Tubular complexes as a source for islet neogenesis in the pancreas of diabetes-prone BB rats

Tubular complexes (TC) in the pancreas contain duct-like structures with low cuboidal or flattened cells surrounding a large lumen and are thought to be a response to pancreatic injury. TC have been studied in animal models of chemical or surgically induced pancreatic damage but their occurrence has not been reported in rodent models of spontaneous autoimmune type I diabetes. We hypothesized that TC would be increased during the active phase of islet destruction in autoimmune diabetes and could contain islet progenitor cells. We analyzed TC in pancreas of Wistar Furth (WF), control (BBc) and diabetes-prone BioBreeding (BBdp) rats using immunohistochemistry and morphometry. TC were observed in all rat strains during active pancreas remodeling ( approximately 13 days). They increased between 60 and 93 days only in BBdp rats coincident with the increase in diabetes cases. Most TC were infiltrated with CD3(+) T-cells. Duct-like cells in the TC had low expression of the exocrine marker amylase, increased expression of epithelial cell markers, keratin and vimentin, and remarkably high cell proliferation and cell death. TC islets contained cells stained positive for insulin, glucagon, somatostatin, pancreatic polypeptide, as well as PDX-1, chromogranin, and hepatocyte-derived growth factor receptor, c-met. Transitional cells that were keratin(+)/insulin(+) and keratin(+)/amylase(+) cells were present in TC. The stem cell marker, nestin was upregulated in the TC region. Duct-like cells in TC of BBdp rats expressed markers of committed endocrine precursors: PDX-1, neurogenin 3 and protein gene product 9.5. This study demonstrates that TC are upregulated during beta-cell destruction and contain potential endocrine progenitors.     

Effects of a protective hydrolyzed casein diet upon the metabolic and secretory responses of pancreatic islets to IL-1beta, cytokine production by mesenteric lymph node cells, mitogenic and biosynthetic activities in Peyer's patch cells, and mitogenic activity in pancreatic lymph node cells from control and diabetes-prone BB rats

The effects of substituting a plant-based control diabetogenic diet (NIH diet) by a protective hydrolyzed casein diet (HC diet) upon selected metabolic and functional variables were recently investigated in Peyer's patch cells, splenocytes, mesenteric lymph node cells, and pancreatic islets from either control (BBc) or diabetes-prone (BBdp) BB rats. In the present work, the plasma d-glucose and insulin concentrations, the protein and insulin content of pancreatic islets, the metabolism of d-glucose, and its insulinotropic action in islets first cultured for 24 h in the absence or presence of IL-1beta, the production of IFN-gamma and IL-10 by mesenteric lymph node cells cultured for 48 h in the absence or presence of concanavalin A, the mitogenic activity of Peyer's patch cells and pancreatic lymph node cells in the absence or presence of the same lectin, and the biosynthetic activity of Peyer's patch cells were measured in the BBc and BBdp rats fed either the NIH or the HC diet. Two major novel findings emerged from this study. First, in immune cells, diet HC increased to a greater extent the responsiveness to concanavalin A of certain metabolic and functional variables in BBdp rats than in BBc rats. Second, pancreatic islet cells of BBdp rats were less sensitive to IL-1beta than those of BBc rats and this difference was further accentuated when the animals were fed the HC rather than the NIH diet. These findings afford further support to the view that, in BB rats, changes in the biological behavior of Peyer's patch cells, mesenteric and pancreatic lymph node cells, and pancreatic islet cells participate in the pathogenesis of insulin-dependent diabetes mellitus and its prevention by a suitable dietary manipulation.     

Quantitative and qualitative alterations of intestinal mucins in BioBreeding rats

In diabetes-prone BioBreeding rats, an enteropathy often precedes the onset of auto-immune insulitis. The present study draws attention to quantitative and qualitative alterations of intestinal mucins in this animal model of type 1 diabetes mellitus. Male and female diabetes-resistant (BBc) and diabetes-prone (BBdp) BioBreeding rats fed, from one to two weeks after weaning onwards, either a plant-based diabetes-promoting diet (NTP) or a hydrolysed casein diabetes-protective diet (HC), were sacrificed at 11-14 weeks of age. Proteins and total mucins, as well as acid and neutral mucins, were measured in a segment of the intestinal tract, located 25-30 cm below the pylorus. No significant difference between BBc and BBdp rats was found when fed the HC diet. However, the NTP diet lowered both total and neutral mucins, whilst failing to affect significantly acidic mucins. The effects of the NTP diet were more pronounced in BBdp rats than in BBc rats. It is speculated that the quantitative and qualitative changes evoked by the NTP diet in BBdp rats may play a role in the alteration of gut permeability found under the same experimental conditions.     

Pronounced cytosolic aggregation of cellular prion protein in pancreatic beta-cells in response to hyperglycemia

Cellular prion protein (PrP(C)), an N-linked glycoprotein, is expressed in a variety of tissues, but its functions remain unclear. PrP(C) is abundantly expressed in the endocrine pancreas, which regulates blood glucose homeostasis. Therefore, we investigated whether the expression of PrP(C) was altered in islets of Langerhans in a model of spontaneous type 1 diabetes, the diabetes-prone BioBreeding (BBdp) rat and a model of beta-cell adaptation to hyperglycemia, the chronic glucose-infused Sprague Dawley rat. Pancreatic sections from animals aged 7-100 days were stained immunohistochemically and evaluated using light, fluorescence and confocal microscopy. PrP(C) was ubiquitously expressed in all four major endocrine cell types within islets. Surprisingly, cytosolic inclusions containing PrP(C) were identified exclusively in a subpopulation of insulin-producing beta-cells. The inclusions exhibited different molecular characteristics from the PrP aggregates previously described in vitro in neurons. The frequency of beta-cells with PrP(C) inclusions increased with age and was threefold greater in diabetes-prone rats than in controls at 100 days. Cytosolic PrP(C) expression in beta-cells was suppressed whereas the number and size of PrP(C) inclusions markedly increased in response to hyperglycemia during the first 2 days of continuous glucose infusion in Sprague Dawley rats. In summary, this is the first report describing in vivo cytosolic PrP(C) aggregation. These unique PrP(C) inclusions were beta-cell specific, more frequent in diabetes-prone animals, and responded to hyperglycemia in glucose-infused Sprague Dawley rats. These data suggest a potential dysfunction in beta-cells of diabetes-prone rats, and point to new avenues for the study of diabetes pathogenesis.     

Identification of T cell subsets and class I and class II antigen expression in islet grafts and pancreatic islets of diabetic BioBreeding/Worcester rats

The BioBreeding/Worcester (BB/Wor) rat develops a spontaneous disorder that closely resembles human insulin-dependent (Type I) diabetes mellitus. The syndrome is preceded by lymphocytic insulitis that destroys pancreatic beta cells. The morphologic features of the spontaneous insulitis lesions are also observed within islets transplanted beneath the renal capsule of diabetes-prone and diabetic animals. This study reports the results of experiments in which immunohistochemical techniques were used to characterize the phenotype of the infiltrating mononuclear cells and detect the expression of class I and class II MHC antigens in native islets and islet transplants in diabetic and diabetes-prone BB/Wor rats. The infiltrates within native pancreatic islets and islet grafts were comprised predominantly of Ia+ cells (dendritic cells and macrophages) CD4+ cells (helper/inducer lymphocytes and macrophages), CD5+ (pan-T) cells and smaller numbers of CD8+ (cytotoxic/suppressor and NK) cells. Pancreatic and graft insulitis were accompanied by markedly enhanced class I antigen expression on islet and exocrine cells. Class II (Ia) antigens were not detected on normal islet cells, islets undergoing insulitis or on islet transplants subjected to immune attack. In islet grafts stained with polymorphic MAbs that distinguish Ia antigens of donor and host origin, Ia antigen expression was limited to infiltrating dendritic cells and macrophages of host origin. It is concluded that the phenotypes of infiltrating mononuclear cells that comprise the insulitis lesion in spontaneous BB/Wor diabetes, and the inflammatory attack on islets transplanted into diabetic BB/Wor rats are the same, that pancreatic islet and graft insulitis occur in the presence of enhanced class I antigen expression but in the absence of class II antigen expression, and that infiltrating Ia+ cells within islet grafts are exclusively of recipient (BB/Wor) origin and may explain the initiation of immune insulitis within grafts derived from donors of incompatible MHC.     

Necrosis is the predominant type of islet cell death during development of insulin-dependent diabetes mellitus in BB rats

Several reports propose that apoptosis of pancreatic beta cells may play a central role in the pathogenesis of both spontaneous and induced insulin-dependent diabetes mellitus (IDDM) in animal models. Whether apoptosis is a major cell death pathway during diabetes development, however, is highly controversial. The aim of this study was to examine the mode of islet cell death in prediabetic diabetes-prone (dp) BB rats, which spontaneously develop diabetes and serve as an animal model for human IDDM. In addition we investigated the cell death pathway of islet cells treated with the widely used diabetogenic compound streptozotocin or with nitric oxide (NO), which during IDDM development has been found to be present in inflamed islets in high concentrations because of the expression of inducible NO synthase. Islets of prediabetic BBdp rats were analyzed for DNA strand breaks and screened by electron microscopy. The mode of islet cell death in vitro after treatment with cytotoxic concentrations of streptozotocin or of NO was investigated using different methods including morphologic analysis by electron microscopy, detection of DNA strand breaks, poly(ADP-ribose) polymerase cleavage, and annexin V staining. Although cells with DNA stand breaks-often accepted as a proof for apoptosis-could be identified, we did not find apoptosis-specific features during islet cell death. Instead we observed massive necrosis as evidenced by disrupted plasma membranes and spilled-out cellular constituents in vitro as well as during disease manifestation in BBdp rats. These results may have serious consequences with regard to the treatment of humans to prevent the development of IDDM.     

IL-1beta,IFN-gamma and TNF-alpha increase vulnerability of pancreatic beta cells to autoimmune destruction

In the pathogenesis of type-1 diabetes insulin-producing beta-cells are destroyed by cellular autoimmune processes. The locality of beta-cell destruction is the inflamed pancreatic islet. During insulitis cytokines released from islet-infiltrating mononuclear cells affect beta-cells at several levels. We investigated whether cytokine-induced beta-cell destruction is associated with changes in the expression of the surface receptors intercellular adhesion molecule (ICAM)-1 and Fas. Islets from diabetes-prone and congenic diabetes-resistant BB rats were exposed to interleukin (IL)-1beta alone or in combination with interferon (IFN)-gamma plus tumour necrosis factor (TNF)-alpha. Cytokines decreased islet insulin content, suppressed glucose stimulated insulin secretion and generated enhanced amounts of nitric oxide and DNA-strand breaks. While no membrane alterations of IL-1beta treated islets cells were detectable, the cytokine combination caused damage of cell membranes. Independent of diabetes susceptibility IL-1beta treated islet beta-cells expressed a significantly increased amount of ICAM-1 on their surfaces which was not further increased by IFN-gamma+TNF-alpha. However, IL-1beta induced Fas expression was significantly enhanced only on beta-cells from diabetes-prone BB rats. From these results we suggest that IL-1beta mediates the major stimulus for ICAM-1 induction which is possibly a necessary but not sufficient step in the process of beta-cell destruction. Obviously, the additional enhancement of Fas expression on the surface of beta-cells is important for destruction. The combined action of all three cytokines induced the expression of Fas on the beta-cell surface independent of diabetes susceptibility, indicating that such a strong stimulus in vitro may induce processes different from the precise mechanisms of beta-cell destruction in vivo.     

Antioxidant enzyme activity and mRNA expression in the islets of Langerhans from the BB/S rat model of type 1 diabetes and an insulin-producing cell line

It has been proposed that low activities of antioxidant enzymes in pancreatic beta cells may increase their susceptibility to autoimmune attack. We have therefore used the spontaneously diabetic BB/S rat model of type 1 diabetes to compare islet catalase and superoxide dismutase activities in diabetes-prone and diabetes-resistant animals. In parallel studies, we employed the RINm5F beta cell line as a model system (previously validated) to investigate whether regulation of antioxidant enzyme activity by inflammatory mediators (cytokines, nitric oxide) occurs at the gene or protein expression level. Diabetes-prone rat islets had high insulin content at the age used (58–65 days) but showed increased amounts of DNA damage when subjected to cytokine or hydrogen peroxide treatments. There was clear evidence of oxidative damage in freshly isolated rat islets from diabetes-prone animals and significantly lower catalase and superoxide dismutase activities than in islets from age-matched diabetes-resistant BB/S and control Wistar rats. The mRNA expression of antioxidant enzymes in islets from diabetes-prone and diabetes-resistant BB/S rats and in RINm5F cells, treated with a combination of cytokines or a nitric oxide donor, DETA-NO, was analysed semi-quantitatively by real time PCR. The mRNA expression of catalase was lower, whereas MnSOD expression was higher, in diabetes-prone compared to diabetes-resistant BB/S rat islets, suggesting regulation at the level of gene expression as well as of the activities of these enzymes in diabetes. The protein expression of catalase, CuZnSOD and MnSOD was assessed by Western blotting and found to be unchanged in DETA-NO treated cells. Protein expression of MnSOD was increased by cytokines in RINm5F cells whereas the expression of CuZnSOD was slightly decreased and the level of catalase protein was unchanged. We conclude that there are some changes, mostly upregulation, in protein expression but no decreases in the mRNA expression of catalase, CuZnSOD or MnSOD enzymes in beta cells treated with either cytokines or DETA-NO. The lower antioxidant enzyme activities observed in islets from diabetes-prone BB/S rats could be a factor in the development of disease and in susceptibility to DNA damage in vitro and could reflect islet alterations prior to immune attack or inherent differences in the islets of diabetes-prone animals, but are not likely to result from cytokine or nitric oxide exposure in vivo at that stage.     

In vitro stimulation by islet antigen facilitates the detectability of beta-cell reactive cells in diabetes-prone BB/OK rats.

It has been supposed that beta-cell destruction in man and animals is due to autoreactive T-cells. We used the [51Cr]-release assay to identify the presence of beta-cell reactive cells in the spleen of diabetes-prone BB/OK rats before and after diabetes manifestation as well as in long-term normoglycaemic rats with a reduced diabetes risk of 3%. Splenic mononuclear cells (MNCs) obtained from diabetes-resistant LEW.1W and the majority of long-term normoglycaemic BB/OK rats (86.4%) showed no reactivity to pancreatic islets in vitro. In contrast, beta-cell reactive cells were identified in dependence on age in 30.4-65.0% of 75-120 days old normoglycaemic rats and in relation to diabetes duration (1 and 20 days) in 75.0% and 16.0% of diabetic BB/OK rats. Islet antigen-specific stimulation of splenic MNCs, that showed no spontaneous islet-directed reactivity, resulted in a concentration-dependent activation of cytolytically reactive cells in BB/OK but not in LEW.1W rats. Splenic MNCs derived from all diabetic, from 82.4% of young normoglycaemic and from 46.2% of long-term normoglycaemic BB/OK rats developed an islet-directed reactivity in vitro. Phenotyping of MNCs showed a significant increase of activated IL2R+ T-lymphocytes in diabetic BB/OK rats, but without any correlation to their cytolytic potential in the [51Cr]-release assay. Despite this fact, IL2R+ cells enriched from the pool of MNCs mediated an enhanced [51Cr]-release from islets, indicating their relevance in the beta-cell destruction. These data suggest, that functional reactivity rather than phenotypic characterization of MNCs is useful to identify the existence of beta-cell reactive cells. Furthermore, for screening diabetes risk in young normoglycaemic BB/OK rats besides the detection of beta-cell reactive cells the occurrence of regulatory cells seems to be decisive.     

Prophylactic insulin treatment of diabetes-prone BB/OK rats by application of a sustained release insulin implant

Normoglycemic diabetes-prone BB/OK rats aged 33, 45 or 75 days were subjected to prophylactic insulin treatment by means of a single subcutaneous application of a sustained release insulin implant. The single application of a sustained release insulin implant decreased the incidence of diabetes or delayed the onset of the disease in BB/OK rats of all treatment groups. Prophylactic insulin administration caused a transient hypoglycemic period accompanied by an inhibition of glucose stimulated insulin secretion and a decrease of the insulin content of Langerhans' islets as detectable in vitro. Compared to islets of normoglycemic controls pancreatic islets isolated from hypoglycemic BB/OK rats within 7-21 days after the insulin application at 45 days of age displayed a decreased susceptibility of the cells to complement-dependent cytotoxicity of the monoclonal islet cell surface antibody (ICSA) K14D10 but not to the cytotoxic effect of the ICSA M3aG8. The appearance of complement-dependent antibody-mediated cytotoxicity to islet cells and pancreatic exocrine cells in serum regarded as a sign of immune dysregulation in BB/OK rats seems not to be affected by insulin prophylaxis and was detectable during hypoglycemia as well as in the subsequent normoglycemic state. In conclusion, BB/OK rats of different age can be protected from diabetes by a single application of a sustained release insulin implant. Insulin and/or hypoglycemia seem to influence the expression of cell surface antigens, thus render the islets of Langerhans less vulnerable to immune cytolysis, whereas the appearance of humoral immunological abnormalites is not affected.     

Increased pancreatic islet mass is accompanied by activation of the insulin receptor substrate-2/serine-threonine kinase pathway and augmented cyclin D2 protein levels in insulin-resistant rats

It is well known that glucocorticoids induce peripheral insulin resistance in rodents and humans. Here, we investigated the structural and ultrastructural modifications, as well as the proteins involved in beta-cell function and proliferation, in islets from insulin-resistant rats. Adult male Wistar rats were made insulin resistant by daily administration of dexamethasone (DEX; 1mg/kg, i.p.) for five consecutive days, whilst control (CTL) rats received saline alone. Structure analyses showed a marked hypertrophy of DEX islets with an increase of 1.7-fold in islet mass and of 1.6-fold in islet density compared with CTL islets ( P <  0.05). Ultrastructural evaluation of islets revealed an increased amount of secreting organelles, such as endoplasmic reticulum and Golgi apparatus in DEX islets. Mitotic figures were observed in DEX islets at structural and ultrastructural levels. Beta-cell proliferation, evaluated at the immunohistochemical level using anti-PCNA (proliferating cell nuclear antigen), showed an increase in pancreatic beta-cell proliferation of 6.4-fold in DEX islets compared with CTL islets ( P <  0.0001). Increases in insulin receptor substrate-2 (IRS-2), phosphorylated-serine-threonine kinase AKT (p-AKT), cyclin D₂ and a decrease in retinoblastoma protein (pRb) levels were observed in DEX islets compared with CTL islets ( P <  0.05). Therefore, during the development of insulin resistance, the endocrine pancreas adapts itself increasing beta-cell mass and proliferation, resulting in an amelioration of the functions. The potential mechanisms that underlie these events involve the activation of the IRS-2/AKT pathway and activation of the cell cycle, mediated by cyclin D₂. These adaptations permit the maintenance of glycaemia at near-physiological ranges.     

Both CD4(+)and CD8(+)T cells are required for IFN-gamma gene expression in pancreatic islets and autoimmune diabetes development in biobreeding rats

To study the relative roles of CD4(+)and CD8(+)T cells and their cytokine products in autoimmune diabetes development, we selectively depleted CD4(+)and CD8(+)T cells in autoimmune diabetes-prone (DP) biobreeding (BB) rats, by administrations of anti-CD2 and anti-CD8 monoclonal antibody (mAb) respectively. We then analysed cytokine mRNA expression, by PCR assay, in mononuclear leukocytes isolated from islets and spleens of control and mAb-treated DP-BB rats. Depletion of CD4(+)T cells (by anti-CD2 mAb) in blood, spleen and islets prevented diabetes development in DP-BB rats, and depletion of CD8(+)T cells (by anti-CD8 mAb) delayed and significantly decreased diabetes incidence. Depletion of either CD4(+)or CD8(+)T cells completely prevented IFN-gamma mRNA upregulation in islets of DP-BB rats above the low level expressed in islets of diabetes-resistant (DR) BB rats. Also, IL-10 mRNA levels in islets of DP-BB rats were significantly decreased by depletion of either CD4(+)or CD8(+)T cells, whereas the effects of the anti-T cell mAb on mRNA levels of other cytokines in islets (IL-2, IL-4, IL-12p40, and TNF-alpha) were discordant. In contrast, both mAb treatments significantly upregulated IL-4 and TNF-alpha mRNA levels in spleens of DP-BB rats. These results demonstrate that islet infiltration by both CD4(+)and CD8(+)T cells is required for IFN-gamma and IL-10 production in islets and beta-cell destruction. Depletion of either CD4(+)or CD8(+)T cells may prevent beta-cell destruction by decreasing IFN-gamma and IL-10 production in islets and increasing IL-4 and TNF-alpha production systemically.     

Investigation of chloroform-methanol soluble wheat proteins and sphingolipids as potential dietary triggers of diabetes in BBdp rats

The present study is a follow-up to the study by Coleman (1990), which found that the incidence of diabetes reduced in non-obese diabetic (NOD) mice when chloroform-methanol soluble substances were removed from a cereal-based diet. We used a 2:1 chloroform-methanol (C-M) mixture to extract C-M soluble sphingolipids and proteins from wheat gluten. The C-M extract was subjected to silica gel chromatography and saponification to remove triglycerides, glycolipids and phospholipids. This sphingolipid-enriched fraction from wheat gluten was subsequently incorporated into a BioBreeding diabetes-prone (BBdp) rat diet. Five diets were fed over a 120-day period to BBdp rats: a negative control hydrolysed casein (HC) diet, a positive control National Toxicology Program 2000 (NTP 2000) diet, a wheat gluten (WG)-based diet, a WGGSLF diet containing the WG residue remaining after chloroform-methanol extraction, and a hydrolysed casein plus sphingolipid-enriched fraction (HCGSL) diet. There was a significant increase in diabetes incidence in rats fed the HC + wheat sphingolipid diet versus the HC diet at days 70 and 80 of the feeding study, but not at the end of the study (120 days). There were no significant differences in diabetes incidence in rats fed the WG diet and WG diet with sphingolipids removed. There were no significant differences in pancreatic insulitis scores or in the numbers of jejunal CD4+ and γδTCR + T cells in BBdp rats due to the dietary addition or absence of CM soluble substances from wheat gluten. Sera IFN-γ levels were significantly higher in the sphingolipid-removed WG diet group compared to the BBdp rats in the other four diet groups. Overall, the results of this study provide some evidence for a slight promotional effect of C-M soluble sphingolipids and proteins in wheat gluten on the incidence of diabetes and up-regulation of immune responses in the BBdp rat.     

Prophylactic Insulin Treatment of Diabetes-prone BB/OK Rats by Application of a Sustained Release Insulin Implant

Normoglycemic diabetes-prone BB/OK rats aged 33, 45 or 75 days were subjected to prophylactic insulin treatment by means of a single subcutaneous application of a sustained release insulin implant. The single application of a sustained release insulin implant decreased the incidence of diabetes or delayed the onset of the disease in BB/OK rats of all treatment groups. Prophylactic insulin administration caused a transient hypoglycemic period accompanied by an inhibition of glucose stimulated insulin secretion and a decrease of the insulin content of Langerhans' islets as detectable in vitro . Compared to islets of normoglycemic controls pancreatic islets isolated from hypoglycemic BB/OK rats within 7-21 days after the insulin application at 45 days of age displayed a decreased susceptibility of the cells to complement-dependent cytotoxicity of the monoclonal islet cell surface antibody (ICSA) K14D10 but not to the cytotoxic effect of the ICSA M3aG8. The appearance of complement-dependent antibody-mediated cytotoxicity to islet cells and pancreatic exocrine cells in serum regarded as a sign of immune dysregulation in BB/OK rats seems not to be affected by insulin prophylaxis and was detectable during hypoglycemia as well as in the subsequent normoglycemic state. In conclusion, BB/OK rats of different age can be protected from diabetes by a single application of a sustained release insulin implant. Insulin and/or hypoglycemia seem to influence the expression of cell surface antigens, thus render the islets of Langerhans less vulnerable to immune cytolysis, whereas the appearance of humoral immunological abnormalites is not affected.     

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.     

Peroxynitrite is a mediator of cytokine-induced destruction of human pancreatic islet beta cells.

The proinflammatory cytokines, interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and interferon gamma (IFNgamma), are cytotoxic to pancreatic islet beta cells, possibly by inducing nitric oxide and/or oxygen radical production in the beta cells. Peroxynitrite, the reaction product of nitric oxide and the superoxide radical, is a strong oxidant and cytotoxic mediator; therefore, we hypothesized that peroxynitrite might be a mediator of cytokine-induced islet beta-cell destruction. To test this hypothesis we incubated islets isolated from human pancreata with the cytokine combination of IL-1beta, TNFalpha, and IFNgamma. We found that these cytokines induced significant increases in nitrotyrosine, a marker of peroxynitrite, in islet beta cells, and the increase in nitrotyrosine preceded islet-cell destruction. Peroxynitrite mimicked the effects of cytokines on nitrotyrosine formation and islet beta-cell destruction. L-N(G)-monomethyl arginine, an inhibitor of nitric oxide synthase, prevented cytokine-induced nitric oxide production but not hydrogen peroxide production, nitrotyrosine formation, or islet beta-cell destruction. In contrast, guanidinoethyldisulphide, an inhibitor of inducible nitric oxide synthase and scavenger of peroxynitrite, prevented cytokine-induced nitric oxide and hydrogen peroxide production, nitrotyrosine formation, and islet beta-cell destruction. These results suggest that cytokine-induced peroxynitrite formation is dependent upon increased generation of superoxide (measured as hydrogen peroxide) and that peroxynitrite is a mediator of cytokine-induced destruction of human pancreatic islet beta cells.     

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.     

Cytotoxic T-lymphocyte-mediated killing of human pancreatic islet cells in vitro

Cytotoxic T lymphocytes (CTL) are believed to play an essential role in beta-cell destruction leading to development of type 1 diabetes and allogeneic islet graft failure. We aimed to identify the mechanisms used by CTL to kill human beta cells. CTL clones that recognize epitopes from influenza virus and Epstein-Barr virus restricted by human leukocyte antigen (HLA)-A0201 and -B0801, respectively, were used to investigate the susceptibility of human beta cells to CTL. In a short-term (5-hour) assay, CTL killed human islet cells of the appropriate major histocompatibility complex (MHC) class I type that had been pulsed with viral peptides. Killing was increased by pretreating islets with interferon gamma that increases MHC class I on target cells. Killing was abolished by incubation of CTL with the perforin inhibitor concanamycin A. The Fas pathway did not contribute to killing because blocking with neutralizing anti-Fas ligand antibody did not significantly reduce beta-cell killing. In conclusion, we report a novel way of investigating the interaction between CTL and human islets. Human islets were rapidly killed in vitro by MHC class I-restricted CTL predominantly by the granule exocytosis pathway.     

Application of an endothelialized modular construct for islet transplantation in syngeneic and allogeneic immunosuppressed rat models

Modular tissue engineering is a novel approach to assemble tissues with an inherent vascularization. In this article, we evaluated whether endothelialized module-driven vascularization enhances islet engraftment in diabetic rats. Two thousand islets were transplanted in the omental pouch of syngeneic and allogeneic immunosuppressed diabetic recipients as free islets, islets in collagen modules, or islets in endothelialized modules. Transplantation of islets in endothelialized modules significantly increased the vessel density compared with controls. Donor green fluorescent protein-positive endothelial cells (ECs) formed vessels in proximity to transplanted islets; donor vessels connected to host vasculature as the vessels included erythrocytes in their lumens and were supported by host smooth muscle cells by 21 days. Transplantation of 2000 islets reversed diabetes in two of five of syngeneic recipients until 60 days, although there was no apparent benefit to islet function of adding ECs relative to collagen modules without EC. However, there was a trend toward increased viability when islets were implanted in endothelialized modules compared with collagen modules at 21 days. Meanwhile, 2000 islets in allogeneic immunosuppressed recipients lowered blood glucose levels short term, but there was graft failure within 1 week. This study explored the simultaneous transplantation of primary ECs with islets in diabetic recipients. The endothelialized modular approach increased vessel density around transplanted islets. Further modulation (i.e., acceleration) of vessel maturation, is presumed necessary to improve islet engraftment.