $138 billion directly to healthcare costs, with an additional $45 billion in lost productivity. The incidence is increasing, and at least one million cases are diagnosed each year. As the fifth deadliest disease in the United States, diabetes kills almost 210,000 people each year (Hogan et al. 2003; Zimmet et al. 2001). It represents a target disease that will have a tremendous impact on society in terms of increasing the quality of life and freeing healthcare resources for other diseases.

The two main forms of diabetes are types 1 and 2. Type 1 diabetes is due primarily to autoimmune-mediated destruction of pancreatic beta-cells, resulting in absolute insulin deficiency. People with type 1 diabetes must take exogenous insulin for survival. Type 2 diabetes, which accounts for approximately 90% of cases, is characterized by insulin resistance and/or abnormal insulin secretion. People with type 2 diabetes are not dependent on exogenous insulin but may require it for control of blood glucose levels if control is not achieved with diet alone or with oral hypoglycemic agents. Type 2 diabetes is beyond the scope of this article, and the discussions below focus on research related to type 1.

The principal determinant of risk for devastating diabetes complications is the time of exposure to hyperglycemia. The only way at present to restore sustained normoglycemia without the risk associated with hypoglycemia (as seen with intensive insulin therapy), and without procedural complications (as seen in pancreas transplantation), is to perform an isolated pancreatic islet transplant (PIT¹) (Bertuzzi et al. 1999; Bretzel et al. 1999; Hering and Ricordi 1999; Inverardi and Ricordi 1996; Markmann et al. 2003; Ricordi 2003; Ryan et al. 2001, 2002; Shapiro et al. 2000; Weir and Bonner-Weir 1997). The recent demonstration of consistent and sustained type 1 diabetes reversal after PIT, using steroid-free immunosuppressive protocols and technical improvements in the isolation process, has generated optimism for wider application of PIT as a cure for diabetes (Ryan et al. 2001, 2002; Shapiro et al. 2000). Insulin-free, long-term islet allograft survival has been demonstrated in a significant number of diabetic patients at different transplant centers (Baidal et al. 2003; Goss et al. 2002; Hering and Ricordi 1999, Hering et al. 2003; Markmann et al. 2003).

Although type 1 diabetes is an autoimmune disease, very low autoimmune recurrence has been reported after PIT, probably related to the chronic administration of powerful immunosuppressive agents (Hering and Ricordi 1999; Ryan et al. 2001). However, there are major obstacles to overcome before PIT can become a routine therapeutic procedure, including the following: (1) the need for chronic immunosuppression; (2) the vulnerability of pancreatic islets to peritransplant cell death, which requires administration of high numbers of islets to achieve exogenous insulin independence; and (3) the shortage of human organs for transplantation (Hering and Ricordi 1999; Weir and Bonner-Weir 1997; Zwillich 2000).

Immunological Graft Loss and the Necessity of Tolerance Induction

Although short-term success rates of human organ transplantation are extraordinary, often exceeding 90% at 1 yr, late failures from chronic graft rejection or adverse complications of lifelong immunosuppressive therapy constrain long-lasting graft function and patient rehabilitation (Cecka 1994). Only tolerance induction can enable indefinite allograft acceptance without the harmful side effects and economic burden of lifelong immunosuppressive therapy. Induction of "prope [almost] tolerance" (Calne 2000) to reduce rather than eliminate lifelong immunosuppressive therapy is an alternative strategy, although to date there is no evidence of superior efficacy in long-term follow-up. Within the goal of clinical tolerance, experimental opportunities are balanced by complementary characteristics of basic mouse and rat models and the preclinical large animal models. Rodent models continually provide unique resources to distill scientific concepts of tolerance that are tested, in turn, in higher animal models.

A perceived requirement for preclinical testing, particularly in nonhuman primates (NHPs¹), is based on the uncertain translation of rodent protocols to outbred NHPs. For example, whereas hundreds of protocols have been successfully applied to tolerance induction in rodents, only a handful of strategies have led to long-term rejection-free intervals without chronic immunosuppressive therapy in NHPs (Kirk 1999). Therefore, in the field of transplantation, NHPs have, for several reasons, long been regarded as the "gold standard" for preclinical studies (Balner 1974) for the following reasons:

NHPs have evolutionary proximity to humans, with whom they exhibit an overall >95% homology at the genome level. Although the outbred nature of NHPs precludes the elegant types of immunobiology models that can be examined in inbred rodents, this disadvantage is balanced by the relevance of NHP studies to the human situation.
Robust alloimmune response patterns are similar in humans and rhesus macaques and include high sensitivity to graft versus host disease, with a propensity for developing alloantibody and chronic graft rejection.
Polyclonal and monoclonal anti-T cell antibodies that cross-react with macaque T cells are remarkably similar in effecting immunosuppression in both human and NHP species.
The organization and molecular sequence of alleles of the major histocompatibility complex (MHC¹) complex of NPHs--particularly Old World monkeys (Macacca mulatto and Macacca fascicularis)--are well known (Bontrop et al. 1999; Jonker 1990; Kirk 1999; Lobashevsky and Thomas 2000; Lobashevsky et al. 1999; Thomas et al. 1995; Torrealba et al. 2003; Van Bekkum 1994; Vogel et al. 1999).

To date, three tolerance-induction protocols appear to hold promise for clinical translation because NHPs have evidenced prolonged allograft survival without rejection, with either minimal (prope tolerance) or no chronic immunosuppressive therapy (operational tolerance). One strategy involves costimulatory molecule blockade combined with immunosuppressive drug therapy, with and without donor bone marrow or donor-specific blood transfusion. Another strategy involves donor bone marrow infusion to induce chimerism combined with nonmyeloablative treatment to deplete T-cells, with and without irradiation. The third strategy, called STEALTH (Specific Tolerance by Early evasion of Antigen-presenting cells-Lymphocyte interactions with T-Helper-2 cytokine deviation) , utilizes a brief peritransplant treatment combination of anti-CD3-immunotoxin and 15-deoxyspergualin (Kirk 1999; Thomas et al. 2001c). This strategy has induced indefinite tolerance to MHC-incompatible islets after transplantation into spontaneous or streptozotocin-induced diabetic primates without chronic immunosuppressive therapy (Contreras et al. 1999, 2000, 2003a; Thomas et al. 1999, 2001, 2001b). Stable functional islet mass has been demonstrated in these recipients years after the transplant (Contreras et al. 2000, 2003). The benefits and limitations of these assorted NHP strategies have been the subject of other reviews and are not discussed in detail herein (Kirk 1999; Thomas et al. 2001c).

Vulnerability of Pancreatic Islets After Transplantation

The first few days after islet transplantation are characterized by substantial islet cell dysfunction and death (Davalli et al. 1996; Hering and Ricordi 1999). In the most successful clinical experience in PIT, two or three islet infusions were necessary to achieve euglycemia without exogenous insulin therapy (Shapiro et al. 2000). Furthermore, insulin-independent PIT recipients present lower functional islet mass compared with healthy people (Ryan et al. 2001, 2002).

Several factors have been identified that increase the vulnerability of isolated islets in the immediate post-transplant period (Figure 1). These factors include islet damage during pancreas procurement and hypothermic preservation, islet isolation, removal of the extracellular matrix and growth factor deprivation, instant blood inflammatory reaction, exposure to proinflammatory cytokines, poor vascularization soon after transplantation, immune-mediated injury, exposure to diabetogenic immunosuppressive drugs, hyperglycemia, and others (Berney et al. 2001a; Bottino et al. 1998; Davalli et al. 1996; Eizirik and Mandrup-Poulsen 2001; Hering and Ricordi 1999; Kaufman et al. 1990; Paraskevas et al. 2000; Weir and Bonner-Weir 1997; Xenos et al. 1994). The main common unifying pathophysiological process for all of these factors is the trigger of apoptotic machinery in insulin-producing cells. Moreover, islet necrosis has been described (Dupraz et al. 1999; Eizirik and Mandrup-Poulsen 2001). In this regard, islet cytoprotection achieved by gene therapy is an attractive approach to prevent loss of functional islet mass (Contreras et al. 2001a,b, 2002, 2003b; Dupraz et al. 1999; Garcia-Ocana et al. 2003; Giannoukakis et al. 1999, 2000; Grey et al. 2003; Rabinovitch et al. 1999; Loez-Talavera et al. 2004; Thomas 2001a).

Figure 1 Vulnerability of isolated pancreatic islets in the peritransplant period. Factors associated with significant reduction in functional islet mass after transplantation include injury during pancreas preservation and islet isolation, removal of extracellular matrix and growth factors, poor vascularization after transplantation, hypoxia, exposure to proinflammatory cytokines and potential cytotoxic immunosuppressive drugs, direct cytotoxic effects of the immune system, contact to blood, and hyperglycemia.

Gene Transfer into NHP Isolated Islets

Current gene delivery strategies are problematic in terms of efficiency and toxicity. Despite rapid advancement of gene therapy approaches to augment islet cell transplantation, concerns related to safety have emerged. For example, adenoviral vectors are attractive candidates for gene delivery due to their relatively high efficiency compared with other vector systems, but there is a clearly established dose-dependent toxicity associated with the use of these agents (Bilbao et al. 2002; Borgland et al. 2000; Bowen et al. 2002; Contreras et al. 2003c; Kay et al. 2001; Liu and Muruve 2003; Tibbles et al. 2002; Weber et al. 1997; Zhang et al. 2003). Yet adenoviral vectors remain the most efficacious gene delivery agents for in vivo use, and further research and development of these vectors are not only warranted but are possibly key to the realization of many gene therapy approaches. Clearly, adenoviral vectors are the most efficient vectors for gene transfer into isolated pancreatic islets (Bartlett et al. 1997; Bilbao et al. 2002; Contreras et al. 2003c; Efrat et al. 1995; Flotte et al. 2001; Levine 1997; Muruve et al. 1997; Pileggi et al. 2001; Prasad et al. 2000; Rilo et al. 1994; Saldeen et al. 1996; Weber et al. 1997).

Other viral vectors, such as adeno-associated virus or lentivirus, transduce islet cells with lower transfection efficiency (Flotte et al. 2001; Kapturczak et al. 2001; Prasad et al. 2000). For cytoprotective strategies, it is imperative to infect the majority of islet cells, especially insulin-producing cells. In this regard, adenoviral vectors are the only gene transfer vector documented to date as having this capability. Nevertheless, high viral dose is required, and the effect is associated with inflammation and toxicity (Bilbao et al. 2002; Borgland et al. 2000; Bowen et al. 2002; Liu and Muruve 2003; Tibbles et al. 2002; Weber et al. 1997). For this reason, safer versions of these agents have now been developed (Bilbao et al. 2002; Contreras et al. 2003c).

The specificity of interactions between viral capsid components and target cell receptors dictates adenovirus tropism. The major proteins of adenoviral capsid, the fiber and the penton base, both play key roles in cellular receptor recognition and entry processes, thereby defining the tropism of the virus (Figure 2). The initial steps of adenoviral infection involve two sequential virus-cell interactions, each of which is mediated by a specific protein component of adenoviral capsid. The primary binding of the virus to the cell surface receptor, coxsackie-adenovirus receptor (Bergelson et al. 1997; Tomko et al. 1997), is mediated by the knob domain of the fiber protein (Defer et al. 1990; Tomko et al. 2000) followed by internalization of the virus within a clathrin-coated endosome. The virus then escapes the endosome by triggering its acidification via a secondary interaction of the arginine-glycine-aspartame (RGD¹) motif of adenovirus penton base protein with cellular integrins, α_νβ and α_νβ₅ (Wickham et al. 1993, 1994). After the endosome escape, partially dismantled virus translocates to the nuclear pore complex and releases its genome into the nucleoplasm, where subsequent steps of viral replication take place.

Figure 2 Cell-entry pathway of the adenoviral vector. The adenovirus vector initially binds to the cell via the specific cellular receptor, coxsackievirus and adenovirus receptor (CAR). After binding, the virion achieves internalization via receptor-mediated endocytosis pathway. This initial entry involves interaction with cellular integrin receptors via an arginine-glycine-aspartame motif in the adenoviral penton capsid protein. After internalization, the virus is localized within the cellular endosomes. Acidification of the endosomes allows the virions to be released within the cytosol and consequently the virion will be translocated into the nucleus to begin gene expression.

Numerous strategies have been developed to alter the tropism of adenovirus to improve its utility as a gene transfer vector. These approaches have included steps to broaden tropism as a means to infect otherwise refractory cell targets (Dmitriev et al. 1998; Wickham et al. 1996, 1997), as well as steps to ablate native tropism (Roelvink et al. 1999) as a means to mitigate ectopic gene delivery. Thus, genetic manipulations to the capsid proteins hexon and fiber have allowed utilization of alternative cell entry mechanisms by the modified vectors (Dmitriev et al. 1998; Wickham et al. 1996).

We explored the utility of the HI loop of the fiber knob of the adenovirus capsid for incorporation of targeting ligands to modify the adenovirus tropism. Results from phage biopanning proved that the RGD motif has in vivo targeting capabilities (Dimitriev et al. 1998). The RGD motif interacts especially with cellular integrins of the α_νβ and α_νβ₅ types, which are present in high concentrations in insulin-producing cells. In our laboratory, we previously demonstrated that incorporation of the RGD motif into the HI loop of the fiber knob (Figure 3) significantly reduces the dose of virus required to infect most islet cells (Bilbao et al. 2002; Contreras et al. 2003c). Furthermore, the level of transgene expression is higher, compared with standard adenoviral vectors. However, the most striking finding is reduced cytotoxicity related to adenoviral infection. Higher capacity to release insulin after a glucose challenge in vitro was demonstrated in NHP pancreatic islets infected with a targeted vector, compared with control nontargeted adenoviral vector (Bilbao et al. 2002). Moreover, after transplantation of a limited islet mass into streptozotocin-induced diabetic (SCID¹) mice, only islet preparations infected with targeted vector were able to maintain stable, long-term euglycemia in murine diabetic recipients (Bilbao et al. 2002).

Figure 3 Retargeting of the adenoviral vector by incorporation of the arginine-glycine-aspartame (RGD) motif into the fiber knob. The RGD peptide CDCRGDCFC incorporated in the HI loop of the fiber knob bind several types of integrins present in high concentrations in isolated pancreatic islets.

Gene Transfer of Bcl-2 into NHP Isolated Islets

Apoptosis, a highly regulated process of cell death, is controlled through the expression of specific genes that are largely conserved from nematodes through mammals. Among these genes, the Bcl-2 gene family is one of the most prominent (Kroemer 1997). The protein encoded by the Bcl-2 gene has pleiotropic antiapoptotic and antinecrotic effects; it acts within the cytosol, endoplasmic-reticulum, and mitochondria (Kroemer 1997). Gene transfer of Bcl-2 has conferred in vitro protection from apoptosis in isolated islets exposed to proinflammatory cytokines and hypoxia (Contreras et al. 2001b, 2002, 2003b; Dupraz et al. 1999; Rabinovitch et al. 1999). Furthermore, overexpression of Bcl-2 protects liver grafts during hypothermic preservation and reperfusion, apoptosis caused by extracellular matrix removal, growth factor deprivation, and immune-mediated injury (Bilbao et al. 1999a,b; Contreras et al. 2001a,b; Lacronique et al. 1996; Thomas et al. 2001a).

Previous studies from our laboratory demonstrated that targeting the apoptosis pathway by adenoviral-mediated gene transfer of Bcl-2 exerts a major cytoprotective effect on isolated macaque pancreatic islets (Contreras et al. 2001b). Overexpression of Bcl-2 confers long-term stable functional islet mass after transplantation into diabetic SCID mice (Contreras et al. 2002). Notably, genetic modification of pancreatic islets also reduced the islet mass required to achieve stable euglycemia. In this regard, all diabetic recipients given 2000 unmodified islet equivalents (IEQ¹) were euglycemic 3 days after transplant. With 1000 IEQ, 58% of the recipients given unmodified islets or 50% of the recipients given islets infected with AdLacZ were euglycemic. None of the recipients that received either 500 IEQ of unmodified islets or islets infected with AdLacZ presented with euglycemia after the transplant. In contrast, 100% of the recipients that received 1000, 750, and 500 AdBcl-2-infected IEQ were euglycemic after transplant (Contreras et al. 2001b).

Shortage of Cadaveric Organs for Transplantation and the Necessity of Alternative Sources of Pancreatic Tissue

In light of the significant benefits of PIT, combined with the extreme shortage of human tissue for transplantation, alternative sources of islets are being considered. Several sources have been suggested, including islets obtained from pigs, fish, human pancreatic duct cells, fetal pancreatic stem cells, embryonic stem cells, and therapeutic cloning (Larsen and Rolin 2004; Serup et al. 2001; Wright et al 2004).

Pigs are an attractive source of islets because they breed rapidly, there is a long history of porcine insulin use in humans, and there is the potential for genetic engineering. However, several immunological obstacles must be overcome before clinical application of islet xenotransplantation. Obstacles include susceptibility of porcine pancreatic islets to destruction by immunological processes, islet injury after exposure to blood during intraportal islet infusion, and problems related to transmission of infectious diseases (Bennet et al. 2000; Cascalho and Platt 2001; Dorling 2002; Knosalla et al. 2002).

Immunological rejection of discordant xenografts (e.g., pig-to-human or pig-to-primate combinations) is caused by activation of complement after deposition on the graft xenoreactive natural antibodies (Miyatake et al. 1998; Satake et al. 1994). Previous studies in primates indicate that xenogeneic porcine islets are susceptible to immediate damage after transplantation (Bennet et al. 2000; Hamelmann et al. 1994). Furthermore, exposure of porcine islets to fresh human serum or blood, both in vivo and in vitro, resulted in acute islet damage mediated primarily by complement (Bennet et al. 2000; Rayat et al. 1998, 2000). Under certain circumstances, when antidonor antibodies and complement-mediated immune responses are inhibited for a few days, grafts can survive indefinitely despite the return of antidonor antibodies and complement, a phenomenon termed "accommodation" (Bach et al. 1991; Soares et al. 1999). Expression in the graft of antiapoptotic or "protective genes," such as Bcl-2, A20 Bcl-xL and heme oxygenase-1, make the graft resistant to antibodies and complement-mediated rejection (Bach et al. 1991; Lin et al. 1999; Soares et al. 1999).

Our approach to islet xenograft protection is the induction of Bcl-2 overexpression by gene transfer of Bcl-2. In this regard, we infected adult isolated porcine islets ex vivo with an adenoviral vector encoding Bcl-2 (Contreras et al. 2001a). After islet exposure to NHP serum, islet viability was significantly increased in islets infected with the adnoviral vector encoding Bcl-2 compared with islet controls. Furthermore, islets genetically modified with Bcl-2 presented greater capacity to release insulin after a glucose challenge in vitro and greater functionality after transplantation into streptozotocin-induced SCID mice (Contreras et al. 2001a). We further analyzed the cytoprotective effects of Bcl-2 in vivo after transplantation by reconstituting NOD-SCID mice with rhesus lymphocytes (Berney et al. 2001b). Two weeks after reconstitution, high numbers of rhesus T and B cells appear in blood, spleen, liver, bone marrow, and other organs. Serum obtained from reconstituted mice was able to induce cytotoxicity in vitro to porcine islets evaluated by intracellular lactate dehydrogenase release and ethidum bromide/acridine orange staining. Gene transfer of Bcl-2 significantly enhanced islet viability after exposure to serum obtained from reconstituted mice. Furthermore, overexpression of Bcl-2 reduced islet loss after intraportal transplantation in reconstituted diabetic mice.

We evaluated the results described above by normalizing glucose levels and glucose disposal rates after the transplant. In contrast, immediate destruction of porcine islets was observed in control animals that received either uninfected islets or islets infected with an adenoviral vector encoding an irrelevant gene (β-galactosidase). Histological analysis confirmed the presence of intrahepatic porcine pancreatic islets only in animals that received islets infected with adenovirus encoding Bcl-2 (Contreras 2001b). Taken together, these results demonstrate the capacity of Bcl-2 to prevent immediate loss of porcine islet mass after exposure to xenoreactive natural antibodies and complement, both in vitro and in vivo, after xenotransplantation.

Conclusions

Interest in pancreatic islet transplantation has increased enormously, because of impressive success rates with newer immunosuppressive regimens and islet preparation techniques. However, major obstacles must be overcome before islet transplantation can become a routine therapeutic procedure. Problems include chronic immunosuppression; the loss of functional islet mass after transplantation, which necessitates multiple islet infusion to achieve euglycemia without exogenous administration of insulin; and the shortage of human tissue for transplantation. With reference to the first obstacle, stable islet allograft function without immunosuppressive therapy has been achieved after tolerance induction in diabetic primates. In regard to the second obstacle, different strategies, including gene transfer of antiapoptotic genes, have been employed to protect isolated islets before and after transplantation. With reference to the third obstacle, xenotransplantation is an attractive strategy, which utilizes pigs as an alternative source of human pancreatic tissue. This strategy is attractive because pigs breed rapidly, there is a long history of porcine insulin use in humans, and there is the potential for genetic engineering. Well-designed preclinical studies in primates can provide the quality of information required to translate islet transplant research into clinical transplantation safely.

Acknowledgments

This work was supported by National Institutes of Health grant RO3 DK064827-01, the Diabetes Trust Fund, the American Society of Transplantation/Fujisawa Faculty Development Award, and the University of Alabama at Birmingham Faculty Development Award.

¹Abbreviations used in this article: IEQ, islet equivalents; MHC, major histocompatibility complex; NHP, nonhuman primate; PIT, pancreatic islet transplant; RGD, arginine-glycine-aspartame; SCID, severe combined immune deficiency.

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