Islet Remodeling in Female Mice with Spontaneous Autoimmune and Streptozotocin-Induced Diabetes

Islet alpha- and delta-cells are spared autoimmune destruction directed at beta-cells in type 1 diabetes resulting in an apparent increase of non-beta endocrine cells in the islet core. We determined how islet remodeling in autoimmune diabetes compares to streptozotocin (STZ)-induced diabetes. Islet cell mass, proliferation, and immune cell infiltration in pancreas sections from diabetic NOD mice and mice with STZ-induced diabetes was assessed using quantitative image analysis. Serial sections were stained for various beta-cell markers and Ngn3, typically restricted to embryonic tissue, was only upregulated in diabetic NOD mouse islets. Serum levels of insulin, glucagon and GLP-1 were measured to compare hormone levels with respect to disease state. Total pancreatic alpha-cell mass did not change as autoimmune diabetes developed in NOD mice despite the proportion of islet area comprised of alpha- and delta-cells increased. By contrast, alpha- and delta-cell mass was increased in mice with STZ-induced diabetes. Serum levels of glucagon reflected these changes in alpha-cell mass: glucagon levels remained constant in NOD mice over time but increased significantly in STZ-induced diabetes. Increased serum GLP-1 levels were found in both models of diabetes, likely due to alpha-cell expression of prohormone convertase 1/3. Alpha- or delta-cell mass in STZ-diabetic mice did not normalize by replacement of insulin via osmotic mini-pumps or islet transplantation. Hence, the inflammatory milieu in NOD mouse islets may restrict alpha-cell expansion highlighting important differences between these two diabetes models and raising the possibility that increased alpha-cell mass might contribute to the hyperglycemia observed in the STZ model.     

Histopathological Changes in Insulin,Glucagon and Somatostatin Cells in the Islets of NOD Mice During Cyclophosphamide-accelerated Diabetes: A Combined Immunohistochemical and Histochemical Study

Summary 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.     

Beta-cell apoptosis in an accelerated model of autoimmune diabetes.

BACKGROUND: The non-obese diabetic (NOD) mouse is a model of human type 1 diabetes in which autoreactive T cells mediate destruction of pancreatic islet beta cells. Although known to be triggered by cytotoxic T cells, apoptosis has not been unequivocally localized to beta cells in spontaneously diabetic NOD mice. We created a model of accelerated beta-cell destruction mediated by T cells from spontaneously diabetic NOD mice to facilitate the direct detection of apoptosis in beta cells. MATERIALS AND METHODS: NOD.scid (severe combined immunodeficiency) mice were crossed with bm1 mice transgenically expressing the costimulatory molecule B7-1 (CD80) in their beta cells, to generate B7-1 NOD.scid mice. Apoptosis in islet cells was measured as DNA strand breakage by the TdT-mediated-dUTP-nick end labeling (TUNEL) technique. RESULTS: Adoptive transfer of splenocytes from spontaneously diabetic NOD mice into B7-1 NOD.scid mice caused diabetes in recipients within 12-16 days. Mononuclear cell infiltration and apoptosis were significantly greater in the islets of B7-1 NOD.scid mice than in nontransgenic NOD.scid mice. Dual immunolabeling for TUNEL and either B-7 or insulin, or the T cell markers CD4 and CD8, and colocalization by confocal microscopy clearly demonstrated apoptosis in beta cells as well in a relatively larger number of infiltrating T cells. The clearance time of apoptotic beta cells was estimated to be less than 6 min. CONCLUSIONS: B7-1 transgenic beta cells undergo apoptosis during their accelerated destruction in response to NOD mouse effector T cells. Rapid clearance implies that beta cells undergoing apoptosis would be detected only rarely during more protracted disease in spontaneously diabetic NOD mice.     

Postnatal development of numbers and mean sizes of pancreatic islets and beta-cells in healthy mice and GIPR(dn) transgenic diabetic mice

The aim of this study was to examine postnatal islet and beta-cell expansion in healthy female control mice and its disturbances in diabetic GIPR(dn) transgenic mice, which exhibit an early reduction of beta-cell mass. Pancreata of female control and GIPR(dn) transgenic mice, aged 10, 45, 90 and 180 days were examined, using state-of-the-art quantitative-stereological methods. Total islet and beta-cell volumes, as well as their absolute numbers increased significantly until 90 days in control mice, and remained stable thereafter. The mean islet volumes of controls also increased slightly but significantly between 10 and 45 days of age, and then remained stable until 180 days. The total volume of isolated beta-cells, an indicator of islet neogenesis, and the number of proliferating (BrdU-positive) islet cells were highest in 10-day-old controls and declined significantly between 10 and 45 days. In GIPR(dn) transgenic mice, the numbers of islets and beta-cells were significantly reduced from 10 days of age onwards vs. controls, and no postnatal expansion of total islet and beta-cell volumes occurred due to a reduction in islet neogenesis whereas early islet-cell proliferation and apoptosis were unchanged as compared to control mice. Insulin secretion in response to pharmacological doses of GIP was preserved in GIPR(dn) transgenic mice, and serum insulin to pancreatic insulin content in response to GLP-1 and arginine was significantly higher in GIPR(dn) transgenic mice vs. controls. We could show that the increase in islet number is mainly responsible for expansion of islet and beta-cell mass in healthy control mice. GIPR(dn) transgenic mice show a disturbed expansion of the endocrine pancreas, due to perturbed islet neogenesis.     

Diabetes Increases the Expression of Hypothalamic Neuropeptides in a Spontaneous Model of Type I Diabetes, the Nonobese Diabetic (NOD) Mouse

1. Synthesis of oxytocin (OT) and arginine-vasopressin(AVP) is increased in induced models of Type I diabetes, such as thestreptozotocin model. However, these parameters have not yet been evaluated inspontaneous models, such as the nonobese diabetic mouse (NOD). Therefore, we studied in the magnocellular cells of the paraventricular nucleus (PVN)of nondiabetic and diabetic 16-week-old female NOD mice and control C57Bl/6mice, the immunocytochemistry of OT and AVP peptides and their mRNA expression, using nonisotopic in situ hybridization (ISH).2. In nondiabetic and diabetic NOD female mice, the number of OT- and AVP-immunoreactive cells were similar to those of the controls, whereas immunoreaction intensity was significantly higher for both peptides in diabetic NOD as compared with nondiabetic NOD and control C57Bl/6 mice.3. ISH analysis showed that the number of OT mRNA-containing cells was in the same range in the three groups, whereas higher number of AVP mRNA expressing cells was found in diabetic NOD mice. However, the intensity of hybridization signal was also higher for both OT and AVP mRNA in the diabetic group as compared with nondiabetic NOD and control mice.4. Blood chemistry demonstrated that haematrocrit, total plasma proteins, urea, sodium, and potassium were within normal limits in diabetic mice. Thus, NODmice were neither hypernatremic nor dehydrated.5. We suggest that upregulation of OT and AVP reflects a high-stress condition in the NOD mice. Diabetes may affect neuropeptide-producing cells of the PVN, with the increased AVP and OT playing a deleterious role on the outcome of the disease.     

Dual Role of VAMP8 in Regulating Insulin Exocytosis and Islet β Cell Growth

Optimal insulin secretion required to maintain glucose homeostasis is the summation of total pancreatic islet β cell mass and intrinsic secretory capacity of individual β cells, which are regulated by distinct mechanisms that could be amplified by glucagon-like-peptide-1 (GLP-1). Because of these actions of GLP-1 on islet β cells, GLP-1 has been deployed to?treat diabetes. We employed SNARE protein VAMP8-null mice to demonstrate that VAMP8 mediates insulin granule recruitment to the plasma membrane, which partly accounts for GLP-1 potentiation of glucose-stimulated insulin secretion. VAMP8-null mice also exhibited increased islet β cell mass from increased β cell mitosis, with β cell proliferative activity greatly amplified by GLP-1. Thus, despite the β cell exocytotic defect, VAMP8-null mice have an increased total insulin secretory capacity, which improved glucose homeostasis. We conclude that these VAMP8-mediated events partly underlie the therapeutic actions of GLP-1 on insulin secretion and β cell growth.     

Duodenal-jejunal bypass protects GK rats from {beta}-cell loss and aggravation of hyperglycemia and increases enteroendocrine cells coexpressing GIP and GLP-1

Dramatic improvement of type 2 diabetes is commonly observed after bariatric surgery. However, the mechanisms behind the alterations in glucose homeostasis are still elusive. We examined the effect of duodenal-jejunal bypass (DJB), which maintains the gastric volume intact while bypassing the entire duodenum and the proximal jejunum, on glycemic control, β-cell mass, islet morphology, and changes in enteroendocrine cell populations in nonobese diabetic Goto-Kakizaki (GK) rats and nondiabetic control Wistar rats. We performed DJB or sham surgery in GK and Wistar rats. Blood glucose levels and glucose tolerance were monitored, and the plasma insulin, glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) levels were measured. β-Cell area, islet fibrosis, intestinal morphology, and the density of enteroendocrine cells expressing GLP-1 and/or GIP were quantified. Improved postprandial glycemia was observed from 3 mo after DJB in diabetic GK rats, persisting until 12 mo after surgery. Compared with the sham-GK rats, the DJB-GK rats had an increased β-cell area and a decreased islet fibrosis, increased insulin secretion with increased GLP-1 secretion in response to a mixed meal, and an increased population of cells coexpressing GIP and GLP-1 in the jejunum anastomosed to the stomach. In contrast, DJB impaired glucose tolerance in nondiabetic Wistar rats. In conclusion, although DJB worsens glucose homeostasis in normal nondiabetic Wistar rats, it can prevent long-term aggravation of glucose homeostasis in diabetic GK rats in association with changes in intestinal enteroendocrine cell populations, increased GLP-1 production, and reduced β-cell deterioration.     

Polyploidy and chromatin remodeling in hepatocytes from insulin-dependent diabetic and normoglycemic aged mice

Changes in polyploidization, chromatin supraorganization, and chromatin accessibility were investigated in hepatocytes collected from adult, nonobese diabetic (NOD) mice with increasing hyperglycemia and compared with adult normoglycemic controls and 56-week-old normoglycemic BALB/c mice. Our goal was to determine the changes in ploidy degrees and chromatin characteristics in mouse hepatocytes that are associated with insulin-dependent diabetes and to detect similarities in these aspects with those verified with aging, with greater accuracy than previous studies. Image analysis of Feulgen-stained nuclei revealed changes in ploidy degrees and chromatin supraorganization. Chromatin accessibility was assessed with micrococcal nuclease (MNase) digestion. Increased polyploidy was associated with increasing levels of glycemia, and this trend toward polyploidy was found even under normoglycemic conditions in NOD mice. Although high degrees of ploidy were also detected in aged BALB/c mice, the magnitude of polyploidy was not the same magnitude as that in the diabetic mice. While there was increased homogeneity of chromatin packaging with increasing polyploidy under conditions of severe hyperglycemia (and even under conditions of normoglycemia) in NOD mice, an inverse relationship was observed in aged BALB/c mice. Chromatin accessibility to MNase increased under severe hyperglycemia and advanced age, but it was much higher in the diabetic mice. In conclusion, although similarities in polyploidy were observed between the hepatocytes from increasingly hyperglycemic adult mice and those from normoglycemic aged mice, the relationship between chromatin remodeling and increases in ploidy degrees was not the same between the hepatocytes of these two groups. These findings demonstrate that strict similarities between diabetes and aging are not always true at the cellular level. This discordance is likely due to differences in the metabolic state of mouse hepatocytes during aging and diabetic conditions consequent to specificities in their gene regulatory programs. ? 2012 International Society for Advancement of Cytometry.     

The Non-Immune RIP-kb Mouse is a Useful Host for Islet Transplantation,as the Diabetes is Spontaneous,Mild and Predictable

Chemically-induced diabetic mice and spontaneously diabetic NOD mice have been valuable as recipients for experimental islet transplantation. However, their maintenance often requires parenteral insulin. Diabetogenic chemicals can be cytotoxic to the host’s immune system and to other organs some of which are often used as the transplant site. Procurement of diabetic cohorts in the NOD mouse is problematic due to variability in the age of disease onset. We show that RIP-K^b mice, which spontaneously develop non-immune diabetes due to over-expression of the H-2K^b heavy chain in beta cells, offer many advantages as islet transplant recipients. Diabetes is predictable with a relatively narrow range of onset (4 wk) and blood glucose levels (23.0± 4.0 mmol/l for 39 males at 6 weeks of age). The diabetes is mild enough so that most diabetic mice can be maintained to 40 weeks of age without parenteral insulin. This consistency of diabetes avails that outcomes of intervention can be interpreted with confidence.     

Alterations in oviductal cilia morphology and reduced expression of axonemal dynein in diabetic NOD mice

In the present study, we examined the morphology of cilia and expression of the dynein intermediate chain 2 (DNAI2) in the oviduct of non-obese diabetic (NOD) mice. Results obtained with immunohistochemistry showed that DNAI2 expression was reduced in oviducts of diabetic NOD (dNOD) mice, as compared to that observed in the normoglycemic NOD (cNOD) group, especially in the acyclic dNOD mice. Oviductal cilia of dNOD mice appeared to be reduced in number. Results obtained with Western blot analysis revealed that the expression of DNAI2 protein was significantly less in oviducts of dNOD mice as compared to that of cNOD mice corroborating the results obtained with immunohistochemistry. Electron microscopic examination and quantitative imaging of thin sections of Epon-embedded oviducts of both dNOD and cNOD mice confirmed the reduction of the number of cilia in the oviduct of the dNOD group which also displayed aberrant axonemal ultrastructure, including disorganization of the axoneme and alteration of microtubule doublets into singlets as well as disruption of the plasma membrane in many cilia. Taken together, the present findings suggest that structural alterations of oviductal cilia in female diabetic NOD mice might be detrimental to the normal function of these particular cell structures in gamete transport.     

Immunohistochemical Demonstration of Leptin in Pancreatic Islets of Non-Obese Diabetic and CD-1 mice: Co-localization in Glucagon Cells and its Attenuation at the Onset of Diabetes

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.     

Islet allograft rejection in nonobese diabetic mice involves the common gamma-chain and CD28/CD154-dependent and -independent mechanisms

Once nonobese diabetic (NOD) mice become diabetic, they are highly resistant to islet transplantation. The precise mechanism of such resistance remains largely unknown. In the present study we tested the hypothesis that islet allograft survival in the diabetic NOD mouse is determined by the interplay of diverse islet-specific T cell subsets whose activation is regulated by CD28/CD154 costimulatory signals and the common gamma-chain (gammac; a shared signaling element by receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21). We found that common gammac blockade is remarkably effective in blocking the onset and the ongoing autoimmune diabetes, whereas CD28/CD154 blockade has no effect in suppressing the ongoing diabetes. However, CD28/CD154 blockade completely blocks the alloimmune-mediated islet rejection. Also, a subset of memory-like T cells in the NOD mice is resistant to CD28/CD154 blockade, but is sensitive to the common gammac blockade. Nonetheless, neither common gammac blockade nor CD28/CD154 blockade can prevent islet allograft rejection in diabetic NOD mice. Treatment of diabetic NOD recipients with CD28/CD154 blockade plus gammac blockade markedly prolongs islet allograft survival compared with the controls. However, allograft tolerance is not achieved, and all CTLA-4Ig-, anti-CD154-, and anti-gammac-treated diabetic NOD mice eventually rejected the islet allografts. We concluded that the effector mechanisms in diabetic NOD hosts are inherently complex, and rejection in this model involves CD28/CD154/gammac-dependent and -independent mechanisms.     

Dendritic Cells Transduced to Express Interleukin 4 Reduce Diabetes Onset in Both Normoglycemic and Prediabetic Nonobese Diabetic Mice

Background

We and others have previously demonstrated that treatment with bone marrow derived DC genetically modified to express IL-4 reduce disease pathology in mouse models of collagen-induced arthritis and delayed-type hypersensitivity. Moreover, treatment of normoglycemic NOD mice with bone marrow derived DC, genetically modified to express interleukin 4 (IL-4), reduces the onset of hyperglycemia in a significant number of animals. However, the mechanism(s) through which DC expressing IL-4 function to prevent autoimmune diabetes and whether this treatment can reverse disease in pre-diabetic NOD mice are unknown.

Methodology/Principal Findings

DC were generated from the bone marrow of NOD mice and transduced with adenoviral vectors encoding soluble murine IL-4 (DC/sIL-4), a membrane-bound IL-4 construct, or empty vector control. Female NOD mice were segregated into normoglycemic (<150mg/dL) and prediabetic groups (between 150 and 250 mg/dL) on the basis of blood glucose measurements, and randomized for adoptive transfer of 10⁶ DC via a single i.v. injection. A single injection of DC/sIL-4, when administered to normoglycemic 12-week old NOD mice, significantly reduced the number of mice that developed diabetes. Furthermore, DC/sIL-4, but not control DC, decreased the number of mice progressing to diabetes when given to prediabetic NOD mice 12–16 weeks of age. DC/sIL-4 treatment also significantly reduced islet mononuclear infiltration and increased the expression of FoxP3 in the pancreatic lymph nodes of a subset of treated animals. Furthermore, DC/sIL-4 treatment altered the antigen-specific Th2:Th1 cytokine profiles as determined by ELISPOT of splenocytes in treated animals.

Conclusions

Adoptive transfer of DC transduced to express IL-4 into both normoglycemic and prediabetic NOD mice is an effective treatment for T1D.     

Molecular mechanisms for gender differences in susceptibility to T cell-mediated autoimmune diabetes in nonobese diabetic mice

Nonobese diabetic (NOD) mice spontaneously develop diabetes with a strong female prevalence; however, the mechanisms for this gender difference in susceptibility to T cell-mediated autoimmune diabetes are poorly understood. This investigation was initiated to find mechanisms by which sex hormones might affect the development of autoimmune diabetes in NOD mice. We examined the expression of IFN-gamma, a characteristic Th1 cytokine, and IL-4, a characteristic Th2 cytokine, in islet infiltrates of female and male NOD mice at various ages. We found that the most significant difference in cytokine production between sexes was during the early stages of insulitis at 4 wk of age. IFN-gamma was significantly higher in young females, whereas IL-4 was higher in young males. CD4(+) T cells isolated from lymph nodes of female mice and activated with anti-CD3 and anti-CD28 Abs produced more IFN-gamma, but less IL-4, as compared with males. Treatment of CD4(+) T cells with estrogen significantly increased, whereas testosterone treatment decreased the IL-12-induced production of IFN-gamma. We then examined whether the change in IL-12-induced IFN-gamma production by treatment with sex hormones was due to the regulation of STAT4 activation. We found that estrogen treatment increased the phosphorylation of STAT4 in IL-12-stimulated T cells. We conclude that the increased susceptibility of female NOD mice to the development of autoimmune diabetes could be due to the enhancement of the Th1 immune response through the increase of IL-12-induced STAT4 activation by estrogen.     

I-Ag7-mediated antigen presentation by B lymphocytes is critical in overcoming a checkpoint in T cell tolerance to islet beta cells of nonobese diabetic mice.

B cell-deficient nonobese diabetic (NOD) mice are protected from the development of spontaneous autoimmune diabetes, suggesting a requisite role for Ag presentation by B lymphocytes for the activation of a diabetogenic T cell repertoire. This study specifically examines the importance of B cell-mediated MHC class II Ag presentation as a regulator of peripheral T cell tolerance to islet beta cells. We describe the construction of NOD mice with an I-Ag7 deficiency confined to the B cell compartment. Analysis of these mice, termed NOD BCIID, revealed the presence of functionally competent non-B cell APCs (macrophages/dendritic cells) with normal I-Ag7 expression and capable of activating Ag-reactive T cells. In addition, the secondary lymphoid organs of these mice harbored phenotypically normal CD4+ and CD8+ T cell compartments. Interestingly, whereas control NOD mice harboring I-Ag7-sufficient B cells developed diabetes spontaneously, NOD BCIID mice were resistant to the development of autoimmune diabetes. Despite their diabetes resistance, histologic examination of pancreata from NOD BCIID mice revealed foci of noninvasive peri-insulitis that could be intentionally converted into a destructive process upon treatment with cyclophosphamide. We conclude that I-Ag7-mediated Ag presentation by B cells serves to overcome a checkpoint in T cell tolerance to islet beta cells after their initial targeting has occurred. Overall, this work indicates that the full expression of the autoimmune potential of anti-islet T cells in NOD mice is intimately regulated by B cell-mediated MHC class II Ag presentation.     

Combination of an Antigen-Specific Therapy and an Immunomodulatory Treatment to Simultaneous Block Recurrent Autoimmunity and Alloreactivity in Non-Obese Diabetic Mice

Restoration of endogenous insulin production by islet transplantation is considered a curative option for patients with type 1 diabetes. However, recurrent autoimmunity and alloreactivity cause graft rejection hindering successful transplantation. Here we tested whether transplant tolerance to allogeneic islets could be achieved in non-obese diabetic (NOD) mice by simultaneously tackling autoimmunity via antigen-specific immunization, and alloreactivity via granulocyte colony stimulating factor (G-CSF) and rapamycin (RAPA) treatment. Immunization with insB_9-23 peptide alone or in combination with two islet peptides (IGRP_206-214 and GAD_524-543) in incomplete Freund’s adjuvant (IFA) were tested for promoting syngeneic pancreatic islet engraftment in spontaneously diabetic NOD mice. Treatment with G-CSF/RAPA alone or in combination with insB_9-23/IFA was examined for promoting allogeneic islet engraftment in the same mouse model. InsB_9-23/IFA immunization significantly prolonged syngeneic pancreatic islet survival in NOD mice by a mechanism that necessitated the presence of CD4⁺CD25⁺ T regulatory (Treg) cells, while combination of three islet epitopes was less efficacious in controlling recurrent autoimmunity. G-CSF/RAPA treatment was unable to reverse T1D or control recurrent autoimmunity but significantly prolonged islet allograft survival in NOD mice. Blockade of interleukin-10 (IL-10) during G-CSF/RAPA treatment resulted in allograft rejection suggesting that IL-10-producing cells were fundamental to achieve transplant tolerance. G-CSF/RAPA treatment combined with insB_9-23/IFA did not further increase the survival of allogeneic islets. Thus, insB_9-23/IFA immunization controls recurrent autoimmunity and G-CSF/RAPA treatment limits alloreactivity, however their combination does not further promote allogeneic pancreatic islet engraftment in NOD mice.     

Reduced neutrophil apoptosis in diabetic mice during staphylococcal infection leads to prolonged Tnfα production and reduced neutrophil clearance

Diabetes is a frequent underlying medical condition among individuals with Staphylococcus aureus infections, and diabetic patients often suffer from chronic inflammation and prolonged infections. Neutrophils are the most abundant inflammatory cells during the early stages of bacterial diseases, and previous studies have reported deficiencies in neutrophil function in diabetic hosts. We challenged age-matched hyperglycemic and normoglycemic NOD mice intraperitoneally with S. aureus and evaluated the fate of neutrophils recruited to the peritoneal cavity. Neutrophils were more abundant in the peritoneal fluids of infected diabetic mice by 48 h after bacterial inoculation, and they showed prolonged viability ex vivo compared to neutrophils from infected nondiabetic mice. These differences correlated with reduced apoptosis of neutrophils from diabetic mice and were dependent upon the presence of S. aureus and a functional neutrophil respiratory burst. Decreased apoptosis correlated with impaired clearance of neutrophils by macrophages both in vitro and in vivo and prolonged production of proinflammatory tumor necrosis factor alpha by neutrophils from diabetic mice. Our results suggest that defects in neutrophil apoptosis may contribute to the chronic inflammation and the inability to clear staphylococcal infections observed in diabetic patients.     

Pancreatic somatostatin contents in spontaneously diabetic KK and non-obese diabetic (NOD) mice

Alterations in the somatostatin (SRIF)-, insulin- and glucagon-containing cells were examined in two strains of spontaneously diabetic mice, KK and newly inbred non-obese diabetic (NOD) mice, using radioimmunoassay and immunohistochemical methods. The total pancreatic content and concentration of SRIF was decreased in male KK mice compared to their male controls aged 12-18 weeks. These results were consistent with the immunohistochemical findings. Pancreatic glucagon concentration and number of glucagon-containing cells were also decreased in KK mice, but pancreatic insulin concentrations were increased in KK mice. On the other hand, NOD mice aged 12-38 weeks within 15 days after onset of diabetes had increased concentrations of pancreatic SRIF. The pancreatic islets in NOD mice were decreased both in number and in size and were characterized by lymphocyte infiltration. SRIF-containing cells occupied the major part of the endocrine cells of the islets. Insulin-containing cells significantly decreased in number, but the number of glucagon-containing cells was fairly well preserved. These results and previous work concerning obob and dbdb mice indicate a parallel relationship between pancreatic SRIF and glucagon. The pancreatic glucagon thus as well as the pancreatic insulin may be an important determinant of pancreatic SRIF concentration in these diabetic animals.