MRI monitoring of transplanted neonatal porcine islets labeled with polyvinylpyrrolidone-coated superparamagnetic iron oxide nanoparticles in a mouse model.

Islet transplantation is a potential treatment option for certain patients with type 1 diabetes; however, it still faces barriers to widespread use, including the lack of tools to monitor islet grafts post-transplantation. This study investigates whether labeling neonatal porcine islets (NPI) with polyvinylpyrrolidone-coated superparamagnetic iron oxide nanoparticles (PVP-SPIO) affects their function, and whether this nanoparticle can be utilized to monitor NPI xenografts with magnetic resonance imaging (MRI) in a mouse model. In vitro, PVP-SPIO-labeled NPI in an agarose gel was visualized clearly by MRI. PVP-SPIO-labeled islets were then transplanted under the kidney capsules of immunodeficient nondiabetic and diabetic mice. All diabetic mice that received transplantation of PVP-SPIO-labeled islets reached normoglycemia. Grafts appeared as hypo-intense areas on MRI and were distinguishable from the surrounding tissues. Following injection of spleen cells from immunocompetent mice, normoglycemic recipient mice became diabetic and islet grafts showed an increase in volume, accompanied by a mixed signal on MRI. Overall, this study demonstrates that PVP-SPIO did not affect the function of NPI that PVP-SPIO-labeled islets were easily seen on MRI, and changes in MRI signals following rejection suggest a potential use of PVP-SPIO-labeled islets to monitor graft viability.

Impact of donor and prolonged cold ischemia time of neonatal pig pancreas on neonatal pig islet transplant outcome.

BACKGROUND: Genetically modified pigs (GMP) have been developed to alleviate the shortage of donors in human islet transplantation and rejection. In this study, we characterized and compared the islets from GalTKO, GalTKO/hCD46, GalTKO/hCD46/hCD39, and wild-type (WT) neonatal pigs. METHODS: Islets were isolated from GMP and WT pig pancreases that have been packaged with ice pack for at least 24 hours. The difference in gene expression and function of islets were evaluated by microarray analysis and transplantation of islets under the kidney capsule of streptozotocin-induced diabetic immune-deficient mice, respectively. Blood glucose levels of these mice were monitored weekly post-transplantation for >100 days, and islet grafts were collected and evaluated for the presence of endocrine cells. RESULTS: The genes involved in extracellular components, cell adhesion, glucose metabolism, and inflammatory response are differentially expressed between GMP and WT pig islets. Variation in the ability of pig islets in correcting the diabetic state of the mouse recipients appears to be dependent on the pig donor. In addition, prolonged cold ischemia time had a negative effect on the transplant outcome. All normoglycemic mice were able to respond well to glucose challenge despite the initial differences in the ability of islet transplants to reverse their diabetic state. Islet xenografts of normoglycemic mice contained abundant insulin- and glucagon-positive cells. CONCLUSION: The effect of GMP and WT neonatal pig islet transplants on hyperglycemia in mice appears to be dependent on the pig donor, and prolonged cold ischemia time negatively affects the neonatal pig islet transplant outcome.

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes--Chapter 3: Porcine islet product manufacturing and release testing criteria.

In the 2009 IXA consensus, the requirements for the quality and control of manufacturing of porcine islet products were based on the U.S. regulatory framework where the porcine islet products fall within the definition of somatic cell therapy under the statutory authority of the U.S. Food and Drug Administration (FDA). In addition, porcine islet products require pre-market approval as a biologic product under the Public Health Services Act and they meet the definition of a drug under the Federal Food, Drug, and Cosmetic Act (FD&C Act). Thus, they are subject to applicable provisions of the law and as such, control of manufacturing as well as reproducibility and consistency of porcine islet products, safety of porcine islet products, and characterization of porcine islet products must be met before proceeding to clinical trials. In terms of control of manufacturing as well as reproducibility and consistency of porcine islet products, the manufacturing facility must be in compliance with current Good Manufacturing Practices (cGMP) guidelines appropriate for the initiation of Phase 1/2 clinical trials. Sponsors intending to conduct a Phase 1/2 trial of islet xenotransplantation products must be able to demonstrate the safety of the product through the establishment of particular quality assurance and quality control procedures. All materials (including animal source and pancreas) used in the manufacturing process of the porcine islet products must be free of adventitious agents. The final porcine islet product must undergo tests for the presence of these adventitious agents including sterility, mycoplasma (if they are cultured), and endotoxin. Assessments of the final product must include the safety specifications mentioned above even if the results are not available until after release as these data would be useful for patient diagnosis and treatment if necessary. In addition, a plan of action must be in place for patient notification and treatment in case the sterility culture results are positive. In terms of the characterization of porcine islet products and product release criteria, the information on the porcine islet products should be acquired from a sample of the final product to be used for transplantation and must include the morphology of the islets, specific identity, purity, viability, and potency of the product. In addition, information on the quantity of the islet products should also be provided in a standardized fashion and this should be in terms of islet equivalents and/or cell numbers. The current consensus was created to provide guidelines that manufacturing facilities may find helpful in the manufacture of and the release criteria for porcine islet products including encapsulated islets and combined islet products. Our intent with the above recommendations is to provide a framework for individual porcine islet manufacturing facilities to ensure a high level of safety for the initiation of Phase 1/2 clinical trials on porcine islet xenotransplantation.

Long-term survival of neonatal porcine islets in nonhuman primates by targeting costimulation pathways.

We evaluated the ability of neonatal porcine islets to engraft and restore glucose control in pancreatectomized rhesus macaques. Although porcine islets transplanted into nonimmunosuppressed macaques were rapidly rejected by a process consistent with cellular rejection, recipients treated with a CD28-CD154 costimulation blockade regimen achieved sustained insulin independence (median survival, >140 days) without evidence of porcine endogenous retrovirus dissemination. Thus, neonatal porcine islets represent a promising solution to the crucial supply problem in clinical islet transplantation.

Yield, cell composition, and function of islets isolated from different ages of neonatal pigs.

The yield, cell composition, and function of islets isolated from various ages of neonatal pigs were characterized using in vitro and in vivo experimental models. Islets from 7- and 10-day-old pigs showed significantly better function both in vitro and in vivo compared to islets from 3- and 5-day-old pigs however, the islet yield from 10-day-old pigs were significantly less than those obtained from the other pigs. Since islets from 3-day-old pigs were used in our previous studies and islets from 7-day-old pigs reversed diabetes more efficiently than islets from other groups, we further evaluated the function of these islets post-transplantation. B6 rag-/- mouse recipients of various numbers of islets from 7-day-old pigs achieved normoglycemia faster and showed significantly improved response to glucose challenge compared to the recipients of the same numbers of islets from 3-day-old pigs. These results are in line with the findings that islets from 7-day-old pigs showed reduced voltage-dependent K+ (Kv) channel activity and their ability to recover from post-hypoxia/reoxygenation stress. Despite more resident immune cells and immunogenic characteristics detected in islets from 7-day-old pigs compared to islets from 3-day-old pigs, the combination of anti-LFA-1 and anti-CD154 monoclonal antibodies are equally effective at preventing the rejection of islets from both age groups of pigs. Collectively, these results suggest that islets from various ages of neonatal pigs vary in yield, cellular composition, and function. Such parameters may be considered when defining the optimal pancreas donor for islet xenotransplantation studies.

Mouse pancreatic islets are resistant to indoleamine 2,3 dioxygenase-induced general control nonderepressible-2 kinase stress pathway and maintain normal viability and function.

Islet transplantation is a promising treatment for diabetes. However, it faces several challenges including requirement of systemic immunosuppression. Indoleamine 2,3-dioxygenase (IDO), a tryptophan degrading enzyme, is a potent immunomodulatory factor. Local expression of IDO in bystander fibroblasts suppresses islet allogeneic immune response in vitro. The aim of the present study was to investigate the impact of IDO on viability and function of mouse islets embedded within IDO-expressing fibroblast-populated collagen scaffold. Mouse islets were embedded within collagen matrix populated with IDO adenovector-transduced or control fibroblasts. Proliferation, insulin content, glucose responsiveness, and activation of general control nonderepressible-2 kinase stress-responsive pathway were then measured in IDO-exposed islets. In vivo viabilities of composite islet grafts were also tested in a syngeneic diabetic animal model. No reduction in islet cells proliferation was detected in both IDO-expressing and control composites compared to the baseline rates. Islet functional studies showed normal insulin content and secretion in both preparations. In contrast to lymphocytes, general control nonderepressible-2 kinase pathway was not activated in islets cocultured with IDO-expressing fibroblasts. When transplanted to diabetic mice, syngeneic IDO-expressing composite islet grafts were functional up to 100 days tested. These findings collectively confirm normal viability and functionality of islets cocultured with IDO-expressing cells and indicate the feasibility of development of a functional nonrejectable islet graft.

Epithelial cells within the human pancreas do not coexpress mesenchymal antigens: epithelial-mesenchymal transition is an artifact of cell culture.

Pancreatic mesenchymal stem cells (MSCs) may be derived from human beta-cells undergoing reversible epithelial-mesenchymal transition (EMT), suggesting that they could be a potential source of new beta-cells. In this study we sought to determine the origin of pancreatic MSCs in the nonendocrine pancreas. Double immunofluorescent (IF) staining and flow cytometry were used to assess the cell phenotype of nonendocrine pancreas tissue following islet procurement, during in vitro expansion of MSCs, and after differentiation. IF staining of paraffin-embedded pancreatic biopsy sections was used to assess cell phenotype in vivo. In this study we demonstrated that: (1) pancreatic epithelial cells do not express MSC antigens in vivo; (2) following islet isolation EpCAM- and CK19-positive epithelial cells coexpressed the MSC antigens CD44 (32+/-8% and 38+/-10%) and CD29 (85+/-4% and 64+/-4%); (3) during in vitro expansion the number of single-positive epithelial and double-positive epithelial/MSCs decreased whereas the number of single-positive MSCs increased and (4) differentiated MSCs do not revert to a true epithelial cell phenotype in our culture conditions, as epithelial cell surface markers (EpCAM, CK19 and E-Cadherin) are not reexpressed, although the MSC phenotype is altered. This study demonstrates that MSCs may be derived in vitro via a pancreatic epithelial cell undergoing EMT, however it is more likely that a small percentage of MSCs that reside in the adult pancreas are proliferating whereas the epithelial cells are negatively selected by the experimental culture conditions.

Prolonged survival of microencapsulated neonatal porcine islet xenografts in immune-competent mice without antirejection therapy.

Several studies have demonstrated that in vitro culture of islets prolonged islet graft survival in immune-competent mice without administration of antirejection drugs. However, we recently showed that in vitro cultured microencapsulated neonatal porcine islets (NPI) were rejected in immune-competent mice not receiving antirejection therapy. The aim of this study was to determine whether culture of microencapsulated NPI in vivo could promote long-term survival of microencapsulated NPI in immune-competent mice without administration of antirejection drugs. Microencapsulated NPI that were cultured in vitro for 7 and 50 days or transplanted initially in immune-deficient C.B.-17 SCID-BEIGE mice for 100 days (in vivo cultured) were characterized and transplanted into streptozotocin-induced diabetic immune-competent BALB/c mice. Day 50 in vitro cultured and day 100 in vivo cultured microencapsulated NPI showed significantly higher insulin and DNA content, indicating maturation of NPI compared to day 7 in vitro cultured microencapsulated NPI. Interestingly, in vivo cultured microencapsulated NPI expressed lower levels of porcine antigens compared to day 7 and day 50 in vitro cultured microencapsulated NPI. Transplantation of day 7 in vitro cultured microencapsulated NPI did not reverse diabetes in immune-competent BALB/c mouse recipients. In contrast, transplantation of day 50 in vitro cultured and in vivo cultured microencapsulated NPI into diabetic immune-competent BALB/c mice resulted in the immediate reversal of hyperglycemia within 2 days posttransplantation. However, all recipients of day 50 in vitro cultured microencapsulated NPI eventually rejected their grafts by day 15 posttransplantation, while 6 of 10 BALB/c mouse recipients of in vivo cultured microencapsulated NPI maintained normoglycemia for 100 days posttransplantation. These results show that in vivo culture of NPI in immune-deficient mice results in the modulation of NPI, which allows for their long-term survival in immune-competent mice without antirejection therapy.

Sertoli cell line lacks the immunoprotective properties associated with primary Sertoli cells.

Sertoli cells are important for maintenance of the immune privileged environment of the testis and prolong survival of cotransplanted cells. The objective of the current study was to examine the immunoprotective properties of a mouse Sertoli cell line (MSC-1) in order to identify a Sertoli cell line that could be used to aid in investigation of the immunoprotective abilities of Sertoli cells. BALB/c islets were cotransplanted with 0-9 million primary BALB/c Sertoli cells or MSC-1 cells into diabetic C3H or BALB/c mice and protection of grafted islets was examined by monitoring blood glucose levels and immunohistochemical analysis. Additionally, expression of potential immunoprotective factors in MSC-1 cells was examined. Cotransplantation of islets with 3 million primary Sertoli cells significantly prolonged islet allograft survival (61.1 +/- 6.9 days; p < 0.05) compared with control mice that received allogeneic islets alone (26.9 +/- 2.1 days). Grafts collected from normoglycemic C3H mice at 100 days posttransplant contained insulin-positive beta-cells adjacent to allogeneic Sertoli cells arranged in tubule-like structures. In contrast, cotransplantation of islet allografts with MSC-1 cells did not prolong islet survival (average 29.8 +/- 3.3 days) regardless of the number of MSC-1 cells transplanted and the rejected grafts contained very few beta-cells and randomly arranged MSC-1 cells. The lack of islet cell survival was not due to detrimental effects of MSC-1 cells because syngneic islets cotransplanted with MSC-1 cells were functional throughout the study. MSC-1 cells were found to express known Sertoli cell-expressed, immunoprotective factors, clusterin, Fas ligand, and transforming growth factor-beta1, suggesting additional factors may be involved in Sertoli cell immune privilege. These data indicate the MSC-1 cell line lacks the immunoprotective properties associated with primary Sertoli cells. Further study of this cell line could be useful in examining the mechanisms that enable Sertoli cells to provide immune privilege.

Monotherapy with anti-LFA-1 monoclonal antibody promotes long-term survival of rat islet xenografts.

Previously we demonstrated that anti-LFA-1 monoclonal (mAb) could promote long-term survival of discordant porcine islet xenografts in mice. The aim of this study, therefore, was to determine whether a shortterm administration of anti-LFA-1 mAb would promote long-term survival of concordant rat islet xenografts in mice, and whether combining short-term administration of anti-LFA-1 mAb therapy with an immunosuppressive drug, rapamycin, would facilitate islet xenograft survival. Streptozotocin-induced diabetic BALB/c mice were transplanted with 500 Wistar-Furth rat islets under the kidney capsule and were either left untreated or treated with short-term administration of rapamycin (0.2 mg/kg) alone, anti-LFA-1 mAb (0.2 mg/ dose) alone, or a combination of rapamycin and anti-LFA-1 mAb using the same doses. All untreated mice rejected their grafts by 24 days posttransplantation with a mean graft survival time of 18.8 +/- 2.5 days posttransplantation (n = 5). All mice treated with rapamycin alone had prolonged islet graft survival but eventually rejected their islet grafts by 81 days posttransplantation. In contrast, the majority of the mice (27/ 28) treated with anti-LFA-1 mAb alone maintained long-term normoglycemia (>100 days). Rapamycin in combination with anti-LFA-1 mAb proved equally effective with 29 of 30 mice maintaining normoglycemia for more than 100 days posttransplantation. Low levels of mouse anti-rat antibodies, as well as a decrease in the degree of mononuclear cell infiltration of the islet graft, closely correlated with long-term islet xenograft survival. These results demonstrate that monotherapy with anti-LFA-1 mAb is highly effective in promoting long-term survival of rat islet xenografts and that combination of anti-LFA-1 mAb with rapamycin does not facilitate nor abrogate the induction of long-term xenograft survival by anti-LFA-1 mAb therapy in BALB/c mice. Our study indicates that immunomodulation through mAb therapy could form a significant component of future antirejection therapies in clinical islet xenotransplantation.

Comparison of successful and unsuccessful islet/Sertoli cell cotransplant grafts in streptozotocin-induced diabetic mice.

Sertoli cells (SC) protect islet allografts from immune destruction in diabetic rodents. In this study, we examined the difference between successful and rejected islet/SC cografts in order to further improve this procedure for optimal extension of islet allograft survival. We cotransplanted 500 BALB/c islets with 1-8 million BALB/c SC under the kidney capsule of diabetic BALB/c, C3H-HeJ, and C57BL/6 mice. Cotransplantation of islets with up to 8 million SC was not detrimental to long-term islet graft function in syngeneic mice. However, large numbers of SC were detrimental to islet graft survival in allogeneic mice with the optimal dose for cotransplantation of 4 or 1 million SC in C3H-HeJ or C57BL/6 mice, respectively. Examination of successful grafts, from euglycemic recipients, revealed the presence of SC arranged in tubule structures with islets surrounding these tubules. Cellular infiltrate in successful grafts revealed CD4 T cells and macrophages along the periphery and within the grafts, and very few CD8 T cells. Conversely, examination of unsuccessful grafts, harvested from hyperglycemic recipients at the time of rejection, revealed the presence of SC arranged randomly with islets adjacent to the Sertoli cells, when present, and massive CD4 and CD8 T cell as well as macrophage cell infiltration. Prolongation of islet allograft survival appeared to be a function of SC transplant mass and recipient genetic background. A consequence of long-term graft acceptance is the formation of SC tubule structures, which may be an additional requirement for optimal protection of islet allografts.

Glutaraldehyde-fixed bioprosthetic heart valve conduits calcify and fail from xenograft rejection.

BACKGROUND: Glutaraldehyde fixation (G-F) decreases but likely does not eliminate the antigenicity of bioprosthetic heart valves. Rejection (with secondary dystrophic calcification) may be why G-F xenograft valves fail, especially in young patients, who are more immunocompetent than the elderly. Therefore, we sought to determine whether rejection of G-F xenograft occurs and to correlate this with graft calcification. METHODS AND RESULTS: Ascending aortas/valves (from rats [syngeneic] or guinea pigs [xenogeneic]) were transplanted (fresh or after 48 hour of G-F) into the infrarenal aortas of young rat recipients for 20 days. A xenogeneic group was also treated with steroids until graft harvest. The valves and media/adventitia were scored blindly for inflammation (0 to 4). Percent graft infiltration by T cells/macrophages was determined (immunohistochemistry), and rat IgG ELISAs were performed. There was >3 times more valve inflammation, >10 times more valve T-cell/macrophage infiltrate, and >3 times antibody rise in the G-F xenogeneic groups compared with the fresh syngeneic or the G-F syngeneic groups (P<0.05). There was >2 times more adventitial inflammation and T-cell/macrophage infiltrate in the xenogeneic groups (P<0.05). Steroid treatment decreased inflammation and antibody rise in the xenogeneic groups (P<0.05). Correlation analysis revealed media/adventitia inflammation (P=0.02) and percent macrophage (P=0.01) infiltration to be predictors of calcification. CONCLUSIONS: G-F xenografts have cellular/humoral rejection and calcify secondarily.

Combination of anti-CD4 with anti-LFA-1 and anti-CD154 monoclonal antibodies promotes long-term survival and function of neonatal porcine islet xenografts in spontaneously diabetic NOD mice.

Type 1 diabetes mellitus (T1DM) is caused by the autoimmune destruction of pancreatic islet beta-cells, which are required for the production of insulin. Islet transplantation has been shown to be an effective treatment option for TIDM; however, the current shortage of human islet donors limits the application of this treatment to patients with brittle T1DM. Xenotransplantation of pig islets is a potential solution to the shortage of human donor islets provided xenograft rejection is prevented. We demonstrated that a short-term administration of a combination of anti-LFA-1 and anti-CD154 monoclonal antibodies (mAbs) was highly effective in preventing rejection of neonatal porcine islet (NPI) xenografts in non-autoimmune-prone B6 mice. However, the efficacy of this therapy in preventing rejection of NPI xenografts in autoimmune-prone nonobese diabetic (NOD) mice is not known. Given that the current application of islet transplantation is for the treatment of T1DM, we set out to determine whether a combination of anti-LFA-1 and anti-CD154 mAbs could promote long-term survival of NPI xenografts in NOD mice. Short-term administration of a combination of anti-LFA-1 and anti-CD154 mAbs, which we found highly effective in preventing rejection of NPI xenografts in B6 mice, failed to promote long-term survival of NPI xenografts in NOD mice. However, addition of anti-CD4 mAb to short-term treatment of a combination of anti-LFA-1 and anti-CD154 mAbs resulted in xenograft function in 9/12 animals and long-term graft (>100 days) survival in 2/12 mice. Immunohistochemical analysis of islet grafts from these mice identified numerous insulin-producing beta-cells. Moreover, the anti-porcine antibody as well as autoreactive antibody responses in these mice was reduced similar to those observed in naive nontransplanted mice. These data demonstrate that simultaneous targeting of LFA-1, CD154, and CD4 molecules can be effective in inducing long-term islet xenograft survival and function in autoimmune-prone NOD mice.

Long-term graft function after allogeneic islet transplantation.

Islet transplants are emerging as a viable option for the treatment of type 1 diabetes mellitus. From 1989 to 1995 we conducted a series of simultaneous islet-kidney transplants in six uremic type 1 diabetic patients. We report two of these patients who have shown persistent islet graft function over many years. Two female patients with duration of diabetes of 27 and 37 years underwent simultaneous islet-kidney transplant under steroid- and cyclosporine-based immunosuppression. Freshly isolated islets were supplemented with cryopreserved islets from our low-temperature bank of frozen islets. A total islet mass of 9,866 and 15,061 islet equivalents/kg body weight, respectively, was transplanted into the liver through portal vein. Reasonable blood glucose control has been achieved for up to 6 years posttransplant in one patient, but there was minimum clinical benefit from the islet graft at 10 years. In contrast, sustained insulin secretion with nearly normal HbA1c at 13 years follow-up was observed in another patient, providing hope for improving long-term graft outcomes for islet transplant recipient.

Reversal of diabetes in pancreatectomized pigs after transplantation of neonatal porcine islets.

Neonatal porcine islets (NPIs) are able to grow and to reverse hyperglycemia after transplantation in immunoincompetent mice. The aim of this study was to demonstrate the feasibility of allogeneic NPI grafts to achieve normoglycemia in a pancreatectomized diabetic pig. NPIs were isolated from pancreases of 1- to 3-day-old pigs, cultured, and then transplanted via the portal vein into the liver of totally pancreatectomized pigs (mean body weight, 20.8 kg). Each pig received NPIs consisting of 3.1 +/- 0.3 x 10(6) beta-cells/kg (12,476 +/- 1,146 islet equivalent/kg). The six pigs that were given cyclosporine and sirolimus achieved normoglycemia by day 14 without insulin therapy. Three pigs died of surgical complications shortly after transplantation, whereas the other three remained insulin independent up to day 69. Of seven nonimmunosuppressed recipients, four pigs became normoglycemic by day 14 without insulin treatment, with two of the animals remaining normoglycemic long term. Well-preserved insulin-positive cells were found in the graft at the end of follow-up with a significant increase in insulin content in long-term survivors of both groups. This study demonstrates for the first time that allogeneic NPIs can reverse hyperglycemia in totally pancreatectomized diabetic pigs.

XIAP overexpression in human islets prevents early posttransplant apoptosis and reduces the islet mass needed to treat diabetes.

The Edmonton Protocol for treatment of type 1 diabetes requires islets from two or more donors to achieve euglycemia in a single recipient, primarily because soon after portal infusion, the majority of the transplanted cells undergo apoptosis due to hypoxia and hypoxia reperfusion injury. X-linked inhibitor of apoptosis protein (XIAP) is a potent endogenous inhibitor of apoptosis that is capable of blocking the activation of multiple downstream caspases, and XIAP overexpression has previously been shown to enhance engraftment of a murine beta-cell line. In this study, human islets transduced with a XIAP-expressing recombinant adenovirus were resistant to apoptosis and functionally recovered following in vitro stresses of hypoxia and hypoxia with reoxygenation (models reperfusion injury). Furthermore Ad-XIAP transduction dramatically reduced the number of human islets required to reverse hyperglycemia in chemically diabetic immunodeficient mice. These results suggest that by transiently overexpressing XIAP in the immediate posttransplant period, human islets from a single donor might be used to effectively treat two diabetic recipients.

Neonatal porcine islets exhibit natural resistance to hypoxia-induced apoptosis.

BACKGROUND: Despite the success of the Edmonton protocol for human islet transplantation, an alternate source of islet tissue must be developed if beta-cell replacement therapy is to see widespread application. Neonatal porcine islets (NPI) represent one potential source of tissue. When human or rodent islets are transplanted, the majority of cells undergo hypoxia-induce apoptosis soon after the grafts are placed in the recipient. In the present study, we investigated whether NPI were similarly sensitive to hypoxia. METHODS: NPI were exposed to hypoxia and hypoxia/reoxygenation using an in vitro hypoxic chamber. Afterwards, viability, frequency of apoptosis, and beta-cell function were evaluated. NPI and adult porcine islets were transplanted into chemically diabetic, immunodeficient mice and graft apoptosis was assessed 24 hours and seven days posttransplant. RESULTS: NPI demonstrated a remarkable capacity to resist apoptosis and maintain insulin secretion despite severe stresses such as hypoxia/reoxygenation. One day after transplantation, NPI grafts showed limited apoptosis, confined to rare strongly insulin positive cells. In contrast, adult porcine islet grafts underwent widespread apoptosis. Western blotting revealed that NPI express high levels of at least one potent endogenous antiapoptotic protein (XIAP). CONCLUSIONS: The majority of cells within transplanted human islets undergo apoptosis soon after portal infusion. In contrast, NPI have the capacity to resist this early posttransplant apoptosis, with likely reduced antigen release and diminished immune stimulation. NPI appear to contain a population of insulin-low to insulin-negative pre-beta-cells, which are resistant to hypoxia-induced apoptosis and still capable of differentiating into mature beta-cells.

Porcine Islet-Specific Tolerance Induced by the Combination of Anti-LFA-1 and Anti-CD154 mAbs Is Dependent on PD-1.

We previously demonstrated that short-term administration of a combination of anti-LFA-1 and anti-CD154 monoclonal antibodies (mAbs) induces tolerance to neonatal porcine islet (NPI) xenografts that is mediated by regulatory T cells (Tregs) in B6 mice. In this study, we examined whether the coinhibitory molecule PD-1 is required for the induction and maintenance of tolerance to NPI xenografts. We also determined whether tolerance to NPI xenografts could be extended to allogeneic mouse or xenogeneic rat islet grafts since we previously demonstrated that tolerance to NPI xenografts could be extended to second-party NPI xenografts. Finally, we determined whether tolerance to NPI xenografts could be extended to allogeneic mouse or second-party porcine skin grafts. Diabetic B6 mice were transplanted with 2,000 NPIs under the kidney capsule and treated with short-term administration of a combination of anti-LFA-1 and anti-CD154 mAbs. Some of these mice were also treated simultaneously with anti-PD-1 mAb at >150 days posttransplantation. Spleen cells from some of the tolerant B6 mice were used for proliferation assays or were injected into B6 rag(-/-) mice with established islet grafts from allogeneic or xenogeneic donors. All B6 mice treated with anti-LFA-1 and anti-CD154 mAbs achieved and maintained normoglycemia until the end of the study; however, some mice that were treated with anti-PD-1 mAb became diabetic. All B6 rag(-/-) mouse recipients of first- and second-party NPIs maintained normoglycemia after reconstitution with spleen cells from tolerant B6 mice, while all B6 rag(-/-) mouse recipients of allogeneic mouse or xenogeneic rat islets rejected their grafts after cell reconstitution. Tolerant B6 mice rejected their allogeneic mouse or xenogeneic second-party porcine skin grafts while remaining normoglycemic until the end of the study. These results show that porcine islet-specific tolerance is dependent on PD-1, which could not be extended to skin grafts.

Combination therapy with sirolimus and interleukin-2 prevents spontaneous and recurrent autoimmune diabetes in NOD mice.

Sirolimus is an immunosuppressant that inhibits interleukin (IL)-2 signaling of T-cell proliferation but not IL-2-induced T-cell apoptosis. Therefore, we hypothesized that administration of IL-2, together with sirolimus, might shift T-cell proliferation to apoptosis and prevent autoimmune destruction of islet beta-cells. We found that sirolimus and IL-2 therapy of female NOD mice, beginning at age 10 weeks, was synergistic in preventing diabetes development, and disease prevention continued for 13 weeks after stopping sirolimus and IL-2 therapy. Similarly, sirolimus and IL-2 were synergistic in protecting syngeneic islet grafts from recurrent autoimmune destruction after transplantation in diabetic NOD mice, and diabetes did not recur after stopping sirolimus and IL-2 combination therapy. Immunocytochemical examination of islet grafts revealed significantly decreased numbers of leukocytes together with increased apoptosis of these cells in mice treated with sirolimus and IL-2, whereas beta-cells were more numerous, and significantly fewer were apoptotic. In addition, Th1-type cells (gamma-interferon-positive and IL-2(+)) were decreased the most, and Th2-type cells (IL-4(+) and IL-10(+)) and Th3-type cells (transforming growth factor-beta1(+)) were increased the most in islet grafts of sirolimus and IL-2-treated mice. We conclude that 1) combination therapy with sirolimus and IL-2 is synergistic in protecting islet beta-cells from autoimmune destruction; 2) diabetes prevention continues after withdrawal of therapy; and 3) the mechanism of protection involves a shift from Th1- to Th2- and Th3-type cytokine-producing cells, possibly due to deletion of autoreactive Th1 cells.