Preventive effects of Cyclosporin on diabetes in NOD mice

Summary Non-obese diabetic mice aged 30 to 60 days were treated orally with Cyclosporin at doses of 25,15 and 2.5 mg/kg every 2 days until 160 days of age. Diabetes developed in 12 out of 18 oil-treated mice (67%), with partial to complete Langerhans' islet destruction associated with lymphocytic infiltration. The non-obese diabetic mice showed a plasma glucose concentration of 6.62 ± 0.92 mmol/l (mean ± SD) at 50 days of age. The plasma glucose level of oil-treated non-obese diabetic mice gradually increased after 130 days of age and reached 14.0 to 19.0 mmol/l at 160 days of age, while Cyclosporin-treated non-obese diabetic mice showed neither clear increase of plasma glucose levels nor development of insulitis. The cumulative incidence of diabetes in Cyclosporin-treated mice was significantly lower than that in oil-treated mice (p < 0.01). Subsequently, Cyclosporin treatment was started after development of glucose intolerance. Twenty-five mg/kg of Cyclosporin was administered every 2 days for 35 days. Cyclosporin appeared to have little therapeutic effect on diabetes in non-obese diabetic mice.     

Lessons from the NOD mouse for the pathogenesis and immunotherapy of human Type 1 (insulin-dependent) diabetes mellitus

Summary Suitable animal models of human Type 1 (insulin-dependent) diabetes mellitus have long been sought, in particular a model that would permit detailed histological and immunological investigation of changes in the islet preceding the metabolic disorder. This would allow hypotheses as to pathogenesis of the condition to be examined and interventions such as immunotherapy to be tested. The most widely studied models include the low-dose streptozotocin induced diabetic mouse and the BB rat, but both differ in important respects from the human disease. In this review we describe one highly successful model, the non obese diabetic mouse. Selected aspects of pathogenesis and immunotherapy are presented and analogies with human Type 1 diabetes discussed.     

Perinatal exposure to high dietary advanced glycation end products in transgenic NOD8.3 mice leads to pancreatic beta cell dysfunction

The contribution of environmental factors to pancreatic islet damage in type 1 diabetes remains poorly understood. In this study, we crossed mice susceptible to type 1 diabetes, where parental male (CD8⁺ T cells specific for IGRP_206-214; NOD8.3) and female (NOD/ShiLt) mice were randomized to a diet either low or high in AGE content and maintained on this diet throughout pregnancy and lactation. After weaning, NOD8.3⁺ female offspring were identified and maintained on the same parental feeding regimen for until day 28 of life. A low AGE diet, from conception to early postnatal life, decreased circulating AGE concentrations in the female offspring when compared to a high AGE diet. Insulin, proinsulin and glucagon secretion were greater in islets isolated from offspring in the low AGE diet group, which was akin to age matched non-diabetic C57BL/6 mice. Pancreatic islet expression of Ins2 gene was also higher in offspring from the low AGE diet group. Islet expression of glucagon, AGEs and the AGE receptor RAGE, were each reduced in low AGE fed offspring. Islet immune cell infiltration was also decreased in offspring exposed to a low AGE diet. Within pancreatic lymph nodes and spleen, the proportions of CD4⁺ and CD8⁺ T cells did not differ between groups. There were no significant changes in body weight, fasting glucose or glycemic hormones. This study demonstrates that reducing exposure to dietary AGEs throughout gestation, lactation and early postnatal life may benefit pancreatic islet secretion and immune infiltration in the type 1 diabetic susceptible mouse strain, NOD8.3.     

Aberrant expression of Class II major histocompatibility complex molecules by B cells and hyperexpression of Class I major histocompatibility complex molecules by insulin containing islets in Type 1 (insulin-dependent) diabetes mellitus

Summary Twenty-three patients with recent onset Type 1 (insulin-dependent) diabetes in whom residual insulin secreting B cells were present and 12 patients with disease of more prolonged duration (maximum 9 years), 8 of whom had residual B cells, were studied. Aberrant expression of Class II major histocompatibility complex molecules was demonstrated immunohistochemically on insulin secreting B cells in 21 out of 23 patients with recent onset disease and 6 of the patients with more prolonged disease. No such expression was seen on glucagon secreting A cells or somatostatin secreting D cells. Islets where there was marked hyperexpression of Class I major histocompatibility complex molecules on islet endocrine cells were seen in all cases in which residual B cells were present. Ninety-two per cent of insulin containing islets but only 1% of insulin deficient islets exhibited this phenomenon (p<0.001, Chi-squared test). There was evidence to suggest that both these abnormalities of major histocompatibility complex expression preceded insulitis within a given islet. They also appeared to be unique to Type 1 diabetes, being absent in pancreases of patients with Type 2 (non-insulin-dependent) diabetes, chronic pancreatitis, cystic fibrosis, graft-versus-host disease and Coxsackie B viral pancreatitis. The development of autoimmunity to B cells in Type 1 diabetes may be a multistep process in which abnormalities of major histocompatibility complex expression on islet endocrine cells are crucial events.     

Induction of class II MHC antigens in vitro on pancreatic B cells isolated from BB/E rats

Summary The ability of recombinant Interferon- to induce class II expression in vitro on pancreatic islet B cells has been investigated by exposing islets isolated from BB/E and normal Wistar rats to Interferon- y and then staining successively with monoclonal antibodies specific for rat class II MHC antigens and insulin. Induction of class 11 expression was never observed on islet cells obtained from either normal Wistar rats or rats from the BB/E low diabetes incidence (< 2%) subline. In contrast, pancreatic B cells from rats from the BB/E high diabetes incidence (60–70%) subline expressed class II antigen following culture with Interferon-.     

Antibodies to pancreatic islet cell antigens in diabetes seen in Southern India with particular reference to fibrocalculous pancreatic diabetes

Fibrocalculous pancreatic diabetes (FCPD) is a type of diabetes secondary to tropical chronic non-alcoholic pancreatitis. Little is known about the aetiopathogenesis of FCPD. We studied glutamic acid decarboxylase antibodies (GAD-Ab) and islet cell antibodies (ICA) in patients with FCPD and compared the results with Type 1 (insulin dependent) diabetes mellitus, Type 2 (non-insulin-dependent) diabetes mellitus and non-diabetic subjects in Southern India. The prevalence of GAD-Ab was 7.0 % (95 % Confidence Interval (CI) 1.9–17.2) in FCPD, 47.5 % (CI 31.4–64.0) in Type 1 (p < 0.001 compared to FCPD), 5.6 % (CI 1.5–13.9) in Type 2 (non-significant (NS) compared to FCPD) and 0 % in controls. The prevalence of ICA was 6.3 % (CI 1.2–17.4) in FCPD, 53.8 % (CI 37.1–70.0) in Type 1 (p < 0.001 compared to FCPD), 9.9 % (CI 4.0–19.4) in Type 2 (NS compared to FCPD) and 4.7 % (CI 0.4–16.1) in controls. The data suggest that in FCPD, the frequency of auto-antibodies is low and its aetiology is probably not linked to autoimmunity in the majority of the patients. © 1998 John Wiley & Sons, Ltd.     

Raised temperature reduces the incidence of diabetes in the NOD mouse

Summary An association between the incidence of childhood Type 1 (insulin-dependent) diabetes mellitus and the average yearly temperature in different countries has been reported, the incidence being higher in countries with a lower mean temperature. We have studied the effect of environmental temperature on the incidence of diabetes in an animal model of Type 1 diabetes, the non-obese diabetic (NOD) mouse. Female NOD mice were divided at weaning, with one group placed at a higher temperature (mean 23.7±1.7° C) and the other at a lower temperature (21.0±1.8° C). At 20 weeks of age 6 of 16 mice at lower temperature and 1 of 17 mice at higher temperature had developed diabetes (p < 0.02); at 30 weeks 10 of 16 and 5 of 17 mice had developed diabetes (p < 0.05). Non-diabetic animals in the low temperature group had a higher food intake than those in the high temperature group between 13–15 weeks of age (28.0±1.2 g/week vs 24.8± 0.7 g/week, P < 0.05). In a parallel experiment, histological examination showed that there were similar degrees of insulitis in the high and low temperature groups at seven weeks of age. We conclude that environmental temperature can affect the incidence of diabetes in the NOD mouse and that this may be related to alterations in food intake.     

Pancreatic NOD beta cells express MHC class II protein and the frequency of I-A(g7) mRNA-expressing beta cells strongly increases during progression to autoimmune diabetes

Aims/hypothesis In the NOD mouse model, attempts to show MHC class II expression by pancreatic beta cells were unsuccessful so far. We readdressed this question by analysing I-A^g7 expression in single pancreatic beta cells.Methods Single-cell multiplex RT PCR and single-cell immunofluorescence were used to study MHC class II expression in NOD and NOD/SCID beta cells.Results Pancreatic beta cells from NOD mice express the I-A^g7 protein as well as the corresponding mRNA. The frequency of MHC class II mRNA-expressing beta cells is drastically increased during the progression to overt diabetes. MHC class II protein is accumulated intracellularly, and invariant chain is co-expressed. Beta cells from 9- to 10-week-old NOD/SCID mice express MHC class II at the same low frequency as beta cells from 3-week-old NOD mice.Conclusion/interpretation NOD beta cells express I-A^g7 and could be a direct target of autoreactive CD4+ T cells. This MHC class II expression is triggered by infiltrating lymphocytes.Abbreviations APC antigen presenting cell - DC dendritic cell - IDDM insulin-dependent diabetes mellitus - Ii invariant chain - NOD non obese diabetic - PPI preproinsulin - SCID severe combined immunodeficiency     

An immunomodulating protein,Ling Zhi-8 (LZ-8) prevents insulitis in non-obese diabetic mice

Summary Ling Zhi-8 (LZ-8), a novel and recently discovered immunomodulatory protein having in vivo immunosuppressive activity, was tested for in vivo effect against Type 1 (insulin-dependent) diabetes mellitus in the non-obese diabetic mouse, the disease having immunologically mediated aetiology in this animal. LZ-8 had mitogenic activity in vitro towards spleen cells of the non-obese diabetic mice as previously shown towards those of DBA/2 mice. Intraperitoneal administration of LZ-8 twice weekly into the mice (10.3–12.6 mg/kg body weight) from 4 weeks of age prevented insulitis and an almost normal number of insulin producing cells were observed. Extreme insulitis and reduction of the number of insulin producing cells were observed in the pancreata of the untreated non-obese diabetic mouse. No cumulative incidence of diabetes mellitus was observed in the LZ-8 treated group, while cumulative incidences of 70% and 60% were observed in an untreated group followed up to 42 weeks of age when the incidence of diabetes was defined as a plasma glucose level of greater than 11 mmol/l and as a urine glucose level of greater than 2 +, respectively. T cell subset population analysis was performed to further investigate the action of LZ-8 on the non-obese diabetic mouse which revealed that LZ-8 treatment increased in L3T4⁺/Lyt-2⁺ ratio.     

Cell-mediated cytotoxic islet cell surface antibodies to human pancreatic beta cells

Summary Sera containing islet cell surface antibodies show a complement-dependent cytotoxic reaction against islet cells, but it has not yet been clarified whether islet cell surface antibodies exhibit cell-mediated cytotoxicity to these cells. By ⁵¹Cr release assay we investigated whether islet cell surface antibodies showed a cytotoxic reaction to human pancreatic B cells (JHPI-1 clone) in the presence of normal human lymphocytes. The sera from 14 islet cell surface antibody-positive, 16 islet cell surface antibody-negative Type 1 (insulin-dependent) diabetic patients and 18 islet cell surface antibody-negative healthy subjects were studied. Four sera containing islet cell surface antibodies showed specific cytotoxicity above the mean +3SD value of healthy subjects, and the mean specific cytotoxicity of islet cell surface antibody-positive sera differed significantly from that of both islet cell surface antibody-negative groups. These results suggest that this cell-mediated cytotoxic mechanism may play an important role in the pathogenesis of Type 1 diabetes.     

Class-II and IL 2 receptor positive cells in the pancreas of NOD mice

Summary The aberrant expression of Class-II molecules on pancreatic B cells in Type 1 (insulin-dependent) diabetes is still a matter of debate. In order to verify if Class-II molecules are expressed on islet cells in the NOD mouse we have studied 21 female mice of different ages (5 to 22 weeks). Serial cryostat pancreas sections were stained with monoclonal rat antibodies against Class-II antigens (P7/7) and the IL2 receptor (AMT-13). Our results show no Class-II expression by endocrine cells at any age, whereas about 25–32% of mononuclear cells infiltrating the islets were Class-II positive, and only 6–9% were IL2 receptor positive. No staining, except of occasional tissue macrophages, was observed in the pancreas of BALB/c, CBA or B10.SCSN mice. Our data are in contrast with those recently published and therefore the reality of expression of Class-II molecules by islet cells of NOD mice should be viewed with caution.     

Modelling of the MHC II allele I-Ag7 of NOD mouse: pH-dependent changes in specificity at pockets 9 and 6 explain several of the unique properties of this molecule

Abstract Aims/hypothesis. We modelled the three-dimensional structure of I-A^g7, the chief genetic component of diabetes in non-obese diabetic mice, to understand the unusual properties of this molecule. Methods. Modelling was done, in complex with established antigenic peptides, based on the structure of I-A^k. Results. The selectivity of the I-A^g7 molecule changes greatly at pockets 9 and 6 but hardly at all at pockets 1, 4 and 7, between endosomal pH (5.0) and extracellular pH (7.0), in agreement with previous results. This selectivity is attributed to the unique combination of β9His, β56His and β57Ser. The positive charges in and around pocket 9 at pH 5, favour binding by negatively charged residues. At pH 7 however, the uncharged α68, β9 and β56 histidines favour the accommodation of the bulky residues lysine, arginine, phenylalanine and tyrosine at pocket 9. The combination of β9His and α66Glu is responsible for the pH-dependent selectivity at pocket 6. Furthermore, the lack of repulsion between β56His and α76Arg at pH 7 leads to a more stable ternary complex. Conclusion/interpretation. These results reconcile previous conflicts over the peptide binding ability of I-A^g7 and its motif. They furthermore provide possible explanations for the short lifetime of cell-surface I-A^g7 complexes in vivo, the higher threshold of thymic negative selection and inherent self-reactivity shown by immunocytes in these mice and the protection from diabetes afforded to them by several transgenically expressed mouse class II alleles. This contributes to an understanding of the pathogenesis of Type I (insulin-dependent) diabetes mellitus in this animal. [Diabetologia (2000) 43: 609–624] Received: 9 August 1999 and in revised form: 27 January 2000     

The life story of the pancreatic B cell

Summary Most research on the pancreatic B cell has so far focussed on the regulation and molecular biology of insulin biosynthesis and release. The present review draws attention to some additional areas of islet research which have become accessible to investigation by recent methodological progress and which may advance our understanding of the role of the B cell in diabetes. There is now evidence to suggest that B cells arise from a pool of undifferentiated precursor cells in the fetal and newborn pancreas. These cells may contribute to islet growth and, if inappropriately stimulated, also to early islet hyperplasia. In the postnatal state, B-cell function is finely tuned by a complex set of incoming signals, one of which is the nutrient supply provided by the blood. Recent studies indicate that a disproportionately high fraction of pancreatic blood is diverted to the islets and that the islet blood flow is increased by glucose. An acute stimulus to insulin release may thus be accompanied by a process which enhances the distribution of the hormone to the target cells. Long-term adjustments of B-cell function are made by changes in B-cell number and total mass. Adaptive growth responses to an increased insulin demand occur in a number of hereditary diabetic syndromes in animals, but in some of these there is an inherited restriction on the capacity for B-cell proliferation leading to further deterioration of the glucose tolerance. Some evidence suggests that a similar mechanism may operate also in human non-insulin-dependent diabetes. A critical appraisal of this hypothesis requires, however, that human B cells should be tested for their growth characteristics. Studies of B-cell proliferation in experimental animals have shown that the B cell passes through the cell cycle at a relatively high rate but that the fraction of proliferating cells is low. Regulation of growth of the total B-cell mass seems to take place by changes in the number of B cells passing through the cell cycle rather than by changes in the rate of the cycle. The number of proliferating B cells also shows a marked decrease with age. It is at present uncertain to what extent these regulatory mechanisms apply also to the human B cell but it can be anticipated that further technical developments will elucidate this problem.The Claude Bernard Lecture 1983 of the European Association for The Study of Diabetes     

Class I and II major histocompatibility complex gene product expression by a rat insulinoma cell line in vitro following exposure to gamma interferon

Summary A study of Class I and II major histocompatibility complex gene product expression by a rat insulinoma cell line (RINm5F) was performed using monoclonal antibodies and immunoperoxidase techniques. RINm5F cells were incubated with different concentrations of gamma interferon. RINm5F cells exhibit low levels of Class I molecules and are normally devoid of Class II gene products. Upon exposure to gamma interferon, RINm5F cells showed a dramatic increase in Class I expression. This expression was homogenous and could be detected on all cells after 18 h of incubation with as little as 1 unit/ml of interferon. In contrast, de novo Class II expression was not homogeneous and required 36 h of incubation with 10 units/ml of interferon. The number of RINm5F cells expressing Class II antigens was dose- and time-dependent. Interferon treatment did not affect the morphology of RINm5F cells as determined by ultrastructural analysis. Withdrawal of interferon from the culture medium for as long as 78 h diminished but did not abolish the expression of Class I and Class II molecules already induced. The ability of interferon to enhance expression of Class I gene products and induce de novo expression of Class II molecules on B-cell-derived RINm5F cells supports the hypothesis that aberrant expression of major histocompatibility complex gene products on pancreatic B cells may be an important factor in triggering the immune response in Type 1 (insulin dependent) diabetes mellitus.     

己酮可可碱预防NOD鼠1型糖尿病的机理研究

目的 探讨己酮可可碱（Pentoxifylline,PTX)对非肥胖糖尿病（NOD）小鼠1型糖尿病发病率，胰岛素的影响及其机制。方法 采用动物模型NOD鼠，注射环磷酰胺（CP）加速其发病。给PTX药物后计算糖尿病发病率，HE染色观察胰岛炎，并用逆转录（RT）PCR法检测脾细胞干扰素γ（IFN－γ），肿瘤坏死因子α（TNF－α），白介素10（IL－10）mRNA的表达。结果 PTX组糖尿病发生率（30．00％）明显低于对照组（67．86％）（P＜0．1）；胰岛炎程度也明显减轻（P＜0．001）；脾细胞IFN－γ，TNF－αmRNA的表达较对照组明显降低（P＜0．05），IL－10mRNA的表达无明显改变。结论 PTX可预防NOD鼠发生糖尿病，其机制可能与纠正Th1与Th2型细胞因子比例失衡有关。     

Prospective prediction of spontaneous but not recurrent autoimmune diabetes in the non-obese diabetic mouse

Aims/hypothesis Type 1 diabetes is a T cell-mediated autoimmune disease with a clinically silent prodrome, during which prediction and treatment of disease are theoretically possible. Using retrospective analysis, spontaneous disease in the non-obese diabetic (NOD) mouse has been correlated with islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-reactive CD8⁺ T cells in the peripheral blood. In this study, we determined prospectively whether IGRP-reactive T cells in peripheral blood could predict disease occurrence. Since recurrent autoimmunity is an important contributor to transplant failure, we also determined whether failure of islet grafts (syngeneic and allogeneic) could be predicted by the presence of circulating autoreactive T cells. Materials and methods Peripheral blood samples were taken weekly from female NOD mice between the ages of 8 and 30 weeks and from NOD mice transplanted with NODscid islets. Peripheral blood cells and islet grafts were analysed for the presence of IGRP-reactive CD8⁺ T cells by flow cytometry. Results Prospective analysis of peripheral blood IGRP-reactive T cells in the prediabetic period predicted disease development with a sensitivity of 100% and a specificity of 60%, resulting in positive and negative predictive values of 85 and 100%, respectively. Significant proportions of IGRP-reactive T cells were found in the grafts, but not in peripheral blood of NOD mice undergoing syngeneic and allogeneic rejection. Conclusions/interpretation The occurrence of spontaneous diabetes can be predicted prospectively by measuring peripheral blood autoreactive T cells. Rejection of syngeneic or allogeneic islets is associated with large populations of autoreactive CD8⁺ T cells within islets, suggesting that immunodominant autoreactive T cells during the prediabetic period are also responsible for autoimmune graft rejection.     

Pancreatic cytokeratin: an antigen of pancreatic exocrine cell autoantibodies in Type 1 (insulin-dependent) diabetes mellitus

Summary Autoantibodies reacting with human pancreatic exocrine cells were investigated by immunofluorescent techniques in 107 patients with Type 1 (insulin-dependent) diabetes mellitus, 20 first-degree relatives of the Type 1 diabetic patients, 347 patients with Type 2 (non-insulin-dependent) diabetes, 34 with alcoholic pancreatitis, 26 with rheumatoid arthritis and 107 normal control subjects. Both immunoblotting analysis and double-immunostaining methods were used to characterize the antigens targeted by the pancreatic exocrine cell autoantibodies. Sera positive for human pancreatic exocrine cell cytoplasm, producing a fine fibrillar pattern, were found in 21% (23/107) of the Type 1 diabetic patients. The autoantibodies were present in 39% (15/38) of Type 1 diabetic patients diagnosed within 3 months, and the prevalence decreased with duration of diabetes. The antibodies were of the IgM class in 87% (13/15) of recent-onset Type 1 diabetes cases, but IgG-autoantibodies became more prevalent with increasing duration of diabetes. Three out of 347 (0.9%) Type 2 diabetic patients and 4 of 20 (20%) first-degree relatives of Type 1 diabetic patients had autoantibodies targeted against pancreatic exocrine cells. None of the patients with alcoholic pancreatitis or rheumatoid arthritis and none of the control subjects had these antibodies. Immunoblotting analysis and double-immunostaining demonstrated that the autoantibodies reacted with 40 kilodalton cytokeratin in pancreatic exocrine cell cytoplasm. The antibody was absorbed by the Triton X-100-insoluble fraction of pancreatic extract. These results indicate the presence of distinct autoantibodies to pancreatic exocrine cells in Type 1 diabetes. This suggests the provocative concept that the cytoskeletal system of pancreatic exocrine cells is involved in the pathogenetic process of Type 1 diabetes.     

Diet can influence the ability of nicotinamide to prevent diabetes in the non‐obese diabetic mouse: a preliminary study

Background

The non‐obese diabetic (NOD) mouse is a widely used model of Type 1 diabetes mellitus (Type 1 DM), which displays many of the characteristics of the disease found in humans. Nicotinamide (NA) is currently being tested in large‐scale, multi‐centre human trials for the prevention of Type 1 DM in subjects considered ‘at risk’ of developing the disease. Human trial populations will certainly differ in their dietary patterns and alterations were made to the diet given to NOD mice to determine if this could alter the effect of NA administration on Type 1 DM incidence.

Methods

The effect of NA in the diet was examined, both with and without carbohydrate in the form of a sucrose supplement, on diabetes incidence and insulitis levels in the NOD mouse. The effects of NA and sucrose were each tested alone as well as in combination.

Results

Diabetes was unaltered using a low dose NA‐supplemented diet (625 mg/kg diet). Diabetes incidence was also unaltered using unmodified diet together with drinking water supplemented with either 5% or 10% w/v sucrose or plain water for controls. However, with mice given NA‐supplemented diet (625 mg/kg diet) together with sucrose‐supplemented or plain water as previously, diabetes was reduced in the NA+10% sucrose group (p<0.001). Finally, a higher dose of NA was given in supplemented diet (1000 mg/kg). Again, neither sucrose nor NA alone altered the incidence of diabetes, but NA treatment combined with a 10% w/v sucrose‐supplemented drinking water reduced diabetes incidence (p<0.001). No mice showed alterations in insulitis, blood‐glucose or insulin levels with respect to controls.

Conclusion

Altering dietary patterns using sucrose can affect the ability of NA to prevent diabetes in the NOD mouse. This finding may be relevant for human studies with NA aimed at preventing Type 1 DM and suggests that diet may need to be monitored or even controlled in these studies. Copyright © 1999 John Wiley & Sons, Ltd.