Gangliosides in vivo reduce diabetes incidence in non-obese diabetic mice

Summary Non-obese diabetic mice were treated daily with a mixture of ganliosides from day 30 until day 250 of life or until the mice became diabetic. Ganglioside treatment reduced diabetes incidence from 80–90% to 47% and from 20–30% to zero in female and male mice respectively. Gangliosides did not affect the frequency of perivasculitis. It is concluded that gangliosides can reduce diabetes incidence in non-obese diabetic mice.Deceased     

In utero undernutrition reduces diabetes incidence in non-obese diabetic mice

AIMS/HYPOTHESIS: Observational studies in humans suggest that low birthweight may decrease the risk of type 1 diabetes, but the mechanism is unknown. We hypothesised that antenatal undernutrition would decrease the incidence of type 1 diabetes in non-obese diabetic (NOD) mice. MATERIALS AND METHODS: A 40% restriction of energy intake was applied to pregnant NOD dams from day 12.5 to day 18.5 of gestation, resulting in intrauterine growth retardation of offspring. All mice were fed a standard diet after weaning. Control and undernourished female offspring were followed to assess diabetes incidence. Male NOD mice were treated with cyclophosphamide to accelerate development of diabetes. Glucose homeostasis, body composition and pancreatic histology were compared in control and undernourished offspring. RESULTS: Mean birthweight was lower in undernourished than in control mice (p = 0.00003). At 24 weeks of age, the cumulative incidence of spontaneous diabetes in female mice was 73% in control and 48% in undernourished mice (p = 0.003). In cyclophosphamide-treated male mice, antenatal undernutrition also tended to reduce the development of diabetes (p = 0.058). Maternal leptin levels were lower in undernourished dams on day 18.5 of pregnancy (p = 0.039), while postnatal leptin levels were significantly higher in undernourished offspring at 4, 20 and 27 weeks of life (p < 0.05). Beta cell mass was similar in both groups (control = 0.4 mg; undernourished = 0.54 mg; p = 0.24). Histological evidence of apoptosis at 20 weeks was greater in control than in undernourished mice (control = 6.3 +/- 1.4%; undernourished = 4.2 +/- 0.3%, p = 0.05). CONCLUSIONS/INTERPRETATION: Antenatal undernutrition reduces the incidence of diabetes in NOD mice, perhaps via alterations in apoptosis.     

血糖与NOD小鼠肝细胞脂肪变性的关系

目的探讨非肥胖性糖尿病(None-obese diabetic NOD)小鼠的肝组织学改变及其与空腹血糖的关系.方法26只NOD小鼠,低脂低蛋白饮食饲养于屏障系统内.观察至发生显性糖尿病或9月龄时处死.分析肝细胞脂肪变性与空腹血糖的关系.结果实验结束时,11只小鼠空腹血糖水平始终正常,15只(57.7%)发生糖尿病.糖尿病组空腹血糖、糖化血红蛋白较血糖正常组显著升高,两组小鼠体重无显著差异.苏木精-伊红染色显示肝组织学改变基本正常者7例(26.9%),其余小鼠均有泡沫样和(或)大泡性肝细胞脂肪变性,8例(30.8%)达到脂肪肝的诊断标准.相关分析显示,随着NOD小鼠血糖水平升高,肝脂肪变性程度呈加剧趋势;糖尿病组肝脂肪变程度显著重于血糖正常组(秩和检验H=14.084,P＜0.001).脂肪肝组(n=8)血糖和糖化血红蛋白显著高于非脂肪肝组(n=17),分别为26.10±7.12对12.31±10.00mmol/L(t=3.057,P＜0.01)和6.90±1.66对3.91±2.11mmol/L(t=3.547,P＜0.01).结论NOD小鼠是研究1型糖尿病与脂肪肝相关性的良好模型,空腹血糖水平与NOD小鼠肝脂肪变性程度密切相关.     

Dissecting autoimmune diabetes through genetic manipulation of non-obese diabetic mice

Type 1 diabetes results from a genetically and immunologically complex autoimmune process that is specifically directed against the pancreatic beta cells. Non-obese diabetic mice spontaneously develop a form of autoimmune diabetes closely resembling the disease in humans. This happens because, like human diabetic patients, non-obese diabetic mice have an unfortunate combination of apparently normal alleles at numerous loci associated with Type 1 diabetes. In isolation, each of these allelic variants affords a small degree of susceptibility to diabetes. In combination, however, they set in motion a series of immunological events that lead to islet inflammation and overt diabetes. Type 1 diabetes is associated with defects in self-tolerance and immunoregulation. It involves presentation of beta cell antigens to autoreactive T lymphocytes by professional antigen-presenting cells, the recruitment of antigen-activated T cells into pancreatic islets, and the differentiation of these antigen-activated lymphocytes into beta cell killers. Understanding the precise sequence of events in the pathogenesis of Type 1 diabetes has been, and remains, a challenging task. Much of our understanding of the immunology of the disease stems from studies of genetically engineered, non-obese diabetic mice. These mice provide reductionist systems, with which the contribution of individual cellular elements, molecules or genes to the disease process can be dissected. This review focuses on the lessons that have been learned through studies of these mice.Abbreviations APCs, antigen-presenting cells - 2m, beta-2 microglobulin - CD62L, L-selectin - CDR3, complementarity-determining region 3 - CTL, cytotoxic T lymphocyte - DC, dendritic cell - IA-2, insulinoma-associated protein 2 - ICA69, islet cell antigen 69 KDa - ICAM-1, intercellular adhesion molecule-1 - IFN-, interferon- - LFA-1, leucocyte function-associated antigen-1 - mAb, monoclonal antibody - NK, natural killer - MIP-1, macrophage inflammatory protein 1 - NOD, non-obese diabetic - 8.3-NOD, 8.3-TCR-transgenic NOD mice - PLN, pancreatic lymph node - rag, recombination-activating gene - RAG-2, recombination-activating gene 2 - RIP, rat insulin promoter - TCR, T cell receptor     

C-peptide constricts pancreatic islet arterioles in diabetic, but not normoglycaemic mice

BACKGROUND: Pancreatic islet blood flow is regulated separately from that of the exocrine pancreas, and a consistent finding during impaired glucose tolerance is an increased blood perfusion. The aim of the present study was to investigate whether C-peptide affects pancreatic islet arterioles in normal and diabetic mice. MATERIALS AND METHODS: Control and diabetic C57-Bl mice were studied after 2 weeks of alloxan-induced diabetes. Islet arterioles were dissected and microperfused with Dulbecco's modified Eagle medium (DMEM) solution. The effect of luminal application of mouse C-peptide was investigated. RESULTS: C-peptide reduced the diameter of islet arterioles from diabetic mice (-10+/-4%, P<0.05) compared to base-line values, whilst arterioles from normoglycaemic animals did not respond to C-peptide (P=0.2). CONCLUSION: These findings suggest a role for C-peptide in the regulation of islet blood flow, especially during conditions with impaired glucose tolerance.     

Th1细胞在NOD小鼠糖尿病早期的应用

目的评价Th1细胞对NOD小鼠糖尿病早期变化的作用。方法选择4 w、8 w和16 w的雌性NOD小鼠,采用流式细胞术测定脾Th1细胞,采用免疫组化法检测胰腺内Th1细胞。结果随年龄增加,雌性NOD小鼠脾和胰腺内Th1细胞逐渐增加。结论 Th1细胞参与NOD小鼠糖尿病早期的病理过程。     

Lymphocyte vaccination protects prediabetic non-obese diabetic mice from developing diabetes mellitus

Summary In the therapeutic manoeuvre termed “lymphocyte vaccination”, activated lymphocytes capable of transferring an autoimmune disease are instead attenuated and given in vaccine form. We have previously shown that such a therapy administered to non-obese diabetic (NOD) mice at 6 weeks of age prevents diabetes mellitus. To assess whether this therapy has potential clinical relevance, in the present study lymphocyte vaccination was applied in NOD mice in 3 weekly doses commencing in the immediate prediabetic period (age 12 weeks), when insulitis is advanced and diabetes incipient. Of 30 NOD mice receiving active vaccine (composed of attenuated lymphocytes from diabetic NOD mice) 13 (43.3 %) remained non-diabetic to the age of 30 weeks, in comparison with 2 of 30 (6.7 %; p < 0.01) mice receiving a control vaccine (composed of attenuated lymphocytes from non-diabetic NOD/B10 mice) and 5 of 26 (19.2 %; p < 0.01) mice receiving saline carrier alone. Moreover, in an additional group of 10 NOD mice receiving active vaccine weekly between 12 and 30 weeks, 8 remained diabetes free at the end of the treatment. The most notable effect of the vaccine was that the delay in diabetes onset was accompanied by a reduction in insulitis and in some cases a complete absence of infiltrating lymphocytes at 30 weeks of age. Immunocytochemistry indicated that when present, islet infiltrating lymphocytes in non-diabetic mice that received active vaccine showed significantly reduced staining for interferon-γ, compared with the infiltrate seen in diabetic mice receiving the control vaccine or saline. This study demonstrates that the rapid progression to diabetes typically seen in 12-week-old NOD mice can be delayed by lymphocyte vaccination, supporting the possibility that a vaccine composed of attenuated autologous peripheral blood lymphocytes could be effective in high risk first degree relatives of patients with insulin dependent diabetes mellitus. [Diabetologia (1997) 40: 1388–1395] Received: 6 May 1997 and in revised form: 24 July 1997     

Infection with Schistosoma mansoni prevents insulin dependent diabetes mellitus in non-obese diabetic mice

The spontaneous development of insulin dependent diabetes mellitus in non-obese diabetic (NOD) mice has been shown to be mediated by a Th1 response against beta cell antigens. It is known that in murine models of Schistosoma mansoni infection, egg production is associated with a switch from a Th1 to Th2 response. This subsequent dominance of a Th2 response in S.mansoni infected mice has been shown to influence the response to other infectious agents or antigens. We therefore determined whether infection with S.mansoni could influence the spontaneous incidence of insulin dependent diabetes mellitus (IDDM) in NOD mice. Infection with this helminth significantly reduced the spontaneous incidence of IDDM. IDDM was also prevented by injecting parasite eggs alone. Because until relatively recently humans might expect to succumb to a variety of infectious agents, the current freedom from infection might permit the expression of a genetic predisposition to autoimmune pathology and be responsible for the increased incidence of IDDM.     

Insulin and glucagon in spontaneously diabetic non-obese mice

Summary To investigate the rôle of glucagon in the development of diabetes mellitus, spontaneously diabetic non-obese mice were studied before (group 1) and after the onset of diabetes mellitus (group 2). In group 1, fasting blood glucose and insulin in plasma and pancreas did not differ significantly, while plasma glucagon was elevated (48.9±10.4 versus control 18.6±6.0pmol/l). In group 2, the insulin content of plasma and the pancreas were markedly reduced, whereas plasma glucagon was elevated (180.9±59.1 pmol/l). When diabetic mice were treated with insulin for 4 weeks (group 3), plasma glucagon was markedly reduced compared with that of group 2 (30.3±9.0 pmol/l). In group 1, glucagon and glucagon-like immunoreactivity of the intestine were reduced. The glucagon content of the intestine was elevated in group 2. Group 3 elicited increased contents of gastric glucagon as well as intestinal glucagon-like immunoreactivity. We conclude that, in addition to insulin deficiency, hypersecretion of glucagon might contribute to the development and clinical course of diabetes mellitus in the non-obese diabetic mouse.     

Vitamin D deficiency in early life accelerates Type 1 diabetes in non-obese diabetic mice

Aims/hypothesis 1,25-dihydroxyvitamin D₃, the active form of vitamin D, prevents Type 1 diabetes in non-obese diabetic (NOD) mice. Epidemiological data show a threefold increase in human Type 1 diabetes when vitamin D deficiency was present in the first months of life. To evaluate whether a similar dietary deficiency affects diabetes incidence in NOD mice, we generated NOD mice with vitamin D deficiency in early life.Methods Breeding pairs of NOD mice, as well as their offspring (test mice), were kept in surroundings devoid of ultraviolet light and were fed a vitamin D-depleted diet for 100 days. Mice were followed for 250 days.Results At 250 days, 35% (12/35) male and 66% (22/33) female vitamin D-deficient mice were diabetic compared to 15% (6/40, p=0.05) and 45% (13/29, p<0.01) of the control mice. At 100 days no difference in insulitis was seen, but more vitamin D-deficient mice were glucose intolerant. Higher IL1 expression was detected in islets of vitamin D-deficient mice and their peritoneal macrophages had an aberrant cytokine profile (low IL1 and IL6, high IL15). Thymus and lymph nodes of vitamin D-deficient mice contained less CD4⁺CD62L⁺ cells.Conclusion/interpretation Vitamin D status increases the expression of Type 1 diabetes in NOD mice. Our data in NOD mice, as well as human epidemiological data, point to the importance of preventing vitamin D deficiency in early childhood. Controlling this dietary factor could be an easy and safe way to reduce the incidence of Type 1 diabetes in subjects who are genetically at risk.Abbreviations 1,25(OH)₂D₃ 1,25-dihydroxyvitamin D₃ - 25(OH)D₃ 25-hydroxyvitamin D₃ - BW body weight - HBSS Hanks balanced salt solution - IP interferon-inducible protein - IPGTT intra-peritoneal glucose tolerance test - iNOS inducible nitric oxide synthase - LPS lipopolysaccharide - MCP monocyte chemo-attractant protein - MIP macrophage inflammatory protein - MLR mixed lymphocyte reaction - NOD non-obese diabetic - RPMI Roswell Park Memorial Institute - UV ultraviolet     

Prevention of diabetes mellitus in non-obese diabetic mice by Linomide,a novel immunomodulating drug

Summary Oral administration of the synthetic immunomodulating drug quinoline-3-carboxamide (Linomide) in the drinking water to 5-week-old female non-obese diabetic (NOD) mice resulted in complete protection from insulitis and maintenance of normal glucose tolerance for over 40 weeks (impaired glucose tolerance: treated n=2 of 18; control n=17 of 18, p<0.0001). Delayed administration of the drug at 16 weeks resulted in slowing of the progression to diabetes when assessed at 42 weeks (treated with diabetes n=7 of 25; control with diabetes 25 of 43, p<0.0234). No gross changes of immune system cell phenotype or function were observed in the Linomide-treated group. Adoptive transfer of spleen and lymph node cells from treated female NOD mice into sub-lethally irradiated male recipients failed to transfer diabetes, whereas a similar transfer of cells obtained from untreated age-matched controls resulted in diabetes in all secondary recipients (diabetes in control group n=12 of 13; in Linomide group n=0 of 11, p<0.0001). Linomide pretreatment of the secondary recipients also inhibited the transfer of diabetes (diabetes in pretreated group n=2 of 9, control group n=12 of 13, p<0.015), as did adoptive co-transfer of cell mixtures obtained from treated female NOD mice, free of diabetes, and from diabetic NOD female mice (diabetes in Linomide group n=4 of 9; in control group 7 of 7, p<0.0337). Our data indicate that Linomide-treated NOD mice generate immune cells with the capacity to downregulate responses to beta-cell antigens, apparently through immunoregulation rather then antigen non-specific immunosuppression. Based on our findings and considering the lack of severe side effects of orally administered Linomide in man, this new compound should be considered as a potential drug for treatment of insulin-dependent diabetes mellitus.Abbreviations IDDM Insulin-dependent diabetes mellitus - NK natural killer - IL interleukin - NOD non-obese diabetic - IPGTT intraperitoneal glucose tolerance test - FITC fluorescein-5-isothiocyanate - DTAF 5(4,6 dichlorotriazinyl) aminofluorescein - FACS fluorescein activated cell sorting - PHA phythaemagglutinin - Con A concanavalin A - SEA staphylococcal extract A - SEB staphylococcal extract B - SRBC sheep erythrocytes - HBSS Hanks buffered salt solution - LPS lipopolysaccharide - MLR mixed lymphocyte reaction - DTH delayed-type, hypersensitivity - BCG Bacillus Calmette Guerrin - APC antigen presenting cell - TNF- tumor necrosis factor-     

Tyrosine kinase inhibitors reverse type 1 diabetes in nonobese diabetic mice

The recent development of small-molecule tyrosine kinase (TK) inhibitors offers increasing opportunities for the treatment of autoimmune diseases. In this study, we investigated the potential of this new class of drugs to treat and cure type 1 diabetes (T1D) in the NOD mouse. Treatment of prediabetic and new onset diabetic mice with imatinib (Gleevec) prevented and reversed T1D. Similar results were observed with sunitinib (Sutent), an additional approved multikinase inhibitor, suggesting that the primary target of imatinib, c-Abl, was not essential in blocking disease in this model. Additional studies with another TK inhibitor, PLX647 (targeting c-Kit and c-Fms) or an anti-c-Kit mAb showed only marginal efficacy whereas a soluble form of platelet-derived growth factor receptor (PDGFR), PDGFRβIg, rapidly reversed diabetes. These findings strongly suggest that inhibition of PDGFR is critical to reverse diabetes and highlight a crucial role of inflammation in the development of T1D. These conclusions were supported by the finding that the adaptive immune system was not significantly affected by imatinib treatment. Finally, and most significantly, imatinib treatment led to durable remission after discontinuation of therapy at 10 weeks in a majority of mice. Thus, long-term efficacy and tolerance is likely to depend on inhibiting a combination of tyrosine kinases supporting the use of selective kinase inhibitors as a new, potentially very attractive approach for the treatment of T1D.     

Diabetes preventive gluten-free diet decreases the number of caecal bacteria in non-obese diabetic mice

BACKGROUND: A gluten-free diet reduces the incidence of diabetes mellitus in non-obese diabetic (NOD) mice, but the mechanism is not known. The aim of this study was to examine the possible influence of the diet on the caecal bacterial flora, which may affect the intestinal physiology and mediate disease prevention. METHODS: Two groups of NOD mice from the age of 3 weeks were fed either a gluten-free diet or a standard diet. Each diabetic mouse, when diagnosed, along with a non-diabetic mouse from the same diet group and two non-diabetic mice from the alternate diet group were euthanized and sampled for classical bacteriological examination. RESULTS: Nine out of 19 (47%) standard-fed mice and 1 out of 19 (5%) gluten-free-fed mice developed diabetes (p < 0.01). Mice on the gluten-free diet had significantly fewer aerobically (p < 0.01) and microaerophilically (p < 0.001) cultivated bacteria in their intestines than standard-fed mice. Non-diabetic mice also had significantly fewer microa erophilic and anaerobic bacteria than diabetic mice (p < 0.05). These differences were primarily due to a difference in the Gram-positive flora. CONCLUSIONS: The gluten-free diet compared to the standard diet both qualitatively and quantitatively substantially altered the composition of the caecal bacterial flora in NOD mice. Although Gram-positive bacteria might influence the beta cells through certain digestive products, it is more likely to assume that any effect on diabetes incidence is immunological.     

Dietary prevention of diabetes in the non-obese diabetic mouse

Summary Diabetes prone NOD female mice were fed diets containing different proteins from just before weaning. Only mice receiving meat meal or casein as the protein source developed diabetes at the rate expected from this colony. Lactalbumin and gluten did not precipitate diabetes except in a small number. Casein hydrolysate in lieu of protein protects against overt diabetes, but only if introduced early. The animals which did not show overt diabetes nevertheless had intermittent trace glycosuria and the majority showed mild degrees of peninsular lymphocytic infiltration.     

Autoantigen-specific protection of non-obese diabetic mice from cyclophosphamide-accelerated diabetes by vaccination with dendritic cells

Aims/hypothesis Dendritic cells (DCs) are professional antigen presenting cells involved in the initiation of primary immune responses and the preservation of peripheral tolerance. The aim of this study was to develop a DC vaccine for autoantigen-specific prevention of autoimmune diabetes.Methods Splenocytes from diabetes-prone NOD mice were cultured in conditioned media to obtain a homogeneous DC sub-population for vaccination experiments. These cells were used to modulate autoimmune responses in NOD mice after synchronization of diabetes with cyclophosphamide. After immunisation with insulin-pulsed DCs the incidence of diabetes, the insulitis grade and the cytokine production was examined.Results The long-term culture of splenocytes resulted in the generation of a cell line, termed NOD-DC1, which have a phenotype of myeloid DCs (CD11c, CD11b, DEC-205), express MHC class II and co-stimulatory molecules (CD40, CD80, CD86). The NOD-DC1 cells have preserved functional activity shown by the detection of a high antigen uptake capacity, the induction of a mixed lymphocyte reaction and stimuli-dependent IL-6 and TNF- secretion. Vaccination with insulin-pulsed NOD-DC1 cells results in an antigen-specific prevention of diabetes. This was mediated by a reduction of the severity of insulitis and a decrease of T helper 1 effector cells.Conclusion/interpretation We describe the generation of a DC line which confers protection from diabetes in an antigen-specific way. Our data shows that autoantigen-loaded DCs can induce strong immunoregulatory effects supporting the hypothesis that DCs are promising candidates to develop novel vaccines for the prevention of autoimmune diabetes.Abbreviations APC antigen-presenting cells - CY cyclophosphamide - DCs, dendritic cells - LPS lipopolysaccharide - mAb monoclonal antibodies - NOD non-obese diabetic - Th T-helper cells     

Fas ligand-mediated mechanisms are involved in autoimmune destruction of islet beta cells in non-obese diabetic mice

Aims/hypothesis. A mechanism implicated in pancreatic islet beta-cell destruction in autoimmune diabetes is the binding of the Fas ligand (FasL) on T cells to Fas receptors on beta cells, causing their destruction. Evidence for this mechanism is, however, controversial. The aim of this study was to find whether the Fas ligand contributes to beta-cell death in autoimmune diabetes. Methods. We transplanted syngeneic islets under the renal capsule in non-obese diabetic (NOD) mice and treated the mice with a neutralizing monoclonal antibody to the Fas ligand. Survival of beta cells in islet grafts and phenotypes of graft-infiltrating cells were investigated. Results. We found 58 % (7 of 12) of mice treated with anti-Fas ligand antibody were normoglycaemic at 30 days after islet transplantation compared with none (0 of 9) of the mice treated with control antibody. Immunohistochemical analysis of islet grafts showed that infiltration of leucocytes (CD4⁺ T cells, CD8⁺ T cells, macrophages and neutrophils) and apoptosis of beta cells in the grafts was significantly decreased in mice treated with anti-Fas ligand antibody. Expression of proinflammatory cytokines (interleukin 1 alpha, tumour necrosis factor alpha and interferon gamma) was not different in islet grafts of mice treated with anti-Fas ligand and control antibodies. Conclusion/interpretation. These findings indicate that Fas ligand-mediated mechanisms play a major part in promoting leucocytic infiltration of islets and beta-cell destruction in autoimmune diabetes. [Diabetologia (2000) 43: 1149–1156] Received: 31 March 2000 and in revised form: 5 June 2000     

Novel T-cell inhibiting peptides delay the onset of Type 1 diabetes in non-obese diabetic mice

The aim of this study was to investigate the effectiveness of immunomodulatory peptides in preventing the spontaneous onset of Type 1 diabetes in NOD mice. Two such peptides, CP and C1, were injected intraperitoneally in NOD mice three times a week starting at two different time points, nine weeks and 11 weeks of age, and blood sugar levels monitored for the development of diabetes. CP was shown to be effective in delaying the onset of diabetes compared to control (P = 0.006). The timing of peptide administration was crucial since delay in treatment did not prevent the onset of diabetes (nine weeks versus 11 weeks of age). C1 was effective in delaying the onset of Type 1 diabetes with borderline significance when given at week 11 (P = 0.05). These findings confirm the efficacy of these peptides in the prevention and possible treatment for Type 1 diabetes and thereby create new opportunities for genetic manipulation.     

Preventive effect of a new immunosuppressant FK-506 on insulitis and diabetes in non-obese diabetic mice

Summary We investigated the effect of an immunosuppressant FK-506 on histological change of islets, the onset of diabetes, and the change of spleen cell subsets in female non-obese diabetic mice. Mice administered intraperitoneally with FK-506 from 5 to 20 weeks of age showed marked suppression of mononuclear cell infiltration (insulitis) at 10 weeks of age. Among the subsets of the spleen cells, a significant decrease in the population of Thyl.2-positive T cells (pan-T), L3T4-positive T cells (mainly helper/inducer), and Lyt2-positive T cells (mainly suppressor/cytotoxic) was observed in FK-506-treated mice. Furthermore, glucose tolerance of the mice at 15 weeks of age was clearly improved. Cumulative incidence observed up to 40 weeks of age was 86% in control mice and 23% in FK-506-treated mice (p<0.01). These data indicate that FK-506 has a preventive effect on insulitis and diabetes by the suppression of cell-mediated autoimmunity in non-obese diabetic mice.