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Exposure of isolated human islets to proinflammatory cytokines leads to up-regulation of inducible nitric oxide synthase (iNOS), raised NO, and beta cell toxicity. These findings have led to increasing interest in the clinical utility of iNOS blockade to mitigate beta cell destruction in human type 1 diabetes (T1D). However, recent studies show that iNOS-derived NO may also confer beta cell protection. To investigate this dichotomy, we compared islet cell distributions and intensity of iNOS immunostaining in pancreatic sections, co-stained for insulin and glucagon, from new-onset T1D donors (group 1), with non-diabetic autoantibody-negative (group 2), non-diabetic autoantibody-positive (group 3) and long-term diabetic donors (group 4). The cellular origins of iNOS, its frequency and graded intensities in islets and number in peri-islet, intra-islet and exocrine regions were determined. All donors showed iNOS positivity, irrespective of disease and presence of beta cells, had variable labelling intensities, without significant differences in the frequency of iNOS-positive islets among study groups. iNOS was co-localised in selective beta, alpha and other endocrine cells, and in beta cell-negative islets of diabetic donors. The number of peri- and intra-islet iNOS cells was low, being significantly higher in the peri-islet area. Exocrine iNOS cells also remained low, but were much lower in group 1. We demonstrate that iNOS expression in islet cells is variable, heterogeneous and independent of co-existing beta cells. Its distribution and staining intensities in islets and extra-islet areas do not correlate with T1D or its duration. Interventions to inactivate the enzyme to alleviate disease are currently not justified.

Peroxynitrite-induced nitration of cellular proteins has been shown to associate with various human pathologies. The expression of pancreatic nitrotyrosine and its cellular source relative to insulitis were analysed in cases with increasing duration of type 1 diabetes and compared with non-diabetic autoantibody-negative and -positive cases. Pancreatic tail sections from non-diabetic autoantibody-negative cases (Group 1; n = 7), non-diabetic autoantibody-positive cases (Group 2; n = 6), recently diagnosed cases (Group 3; n = 6), 0.25-5 years of diabetes (Group 4; n = 8) and 7-12 years of diabetes (Group 5; n = 6) were immunostained sequentially for nitrotyrosine, insulin and leucocytes. Nitrotyrosine expression was observed in selective beta cells only. In group 1, the percentage of insulin-positive islets with nitrotyrosine ranged from 7.6 to 58.8%. In group 2, it was minimally expressed in 2 cases and was present in 4.7-19.3% of insulin-positive islets in 3 cases and in all islets in 1 case. In group 3, it was absent in 1 case and in the remaining 5 cases, the values were 17.4-85.7%. In group 4, nitrotyrosine was absent in 6 cases and positive in 1.8 and 22.2% of insulin-positive islets in 2 cases. In group 5, the values were 60% (1 case) and 100% (2 cases), being absent in 3 cases, consistent with insulin-negativity. This case analysis shows that nitrotyrosine immunostaining is independent of the presence and severity of insulitis. Variable nitrotyrosine expression is present in some non-diabetic cases. Its increased expression in beta cells of recent-onset and long-standing disease requires further studies to determine whether beta cell nitration plays a pathogenic role during T1D.

In transplantation, there is a critical need for noninvasive biomarker platforms for monitoring immunologic rejection. We hypothesized that transplanted tissues release donor-specific exosomes into recipient circulation and that the quantitation and profiling of donor intra-exosomal cargoes may constitute a biomarker platform for monitoring rejection. Here, we have tested this hypothesis in a human-into-mouse xenogeneic islet transplant model and validated the concept in clinical settings of islet and renal transplantation. In the xenogeneic model, we quantified islet transplant exosomes in recipient blood over long-term follow-up using anti-HLA antibody, which was detectable only in xenoislet recipients of human islets. Transplant islet exosomes were purified using anti-HLA antibody-conjugated beads, and their cargoes contained the islet endocrine hormone markers insulin, glucagon, and somatostatin. Rejection led to a marked decrease in transplant islet exosome signal along with distinct changes in exosomal microRNA and proteomic profiles prior to appearance of hyperglycemia. In the clinical settings of islet and renal transplantation, donor exosomes with respective tissue specificity for islet β cells and renal epithelial cells were reliably characterized in recipient plasma over follow-up periods of up to 5 years. Collectively, these findings demonstrate the biomarker potential of transplant exosome characterization for providing a noninvasive window into the conditional state of transplant tissue.

Nonspecific inflammatory response is the major cause for failure of islet grafts at the early phase of intraportal islet transplantation (IPIT). Bilirubin, a natural product of heme catabolism, has displayed anti-oxidative and anti-inflammatory activities. The present study has demonstrated that bilirubin protected islet grafts by inhibiting nonspecific inflammatory response in a syngeneic rat model of IPIT. The inflammation-induced cell injury was mimicked by exposing cultured rat insulinoma INS-1 cells to cytokines (IL-1β, TNF-α and IFN-γ) in in vitro assays. At appropriate lower concentrations, bilirubin significantly attenuated the reduced cell viability and enhanced cell apoptosis induced by cytokines, and protected the insulin secretory function of INS-1 cells. Diabetic inbred male Lewis rats induced by streptozotocin underwent IPIT at different islet equivalents (IEQs) (optimal dose of 1000, and suboptimal doses of 750 or 500), and bilirubin was administered to the recipients every 12 h, starting from one day before transplantation until 5 days after transplantation. Administration of bilirubin improved glucose control and enhanced glucose tolerance in diabetic recipients, and reduced the serum levels of inflammatory mediators including IL-1β, TNF-α, soluble intercellular adhesion molecule 1, monocyte chemoattractant protein-1 and NO, and inhibited the infiltration of Kupffer cells into the islet grafts, and restored insulin-producing ability of transplanted islets.

Objectives: To determine if there is an abrupt change in the frequency of intraislet T regulatory (Treg) cells in female nonobese diabetic (NOD) mice preceding and following spontaneous diabetes and during cyclophosphamide-accelerated disease., Methods: The frequency of intraislet CD4-positive Treg cells was analyzed between days 21 and 250, at onset, and at 1, 2, and 3 weeks of diabetes by dual-label immunohistochemistry. Tissues were also analyzed between days 0 and 14 after injection of cyclophosphamide or diluent., Results: In the spontaneous group, intraislet Treg cells were first observed on day 30 in CD4 T cells and increased from day 45. There was no statistical difference in the frequency of Treg cells in nondiabetic NOD mice on days 45, 60, and 90. A sustained frequency at and after 1, 2, and 3 weeks of diabetes was also observed. In the cyclophosphamide group, there was a sharp decline in the frequency of Treg cells on day 4, which remained lower on day 7 but increased by days 11 and 14., Conclusions: During spontaneous diabetes and after onset, the frequency of intraislet CD4-positive Treg cells remains unchanged. They may possess diminished immunoregulatory function and thus unable to counteract the increasing tempo of immune-mediated beta-cell destruction.

The precise fate of beta cells and the presence of islet infiltrates after onset of type 1 diabetes have not yet been fully characterized. Recently we showed that in newly diabetic NOD mice an appreciable number of beta cells remain. This was also observed during the first 2 weeks of diabetes in NOD mice without treatment with insulin. However, the mean number of beta cells per unit islet cross-sectional area decreased with increasing duration of disease. In contrast, glucagon and somatostatin cell numbers showed an increase. The persistence of insulitis in several islets until 4 weeks of diabetes suggests ongoing beta cell autoimmunity over a protracted phase. Combined daily treatment of newly diabetic NOD mice with epidermal growth factor (EGF) and gastrin for the first 14 days of diabetes resulted in temporary restoration of normoglycemia in 7 of 15 mice. We speculate that the residual beta cells present soon after onset of diabetes may respond to experimental regeneration. Treatment of newly diabetic NOD mice with the bioactive peptides EGF and gastrin resulted in partial and temporary reversal of diabetes. We propose that peptide therapies combined with other benign immunomodulatory approaches to rescue and preserve beta cells in the long term and to prevent recurring autoimmunity may be more effective than peptide therapy alone in reversing diabetes in NOD mice.

Objectives: We determined if newly weaned female nonobese diabetic (NOD) mice show greater diabetes sensitivity to dose-adjusted regimens of multiple low doses of streptozotocin (Stz) than nondiabetes-prone CD-1 mice., Methods: Female NOD mice received 5 daily doses of Stz from day 21 (0, 5, 10, 15, 20, 30, and 40 mg/kg body weight) and CD-1 mice 20, 30, and 40 mg., Results: : Streptozotocin, at the 15-, 20-, 30-, and 40-mg dose, induced rapid diabetes in NOD mice. By day 100, 90% to 95% of NOD mice became diabetic after the 40- and 30-mg dose and 33% to 40% with the 15- and 20-mg dose. In comparison, only about 50% and 33% of CD-1 mice developed diabetes with the 40- and 30-mg dose, respectively, and 5.5% with the 20-mg dose. In NOD mice, the 20-mg dose also partially suppressed spontaneous diabetes. All diabetic mice displayed insulitis, variable immunostaining for insulin, and redistribution of glucagon and somatostatin cells. Glucose transporter-2 was markedly attenuated in selective beta cells., Conclusions: Newly weaned female NOD mice show heightened early sensitivity to low doses of Stz than CD-1 mice. At diabetes, several beta cells remain and show variable immunostaining for insulin and an attenuated expression for glucose transporter-2. Specific low doses of Stz may also suppress spontaneous diabetes.

The molecular processes that initiate insulitis in type 1 diabetes remain unclear. Chemokines, such as monocyte chemoattractant protein-1 (MCP-1), mediate chemotaxis and leukocyte migration to inflammatory sites. Although MCP-1 mRNA has been shown in islets isolated from NOD mice at 2 weeks of age and at later stages, the cellular sources of this chemokine, at the protein level, and its role in insulitis are unclear. The aims of the present study were to employ immunohistochemical techniques to examine the expression of MCP-1 and quantify its cellular sources in islets of NOD mice both after cyclophosphamide (Cy) administration and in spontaneous diabetes. Tissues were examined at days 1 (=day 73, first day of Cy), 4, 7, 11, and 14 and in age-matched control NOD mice. Pancreatic sections from NOD mice without Cy administration were also studied between days 21-65 and at onset of diabetes and from adult CD-1 mice. In the Cy group, a small number of peri-islet macrophages were immunopositive for MCP-1 at day 1 whereas at day 4, the number declined but increased subsequently at day 7. In the same group, it increased markedly at days 11 and 14 compared with age-matched control NOD mice. In young NOD mice, MCP-1 was present in selective macrophages in islets with early insulitis (day 45) but was absent at diabetes onset. MCP-1 was undetectable in beta cells and in most T cells. Islets from adult CD-1 mice did not show immunostaining for MCP-1. We conclude that MCP-1 is expressed in a proportion of islet and exocrine macrophages. This expression increases during the later stages of Cy-induced diabetes. Thus, MCP-1 positive macrophages that migrate to the islet periphery during the early stages of Cy-induced diabetes and preceding spontaneous diabetes may augment insulitis by further attracting macrophages and T cells.

The cyclophosphamide model of accelerated diabetes in the NOD mouse is a useful model of insulin-dependent diabetes mellitus (IDDM). Knowledge on the progressive destruction of beta cells and the fate of other islet endocrine cell-types in this model is sparse. We employed immunohistochemistry and histochemistry, to study temporal changes in islet cell populations, insulitis and glucose transporter-2 expression during cyclophosphamide administration. Cyclophosphamide was administered to day 95 female NOD mice and the pancreas studied at days 0 ( = day 95), 4, 7, 11 and 14 after treatment and in age-matched control mice. At day 0, a majority of the endocrine cells were insulin-positive. Glucagon and somatostatin cells were mostly in the islet periphery and also internally. In the cyclophosphamide group, insulitis was moderate at day 0, declined at day 4 but increased progressively from day 7. The extent of insulitis in treated mice which were diabetes-free at day 14 was comparable to age-matched control mice. From day 11, the marked increase in insulitis correlated with a reciprocal decline in the extent of insulin immunostained islet area. At day 14, the mean insulin area per islet was markedly less in diabetic mice than in age-matched non-diabetic treated and controls. At diabetes, some islets showed co-expression of glucagon and insulin. Our studies suggest that the mean number of glucagon or somatostatin cells per islet does not vary during the study. Glucose transporter-2 immunolabelling was restricted to beta cells but declined in those adjacent to immune cells. We conclude that in the cyclophosphamide model, there is specific and augmented destruction of beta cells immediately prior to diabetes onset. We speculate that the selective loss of glucose transporter-2 shown in this study suggests the existence of a deleterious gradient close to the immune cell and beta cell surface boundary.

Reactive oxygen species and nitric oxide generated within the beta cell or by intraislet immune cells may be major mediators of beta cell death during insulin-dependent diabetes mellitus. Here, the intraislet expression of nitrotyrosine, a biomarker of oxidative damage, and its cellular sources were examined in the islets of NOD mice at various stages of spontaneous and cyclophosphamide-accelerated diabetes. At day 30, nitrotyrosine-positive cells were undetectable; they were rare at day 40, being expressed in only a few beta cells and in macrophages located in the periphery of some islets. At day 90 and at onset of diabetes, an increasing number of macrophages and beta cells were nitrotyrosine positive. In the cyclophosphamide group at day 4, the number of beta cells and macrophages with positive immunolabeling declined slightly from day 0 (day of cyclophosphamide injection = day 90). This pattern of immunolabeling increased gradually by day 7 but increased markedly by days 11 and 14 after cyclophosphamide administration. In the spontaneous and cyclophosphamide groups, macrophages were the predominant source of nitrotyrosine and were present within the intraislet and periislet regions. Nitrotyrosine labeling was observed in a significant number of beta cells but less than in immunolabeled macrophages. We conclude that protein nitration in beta cells and macrophages is an important feature of the diabetogenic process in the NOD mouse. Whether this deleterious process also occurs during the very early stages of the disease and preceding insulitis is unclear.

Leptin is a 16 kD polypeptide hormone produced predominantly by white adipose tissue and exerts profound effects on food intake and energy balance. More recent studies have shown extra sites of leptin production in human and rodent tissues and have ascribed additional roles for the hormone, e.g., in immune and reproductive functions. A role for the hormone has also been implicated in insulin-dependent diabetes mellitus in the non-obese diabetic (NOD) mouse. However, whether leptin originates from islet cells of the mouse is not known. Here dual-label immunohistochemistry was employed to examine leptin expression in islet cells, and its distribution and cellular sources in pancreatic sections of female NOD/Ak and CD-1 mice of various ages. For comparison, leptin immunolabelling was examined in adult pancreatic sections from male NOD/Ak CD-1, Balb/c and FVB/N mice and female severe combined immunodeficient CB. 17 mice. Pancreatic tissues from adult female guinea pig, sheep and cattle and neonatal pigs were also studied. Our results show that in the day 1 NOD and CD-1 mice, leptin immunolabelling was observed in selective glucagon cells within the developing islets while at days 15 and 22, it became more intense and co-incident. This pattern of staining was maintained at days 40, 90, 150 and 250. In the female NOD mouse, leptin was absent in intra-islet immune cells. Its expression was variable in islets from male NOD and CD-1 mice. In spontaneously diabetic female NOD mice and following acceleration of diabetes with cyclophosphamide, despite the persistence of strong immunolabelling for glucagon in the re-distributed alpha cells, leptin expression was either absent, diminished or present in only a proportion of alpha cells. The reduction in leptin labelling was often associated with diabetic islets which had insulitis in association with only a small number of residual beta cells. Leptin expression was absent in guinea pig, ovine, bovine and neonatal porcine islet cells, despite the expression of intensely labelled glucagon cells. The present results demonstrate leptin co-localization in glucagon cells of the mouse islet. Its expression diminishes in the presence of inadequate insulin. Leptin produced within the mouse islet may have bi-directional influences on leptin and insulin regulation and may play local functions in islet development and metabolism.

Apoptosis may be a major mechanism of beta cell loss during insulin-dependent diabetes mellitus. Caspase-3 is a key enzyme involved in the terminal steps of this death process. Here, the intra-islet expression of caspase-3 in the NOD mouse was examined immunohistochemically following acceleration of the disease with cyclophosphamide. Female NOD mice were treated at day 95 with cyclophosphamide, and caspase-3 expression in pancreatic sections was studied at days 0, 4, 7, 11, and 14 and compared with age-matched control tissue. In the treated group at day 0, caspase-3 labeling was seen in several peri-islet macrophages and only extremely rarely in beta cells. At day 4, only a few beta cells weakly expressed the enzyme. From day 7, caspase-3 expression began to increase in intra-islet macrophages and reached a peak at days 11 and 14, when a small number of CD4 and CD8 T cells also showed positive labeling. Beta cell expression of caspase-3 at days 11 and 14 was rare. At this stage, several intra-islet immune cells with positive labeling for the enzyme coexpressed either Fas or interleukin-1beta. Only a small proportion of intra-islet caspase-3 cells showed apoptotic nuclei judged by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). We conclude that, during cyclophosphamide-accelerated diabetes, the predominant caspase-3 immunolabeling in intra- and extra-islet macrophages suggests that apoptosis of macrophages may be an important mechanism for their elimination. The virtual absence of caspase-3 immunolabeling in most beta cells even during the height of beta cell loss supports the need for developing other markers of early beta cell apoptosis in the NOD mouse.

During insulin-dependent diabetes mellitus, beta cell destruction may involve activation of the Fas-Fas ligand (Fas-FasL) system. Here, we employed dual-label immunohistochemistry to examine the intra-islet expression, distribution, and cellular sources of Fas and FasL in the NOD mouse. Pancreatic tissues were studied during spontaneous diabetes (days 21, 40, and 90) and following acceleration of diabetes with cyclophosphamide (days 0, 4, 7, 11, and 14 after cyclophosphamide administration). Our results show that FasL was expressed constitutively in most beta cells of NOD mice and in nondiabetes-prone mice, but not in glucagon or somatostatin cells or in islet inflammatory cells. It paralleled the loss of insulin immunolabeling with advancing disease. Immunolabeling for Fas was first observed in extra-islet macrophages and those close to the islet in NOD and nondiabetes-prone mice. During spontaneous and cyclophosphamide diabetes, it was observed in a higher proportion of islet infiltrating macrophages than in CD4 and CD8 cells. In the cyclophosphamide group, Fas expression in intra-islet CD4 and CD8 cells showed an increase close to the onset of diabetes. At days 11 and 14, several intra-islet macrophages with immunolabeling for Fas also coexpressed interleukin-1beta and inducible nitric oxide synthase. Fas was not detected in beta cells and other endocrine cells during spontaneous and cyclophosphamide diabetes. We show constitutive expression of FasL in beta cells in the NOD mouse and predominant expression of Fas in intra-islet macrophages and to a lesser extent in T cells prior to diabetes onset. The role of Fas-FasL in beta cell destruction in the NOD mouse requires further clarification.

During insulin-dependent diabetes mellitus, immune cells infiltrate pancreatic islets progressively and mediate beta cell destruction over a prolonged asymptomatic prediabetic period. Apoptosis may be a major mechanism of beta cell loss during the disease. This process involves a proteolytic cascade in which upstream procaspases are activated which themselves activate downstream caspases, including caspase-3, a key enzyme involved in the terminal apoptotic cascade. Here dual-label immunohistochemistry was employed to examine the intra-islet expression, distribution and cellular sources of active caspase-3 in the non-obese diabetic (NOD) mouse given cyclophosphamide to accelerate diabetes. NOD mice were treated at day 95 and caspase-3 expression was studied at days 0, 4, 7, 11 and 14. Its expression was also correlated with advancing disease and compared with age-matched NOD mice treated with diluent alone. At day 0 (=day 95), caspase-3 immunolabelling was observed in several peri-islet and intra-islet macrophages, but not in CD4 and CD8 cells and only extremely rarely in beta cells. At day 4, only a few beta cells weakly expressed the enzyme, in the absence of significant insulitis. At day 7, caspase-3 expression was observed in a small proportion of intra-islet macrophages. At day 11, there was a marked increase in the number of intra-islet macrophages positive for caspase-3 while only a few CD4 cells expressed the enzyme. At day 14, caspase-3 labelling became prominent in a significant proportion of macrophages. Only a few CD4 and CD8 cells expressed the enzyme. Capase-3 labelling was also present in a proportion of macrophages in perivascular and exocrine regions. Surprisingly, beta cell labelling of caspase-3 at days 11 and 14 was rare. At this stage of heightened beta cell loss, a proportion of intra-islet interleukin-1beta-positive cells coexpressed the enzyme. Caspase-3 was also observed in numerous Fas-positive cells in heavily infiltrated islets. During this late stage, only a proportion of caspase-3-positive cells contained apoptotic nuclei, as judged by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). We conclude that during cyclophosphamide-accelerated diabetes in the NOD mouse, the predominant immunolabelling of caspase-3 in intra-islet macrophages suggests that apoptosis of macrophages may be an important mechanism for its elimination. The virtual absence of caspase-3 immunolabelling in most beta cells even during heightened beta cell loss supports their rapid clearance following their death during insulin-dependent diabetes mellitus.

The expression of IL-1beta in the NOD mouse pancreas was examined following disease acceleration with cyclophosphamide (Cy). Female NOD mice were injected with Cy at day 95 and the pancreas examined immunohistochemically at days 0, 4, 7, 11, and 14 (Cy group). Cyclophosphamide was also administered to NOD mice that were given oral nicotinamide from day 21. At day 0 (Cy group), IL-1beta was expressed in selective intraislet macrophages but showed an increase from day 7 onwards in macrophages, a few beta cells, and somatostatin cells. Peak expression was seen at day 11, when it was significantly higher than in day-11 mice given nicotinamide. In the Cy group a proportion of macrophages coexpressed IL-1beta and inducible nitric oxide synthase (iNOS). IL-1beta expressed within the islet macrophages may act in concert with other cytokines, promote free radical generation including NO, and promote beta cell death during IDDM.

Leukocytes infiltrate the pancreatic islets of nonobese diabetic mice, causing beta-cell destruction and autoimmune Type I diabetes. Here, we completely blocked adoptive transfer of diabetes and reduced spontaneous disease incidence from 71% to 17% by simultaneously administering a combination of antibodies directed against alpha4, beta2, and beta7 integrins and their ligands VCAM-1, MAdCAM-1, and ICAM-1 for 52 and 28 days, respectively. CD4 and CD8 T cells and macrophages were excluded from islets and remained entrapped in a peri-islet location as inactive exiles, no longer expressing normal levels of interferon-gamma, interleukin-4, and iNOS. Only IL-10 expression was retained, which could aid immunosuppression. Infiltrating leukocytes retained a peri-islet location, even 215 days following suspension of antibody treatment, potentially forming a barrier to the entry of active, autoantigen-reactive T cells. Combination treatment was effective against spontaneous disease when administered from 7 days of age but ineffective when initiated late in the prediabetic period (day 40 or 70). Nevertheless, anti-alpha4 subunit mAb monotherapy alone was very effective, reducing insulitis to levels similar to those obtained with combinational antibody treatment, suggesting that alpha4 integrins are major receptors contributing to leukocyte infiltration. Treatment with anti-alpha4 integrin antibody retained some therapeutic benefit when administered from days 7, 40, or 70 of age. The results have implications for the treatment of diabetes and provide a unique insight into the fate of disease-forming leukocytes following anti-CAM therapy.

During insulin-dependent diabetes mellitus, islet invading immune cells destroy beta cells over a prolonged asymptomatic pre-diabetic period. Cytokines synthesised and secreted by specific immune cells within the islet infiltrate may be crucial effectors of beta cell destruction or protection during the disease. Interleukin-1beta may be a key cytokine which may act in concert with other cytokines in initiating and/or promoting beta cell destruction. We have examined this hypothesis in NOD mice by assessing the intra-islet expression and co-localization of interleukin-1beta at different time-points following cyclophosphamide administration. We have also tested the effects of long-term oral nicotinamide given to NOD mice in suppressing intra-islet expression of the cytokine in this accelerated model. Cyclophosphamide was administered to day 95 female NOD mice. Pancreatic tissues were examined by dual-label confocal immunofluorescence microscopy for the expression and co-localization of interleukin-1beta at days 0, 4, 7, 11 and at onset of diabetes (day 14). Diabetes developed in 7/11 mice 14 days after administration of cyclophosphamide while nicotinamide completely prevented the disease. At day 0, interleukin-1beta immunolabelling was observed in selective intra-islet macrophages, several somatostatin cells and in a few beta cells. However, at day 4, it was seen mostly in somatostatin and some beta cells. At day 7, an increasing number of interleukin-1beta cells were observed within the islets and co-localized to several somatostatin cells, beta cells and macrophages. The mean number of intra-islet interleukin-1beta cells reached a peak at day 11 and was significantly higher than at day 7 (p = 0.05) and at day 14 (onset of diabetes; p = 0.03). At day 11, interleukin-1beta immunolabelling was also present in selective macrophages which co-expressed inducible nitric oxide synthase. At onset of diabetes, some macrophages, residual beta cells and somatostatin cells showed immunolabelling for the cytokine. Exposure of NOD mice to oral nicotinamide was associated with a considerably reduced expression of interleukin-1beta cells within the islet at day 11 (p = 0.002). We conclude that cylophosphamide treatment enhances the expression of interleukin-1beta in selective macrophages, somatostatin and beta cells during the course of the disease. Its expression reaches a maximum immediately prior to onset of diabetes. Interleukin-1beta present in intra-islet macrophages, somatostatin and beta cells may influence its expression by autocrine and paracrine means. Interleukin-1beta expression within islet macrophages may also up-regulate inducible nitric oxide synthase within the same macrophage or adjacent macrophage populations. These intra-islet molecular events may corroborate with other local cytotoxic processes leading to beta cell destruction. Oral nicotinamide may attenuate intra-islet expression of interleukin-1beta and thus inducible nitric oxide synthase during prevention of Type 1 diabetes in this animal model. The expression of interleukin-1beta in specific islet endocrine cell-types shown in this study requires further investigation.

Beta cell destruction has been shown to occur when rodent or human islets are exposed in vitro to inflammatory cytokines, such as interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). Other cytokines such as interleukin-4 (IL-4) or interleukin-10 (IL-10), when given to NOD mice, prevent insulin-dependent diabetes mellitus (IDDM). In this study, we have employed immunofluorescence histochemistry to study the expression of IFN-gamma and IL-4 in the pancreas of female NOD mice at various time-points (days 0, 4, 7, 11 and at onset of diabetes) following disease acceleration with cyclophosphamide (Cy). Dual-label confocal and light microscopy were employed to determine the precise cellular sources of the two cytokines. IL-4 immunolabelling was observed in a few immune cells at days 0, 4, and 7 within the pancreatic islets but in larger numbers at day 11 and at onset of diabetes. The cytokine was co-localized predominantly in CD4 cells, while only a small minority of CD8 cells and macrophages also expressed IL-4. At days 0, 4, 7 and 11, weak to moderate immunolabelling for IL-4 was also observed in beta cells. In contrast, immunolabelling for IFN-gamma within the islets was not observed until day 11 and this labelling persisted at onset of diabetes. It was immunolocalized in macrophages and to a lesser extent in CD4 cells. Only a few CD8 cells were immunopositive for IFN-gamma. At day 11, a proportion of beta cells showed weak immunolabelling for IFN-gamma. During the study period, immunolabelling for IFN-gamma was also observed in a proportion of endothelial cells located in the intra-islet and exocrine regions of Cy and diluent-treated mice. From day 11 onwards, both the cytokines were observed in some of the peri-vascular regions. Our results demonstrate that during Cy-induced diabetes, there is increasing expression of both IL-4 and IFN-gamma in specific immune cells within the inflamed islets in the late prediabetic stage and at onset of diabetes. Further studies are required to correlate our protein immunohistochemical findings with in situ cytokine gene expression and to determine whether there is a clear Th1 cytokine protein bias at clinical onset of diabetes and immediately preceding it.

Beta cell destruction in NOD mice can be accelerated by adoptive transfer of diabetic spleen cells into irradiated adult NOD mice. Here mice receiving diabetic spleen cells were examined at days 0, 7, 14, 21 and at onset of diabetes for the resulting insulitis and the number of intra-islet CD4 and CD8 cells and macrophages. The progression of insulitis and the number of intra-islet CD4 and CD8 cells and macrophages were correlated with the expression and co-localization of inducible nitric oxide synthase, interferon-gamma and interleukin-4 by dual-label light and confocal immunofluorescence microscopy. Diabetes developed in 7/8 mice by 27 days following cell transfer. The insulitis score increased slightly by day 7 but rose sharply at day 14 (p = 0.001) and was maintained until diabetes. The mean number of intra-islet CD4 and CD8 cells and macrophages showed a similar trend to the insulitis scores and were present in almost equal numbers within the islets. Immunolabelling for inducible nitric oxide synthase was observed at day 7 in only some cells of a few islets but increased sharply from day 14. It was restricted to islets with insulitis and was co-localized in selective macrophages. Weak intra-islet interleukin-4 labelling was observed at days 7 and 14 but became more pronounced at day 21 and at onset of diabetes, being present in selective CD4 cells. Intra-islet labelling for interferon-gamma was first observed at day 21, but became more intense at onset of diabetes and was co-localized in a proportion of macrophages. Both cytokines were expressed in islets with advanced insulitis. Interferon-gamma staining was also observed within endothelial cells located in the exocrine pancreas. We conclude that transfer of diabetic spleen cells results in a rapid influx of CD4 and CD8 cells and macrophages within the pancreas of recipient mice. During the period of heightened insulitis, selective immune cells begin to express inducible nitric oxide synthase and the opposing cytokines, interferon-gamma and interleukin-4. Expression of these molecules becomes more pronounced immediately prior to and during the onset of diabetes.

Glucose transporter-2 (glut2) is underexpressed in beta cells of several rodent models of non-insulin-dependent diabetes mellitus (NIDDM). This may also be true for rodent models of insulin-dependent diabetes mellitus (IDDM). The present study examines two murine models of autoimmune IDDM, the nonobese diabetic (NOD) and the low-dose streptozotocin (stz) murine models for changes in the expression of glut2 by double-label light and confocal microscopy during various stages of the disease. The spatial distribution of glut2 cells was also examined in relation to insulin immunoreactive cells and the islet inflammatory cells during these stages. In both the female NOD mouse and the female Swiss mouse without stz treatment, glut2 colocalized with insulin in virtually all the beta cells. In the NOD mouse, islets with moderate to advanced insulitis showed either an absence or considerably reduce expression of glut2 in insulin-containing beta cells. Cells with reduced glut2 expression were usually located adjacent to the region of insulitis. At onset of diabetes, glut2 immunolabeling was reduced despite the preservation of weak insulin immunoreactivity. In Swiss mice treated repeatedly with stz, glut2 labeling began to decline in select Beta cells after the fourth injection in approximately 50% of the islets, despite the lack of insulitis. At this stage expression of glut2 fell in a small number of islets with evidence of early macrophage infiltration. Loss of glut2 became more pronounced in nondiabetic Swiss mice after the fifth injection. At this stage glut2 labeling in the plasma membrane appeared diffuse and variable. At onset of stz-induced diabetes, glut2 expression significantly fell, despite weak immunoreactivity for insulin. This loss was associated with an enhanced influx of both macrophages and T lymphocytes within the islets of diabetes mice. In both the NOD and the low-dose stz mouse models, loss of glut2 thus occurs from an early stage and precedes hyperglycaemia. This loss may be mediated by immune and nonimmune mechanisms.

Glutamic acid decarboxylase (GAD) is present in the central nervous system and in several nonneuronal tissues including the pancreatic islets. There are two isoforms with molecular weights of 65 kDa (GAD65) and 67 kDa (GAD67). The cellular specificity of the two molecular forms of GAD and their levels within the mammalian islets may be species-dependent, being coexpressed in both beta and in non-beta cells. We have examined the ovine pancreas, from the adult and fetal stages of late gestation, for the expression of GAD65 within the islet cells by double-label immunofluorescence light and confocal microscopy. In the adult tissue, GAD65 was colocalized in a majority of the beta cells (> 95%), with only a few glucagon and somatostatin cells (< 5%) showing immunolocalization. During the fetal stages GAD65 also showed a similar predominant beta-cell coexpression. The enzyme was also detected in a few fetal glucagon (< 5%) but not somatostatin cells. In the degenerating large fetal islets, GAD65 was also observed in the majority of the residual beta cells. These results demonstrate that in the ovine pancreas GAD65 is expressed during fetal development and is predominantly beta-cell-restricted. This pattern of expression is maintained during adult life. However, the physiological role of pancreatic GAD and/or its biosynthetic product, gamma-aminobutyric acid, in islet function in the sheep and in other ruminants remains unclear.

Nitric oxide has been shown to mediate beta-cell destruction in rodent islets exposed to interleukin 1 beta in culture. The inhibitory effect is potentiated by tumour necrosis factor-alpha and interferon-gamma. Cytokine stimulation leads to gene transcription and translation of inducible nitric oxide synthase, the biosynthetic enzyme of nitric oxide. In the non-obese diabetic mouse, progressive invasion of pancreatic islets by immune cells may lead to local production of inflammatory cytokines, resulting in inducible nitric oxide synthase expression within the islets. In this study, the ontogeny of this enzyme and its cellular expression were examined in pancreatic sections of female non-obese diabetic mice by double-label immunofluorescence. Light and confocal microscopy were employed to study the up-regulation, co-localization and immunocytoplasmic distribution of the enzyme in female non-obese, diabetic and Swiss mice following cytokine treatment. From day 40 to day 220 a small number of beta-cells and a proportion of macrophages, usually in peri-islet and exocrine areas, expressed the enzyme. At onset of diabetes, an increasing number of macrophages within and surrounding the islets were positive for the enzyme. Treatment of day 60 female non-obese diabetic mice with interleukin 1 beta alone and in combination with tumour necrosis factor-alpha and/or interferon-gamma resulted in a significant influx of macrophages into the pancreas, while this was lower in female Swiss mice treated similarly. Cytokine administration led to intense but sometimes eccentric immunocytoplasmic labelling for the enzyme in a considerable proportion of macrophages and beta-cells. Macrophages positive for inducible nitric oxide synthase were located in peri- and intra-islet areas, being distal and adjacent to enzyme-positive and negative beta-cells. Treatment with tumour necrosis factor-alpha and/or interferon-gamma did not lead to enzyme up-regulation. These results show that in the non-obese diabetic mouse there is low and sustained expression of islet inducible nitric oxide synthase in the prediabetic period, which is followed by an increase around onset. However, treatment of female non-obese diabetic and Swiss mice with interleukin-1 beta, alone or together with tumour necrosis factor-alpha and/or interferon-gamma leads to a marked expression of this enzyme within macrophages and beta-cells.

Glutamic acid decarboxylase (GAD) has been shown to exist as two isoforms with molecular weights of 65 kD (GAD65) and 67 kD (GAD67) in the central nervous system as well as in several non-neuronal tissues, including the pancreatic islets. Recently, this enzyme has been proposed as a key beta-cell autoantigen in insulin-dependent diabetes mellitus (IDDM). In the present study, we used double label light and confocal microscopy to examine the expression of the two GAD isoforms in islet cells of fetal, neonatal and adult porcine pancreas. We also aimed to identify the islet cell-type(s) which co-express GAD. In the adult pig, GAD65 was localized exclusively in most of the beta cells, whereas GAD67, in addition to being present in a majority of the beta cells, was also seen in a proportion of glucagon and somatostatin labelled cells. In the 90-day fetus and the 7-day neonate, while GAD65 was also observed in a majority of beta cells, a proportion of glucagon cells also co-expressed this isoform. The cellular expression of GAD67 in the fetal and neonatal stages was similar to that in the adult. Detailed confocal analysis of GAD65 immunoreactive cells showed a granular cytoplasmic staining, with labelled granules often concentrated in specific perinuclear regions, possibly the Golgi apparatus. In contrast, GAD67 positive cells showed more diffuse cytoplasmic staining. The predominant expression of both the isoforms in porcine beta cells suggests that islet cells from this species may act as a suitable cellular model for study of GAD autoreactivity during the early stages of IDDM.

We have previously shown that diabetes in the NOD mouse can be prevented if mice are placed from weaning on an infant formula diet in which the protein source is replaced with casein hydrolysate (Pregestimil) or soy protein (Prosobee), or if 1% nicotinamide is given in the drinking water. Nicotinamide somewhat suppresses insulitis but the hydrolysed casein formula does not. In this study, Prosobee was given concurrently with oral nicotinamide from weaning and their effects on the development of insulitis and diabetes measured. These effects were also assessed in mice given Prosobee alone from conception (day -20) or from weaning. Unlike the earlier experiments, a marked suppression of insulitis was observed when the diets and nicotinamide were given concurrently (mean insulitis scores +95% confidence intervals (back transformed): day 40 = 0.4% [0.03, 1.17] vs. 12.5% [2.52, 28.40] and at day 90 = 8.8% [3.65, 15.68] vs. 48.1% [33.89, 62.49], P = 0.0001). A similar suppression was observed on day 90 with Pregestimil combined with nicotinamide 7.3% [3.88, 11.70] vs. 43.8% [32.59, 55.35] (P = 0.0001). Qualitatively, introduction of Prosobee from conception appeared to elicit a greater degree of suppression of insulitis than when introduced from day 21. Insulitis lesions were examined immunohistochemically for CD4, CD8 and MAC-1 cells. The proportion of these cells was not different for any regime despite the great differences in total number of inflammatory cells in and around the islets of mice fed the combined diet. All the three dietary treatments (Prosobee from day -20, Prosobee from day 21, Prosobee+nicotinamide from day 21) resulted in substantial protection from diabetes in mice followed until 250 days. We conclude that the complete prevention of diabetes in the NOD mouse fed a casein-free diet together with nicotinamide is accompanied by marked inhibition of insulitis, which is not seen when either dietary agent is introduced alone. The somewhat greater suppression of insulitis in mice given the soy diet from conception compared to those fed from day 21 may indicate that even maternal diet during gestation may influence diabetes outcome in the offspring.

CD4 and CD8 T cells and macrophages have been implicated as cellular mediators of beta cell destruction in insulin-dependent diabetes mellitus (IDDM). The ratios of the two T cell subsets were, therefore, quantified in nonobese diabetic (NOD) mouse islets prior to IDDM, at the onset of the disease, and following onset by immunohistochemistry. The number of periislet-, intraislet-, and exocrine-located macrophages were also determined during these stages. At all time points studied (day 90, day 250, at diabetes onset, 4-6 weeks after diabetes), CD4 cells were 2.5 times higher than CD8 cells except at day 250, on which the CD4:CD8 ratio was 3.8. At days 90 and 250, islets were heavily infiltrated with both the T cell subsets associated with lower numbers of macrophages. At onset of the disease and after insulin treatment, although the CD4:CD8 ratios were similar, the absolute numbers of the two subsets were reduced-considerably. At these stages a majority of the islets was atrophied, some were still surrounded by T cells and macrophages and were enriched with glucagon cells. CD4 and CD8 cells were also observed in the exocrine region at the two stages. Macrophages located in the periislet areas were significantly higher in number than in the intraislet positions in all study groups (p = 0.001). They also showed a gradual decline from day 90 to clinical diabetes. Periislet-located macrophages were significantly higher at day 90 than after onset and in control Swiss mice (p < 0.05). The numbers of macrophages in the exocrine areas were similar in all groups of NOD mice. In control Swiss mice they were significantly lower in the periislet, intraislet, and exocrine regions.

Nicotinamide (NIC) is presently extensively studied as a potential agent that might prevent the development of insulin-dependent diabetes mellitus. This study aimed to examine the consequence of exposing isolated rat pancreatic islets to various concentrations of NIC (0, 0.5, 1.0, 5.0, 10, and 25 mM) over a prolonged period (6 days) in tissue culture and also to assess the efficacy of NIC to counteract interleukin-1 beta (IL-1; 25 U/ml)-induced beta-cell dysfunction. Except for a 30-40% increase at 5.0 mM NIC, the insulin content of islets was not affected by NIC. Also, the islet DNA content remained essentially unchanged. The insulin accumulation in the culture medium declined at 5-25 mM NIC between days 4-6. The insulin release in response to 16.7 mM glucose on day 6 was enhanced after culture with the addition of 0.5 mM NIC, but 25 mM NIC caused a strong inhibition of insulin secretion. However, neither the (pro)insulin nor the total protein biosynthesis rate of the islets was affected by NIC. A significant inhibition of the islet poly(ADP-ribose) polymerase activity by 40-80% was observed at 5-25 mM NIC. IL-1 was then tested together with 1.0 and 10 mM NIC. The islet DNA content was markedly reduced in all groups treated with IL-1, as was the medium insulin accumulation. Moreover, NIC failed to prevent IL-1 induced impairment of islet insulin release on day 6. The cytokine induced a very pronounced and sustained increase in the medium nitrite accumulation, and NIC could not influence this elevation. Thus, these data show that prolonged exposure to elevated NIC levels impaired the function of rat beta-cells, and NIC failed to counteract IL-1 actions. Whether these events are mimicked in the ongoing clinical trials with NIC remain to be established.

Streptozotocin (stz) given in low doses (40 mg/kg body weight) on 5 consecutive days to susceptible strains of mice causes diabetes. Previous studies have shown that the induction of diabetes is associated with inflammatory infiltrates within the pancreatic islets. However, it is unclear whether stz causes limited beta cell destruction followed by insulitis or whether the diabetogen promotes immune cell influx into the pancreatic islets, followed by immune-mediated beta-cell destruction. It is also unclear whether stz given in sub-diabetogenic doses is capable of causing diabetes independent of cell-mediated processes. Here we have examined these possibilities in CB.17 Scid mice which lack functional T and B cells but have immunocompetent macrophages and NK cells. Low dose stz given to Scid mice caused diabetes in approximately 50% of mice of both sexes by 21 days (14/24 males; 10/18 females). Sections of pancreas were examined immunohistochemically for the presence of MAC-1 positive cells (macrophages and natural killer cells) in the exocrine, peri- and intra-islet regions at different time points following the administration of stz. There were no statistically significant differences in the number of immunoreactive cells in the three locations between tissues obtained from stz-injected mice (3, 7, 14 and 21 days after stz injection and at onset of diabetes) and buffer-injected Scid mice. Although diabetic Scid mice showed a reduced number of insulin immunoreactive cells and peri- and intra-islet distributed glucagon cells, no insulitis was seen histochemically. In parallel studies, normal Swiss male mice given stz at a similar dose developed diabetes (10/10) associated with insulitis which consisted predominantly of CD4, CD8 and MAC-1 cells. Balb/c mice given stz similarly, also developed diabetes (5/8) without showing insulitis, although a moderate increase in the number of macrophages were observed within several islets. These studies demonstrate that stz administered in multiple low doses to Scid mice can cause beta cell destruction and diabetes in the absence of immune cell infiltrate within the pancreatic islets.

Streptozotocin (STZ), a selective beta-cell cytotoxin, given in multiple low doses to susceptible mouse strains causes insulin-dependent diabetes mellitus (IDDM) with an autoimmune pathology. Studies in the human suggest that environmental factors such as viruses and certain toxins may modulate the expression of the disease in genetically-prone individuals and the effect may also be age-dependent. Here we have examined the effects of graded, low doses of STZ on beta-cell function and insulin and DNA contents in vitro in cultured islets isolated from female IDDM-prone NOD mice at 4 weeks and at 8 weeks. Results were compared with islets from age and sex-matched non-diabetes prone C57BL/Ks mice. No changes in islet DNA or insulin contents were observed after an acute 30 min exposure to STZ (0, 1.1, 2.2 and 4.4 mM) in the two strains at each of the age groups. However, the DNA content in the NOD mouse islets tended to be lower at 8 weeks, being significant at 1.1 mM STZ. At 4 weeks, islets from NOD mice had a higher insulin content than the control mice but this declined at 8 weeks when it became comparable to the control strain. STZ caused a dose-dependent inhibition of islet glucose oxidation rates in all groups. However, at 4 weeks, exposure to 2.2 mM STZ resulted in a significantly greater inhibition in NOD mice than in age-matched control mice. This was reversed at 8 weeks when the islets from NOD mice showed a greater resistance to oxidative impairment than from C57BL/Ks mice. In the presence of 16.7 mM glucose, an inhibitory pattern, similar to the glucose oxidation rate, was also observed for insulin release. In the control mice the relative inhibition of insulin release and glucose oxidation rate was similar at 4 and 8 weeks. These results suggest that islets from the NOD mouse at 4 weeks and prior to insulitis are more sensitive to STZ-induced functional impairment. This enhanced sensitivity suggests that cumulative exposure of diabetes-prone islets to low doses of selective beta cell toxins may be a determinant for later development of IDDM.

The precise role of immune cells in beta cell killing and their manner of invasion of pancreatic islets in insulin-dependent diabetes mellitus (IDDM) are unclear. We have attempted to target pancreatic islets of severe-combined immunodeficient (SCID) mice with spleen cells from diabetic and non-diabetic female non-obese diabetic (NOD) mice given i.p. or i.v. Pancreatic, liver and kidney sections of SCID mice were assessed histologically for the presence of donor cells. The presence of raised levels of serum Ig was also used as an index of engraftment of donor cells in the periphery of SCID mice. All six SCID mice which received i.v. spleen cells from normal Swiss mice died within 2 weeks from graft versus host disease (GVHD) whereas five out of nine mice survived for 30 days after i.p. injection. No deaths were recorded after i.v. or i.p. injection of spleen cells from NOD mice. Pancreatic islets of four out of six SCID recipients of diabetic and three out of five recipients of non-diabetic spleen cells following i.p. injection showed lymphocytic infiltrates in the peri-islet and perivascular regions. All SCID mice which received i.v. spleen cells from diabetic (six SCID recipients) and non-diabetic NOD mice (seven SCID recipients) showed peri-islet and perivascular infiltrates in their pancreas. Immunohistochemical analysis showed that the islet engrafted cells were of CD4 and CD8 phenotype. Donor cells were also observed in the exocrine pancreas of some recipients. A majority of mice showed various degrees of lymphocytic aggregates in the perivascular regions of the liver but not in the kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Indirect evidence suggests there may be early influx of beta-cell-directed macrophages into the islets of NOD mice, and before the onset of T-cell insulitis. We have therefore examined immunohistochemically the pancreas of young female NOD mice with monoclonal antibody F4/80 for the presence of macrophages in intraislet, peri-islet, exocrine, and perivascular regions preceding insulitis (days 18 and 22) and during early insulitis (days 30 and 40) and compared their distribution in age-matched normal Swiss mouse pancreas. In the absence of insulitis (day 18 and day 22) and during very early insulitis (day 30) macrophage-positive cells had a predominantly exocrine and perivascular distribution with reduced numbers in the peri-islet area. Intraislet macrophages usually occurred singly and were sparse. At day 40, an enhanced influx of the immune cells was observed in intra- and peri-islet locations and in parallel with increased numbers in the exocrine areas. At this age, higher numbers of macrophages were observed within the islet, distributed among the T-cell infiltrate and also scattered among the endocrine cells. During the study period, the number of macrophages in the peri-, intraislet, and exocrine regions was significantly higher among NOD mice than in the Swiss mouse group (p = 0.003, p = 0.005, and p = 0.0009, respectively). In the absence of insulitis (days 18 and 22), although the number of positive cells tended to be higher in NOD mice, this difference reached statistical significance in the peri-islet area (p = 0.004).(ABSTRACT TRUNCATED AT 250 WORDS)

The ontogenic variation of beta cell function and its relationship with the degree of islet damage and levels of autoantibodies have been studied in the non-obese diabetic (NOD) mouse model. We conducted in vivo first phase insulin release (FPIR) in response to intravenous glucose and studied its correlation with the degree of insulitis, islet cell antibody (ICA) and insulin autoantibody (IAA) levels in female NOD mice cross-sectionally at days 40 (n = 19), 90 (n = 21), 150-160 (n = 21) and day 250 (n = 20). The mean +/- SEM FPIR values showed an age-related decline from day 40 (46.2 +/- 5.3 microU/ml) to day 150-160 (17.8 +/- 2.5 microU/ml) and then doubled at day 250 (34.5 +/- 5 microU/ml), while the mean +/- SEM insulitis scores increased progressively until day 150-160 (61.7 +/- 6.1%) and then declined slightly at day 250 to 50.2 +/- 6.2%. In female NOD mice with spontaneous diabetes (n = 4) and streptozotocin-induced diabetic Swiss mice (n = 5) FPIR was either absent or greatly attenuated. A statistically significant inverse correlation between FPIR and insulitis was found among NOD mice at days 90 (P = 0.02; r = -0.52) and 150-160 (P = 0.03; r = -0.48). However, no statistically significant correlation was observed at days 40 and 250. Morphometric techniques applied to day 150-160 pancreatic sections showed a statistically significant negative correlation between insulitis and beta cell number per unit area of islet tissue (P = 0.0001; r = - 0.75). At this age some islet beta cells showed different intensities of staining by immunofluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

We have previously shown the presence of circulating islet cell cytoplasmic antibodies (ICA) and insulin autoantibodies (IAA) in the NOD mouse before onset of insulin-dependent diabetes mellitus (IDDM). Here we have determined the levels of the two autoantibodies in 28 female NOD mice longitudinally from approximately day 40 to day 250, to examine their ontogeny, association and predictive value for diabetes. All animals (11 diabetic, 17 non-diabetic) showed varying levels of ICA at some stage, while IAA activity was found in 21 out of 28 mice. Expression of both the markers was seen in more than half of the animals by day 60, with higher levels and rates occurring subsequently in both diabetic and non-diabetic groups. The expression of ICA did not always correlate with that of IAA. There was no apparent difference in the ontogeny of ICA and IAA between the two groups. During the study period the number of animals with ICA was similar in the two groups, while the number of those with IAA was higher in the diabetic animals. In this group declining and rising levels of ICA were seen just before clinical diabetes with frequent peaks of IAA. In the same animals, eight out of 11 mice showed co-expression of high levels of both markers either intermittently or persistently prior to onset, whereas only one non-diabetic animal showed this. We conclude that the ontogeny and serum level of ICA or IAA alone could not be used to predict the clinical onset of diabetes in these animals. However, co-expression of high levels of both markers prior to onset may suggest a strong predisposition to clinical diabetes. This may have relevance to attempts to predict the onset of IDDM in humans who have one or both of these immunological markers.

Antibodies to insulin, glucagon, pancreatic polypeptide hormone and somatostatin were utilized to demonstrate the cellular localization of the hormones in pancreatic tissue of fetal guinea pig of advanced gestation by immunofluorescence histochemistry. The topographical distribution of the 4 endocrine cell types was compared with those of the adult pancreas and was found to be significantly different particularly for cells immunostaining for insulin, glucagon and somatostatin. These observations suggest changes in histogenesis of pancreatic endocrine cells during transition from fetal to postnatal and adult life. The presence of the 4 islet hormones in the fetal pancreas of this species implies that they may be important in fetal metabolism and growth.

Antibodies to insulin, glucagon, pancreatic polypeptide hormone (PP) and somatostatin were used in the immunofluorescence histochemical procedure to study the ontogeny of pancreatic endocrine cells containing the four hormones in the bovine fetus of approximately 100 days gestation to term. Pancreatic sections from the bovine neonate and adult were also examined for the cellular distribution of the four hormones. Immunoreactive cells staining for insulin, glucagon, PP and somatostatin were present in the pancreas of all fetuses studied. Each endocrine cell type displayed a characteristic distribution within the developing pancreas and in the neonate and adult. The presence of the four islet hormones relatively early in bovine fetal life suggests that they may be important in intra- and extra-islet metabolism in the fetus.

We report herein a case of histologically identified peliosis of pancreatic islets in the surgically removed portion of the pancreas of a 30-year-old woman with multiple endocrine neoplasm, type 1 (MEN-1) syndrome. In addition to microscopic peliosis, the pancreas contained multiple endocrine tumors producing insulin, glucagon, somatostatin, and growth hormone-releasing factor and showed evidence of widespread nesidioblastosis. It is uncertain whether peliosis of pancreatic islets and MEN-1 syndrome were coincidental or whether the two diseases were causally related. Since hormonal factors can result in hepatic peliosis, it is tempting to speculate that the endocrine imbalance secondary to MEN-1 syndrome might have played a role in the genesis of peliosis in this case. Although no direct proof of vascular damage was encountered, it is conceivable that escape of red blood cells from the circulation and their accumulation in tissue spaces was due to abnormal islet blood flow and increased capillary permeability.

Insulin, glucagon, pancreatic polypeptide hormone (PP) and somatostatin immunoreactive cells were demonstrated in the islet of the goat pancreas by the immunofluorescence procedure. Islet cells showing immunostaining for the hormones appeared to have a characteristic distribution. The demonstration of PP and somatostatin within the pancreas of the goat suggests they may be significant in modulating intra- and extra-islet function in this ruminant species.

Antibodies to insulin, glucagon, pancreatic polypeptide (PP), and somatostatin were used in the immunofluorescence procedure to demonstrate localization of the four hormones in cells of the pancreatic islets of the brushtailed possum, Trichosurus vulpecula. Most pancreatic islets revealed some differences in the topographical distribution and cell number of each endocrine cell type. Insulin immunoreactive cells were observed in most islets where they occurred as groups of cells peripherally and within the islet. In several islets glucagon cells were the predominant cell population and were distributed peripherally as well as centrally. Pancreatic polypeptide cells were fewer in number and usually occurred as single cells within the islet. Cells immunoreactive to antisomatostatin serum were observed in varying numbers in the peripheral and central regions of the islet. The present immunofluorescence study demonstrates differences in the topographical distribution of the four major pancreatic hormones between a marsupial species and several of the eutherian mammals.

The predictive value of insulitis, islet cell cytoplasmic antibodies and insulin autoantibodies for insulin-dependent diabetes was studied in young female non-obese diabetic mice. The ontogeny of the three markers was examined cross-sectionally at days 15, 25, 40 and 90 while islet cell antibodies and insulin autoantibodies were studied longitudinally from day 35 or day 144-168 until approximately day 250. Insulitis was first observed at day 40 (50%) and subsequently at day 90 (70%). Islet cell antibodies and insulin autoantibodies were present at day 15 in 46% and 54% of the animals respectively. The rate of islet cell antibodies was slightly higher at day 25 (60%) than at day 40 (40%) and day 90 (54%) whereas antibodies to insulin were present in all samples from day 25-90. At day 40 and day 90 insulitis and insulin autoantibodies were present together in 42% and 70% of the animals, respectively, while insulitis and islet cell antibodies had a lower rate of concordance (17% and 42%, respectively; diabetes rate, 30%). The concordance rates for islet cell antibodies and insulin autoantibodies were 42% at day 40 and 54% at day 90. Concordance for all three markers was first observed at day 40 (17%) which increased to 38% at day 90. In longitudinal studies, islet cell antibodies and insulin autoantibodies were often present together whether or not diabetes supervened. In the islet cell antibody procedure, immunoreactive cells were shown immunohistochemically to correspond with insulin and/or glucagon cells. However, this staining was not suppressible with insulin- or glucagon- absorbed sera, implying the presence of non-hormonal autoantigens. We conclude that the three markers investigated are expressed early after birth and well before clinical symptoms appear in this animal model. Both islet cell antibodies and insulin autoantibodies preceded insulitis but the prevalence rate for each marker or their degree of concordance was different from the anticipated rate of diabetes in our colony. Consequently, the early expression of the three markers alone is not predictive of diabetes although concordance for the two, or all three markers may be of some value. However, no animal developed diabetes without the prior appearance of both islet cell antibodies and insulin autoantibodies.