Revascularization and remodelling of pancreatic islets grafted under the kidney capsule

The revascularization and the structural changes resulting from interactions between the graft and the host were investigated in transplanted pancreatic islets under the kidney capsule. Islets were isolated from mice pancreata and transplanted in syngeneic diabetic animals. Graft-bearing kidneys were collected on different days post-transplant and processed for light microscopy, immunohistochemistry and transmission electron microscopy. A numerical analysis was performed in order to compare the percentage number of the different types of cells in native islets and at different time points after the transplant. Recipient animals reversed diabetes within 4 days. An intraperitoneal glucose tolerance test was performed to determine islet functionality under stressful conditions. During the initial few days post-transplant, the islets showed peculiar shapes and the graft tended to aggregate along the vessels. Starting at days 4-7 post-transplant, islets were revascularized from vessels connected to both the cortical and the capsular vascular network of the kidney. From day 7-14 post-transplant, the vessels progressively appeared more similar in features and size to those of in situ pancreatic islets. Both the percentage number of the different cell types and the distribution of Alpha, Beta and Delta cells inside the graft were significantly different as compared with intact islets, demonstrating quantitative and structural changes after the engraftment. No concomitant proliferation of Beta cells was detected using a bromodeoxyuridin staining method. Despite the fact that quick revascularization preserved a large mass of tissue, the remodelling process of the graft and the newly formed vascularization led to a different organization of the endocrine tissue as compared with intact in situ islets. This constitutes the morphological basis for alterations of the normal intercellular interactions and may explain the altered secretory cell function often observed in transplant.     

Extrahepatic islet transplantation with microporous polymer scaffolds in syngeneic mouse and allogeneic porcine models

Intraportal transplantation of islets has successfully treated select patients with type 1 diabetes. However, intravascular infusion and the intrahepatic site contribute to significant early and late islet loss, yet a clinical alternative has remained elusive. We investigated non-encapsulating, porous, biodegradable polymer scaffolds as a vehicle for islet transplantation into extrahepatic sites, using syngeneic mouse and allogeneic porcine models. Scaffold architecture was modified to enhance cell infiltration leading to revascularization of the islets with minimal inflammatory response. In the diabetic mouse model, 125 islets seeded on scaffolds implanted into the epididymal fat pad restored normoglycemia within an average of 1.95 days and transplantation of only 75 islets required 12.1 days. Increasing the pore size to increase islet-islet interactions did not significantly impact islet function. The porcine model was used to investigate early islet engraftment. Increasing the islet seeding density led to a greater mass of engrafted islets, though the efficiency of islet survival decreased. Transplantation into the porcine omentum provided greater islet engraftment than the gastric submucosa. These results demonstrate scaffolds support murine islet transplantation with high efficiency, and feasibility studies in large animals support continued pre-clinical studies with scaffolds as a platform to control the transplant microenvironment.     

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.     

Synergistic effect of microencapsulation and immunoalteration on islet allograft survival in bioartificial pancreas

Recently, we reported successful transplantation (Tx) of microencapsulated (mc) islets. However, graft failure observed in several cases was associated with an increased foreign body reaction compared to long-term functioning grafts. This study was performed to investigate the impact of an immunoalterating islet pretreatment (12–14 days culture at 22°C) on graft function. After microencapsulation in barium alginate beads the islets were cultured for another day. Diabetic LEWIS rats (blood glucose >19 mM) were transplanted with 3500 immunoaltered mc-Wistar islets intraperitoneally. Controls were transplanted with 3500 non-cultured syngeneic or allogeneic mc-islets. Additional syngeneic and allogeneic controls were transplanted with 6000 non-cultured, non-encapsulated islets intraperitoneally. Seventy percent of the recipients of microencapsulated, long-term low temperature cultured islets maintained normoglycemia at least for 15 weeks, while this was true in only 17% of those animals receiving microencapsulated non-pretreated allogeneic islets. Islets in non-encapsulated controls were rejected within several days. Graft function correlated with histologically proven viable islets within the capsules. Microencapsulation of islets markedly prolonged allograft survival compared to non-encapsulated islets; application of an immunoaltering low-temperature culture further improved graft function significantly. These data may support the hypothesis of induction of a reaction against microcapsules by the antigen release from the graft which may be avoided by immunoaltering islet pretreatment.     

The fate of transplanted pancreatic islets in the rat.

Syngeneic pancreatic islets transplanted into the liver or the spleen reverse streptozotocin-induced diabetes in the rat, but allogeneic islets function only briefly and are rejected. Shortly after transplantation, thrombi often form around transplanted tissue, particularly around nonislet tissue that contaminates islet preparations. These thrombi are a source of transient liver injury in recipients of intrahepatic grafts. A few days after transplantation, syngeneic islets injected into the portal vein are found at the periphery of portal tracts in direct contact with periportal hepatocytes, some of which become hypertrophied. Isografts remain situated in the portal tracts for prolonged periods without adverse effect on the surrounding liver. In contrast, allogeneic islets injected into the portal vein are infiltrated by small lymphocytes within 2 days of transplantation and are rapidly destroyed by the host. Syngeneic islets injected into the splenic pulp localize in the sinusoids and, 1 month or more after transplantation, are often surrounded by connective tissue or local collections of hemosiderin-laden macrophages. Allogeneic islets injected into the spleen are rejected with the same intensity and at approximately the same rate as allogeneic islets injected into the portal vein. Transplant rejection leaves no significant lasting morphologic effect on the host liver or spleen.     

Islet‐expressed TLR2 and TLR4 sense injury and mediate early graft failure after transplantation

Although islet transplantation is an effective treatment for Type 1 diabetes, primary engraftment failure contributes to suboptimal outcomes. We tested the hypothesis that islet isolation and transplantation activate innate immunity through TLR expressed on islets. Murine islets constitutively express TLR2 and TLR4, and TLR activation with peptidoglycan or LPS upregulates islet production of cytokines and chemokines. Following transplantation into streptozotocin‐induced diabetic, syngeneic mice, islets exposed to LPS or peptidoglycan had primary graft failure with intra‐ and peri‐islet mononuclear cell inflammation. The use of knockout mice showed that recipient CD8⁺ T cells caused engraftment failure and did so in the absence of islet‐derived DC. To mimic physiological islet injury, islets were transplanted with exocrine debris. Transplantation of TLR2/4^?/? islets reduced proinflammatory cytokine production and improved islet survival. Stressed islets released the alarmin high‐mobility group box protein 1 (HMGB1) and recombinant HMGB1 (rHMGB1) induced NFkB activation. NFkB activation was prevented in the absence of both TLR2 and TLR4. rHMGB1 pretreatment also prevented primary engraftment through a TLR2/4‐dependent pathway. Our results show that islet graft failure can be initiated by TLR2 and TLR4 signaling and suggest that HMGB1 is one likely early mediator. Subsequent downstream signaling results in intra‐islet inflammation followed by T‐cell‐mediated graft destruction.     

Bone marrow-derived mesenchymal stromal cells enhance chimeric vessel development driven by endothelial cell-coated microtissues

Adding bone marrow-derived mesenchymal stromal cells (bmMSCs) to endothelialized collagen gel modules resulted in mature vessel formation, presumably caused in part by the observed display of pericyte-like behavior for the transplanted GFP(+) bmMSCs. A previous study determined that rat aortic endothelial cells (RAECs) delivered on the surface of small (～0.8?mm long×0.5?mm diameter) collagen gel cylinders (microtissues, modular tissue engineering) formed vessels after transplantation into immunosuppressed Sprague-Dawley (SD) rats. Although the RAECs formed vessels in this allogeneic transplant model, there was a robust inflammatory response and the vessels that formed were leaky as shown by microcomputed tomography (microCT) perfusion studies. In vitro assays showed that SD rat bmMSCs embedded into the collagen gel modules increased the extent of EC proliferation and enhanced EC sprouting. In vivo, although vessel number was not affected, the new vessels formed by the bmMSCs and RAECs were more stable and leaked less in the microCT perfusion analysis than vessels formed by implanted RAECs alone. Addition of the bmMSCs also decreased the total number of CD68(+) macrophages that infiltrated the implant and changed the distribution of CD163(+) (M2) macrophages so that they were found within the newly developed vascularized tissue. Most interestingly, the bmMSCs became smooth muscle actin positive and migrated to surround the EC layer of the vessel, which is the location typical of pericytes. The combination of these two effects was presumed to be the cause of improved vascularity when bmMSCs were embedded in the EC-coated modules. Further exploration of these observations is warranted to exploit modular tissue engineering as a means of forming large vascularized functional tissues using microtissue components.     

MRI of transplanted surface-labeled pancreatic islets with heparinized superparamagnetic iron oxide nanoparticles

Transplantation of insulin-secreting pancreatic islets can provide real-time regulation of blood glucose in patients with type 1 diabetes mellitus. Currently, noninvasive and repetitive monitoring of islet engraftment and function is an emerging and promising modality for successful islet transplantation. Here we report a new technique for highly sensitive in?vivo magnetic resonance (MR) imaging of transplanted pancreatic islets. To this end, heparinized superparamagnetic iron oxide (heparin-SPIO) nanoparticle was newly synthesized for chemical conjugation onto islet surface. Compared to typical cellular labeling of Feridex(?) via random endocytosis, chemical conjugation of heparin-SPIO was stable and improved the hypointensity of transplanted islets due to surface modification of every islet. These heparin-SPIO-conjugated islets showed normal viability and insulin secretion, and were quantified by spin echo T(2)-weighted MR imaging with linear correlation depending on transplanted islet mass in?vitro and in?vivo for 30 days. Also, from the immunohistochemistry, we confirmed the existence of heparin-SPIO and insulin biosynthesis in transplanted islets. However, Feridex-uptake islets showed late glucose responsiveness according to changing glucose concentration although they could normally control the blood glucose levels in diabetic mouse. Thus, we anticipate that this surface labeling with heparin-SPIO can be directly applicable for MR imaging of transplanted islets.     

Plasma-fibroblast gel as scaffold for islet transplantation

The transplant of pancreatic islets into the liver can restore normal blood glucose levels in patients with type I diabetes. However, long-term results have indicated that the site and method of transplantation still need to be optimized to improve islet engraftment. This study was designed to assess the efficiency of the use of clotted blood plasma containing fibroblasts ("plasma-fibroblast gel") as a scaffold for subcutaneous islet transplantation in diabetic athymic mice. Islets embedded in the plasma-fibroblast gel were able to resolve hyperglycemia in transplanted mice, restoring normoglycemia over a 60-day period and allowing gradual body weight recovery. Glucose clearances were significantly improved when compared to those recorded in diabetic animals and similar to those observed in the control group (free islets transplanted beneath the kidney capsule). Histological evaluation revealed functional islets within a subcutaneous tissue rich in collagen fibers that was well vascularized, with blood vessels observed around and inside the islets. These findings suggest that this approach could be used as an alternative option for the treatment of type I diabetes in human clinical practice.     

Islet Transplantation in Mice Differing in the I and S Subregions of the H-2 Complex

Pancreatic islets from A.TH mice were transplanted into the spleen or streptozotocin (SZ)-diabetic A.TL mice. The two strains of mice are congenic inbred strains, differing only in the I and S subregions of the H-2 complex. The allogeneic islet grafts decreased blood glucose temporarily, but the islets were rejected after 21 ± 7 days (mean ± SD). The effect of skin presensitization was tested by giving both allogeneic and syngeneic skin grafts to each of a second set of A.TL mice before streptozotocin treatment and islet transplantation. The time course of rejection of the allogeneic islets in animals that received initial skin grafts was decreased to 8 ± 3 days. In both skin-presensitized and non-presensitized mice syngeneic islet grafts were able to restore normoglycaemia, even in animals that had previously rejected an islet allograft. These observations demonstrate that transplantation of pancreatic islet allografts across the I and S subregions of the H-2 complex is sufficient to induce rejection of the islets. The islet rejection was markedly accelerated by prior sensitization with allogeneic skin grafting. It is suggested that elements in allogeneic skin grafts serve as inducers of cytotoxic T-cell responses directed against gene products of the I and/or S subregions present on cells in the allogeneic islets.     

Highly poly(ethylene) glycolylated islets improve long-term islet allograft survival without immunosuppressive medication

The surface modification of islets using poly(ethylene glycol) (PEG) is being studied as a means of preventing host immune responses against transplanted islets. In this study, to completely shield islets with PEG molecules, we increased the amount of PEG conjugated to islet surfaces, by multiple PEGylation or amplified PEGylation using poly-L-lysine, poly(allylamine), or poly(ethyleneimine), respectively. Amplified PEGylation was associated with islet cytotoxicity and functional impairment, but multiple PEGylation affected neither islet viability nor functionality. In addition, when triply PEGylated islets were allotransplanted into diabetic recipients, these islets survived in 3 of the 7 recipients for more than 100 days without any immunosuppressive treatment. Moreover, the blood glucose levels of these 3 recipients were stable and in the normal range. Immunohistochemical analysis showed that 3 of 7 triply PEGylated islets transplants survived for 100 days and that 4 that were rejected before day 20 were all immunologically protected from immune cells. However, unmodified islets were completely destroyed within 1 week. Consequently, we suggest that multiple PEGylation offers an effective means of reducing the immunogenicity of transplanted islets by increasing the amount of surface-bound PEG.     

Early islets and mesenchyme from an injured adult pancreas improve syngeneic engraftments and islet graft function in diabetic rats

A decrease in mass of isografts and a decline in islet function are major challenges in islet transplantations. Despite this, transplantation of 84?h harvested pancreatic duct ligation (PDL) tissues have been shown to have the same functional ability to foetal pancreata, but there was only 40% success in reverting hyperglycaemia. We tested the potential of early islets with mesenchymal stromal cells (MSCs) to promote isogeneic grafts survival and to restore normoglycemia in diabetic rats, in comparison with late islets. Islets were isolated from injured adult pancreata of donor rats at 24?h post ligation either with MSCs (24?h islet/MSC+) or without MSCs (24?h islet/MSC-), and at 84?h without MSCs (84?h islet/MSC-). These cells were transplanted under the renal capsule of syngeneic STZ-diabetic recipient rats. The islet grafts were monitored using the BGLs of recipients and the immunohistomorphology of the grafts were analysed using anti-insulin and anti-Ki67 antibodies. The mean BGL in 24?h islet/MSC+ recipients was reduced over time toward the control value. The curves of the mean BGLs in the control islet/MSC- and the 24?h islet/MSC- recipients dropped significantly below the control normal glucose group’s levels to reach their nadirs on weeks 4 and 6, respectively. Both curves had a peak overshoot on week 9, with no statistical significant difference between them. Engrafted islets were evident in these recipients, lasted for 5 and 6 weeks and correspondingly survived failure. However, insulin+ cells were present in the isografts of all recipients; but, only isografts in the 24?h islet/MSC+ presented with a homogenous subcapsular beta cell mass. In addition, the tendency of 24?h islet/MSC- to restore normoglycaemia with its survival capacity was statistically highly significant compared to the 84 islet/MSC- recipients (80%; 20%; p?=?0.001). Transplantation of early islets with MSCs from injured adult pancreata prolongs islet graft survival and improves isograft function in diabetic rats. This novel observation requires much further exploration for its clinical application, but this model already provides hope for new sources of donor islets for transplantation.     

Pseudoislet vascularization. Induction of diaphragm-fenestrated endothelia from the hepatic sinusoids

The avascular islet tissue preparation, the pseudoislet, was transplanted into the liver of streptozotocin diabetic rats. The vascularization of the islet tissue was studied ultrastructurally over a 2-week period. At 2 days, proliferations of fibroblast-like cells and large amounts of collagen fibers were present at the periphery of grafts. By 7 days, vascular buds were present at the edge of the graft and invaginating into the islet tissue. At this time, diaphragmed fenestrae were present in the endothelial cells of these forming vessels and they were surrounded by a thin continuous basal lamina. By 12 to 14 days, vascularization was complete as determined by the presence of perfused vessels. At this time the mural architecture of the capillary endothelium was identical to that found in pancreatic islets in situ, i.e., diaphragmed fenestrae, transendothelial channels, and a continuous basal lamina. This contrasted sharply with normal hepatic sinusoidal endothelium which has larger open fenestrae, no channels, and a discontinuous or absent basal lamina. In animals that had been labeled with colloidal carbon before pseudoislet transplantation, the fenestrated endothelium in the grafts contained carbon-filled phagocytic vacuoles indicating the hepatic origin of these cells. Also present at this time was a change in the hepatic sinusoids near the graft sites to a near continuous endothelium. This study demonstrates that isolated avascular adult islet cells are capable of inducing a diaphragm-fenestrated endothelium.     

Permanent protection of PLG scaffold transplanted allogeneic islet grafts in diabetic mice treated with ECDI-fixed donor splenocyte infusions

Allogeneic islet cell transplantation is a promising treatment for human type 1 diabetes. Currently, human islets are transplanted via intra-portal infusions. While successful, it leads to significant early islet attrition from instant blood-mediated inflammatory reaction. An extra-hepatic site was established by transplanting islet-loaded microporous poly(lactide-co-glycolide) (PLG) scaffolds into the epididymal fat pad in syngeneic islet transplant models. This study examined this technology in allogeneic islet transplantation and determined whether transplant tolerance could be effectively induced to protect PLG scaffold transplanted allogeneic islets. The efficacy of an established tolerance induction strategy using donor splenocytes treated with ethylcarbodiimide(ECDI) was tested. ECDI-fixed donor splenocytes were infused 7 days before and 1 day after islet transplantation. Immediate normoglycemia was restored, and treated mice maintained indefinite normoglycemia whereas untreated mice rejected islet grafts within 20 days of transplantation. Interestingly, efficacy of tolerance induction was superior in PLG scaffold compared with intra-portal transplanted islets. Protection of PLG scaffold islet allografts was associated with several mechanisms of immune regulation. In summary, PLG scaffolds can serve as an alternative delivery system for islet transplantation that does not impair tolerance induction. This approach of combining tolerance induction with scaffold islet transplantation has potential therapeutic implications for human islet transplantation.     

Different islet protein expression profiles during spontaneous diabetes development vs. allograft rejection in BB-DP rats

Type 1 diabetes (T1D) is characterized by selective autoimmune destruction of the insulin producing beta-cells in the islets of Langerhans. When the beta-cells are destroyed exogenous administration of insulin is necessary for maintenance of glucose homeostasis. Allogeneic islet transplantation has been used as a means to circumvent the need for insulin administration and has in some cases been able to restore endogenous insulin production for years. However, long life immunosuppression is needed to prevent the graft from being rejected and destroyed. Changes in protein expression pattern during spontaneous diabetes development in the diabetes prone BioBreeding rat (BB-DP) have previously been described. In the present study, we have investigated if any of the changes seen in the protein expression pattern during spontaneous diabetes development are also present during allograft rejection of BB-DP rat islets. Two hundred neonatal islets were syngeneically transplanted under the kidney capsule of 30 day old BB-DP rats and removed prior to and at onset of diabetes. Allogeneically transplanted islets from BB-DP rats were removed before onset of allograft rejection and at maximal islet graft inflammation (rejection). The protein expression profiles of the transplants were visualised by two-dimensional gel (2-DG) electrophoresis, analysed and compared. In total, 2590 protein spots were visualised and of these 310 changed expression (p < 0.01) in syngeneic islet transplants in the BB-DP rats from 7 days after transplantation until onset of diabetes. In BB-DP islets transplanted to WK rats 53 protein spots (p < 0.01) showed changes in expression when comparing islet grafts removed 7 days after transplantation with islet grafts removed 12 days after transplantation where mononuclear cell infiltration is at its maximum. Only four protein spots (1%) were significantly changed in both syngeneic (autoimmune) and allogeneic islet destruction. When comparing protein expression changes in syngeneic BB-DP islet transplants from 37 days after transplantation to onset of diabetes with protein expression changes in allografts from day 7 to 12 after transplantation only three spot were found to commonly change expression in both situations. In conclusion, a large number of protein expression changes were detected in both autoimmune islet destruction and allogeneic islet rejection, only two overlaps were detected, suggesting that autoimmune islet destruction and allogeneic islet rejection may result from different target cell responses to signals induced by the cellular infiltrate. Whether this reflects activation of distinct signalling pathways in islet cells is currently unknown and need to be further investigated.     

The influence of immune system stimulation on encapsulated islet graft survival

INTRODUCTION: The aim of this study was to determine the influence activating of the recipient immune system on the function of microencapsulated islet xenografts. MATERIAL/METHODS: The skin of WAG or Fisher rats and WAG free or encapsulated (APA) Langerhans islets were transplanted to healthy or to streptozotocin diabetic BALB/c mice. Skin grafts were performed following the method of Billingham and Medawar. Rat islets were isolated from pancreas by the Lacy and Kostianovsy method and encapsulated with calcium alginate-poly-L-lysine-alginate according to the 3-step coating method of Sun. RESULTS: The transplantation of encapsulated WAG islets, despite activation of the host immune system, restored euglycemia for over 180 +/-100 days. A subsequent skin graft taken from the same donor was rejected in the second set mode, but euglycemia persisted. In diabetic recipients, impaired immune response was corrected by successful encapsulated islet transplantation. In diabetic mice, strong stimulation with 2-fold skin transplantation induced primary non-function of grafted islets despite their encapsulation. CONCLUSIONS: The survival of an islet xenograft depends on the level of activation of the recipient immune system. The immune response of diabetic mice was impaired, but increased after post-transplant restitution of euglycemia. Microencapsulation sufficiently protected grafted islets, and remission of diabetes was preserved. However, after strong specific or non-specific stimulation of the host immune system, non-function of xenografted islets developed despite their encapsulation. Therefore, islet graft recipients should avoid procedures which could stimulate their immune systems. If absolutely necessary, the graft should be protected by exogenous insulin therapy at that time.     

Diabetes induces rapid suppression of adaptive immunity followed by homeostatic T-cell proliferation

Surprisingly, the effect of acute diabetes on immunity has not been examined in detail. We, herein, show for the first time that untreated acute diabetes causes rapid lymphopenia followed by homeostatic T-cell proliferation. The diabetes-induced lymphopenia was associated with an immunosuppressed state that could be sufficiently strong to allow engraftment of fully allogeneic beta-cells or block rejection of islet transplants. In contrast, homeostatic proliferation and recovery of T-cell numbers were associated with islet rejection. Thus, the timing of islet transplant challenge in relation to diabetes induction was critical in determining whether islets were accepted or rejected. In addition, we tested whether diabetes-related immunosuppression could result in an overestimation of the efficacy of a tolerance-inducing protocol. Consistent with this possibility, a protocol targeting CD40L and ICOS that we have shown induces tolerance in diabetic recipients was unable to induce tolerance in non-diabetic recipients. The data uncover a previously unrecognized suppressive effect of diabetes on adaptive immunity. Furthermore, they suggest that the standard methods of testing new tolerance-inducing protocols in islet transplantation require modification and that diabetes itself can contribute to homeostatic proliferation, a process associated with autoimmunity and a resistance to tolerance induction.     

Different islet protein expression profiles during spontaneous diabetes development vs. allograft rejection in BB-DP rats

Type 1 diabetes (T1D) is characterized by selective autoimmune destruction of the insulin producing β-cells in the islets of Langerhans. When the β-cells are destroyed exogenous administration of insulin is necessary for maintenance of glucose homeostasis. Allogeneic islet transplantation has been used as a means to circumvent the need for insulin administration and has in some cases been able to restore endogenous insulin production for years. However, long life immunosuppression is needed to prevent the graft from being rejected and destroyed. Changes in protein expression pattern during spontaneous diabetes development in the diabetes prone BioBreeding rat (BB-DP) have previously been described. In the present study, we have investigated if any of the changes seen in the protein expression pattern during spontaneous diabetes development are also present during allograft rejection of BB-DP rat islets.

Two hundred neonatal islets were syngeneically transplanted under the kidney capsule of 30 day old BB-DP rats and removed prior to and at onset of diabetes. Allogeneically transplanted islets from BB-DP rats were removed before onset of allograft rejection and at maximal islet graft inflammation (rejection). The protein expression profiles of the transplants were visualised by two-dimensional gel (2-DG) electrophoresis, analysed and compared.

In total, 2590 protein spots were visualised and of these 310 changed expression (p < 0.01) in syngeneic islet transplants in the BB-DP rats from 7 days after transplantation until onset of diabetes. In BB-DP islets transplanted to WK rats 53 protein spots (p < 0.01) showed changes in expression when comparing islet grafts removed 7 days after transplantation with islet grafts removed 12 days after transplantation where mononuclear cell infiltration is at its maximum. Only four protein spots (1%) were significantly changed in both syngeneic (autoimmune) and allogeneic islet destruction. When comparing protein expression changes in syngeneic BB-DP islet transplants from 37 days after transplantation to onset of diabetes with protein expression changes in allografts from day 7 to 12 after transplantation only three spot were found to commonly change expression in both situations.

In conclusion, a large number of protein expression changes were detected in both autoimmune islet destruction and allogeneic islet rejection, only two overlaps were detected, suggesting that autoimmune islet destruction and allogeneic islet rejection may result from different target cell responses to signals induced by the cellular infiltrate. Whether this reflects activation of distinct signalling pathways in islet cells is currently unknown and need to be further investigated.     

Islet Mass Plays a Critical Role in Initiation, but not Progression, of the Diabetogenic Process in NOD Mice

Pancreatectomy (90%) at a preinsulitis age (7 weeks) protects NOD mice from diabetes while pancreatectomy at a mid-insulitis age (13 weeks) has no such protective effect. The present study examined the effects of islet transplantation in pancreatectomized diabetes-free NOD mice. Transplantation of syngeneic NOD islets as well as allogeneic C3H/He and C57BL/6 islets 3 weeks after pancreatectomy-induced spontaneous diabetes whereas transplantation of xenogeneic Sprague-Dawley rat islets or allogeneic C3H/He skin failed to induce diabetes, demonstrating that the diabetogenic antigen(s) of NOD islets is also expressed by islets of diabetes-resistant mouse strains but not by xenogeneic rat islets. Removal of NOD islet grafts by nephrectomy 7-14 days after transplantation had no effect on the subsequent chronic development of diabetes, while graft removal 3 days after transplantation completely abolished the diabetogenic effect of islet transplantation. Thus, activation of the diabetogenic response by islet isografting takes less than 7 days and the continuous presence of a large islet mass is not required for progression to diabetes. While islet transplantation at 10 and 15 weeks of age caused diabetes, delayed islet transplantation at 23 and 35 weeks of age failed to induce diabetes in pancreatectomized diabetes-free NOD mice, suggesting that initiation of the diabetogenic autoimmune process must take place within a certain window of time. The pancreatectomy/islet transplantation model is excellent for studying the immunological events surrounding activation and progression of the diabetogenic autoimmune process and for identifying the diabetogenic islet antigen(s).     

Enhancement of rat islet tolerance with bone marrow transplantation using a non-myeloablative procedure II: failure despite the presence of lymphocyte microchimerism in the fully allogeneic Lewis/Brown-Norway model

Transplantation of donor bone marrow cells (BMTx) has been proven to be capable of including allogeneic transplant tolerance. In our previous experiments we reported the positive effect of BMTx together with short-term tacrolimus/hydrocortisone therapy on pancreatic islet survival in recipients haploidentical with donors. In this project we used the same transplant protocol to further investigate this effect in a fully mean histocompatibility system-mismatched model. Lewis male rats and Brown-Norway female rats were used as donors and recipients, respectively. Diabetic animals were treated according to four different protocols. Recipients in group I (n = 12) underwent islet transplantation (ITx) only. Group II (n = 12) and group III (n = 11) included animals treated for 52 days with tacrolimus (0.5 mg/kg) and hydrocortisone (2 mg/kg). Diabetes was induced by intravenously applied streptozocin (50 mg/kg). Seven days later islets were injected intrahepatically through the portal vein. In addition, rats in group III underwent BMTx on day 10. In group IV (n = 6) tacrolimus therapy, ITx and BMTx were used according to the previously published protocol of Ricordi et al. After more than 120 days, cumulative survival rates were 56% and 64% for recipients in groups II and III, respectively (p > 0.05). All animals in group I became hyperglycemic by day 11 following transplantation. Despite positive detection of lymphocyte microchimerism, we did not observe improved survival of allogeneic islets in animals treated with BMTx. Surprisingly, better islet survival was not found in group IV either (survival rate at 100 days: 33%). We conclude that demonstration of lymphocyte microchimerism, as detected by a sensitive polymerase chain reaction method, did not improve allogeneic islet survival in vivo and was not able to block mixed lymphocyte reaction in vitro. Whether a larger amount of transplanted hematopoietic cells could induce tolerance in this model remains to be evaluated.