The Dual Role of Scavenger Receptor Class A in Development of Diabetes in Autoimmune NOD Mice

Human type 1 diabetes is an autoimmune disease that results from the autoreactive destruction of pancreatic β cells by T cells. Antigen presenting cells including dendritic cells and macrophages are required to activate and suppress antigen-specific T cells. It has been suggested that antigen uptake from live cells by dendritic cells via scavenger receptor class A (SR-A) may be important. However, the role of SR-A in autoimmune disease is unknown. In this study, SR-A^−/− nonobese diabetic (NOD) mice showed significant attenuation of insulitis, lower levels of insulin autoantibodies, and suppression of diabetes development compared with NOD mice. We also found that diabetes progression in SR-A^−/− NOD mice treated with low-dose polyinosinic-polycytidylic acid (poly(I∶C)) was significantly accelerated compared with that in disease-resistant NOD mice treated with low-dose poly(I∶C). In addition, injection of high-dose poly(I∶C) to mimic an acute RNA virus infection significantly accelerated diabetes development in young SR-A^−/− NOD mice compared with untreated SR-A^−/− NOD mice. Pathogenic cells including CD4⁺CD25⁺ activated T cells were increased more in SR-A^−/− NOD mice treated with poly(I∶C) than in untreated SR-A^−/− NOD mice. These results suggested that viral infection might accelerate diabetes development even in diabetes-resistant subjects. In conclusion, our studies demonstrated that diabetes progression was suppressed in SR-A^−/− NOD mice and that acceleration of diabetes development could be induced in young mice by poly(I∶C) treatment even in SR-A^−/− NOD mice. These results suggest that SR-A on antigen presenting cells such as dendritic cells may play an unfavorable role in the steady state and a protective role in a mild infection. Our findings imply that SR-A may be an important target for improving therapeutic strategies for type 1 diabetes.     

An Immunohistochemical Study of Macrophage Influx and the Co-localization of Inducible Nitric Oxide Synthase in the Pancreas of Non-obese Diabetic (NOD) Mice During Disease Acceleration with Cyclophosphamide

Cyclophosphamide has been used to accelerate and synchronize diabetes in non-obese diabetic (NOD) mice. It was injected to 70-day-old female NOD mice and its effect on the progression of insulitis studied at days 0, 4, 7, 11 and at onset of diabetes. Pancreatic sections were also examined for the influx of CD4 and CD8 T cells and macrophages following immunofluorescence staining. The kinetics of macrophage immunoreactive cells in the exocrine and intra-islet areas were also investigated. Light and confocal microscopy were employed to examine the expression and co-localization of inducible nitric oxide synthase following dual- and triple-label immunofluorescence histochemistry. After cyclophosphamide administration, the severity of insulitis remained similar from days 0 to 4 but began to rise at day 7 and markedly by day 11 and at onset of diabetes. At these two later stages, the insulitis scores were close to 100% while in age-matched control groups the insulitis scores were considerably lower. Immunohistochemical staining showed increasing numbers of CD4 and CD8 T cell subsets and macrophages within the islets and in exocrine, sinusoidal and peri-vascular regions. At onset of diabetes, several islets contained prominent clusters of macrophage immunoreactive cells. Macrophage influx into the islets increased sharply from day 7 (mean number per islet: 119±54 SEM), peaked at day 11 (mean number per islet: 228±42), and then declined at onset of diabetes (mean number per islet: 148±49). Several cells with immunolabelling for inducible nitric oxide synthase were detectable from day 7 onwards until the onset of diabetes. Dual- and triple-label immunohistochemistry showed that a significant proportion of macrophages and only a few beta cells contained the enzyme. Macrophages positive for the enzyme were located as clusters or occasionally contiguously, in the peri-islet and intra-islet areas but rarely in the exocrine region. Islets with minimal distribution of macrophages in the peri-islet areas were not positive for inducible nitric oxide synthase. Beta cells positive for the enzyme were observed in islets with significant macrophage infiltration in locations close to macrophages. The present results show that cyclophosphamide administration to female NOD mice results in a rapid influx of CD4 and CD8 cells and macrophages. The marked up-regulation of inducible nitric oxide synthase in a selective proportion of macrophages, within the islets, immediately preceding and during the onset of diabetes suggests that nitric oxide released by islet macrophages may be an important molecular mediator of beta cell destruction in this accelerated model of insulin-dependent diabetes mellitus.     

Histological study of pancreatic beta-cell loss in relation to the insulitis process in the non-obese diabetic mouse

Non-obese diabetic (NOD) mice spontaneously develop an autoimmune diabetes with higher incidence in females than in males. In order to elucidate possible factors involved in the different incidence of diabetes between male and female mice, we studied the progression of pancreatic beta-cell loss in relation to mononuclear cell infiltration of the pancreas (insulitis). We examined the pancreas of 76 NOD mice (39 males and 37 females) of different ages. The beta-cell content was assessed by immunoperoxidase staining of sections with an anti-insulin serum and the severity of insulitis was determined by haematoxylin staining of the same sections. A semi-quantitative criterion was used to grade both parameters. The results showed that females have a faster loss of beta-cell mass, which progressively decreases with the increase of severity of insulitis. In males, a medium to severe degree of insulitis is required before initial loss of beta cells occurs. Under the age of 10 weeks there was a significantly lower content of beta cells in females than males (2.84±0.03 vs 2.67±0.07; P=0.02). Since we never observed a significant difference in the degree of mononuclear cell infiltration in age-matched males and females, these data support the hypothesis of weaker beta-cell resistance to immunological attack in female mice. Thus beta-cell sensitivity, in addition to immunological activity, is an important factor in the pathogenesis of insulin dependent diabetes.     

Protective role for cytosolic phospholipase A2alpha in autoimmune diabetes of mice

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) plays an important role in arachidonate pathway. To investigate the contribution of cPLA(2)alpha to autoimmune diabetes, we established non-obese diabetic (NOD) mouse, an excellent model for human type 1 diabetes, deficient in cPLA(2)alpha. These mice showed severe insulitis and a higher incidence of diabetes. In their macrophages, decreased prostaglandin E(2) (PGE(2)) induced by cPLA(2)alpha deficiency, and the increase in production of tumor necrosis factor (TNF)-alpha were observed. These results suggested that cPLA(2)alpha plays a protective role in progression of insulitis and development of autoimmune diabetes by suppression of TNF-alpha production from macrophages.     

Preventive effect of Ninjin-to (Ren-Shen-Tang), a Kampo (Japanese traditional) formulation, on spontaneous autoimmune diabetes in non-obese diabetic (NOD) mice

We previously found that ingestion of an extract of Ninjin-to (NJT; Ren-Shen-Tang) suppressed the development of autoimmune diabetes in C57BL/KsJ mice induced by multiple low doses of streptozotocin. To verify this effects on spontaneous autoimmune diabetes, the effects of NJT on NOD mice were investigated in the present study. NJT, provided in drinking water (0.25%, 450 mg/kg/day) from 6 weeks of age, significantly prevented the incidence of spontaneous diabetes in female NOD mice at 30 weeks of age (2/10) compared with that of the controls (7/10), with no effects on body growth or food intake. Even in non-diabetic mice, the blood glucose levels of the NOD controls gradually increased with age, while such increase in NJT-treated mice was significantly suppressed by preventing any deficiency of glucose tolerance. NJT also significantly suppressed the progression of insulitis, which causes insulin deficiency and diabetes. It is well known that NOD mice develop insulitis and diabetes because of their Th1-dominant autoimmune response. IFN-gamma production from splenic T lymphocytes stimulated with anti-CD3 monoclonal antibodies was increased, whereas IL-4 production was decreased in NOD controls compared to age- and sex-matched normal ICR mice. NJT-treatment reduced these deviations of cytokine production in NOD mice. These data all suggest that NJT can prevent spontaneous insulitis and diabetes by the modification of deviated cytokine production in NOD mice.     

The Gut Microbiota Modulates Glycaemic Control and Serum Metabolite Profiles in Non-Obese Diabetic Mice

Islet autoimmunity in children who later progress to type 1 diabetes is preceded by dysregulated serum metabolite profiles, but the origin of these metabolic changes is unknown. The gut microbiota affects host metabolism and changes in its composition contribute to several immune-mediated diseases; however, it is not known whether the gut microbiota is involved in the early metabolic disturbances in progression to type 1 diabetes. We rederived non-obese diabetic (NOD) mice as germ free to explore the potential role of the gut microbiota in the development of diabetic autoimmunity and to directly investigate whether the metabolic profiles associated with the development of type 1 diabetes can be modulated by the gut microbiota. The absence of a gut microbiota in NOD mice did not affect the overall diabetes incidence but resulted in increased insulitis and levels of interferon gamma and interleukin 12; these changes were counterbalanced by improved peripheral glucose metabolism. Furthermore, we observed a markedly increased variation in blood glucose levels in the absence of a microbiota in NOD mice that did not progress to diabetes. Additionally, germ-free NOD mice had a metabolite profile similar to that of pre-diabetic children. Our data suggest that germ-free NOD mice have reduced glycaemic control and dysregulated immunologic and metabolic responses.     

Presence of residual beta cells and co-existing islet autoimmunity in the NOD mouse during longstanding diabetes: a combined histochemical and immunohistochemical study

During type 1 diabetes, most beta cells die by immune processes. However, the precise fate and characteristics of beta cells and islet autoimmunity after onset are unclear. Here, the extent of beta cell survival was determined in the non-obese diabetic (NOD) mouse during increasing duration of disease and correlated with insulitis. Pancreata from female NOD mice at diagnosis and at 1, 2, 3 and 4 weeks thereafter were analysed immunohistochemically for insulin, glucagon and somatostatin cells and glucose transporter-2 (glut2) and correlated with the degree of insulitis and islet immune cell phenotypes. Insulitis, although variable, persisted after diabetes and declined with increasing duration of disease. During this period, beta cells also declined sharply whereas glucagon and somatostatin cells increased, with occasional islet cells co-expressing insulin and glucagon. Glut2 was absent in insulin-containing cells from 1 week onwards. CD4 and CD8 T cells and macrophages persisted until 4 weeks, in islets with residual beta cells or extensive insulitis. We conclude that after diabetes onset, some beta cells survive for extended periods, with continuing autoimmunity and expansion of glucagon and somatostatin cells. The absence of glut2 in several insulin-positive cells suggests that some beta cells may be unresponsive to glucose.     

The countervailing actions of myeloid and plasmacytoid dendritic cells control autoimmune diabetes in the nonobese diabetic mouse

Islet Ag-specific CD4(+) T cells receive antigenic stimulation from MHC class II-expressing APCs. Herein, we delineate the direct in vivo necessity for distinct subsets of macrophages and dendritic cells (DC) in type 1 diabetes mellitus of the NOD mouse by using diphtheria toxin-mediated cell ablation. The ablation of macrophages had no impact on islet Ag presentation or on the induction of insulitis or diabetes in either transfer or spontaneous models. However, the ablation of CD11b(+)CD11c(+) DC led to the loss of T cell activation, insulitis, and diabetes mediated by CD4(+) T cells. When the specific myeloid DC subset was "added-back" to mice lacking total DC, insulitis and diabetes were restored. Interestingly, when NOD mice were allowed to progress to the insulitis phase, the ablation of DC led to accelerated insulitis. This accelerated insulitis was mediated by the loss of plasmacytoid DC (pDC). When pDC were returned to depleted mice, the localized regulation of insulitis was restored. The loss of pDC in the pancreas itself was accompanied by the localized loss of IDO and the acceleration of insulitis. Thus, CD11c(+)CD11b(+) DC and pDC have countervailing actions in NOD diabetes, with myeloid DC providing critical antigenic stimulation to naive CD4(+) T cells and pDC providing regulatory control of CD4(+) T cell function in the target tissue.     

Combined insulin B:9-23 self-peptide and polyinosinic-polycytidylic acid accelerate insulitis but inhibit development of diabetes by increasing the proportion of CD4+Foxp3+ regulatory T cells in the islets in non-obese diabetic mice

Insulin peptide B:9-23 is a major autoantigen in type 1 diabetes. Combined treatment with B:9-23 peptide and polyinosinic-polycytidylic acid (poly I:C), but neither alone, induce insulitis in normal BALB/c mice. In contrast, the combined treatment accelerated insulitis, but prevented diabetes in NOD mice. Our immunofluorescence study with anti-CD4/anti-Foxp3 revealed that the proportion of Foxp3 positive CD4⁺CD25⁺ regulatory T cells (Tregs) was elevated in the islets of NOD mice treated with B:9-23 peptide and poly I:C, as compared to non-treated mice. Depletion of Tregs by anti-CD25 antibody hastened spontaneous development of diabetes in non-treated NOD mice, and abolished the protective effect of the combined treatment and conversely accelerated the onset of diabetes in the treated mice. These results indicate that poly I:C combined with B:9-23 peptide promotes infiltration of both pathogenic T cells and predominantly Tregs into the islets, thereby inhibiting progression from insulitis to overt diabetes in NOD mice.     

Differential expression of CC chemokines and the CCR5 receptor in the pancreas is associated with progression to type I diabetes

We investigated the biological role of CC chemokines in the Th1-mediated pathogenesis of spontaneous type I diabetes in nonobese diabetic (NOD) mice. Whereas an elevated ratio of macrophage inflammatory protein-1alpha (MIP-1alpha):MIP-1beta in the pancreas correlated with destructive insulitis and progression to diabetes in NOD mice, a decreased intrapancreatic MIP-1alpha:MIP-1beta ratio was observed in nonobese diabetes-resistant (NOR) mice. IL-4 treatment, which prevents diabetes in NOD mice by polarizing intraislet Th2 responses, decreased CCR5 expression in islets and potentiated a high ratio of MIP-1beta and monocyte chemotactic protein-1 (MCP-1): MIP-1alpha in the pancreas. Furthermore, NOD.MIP-1alpha-/- mice exhibited reduced destructive insulitis and were protected from diabetes. Neutralization of MIP-1alpha with specific Abs following transfer of diabetogenic T cells delayed the onset of diabetes in NOD.Scid recipients. These studies illustrate that the temporal expression of certain CC chemokines, particularly MIP-1alpha, and the CCR5 chemokine receptor in the pancreas is associated with the development of insulitis and spontaneous type I diabetes.     

Temporal relationship between immune cell influx and the expression of inducible nitric oxide synthase, interleukin-4 and interferon-gamma in pancreatic islets of NOD mice following adoptive transfer of diabetic spleen cells

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- 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- 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- 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- and interleukin-4. Expression of these molecules becomes more pronounced immediately prior to and during the onset of diabetes.     

Induction of glutamic acid decarboxylase 65-specific Th2 cells and suppression of autoimmune diabetes at late stages of disease is epitope dependent.

Peptide-based immunotherapy is one strategy by which to selectively suppress the T cell-mediated destruction of beta cells and treat insulin-dependent diabetes mellitus (IDDM). Here, we investigated whether a panel of T cell epitopes derived from the beta cell autoantigen glutamic acid decarboxylase 65 (GAD65) differ in their capacity to induce Th2 cell function in nonobese diabetic (NOD) mice and in turn prevent overt IDDM at different preclinical stages of disease development. The panel consists of GAD65-specific peptides spanning aa 217-236 (p217), 247-265 (p247), 290-309 (p290), and 524-543 (p524). Our studies revealed that all of the peptides effectively prevented insulitis and diabetes when administered to NOD mice before the onset of insulitis. In contrast, only a mixture of p217 and p290 prevented progression of insulitis and overt IDDM in NOD mice exhibiting extensive beta cell autoimmunity. Immunization with the GAD65-specific peptides did not block IDDM development in NOD mice deficient in IL-4 expression. These findings demonstrate that GAD65-specific peptide immunotherapy effectively suppresses progression to overt IDDM, requires the production of IL-4, and is dependent on the epitope targeted and the extent of preexisting beta cell autoimmunity in the recipient.     

An autoimmune diabetes locus (Idd21) on mouse Chromosome 18

Twenty-four named Idd loci that contribute to the development of autoimmune diabetes in the nonobese diabetic (NOD) mouse have been mapped by linkage and congenic analysis. Previously, meta-analysis of genome-wide linkage scans supported the existence of a locus for susceptibility to autoimmune phenotypes on rodent Chromosome (Chr) 18, in a position orthologous to the human type 1 diabetes susceptibility locus IDDM6 (human Chr 18q12-q23). However, an autoimmune diabetes susceptibility locus has not previously been reported on mouse Chr 18. In this study, we demonstrate linkage of the majority of mouse Chr 18 to diabetes in a (ABH × NOD)F₁ × NOD backcross. Congenic analysis, introgressing at least 92% of Biozzi ABH Chr 18 onto the NOD background, confirmed the presence of a diabetes locus. The chromosome substitution strain (NOD.ABH-Chr18) had reduced diabetes incidence compared with NOD mice (P < 0.0001). We have named the Chr 18 diabetes locus Idd21.     

B lymphocytes not required for progression from insulitis to diabetes in non-obese diabetic mice

Previous studies have implicated B lymphocytes in the pathogenesis of diabetes in the non-obese diabetic (NOD) mouse. While it is clear that B lymphocytes are necessary, it has not been clear at which stage of disease they play a role; early, late or both. To clarify when B lymphocytes are needed, T lymphocytes were transferred from 5-week-old NOD female mice to age-matched NOD/severe combined immunodeficiency (SCID) recipient mice. NOD/SCID mice, which lack functionally mature T and B lymphocytes, do not normally develop insulitis or insulin-dependent diabetes melitus (IDDM). The NOD/SCID mice that received purified T lymphocytes from 5-week-old NOD mice subsequently developed insulitis and diabetes even though they did not have detectable B lymphocytes. This suggests that while B lymphocytes may be essential for an initial priming event they are not requisite for disease progression in the NOD mouse.     

Contribution of different mechanisms to pancreatic beta-cell hyper-secretion in non-obese diabetic (NOD) mice during pre-diabetes

The development of insulin-dependent diabetes mellitus (IDDM) results from the selective destruction of pancreatic beta-cells. Both humans and spontaneous models of IDDM, such as NOD mice, have an extended pre-diabetic stage. Dynamic changes in beta-cell mass and function during pre-diabetes, such as insulin hyper-secretion, remain largely unknown. In this paper, we evaluated pre-diabetic female NOD mice at different ages (6, 10, and 14 weeks old) to illustrate alterations in beta-cell mass and function as disease progressed. We found an increase in beta-cell mass in 6-week-old NOD mice that may account for improved glucose tolerance in these mice. As NOD mice aged, beta-cell mass progressively reduced with increasing insulitis. In parallel, secretory ability of individual beta-cells was enhanced due to an increase in the size of slowly releasable pool (SRP) of vesicles. Moreover, expression of both SERCA2 and SERCA3 genes were progressively down-regulated, which facilitated depolarization-evoked secretion by prolonging Ca(2+) elevation upon glucose stimulation. In summary, we propose that different mechanisms contribute to the insulin hyper-secretion at different ages of pre-diabetic NOD mice, which may provide some new ideas concerning the progression and management of type I diabetes.     

Possible mechanism of the preventive effect of BCG against diabetes mellitus in NOD mouse. II. Suppression of pathogenesis by macrophage transfer from BCG-vaccinated mice.

Our previous reports showed that a single injection of live BCG, one of the biological response modifiers, prevents insulitis and overt diabetes in NOD mice and that the suppression could be due to the generation of some type of suppressor cells in the BCG-treated mice. Furthermore, a more recent study has revealed that macrophages suppressive against a variety of lymphocyte functions can be induced by BCG, which suggests that these macrophages are involved in the suppression of the pathogenesis. To obtain valid evidence for this speculation, the effects of transfer of macrophage and T-cell fractions on the pathogenesis were examined in the present study. Transfer of macrophage-enriched spleen cell fraction harvested from the BCG-treated females to young females abolished the occurrence of spontaneous diabetes up to the age of 25 to 30 weeks. Also, macrophage transfer prevented the progress of insulitis. In contrast, transfer of a T-cell-enriched fraction did not suppress insulitis and overt diabetes. From these results, it could be concluded that the suppression of the autoimmune pathogenesis of diabetes by BCG is due to the generation of suppressor macrophages.     

Immunoregulation during disease progression in prediabetic NOD mice: inverse expression of arginase and prostaglandin H synthase 2 vs. interleukin-15.

Non-obese diabetic (NOD) mice spontaneously develop insulin dependent diabetes due to autoimmune destruction of beta-cells. The progression of insulitis can be accelerated and synchronized in the pancreas by a single injection of 250 mg/kg cyclophosphamide. In this study, we will report on three immune mediators that were not known to be expressed during insulitis until now. Early insulitis in ten-week-old female NOD mice was associated with strong expression of prostaglandin H synthase 2 in the pancreas and of arginase, an antagonist enzyme of the inducible NO synthase. After acceleration of insulitis progression by cyclophosphamide, expression of the two enzymes was downregulated within 24 h. There was strong concomitant upregulation of IL-15 gene expression that preceded lymphocyte invasion of islets and a rise of IFN-gamma mRNA levels by several days. The comparison of individual pancreata showed that the expression of IL-12 and IL-18 mRNA closely correlated with levels of IL-15 gene expression. We conclude that arginase and prostaglandin H synthase 2 expression is associated with peri-insulitis, while IL-15 is a candidate cytokine in driving destructive insulitis, as it elicits Th1-cytotoxic responses in lymphoid as well as in non-lymphoid immune cells and is unusually resistant to downregulation by antagonistic cytokines. This is the first report on arginase, prostaglandin H synthase 2 and IL-15 expression in pancreatic lesions of prediabetic NOD mice.     

Reovirus delays diabetes onset but does not prevent insulitis in nonobese diabetic mice

Mice infected with reovirus develop abnormalities in glucose homeostasis. Reovirus strain type 3 Abney (T3A) was capable of systemic infection of nonobese diabetic (NOD) mice, an experimental model of autoimmune diabetes. Reovirus antigen was detected in pancreatic islets of T3A-infected mice, and primary cultures of pancreatic islets from NOD mice supported T3A growth. Significantly fewer T3A-infected animals compared to uninfected controls developed diabetes. However, despite the alteration in diabetes penetrance, insulitis was evident in T3A-infected mice. These results suggest that viral infection of NOD mice alters autoimmune responses to beta-cell antigens and thereby delays development of diabetes.     

Changes in the erythrocyte glutathione concentration in the course of diabetes mellitus

Abstract

This study aims to evaluate the significance of the changes of erythrocyte reduced glutathione (GSH) in the course of diabetes mellitus including the pre-diabetes stage and cardiovascular disease co-morbidity. A total of 222 participants (female:male, 107:115) were selected and their erythrocyte GSH levels were measured. The participants were divided into four groups: (i) control; (ii) those with blood glucose level ≥5.6 mmol/l but < 6.9 mmol/l as pre-diabetes mellitus with no other pathology; (iii) diabetes without co-morbidity; and (iv) those with diabetes mellitus and cardiovascular disease. Statistical analysis was by ANOVA followed by a Fisher's LSD post hoc test. We observed that GSH concentration was significantly different between groups (P < 0.04). The Fisher's post hoc test indicated significant differences in erythrocyte GSH levels between the pre-diabetes mellitus and diabetes mellitus groups compared to control (P < 0.005 and P < 0.05, respectively). A statistically significant change (P < 0.001) involving an initial fall followed by a rise in erythrocyte GSH levels was observed when diabetes mellitus and diabetes mellitus+cardiovascular disease groups were combined and assessed with respect to period of diabetes. We conclude that oxidative stress is already present in the pre-diabetes stage as determined by the fall in GSH, representing the initial phase of oxidative stress in diabetes mellitus progression. This finding provides evidence that antioxidant markers such as GSH could be a useful tool for pre-diabetes mellitus screening.     

Rotavirus infection accelerates type 1 diabetes in mice with established insulitis

Infection modulates type 1 diabetes, a common autoimmune disease characterized by the destruction of insulin-producing islet beta cells in the pancreas. Childhood rotavirus infections have been associated with exacerbations in islet autoimmunity. Nonobese diabetic (NOD) mice develop lymphocytic islet infiltration (insulitis) and then clinical diabetes, whereas NOD8.3 TCR mice, transgenic for a T-cell receptor (TCR) specific for an important islet autoantigen, show more rapid diabetes onset. Oral infection of infant NOD mice with the monkey rotavirus strain RRV delays diabetes development. Here, the effect of RRV infection on diabetes development once insulitis is established was determined. NOD and NOD8.3 TCR mice were inoculated with RRV aged > or = 12 and 5 weeks, respectively. Diabetes onset was significantly accelerated in both models (P < 0.024), although RRV infection was asymptomatic and confined to the intestine. The degree of diabetes acceleration was related to the serum antibody titer to RRV. RRV-infected NOD mice showed a possible trend toward increased insulitis development. Infected males showed increased CD8(+) T-cell proportions in islets. Levels of beta-cell major histocompatibility complex class I expression and islet tumor necrosis factor alpha mRNA were elevated in at least one model. NOD mouse exposure to mouse rotavirus in a natural experiment also accelerated diabetes. Thus, rotavirus infection after beta-cell autoimmunity is established affects insulitis and exacerbates diabetes. A possible mechanism involves increased exposure of beta cells to immune recognition and activation of autoreactive T cells by proinflammatory cytokines. The timing of infection relative to mouse age and degree of insulitis determines whether diabetes onset is delayed, unaltered, or accelerated.