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.

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.

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.

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.

Cyanidin-3-O-glucoside enhanced the function of syngeneic mouse islets transplanted under the kidney capsule or into the portal vein.

BACKGROUND: It was previously shown that islets are susceptible to oxidative stress due to their inherent low antioxidant capacity. Therefore, in this study, we determined whether treatment of mouse islets with an antioxidant cyanidin-3-O-glucoside (C3G) could enhance their function after transplantation under the kidney capsule or into the portal vein. METHODS: B6 mouse islets were treated with various concentrations of C3G, their viability, and the expression of antioxidant (HO-1) and antiapoptotic (Bcl-2 and Survivin) genes were determined. The C3G-treated (1.0 µM) or untreated B6 mouse islets (100, 200, and 400 IEQ) were transplanted under the kidney capsule or into the portal vein of diabetic B6 mice, and their blood glucose levels were monitored for more than 100 days after transplantation. RESULTS: The C3G-treated islets showed higher cell viability compared to untreated control and the expression of HO-1; Bcl-2 and Survivin genes were enhanced in a concentration-dependent manner. All mice that were transplanted with C3G-treated islets achieved normoglycemia faster than recipients of untreated islets. Mice that received 400, 200, or 100 treated islets transplanted under the kidney capsule achieved normoglycemia, whereas only mice that were transplanted with 400 and 200 treated islets into the portal vein achieved normoglycemia. The mean blood glucose levels of mice that received C3G-treated islets were lower compared to those observed in mouse recipients of untreated islets transplanted under the kidney capsule or into the portal vein. CONCLUSIONS: Our results show that C3G could enhance the viability of mouse islets and improve their function after transplantation.

Failure of neonatal B-cell tolerance induction by ABO-incompatible kidney grafts in piglets.

BACKGROUND: ABO-incompatible (ABOi) infant heart transplantation results in B-cell tolerance to graft A/B antigens, confirming human susceptibility to acquired immunologic or "neonatal" tolerance as described originally in murine models. Starting with this clinical observation, we sought to model neonatal ABOi organ transplantation to allow mechanistic studies of tolerance. METHODS: Plasma anti-A/B antibodies were measured over time in piglets to establish developmental antibody kinetics. Blood group O piglets received kidney allografts from group A (AO-incompatible) or group O (AO-compatible) donors under cyclosporine immunosuppression. Anti-A antibodies were measured serially after transplantation; A/H antigen expression and allograft rejection were assessed in graft biopsies. RESULTS: Anti-A antibodies developed in naïve piglets in a kinetic pattern analogous to human infants; anti-B remained low. After transplantation, anti-A antibodies developed similarly in AO-incompatible and AO-compatible groups and were not suppressed by cyclosporine. A/H antigen expression was persistent in all graft biopsies; however, A/H antigens were not detected in vascular endothelium. Cellular and antibody-mediated rejection was absent or minimal in early and late biopsies in both groups, with one exception. CONCLUSIONS: Naturally delayed isohemagglutinin production in piglets is analogous to the developmental kinetics in human infants. However, in contrast to deficient anti-A antibody production as seen long-term after "A-into-O" infant heart transplant recipients, normal anti-A antibody production after "A-into-O" piglet kidney transplantation indicates that tolerance did not develop despite graft A antigen persistence. These findings suggest that the impact on the host immune system of exposure to nonself ABH antigens during early life in human heart versus porcine kidney grafts may depend on expression in vascular endothelium.

Protection of porcine islet xenografts in mice using sertoli cells and monoclonal antibodies.

BACKGROUND: To remedy the shortage of human donor islets, xenotransplantation of neonatal porcine islets (NPI) provides an attractive alternative source of donor tissue so long as graft rejection can be circumvented. Thus, in this study, we sought to determine whether cotransplantation of NPI with Sertoli cells (SC) combined with a short-course treatment of monoclonal antibody (mAb) could provide long-term islet xenograft survival. METHODS: NPI alone or NPI cotransplanted with neonatal porcine SC were transplanted into diabetic C57BL/6 mice. These mice were left untreated or were treated with a short course of antileukocyte function associated antigen-1 (LFA-1), anti-CD154, or anti-CD45RB mAb. Blood glucose levels were monitored twice a week to assess graft function. At more than 100 days posttransplantation or on the day of rejection, graft-bearing kidneys were collected for characterization using immunohistochemistry. RESULTS: None of the untreated control mice transplanted with NPI alone (0/5) or NPI cotransplanted with SC (0/8) achieved normoglycemia. However, of the mice receiving NPI alone, 3 of 7 treated with anti-LFA-1 mAb, 2 of 7 treated with anti-CD154 mAb, and 1 of 7 treated with anti-CD45RB mAb achieved long-term graft survival (>100 days). These proportions improved considerably when NPI were cotransplanted with SC, as 15 of 15 mice treated with anti-LFA-1 mAb, 7 of 8 mice treated with anti-CD154 mAb, and 4 of 9 mice treated with anti-CD45RB mAb achieved long-term graft survival. CONCLUSIONS: These results show that transient administration of anti-LFA-1 mAb or anti-CD154 mAb is efficacious in prolonging NPI xenograft survival when islets are cotransplanted with SC. Interleukin-4 and Serpina3n may be important mediators of protection observed in this model.

Short-term administrations of a combination of anti-LFA-1 and anti-CD154 monoclonal antibodies induce tolerance to neonatal porcine islet xenografts in mice.

OBJECTIVE: The objective of this study was to determine whether tolerance to neonatal porcine islet (NPI) xenografts could be achieved by short-term administrations of anti-LFA-1 and anti-CD154 monoclonal antibodies (mAbs). RESEARCH DESIGN AND METHODS: Diabetic B6 mice received NPI transplants and short-term injections of combined anti-LFA-1 and anti-CD154 mAbs. Mice with long-term islet graft function were treated with depleting anti-CD25 mAb or re-transplanted with a second-party NPI. At the end of the study, grafts from mice with long-term islet function were examined. Their spleen cells were characterized and used for in vitro proliferation and adoptive transfer studies. RESULTS: All mAb-treated NPI recipients maintained normoglycemia for >100 days post-transplantation. Only 5 of 50 mice rejected their grafts before 300 days post-transplantation. Intact islets, foxp3(+) immune cells, as well as interleukin (IL)-10 and transforming growth factor (TGF)-beta regulatory cytokine transcripts were detected in the NPI xenografts from tolerant mice. A higher percentage of CD4(+) T-cell population from these mice expressed regulatory markers, suggesting that tolerance to NPI xenografts may be mediated by T regulatory cells. This was confirmed when tolerant mice treated with depleting anti-CD25 mAb became diabetic. Lymphocytes from tolerant mice inhibited the proliferation of lymphocytes from B6 mice immunized with porcine cells and they displayed limited proliferation when adoptively transferred. All protected B6 mice transplanted with a second-party NPI xenograft maintained long-term normoglycemia even after removal of the first NPI graft-bearing kidney. CONCLUSIONS: These results demonstrate that tolerance to NPI xenografts can be achieved by transient administrations of combined anti-LFA-1 and anti-CD154 mAb therapy.

Portal vein embolization with radiolabeled polyvinyl alcohol particles in a swine model: hepatic distribution and implications for pancreatic islet cell transplantation.

The distribution of radiolabeled polyvinyl alcohol microspheres (PVAMs) when infused into the portal vein of domestic swine was investigated, with the purpose of assessing implications for pancreatic islet cell transplantation. PVAMs measuring 100-300 microm (Contour SE) and labeled with (99m)Tc were infused into the main portal vein of 12 swine, with intermittent portal venous pressure measurements. The infusion catheter was introduced antegradely via direct or indirect cannulation of the portal vein. The liver was subsequently divided into anatomical segments. Radioactivity (decay corrected) was measured for (99m)Tc microsphere synthesis, dose preparation, gross organ activities, tissue samples, and blood. Particulate labeling, catheter positioning, and infusion were successful in all cases. The number of particles used was (185,000 +/- 24,000) with a volume of 1 ml. Mean portal pressure at 5 min was significantly higher than baseline, but without a significant difference at 15 min. Extrahepatic tissue and serum radioactivity was negligible. A significant difference in number of radioactive particles per gram was detected between segments 6/7 and segments 5/8. Intrasegmental activity was analyzed, and for segments 2/3 a significant difference in the percentage dose per gram across samples was demonstrated (P = 0.001). Effective and stable radiolabeling of PVAMs with (99m)Tc-sulfur colloid was demonstrated. Portal venous infusion of 100- to 300-microm particles showed entrapment in the sinusoidal hepatic system with transient portal pressure elevation. Preferential embolization into the right lateral and posterior segments occurs, suggesting that flow dynamics/catheter tip position plays a role in particle distribution.

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.

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.

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.

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.

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.

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.

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.