Diabetes-prone BB Rats Research Articles

Defects in differentiation of bone-marrow derived dendritic cells of the BB rat are partly associated with IDDM2 (the lyp gene) and partly associated with other genes in the BB rat background

BB rats develop various organ-specific autoimmune diseases, e.g. autoimmune diabetes and thyroiditis and have proven important to dissect genetic factors that govern autoimmune disease development. The lymphopenia ( lyp) gene ( iddm2) is linked to autoimmune disease development and is a major genetic difference between diabetes-resistant (DR) and diabetes-prone (DP) BB rats. To study the effects of the lyp gene and other genes on dendritic cell (DC) differentiation from bone-marrow precursors, such differentiation was studied in BB-DP, BB-DR, Wistar and F344 control rats. DC of BB-DP rats showed a lower MHC class II expression as compared to BB-DR, Wistar and F344 rats. LPS-maturation did not restore this low MHC class II expression. DC of BB-DP rats also showed a poor capability to terminally differentiate into mature T cell stimulatory DC under the influence of LPS and produced significantly lower quantities of IL-10, yet these aberrancies were also found in BB-DR rats but did not occur in control rats. This study thus shows that various aberrancies exist in the differentiation of myeloid DC from bone-marrow precursors in the BB rat model of organ-specific autoimmunity. These aberrancies are multigenically determined and partly associated with iddm2 ( lyp gene) and partly associated with other genes in the BB rat.

Diabetes-prone BioBreeding rats do not have a normal immune response when weaned to a diet containing fermentable fibre

Diet is known to modulate the development of diabetes in diabetes-prone BioBreeding (BBdp) rats. The objective of the present study was to determine the effect of fermentable fibre (FF) on immune function in BBdp and diabetes-resistant BioBreeding (BBdr) rats after weaning. Weanling BBdp (thirty-six to thirty-eight per diet) and BBdr rats (thirty to thirty-two per diet) were fed a nutritionally complete, semi-purified, casein-based diet containing either cellulose (control diet, 8 % w/w) or FF (3.2 % cellulose+4.8 % w/w inulin). At 35 d, the small intestine was excised and lymphocytes isolated from spleen, mesenteric lymph nodes and Peyer's patches. Feeding FF to both BBdr and BBdp rats affected the production of anti-inflammatory cytokines (P=0.02). In BBdr rats, feeding FF compared with cellulose resulted in an increased small intestinal length (P=0.0031), higher proliferative (stimulation) index from both splenocytes (P=0.001) and mesenteric lymph nodes (P=0.04), and an increased proportion of CD8+ T-cells in the Peyer's patches (P=0.003). We did not observe an effect of diet on the number of IgA-bearing cells in the jejunum from BBdr rats. Feeding FF to BBdp rats did not affect the same parameters. BBdp rats had both a higher proportion of B-cells in the Peyer's patches (P=0.01) and a higher number of IgA+ cells in the jejunum (P=0.0036) when fed a diet containing FF, a response not observed in BBdr rats. We demonstrate that several aspects of the BBdp immune system respond differently than that of BBdr rats when challenged at weaning with FF.

Neonatal Nutritional Interventions in the Prevention of Type 1 Diabetes

After completing this article, readers should be able to: 1. Outline the current concepts of the pathogenesis of type 1 diabetes (T1D) with regard to dietary triggers and genetic susceptibility. 2. Discuss the evidence, both epidemiologic and that derived from studies of animal models, that supports the association between infant feeding practices and T1D. 3. Describe the roles for intestinal immunity and permeability in supporting the hypothetical model of cow milk protein mediation of T1D autoimmunity. 4. Delineate the goals and rationale for prospective clinical trials evaluating neonatal nutrition and the development of T1D. 5. List the putative dietary modulators of T1D. Type 1 diabetes (T1D), a disease that has unacceptably high morbidity and mortality, is increasing in incidence, prompting the redoubling of efforts toward its prevention. Progress toward prevention and cure relies on elucidation of the disease’s pathogenesis, which, to date, has remained poorly defined. The defining features of T1D, insulin deficiency and hyperglycemia, result from an immune-mediated destruction of insulin-secreting beta cells in the pancreatic islets. The loss of beta cell mass is believed to be gradual for most individuals, accounting for the sometimes prolonged asymptomatic periods of autoimmunity preceding overt diabetes (Fig. 1). Indeed, the chronic autoimmune nature of the disease is well established, as is the genetic predisposition. Specific associations with molecules of the human lymphocyte antigen (HLA) define both susceptibility to and protection from T1D. However, T1D is a polygenic disorder with more than 20 loci associated with susceptibility or resistance to the disease, of which the HLA may account for less than 50% of the genetic predisposition. Genetics clearly comprises a major component of the development of T1D, but the interaction between the environment and the immune system abnormalities is believed to weigh heavily in disease development. Indeed, trends in T1D incidence, both geographic and temporal, suggest a strong …

Tubular complexes as a source for islet neogenesis in the pancreas of diabetes-prone BB rats

Tubular complexes (TC) in the pancreas contain duct-like structures with low cuboidal or flattened cells surrounding a large lumen and are thought to be a response to pancreatic injury. TC have been studied in animal models of chemical or surgically induced pancreatic damage but their occurrence has not been reported in rodent models of spontaneous autoimmune type I diabetes. We hypothesized that TC would be increased during the active phase of islet destruction in autoimmune diabetes and could contain islet progenitor cells. We analyzed TC in pancreas of Wistar Furth (WF), control (BBc) and diabetes-prone BioBreeding (BBdp) rats using immunohistochemistry and morphometry. TC were observed in all rat strains during active pancreas remodeling (∼13 days). They increased between 60 and 93 days only in BBdp rats coincident with the increase in diabetes cases. Most TC were infiltrated with CD3+ T-cells. Duct-like cells in the TC had low expression of the exocrine marker amylase, increased expression of epithelial cell markers, keratin and vimentin, and remarkably high cell proliferation and cell death. TC islets contained cells stained positive for insulin, glucagon, somatostatin, pancreatic polypeptide, as well as PDX-1, chromogranin, and hepatocyte-derived growth factor receptor, c-met. Transitional cells that were keratin+/insulin+ and keratin+/amylase+ cells were present in TC. The stem cell marker, nestin was upregulated in the TC region. Duct-like cells in TC of BBdp rats expressed markers of committed endocrine precursors: PDX-1, neurogenin 3 and protein gene product 9.5. This study demonstrates that TC are upregulated during β-cell destruction and contain potential endocrine progenitors.

Gut Permeability and Intestinal Mucins, Invertase, and Peroxidase in Control and Diabetes-Prone BB Rats Fed Either a Protective or a Diabetogenic Diet

This study deals with the enteropathy recently identified in diabetes-prone BB rats (BBdp). Diabetes-resistant BB rats (BBc) and BBdp rats were fed from days 32-39 onward either a protective diabetes-retardant hydrolyzed casein diet (HC) or a plant-based diabetogenic (NTP) diet. The NTP diet decreased body weight and plasma insulin in BBc and BBdp rats. The BBdp rats displayed low intestinal invertase and increased intestinal peroxidase activity. In the BBdp rats fed the HC diet, the mucin content 30-35 cm below the pylorus was higher and the gut permeability lower than in the other three rat groups. There was a significant inverse correlation between gut permeability and the insulinogenic index in the BBdp rats fed the HC or NTP diet. Thus, in BBdp rats, the HC diet somehow prevents the increase in gut permeability and the decrease in the insulinogenic index otherwise found in some of these diabetes-prone animals.

Quantitative and qualitative alterations of intestinal mucins in BioBreeding rats

In diabetes-prone BioBreeding rats, an enteropathy often precedes the onset of auto-immune insulitis. The present study draws attention to quantitative and qualitative alterations of intestinal mucins in this animal model of type 1 diabetes mellitus. Male and female diabetes-resistant (BBc) and diabetes-prone (BBdp) BioBreeding rats fed, from one to two weeks after weaning onwards, either a plant-based diabetes-promoting diet (NTP) or a hydrolysed casein diabetes-protective diet (HC), were sacrificed at 11-14 weeks of age. Proteins and total mucins, as well as acid and neutral mucins, were measured in a segment of the intestinal tract, located 25-30 cm below the pylorus. No significant difference between BBc and BBdp rats was found when fed the HC diet. However, the NTP diet lowered both total and neutral mucins, whilst failing to affect significantly acidic mucins. The effects of the NTP diet were more pronounced in BBdp rats than in BBc rats. It is speculated that the quantitative and qualitative changes evoked by the NTP diet in BBdp rats may play a role in the alteration of gut permeability found under the same experimental conditions.

Insulin-dependent diabetes and gut dysfunction: the BB rat model.

Accumulating data indicate that intestinal dysfunction and dysregulation of the gut immune system may play a role in the development of type 1 diabetes. This review deals with the occurrence of gut damage and dysfunction in BB rats, an animal model of spontaneous immune type 1 diabetes, placing special emphasis on the effect of diet on the incidence of diabetes in BB rats, the identification of a type 1 diabetes-related protein from wheat, and preliminary observations documenting anomalies in the inductive tissues of the gut immune system (Peyer's patch cells and mesenteric lymph node cells) and pancreatic lymph node cells of diabetes-prone BB rats. In addition to histological evidence of gut damage, the review will also draw attention to altered intestinal disaccharidase activity, changes in intestinal peroxidase activity, glucagon-like peptide 1 anomalies, and perturbation of both intestinal permeability and mucin content in BB rats. In all these cases, the findings in rats fed a diabetes-promoting diet are compared to those collected in animals receiving a protective diabetes-retardant diet.

Partial activation precedes apoptotic death in T cells harboring an IAN gene mutation.

The Biobreeding diabetes-prone rat suffers from a profound peripheral lymphopenia and yet succumbs to a T cell-dependent autoimmune disease. Lymphopenia segregates with a mutated chromosomal locus, termed lyp, recently identified as a frameshift mutation in IAN4. Others have correlated loss of IAN4 function with decreased mitochondrial integrity resulting in T cell apoptosis. Here we report that IAN4-/- T cells enter a state similar to that of partial activation wherein they down-regulate CD62L and undergo incomplete blasting yet do not progress through mitosis. When given a strong stimulus, this partial activation phenotype can be overcome. This phenotype can be recapitulated in wild-type T cells through suboptimal stimulation. The phenotype is not simply a reaction to the lymphopenic environment, as spontaneous CD62L down-regulation occurs in mature single-positive medullary thymocytes that develop within a non-lymphopenic environment, and normal T cells do not undergo similar blasting when parked in a lymphopenic environment. Finally, we show that IAN4-/- T cells are more readily triggered via TCR stimulation. Thus, in addition to their role in apoptosis, IAN family members may also play a role in regulating the T cell activation state through modulation of TCR signaling strength.

Changes in expression of IL-1 beta influenced proteins in transplanted islets during development of diabetes in diabetes-prone BB rats.

Type 1 diabetes mellitus is a multifactorial autoimmune disease characterised by selective destruction of beta cells in the islets of Langerhans. We have previously shown that IL-1 beta modulates beta cell function, causes beta cell death and induces expression changes in 82 out of 1815 protein spots detected by two-dimensional gel electrophoresis (2-DGE) in diabetes-prone bio-breeding (BB-DP) rat islets in vitro. The aim of this study was to describe the relevance of these proteins in the development of diabetes in vivo. Syngeneic neonatal islets ( n=200) were transplanted under the kidney capsule of 30-day-old BB-DP and control rats, removed to different time points after transplantation or at the onset of diabetes, and metabolically labelled with S(35)-methionine for 2-DGE. The 82 proteins were re-localised and followed. In addition, transplants were examined for expression of IL-1 beta mRNA by in situ hybridisation. All 82 proteins could be re-localised in all syngeneic transplants from BB-DP and control rats. A total of 60 of the 82 proteins were changed during development of diabetes. Of the 82 proteins, 32 were changed in expression at the onset of diabetes compared to non-diabetic BB-DP rats, and 25 of these were changed as by IL-1 beta in vitro. Highest expression of IL-1 beta mRNA was found at the onset of diabetes. IL-1 beta-induced protein expression changes in islets in vitro also occur in vivo and change in a complex pattern during the development of diabetes in the BB-DP rat. No single protein seems to be responsible for the development of diabetes, but rather the cumulative numbers of changes seem to interfere with the intracellular stability of the beta cell.

Animal models of spontaneous autoimmune diabetes: notes on their relevance to the human disease.

The spontaneous autoimmune diabetes of the nonobese diabetic mouse and the BioBreeding diabetes-prone rat bears a striking similarity to human type 1 diabetes. For this reason it comes as a disappointment that interventions known to prevent the disease in the animal models proved ineffective in human trials. For an explanation I propose that, although the mechanism of beta-cell destruction is the same in the three species, this common mechanism may be the end point of different loss-of-tolerance pathways in different human patients, only two of which are represented by the two animal models. Research strategies to address this problem will require more attention to immune subphenotypes or their corresponding subgenotypes in the human and, possibly, the generation of additional mouse models of spontaneous disease.

Disaccharidase activity in the intestinal tract of Wistar-Furth, diabetes-resistant and diabetes-prone BioBreeding rats.

Diabetes-prone BioBreeding (BBdp) rats often present an enteropathy that may precede the onset of autoimmune insulitis. The aim of the present study was to assess the influence of sex, the time course, the strain specificity, the distribution along the intestinal tract and the effect of diet for the changes in the activity of gut invertase, maltase and lactase found in BBdp rats, as compared with both Wistar-Furth (WF) and diabetes-resistant BioBreeding (BBc) rats. These hydrolases were measured, therefore, at day 10, 30, 45, 70, 95 and 120 in three intestinal segments of WF, BBc and BBdp rats fed, after weaning, either a protective hydrolysed casein diet, which decreases the incidence of diabetes in the BBdp rats, or one of two diabetogenic diets (National Toxicology Program; NTP or wheat-gluten-based; WG) [corrected]. Except for a somewhat lower lactase activity in the BBdp rats, no obvious difference in hydrolyase activity between the three strains of rats was observed at day 10. Between days 30 and 120, however, the activity of the hydrolases, especially that of invertase and lactase, was lower in the BBdp rats than in either the WF or BBc rats, at least when considering the animals fed either the NTP or WG diet. These findings support the view that BBdp rats exposed to a diabetogenic diet develop an enteropathy well before the onset of autoimmune insulitis, in a manner somehow comparable with the situation found in some type 1 diabetic patients, in whom coeliac disease may be diagnosed before diabetes onset.

Haplotype tag single nucleotide polymorphism analysis of the human orthologues of the rat type 1 diabetes genes Ian4 (Lyp/Iddm1) and Cblb.

The diabetes-prone BioBreeding (BB) and Komeda diabetes-prone (KDP) rats are both spontaneous animal models of human autoimmune, T-cell-associated type 1 diabetes. Both resemble the human disease, and consequently, susceptibility genes for diabetes found in these two strains can be considered as potential candidate genes in humans. Recently, a frameshift deletion in Ian4, a member of the immune-associated nucleotide (Ian)-related gene family, has been shown to map to BB rat Iddm1. In the KDP rat, a nonsense mutation in the T-cell regulatory gene, Cblb, has been described as a major susceptibility locus. Following a strategy of examining the human orthologues of susceptibility genes identified in animal models for association with type 1 diabetes, we identified single nucleotide polymorphisms (SNPs) from each gene by resequencing PCR product from at least 32 type 1 diabetic patients. Haplotype tag SNPs (htSNPs) were selected and genotyped in 754 affected sib-pair families from the U.K. and U.S. Evaluation of disease association by a multilocus transmission/disequilibrium test (TDT) gave a P value of 0.484 for IAN4L1 and 0.692 for CBLB, suggesting that neither gene influences susceptibility to common alleles of human type 1 diabetes in these populations.

A dithiocarbamate analogue decreases intraislet cell infiltration and the incidence of diabetes mellitus in the genetic diabetes-prone BB rat.

Dithiocarbamates are a class of agents that have interesting biologic properties including the ability to limit the production and/or action of nitric oxide (NO). These agents are also potential immunosuppressant agents. Since immunosuppressant agents have been examined for remission of disease in clinical trials, we wanted to examine whether a dithiocarbamate analogue, NOX-200, might inhibit diabetogenesis in the genetic diabetes-prone BB rat model. Immunohistochemical analysis revealed inducible NO synthase (iNOS) gene expression in pancreatic islets of both normoglycemic and hyperglycemic diabetes-prone BB rats but not in diabetes-prone BB rats at the early age of 30 days or in diabetes-resistant BB rats. A qualitative decrease in immunostaining for iNOS was also observed in the pancreata of drug-treated animals. Long-term treatment with NOX-200, used alone or in combination with low-dose cyclosporine (CsA), significantly reduced the incidence of diabetes mellitus. In the subset of animals that became diabetic, NOX-200 did not alter either the time to onset of hyperglycemia or the level of hyperglycemia, insulinopenia, or lymphocytic cell infiltration into the pancreas. In contrast, in animals that did not develop hyperglycemia, treatment with NOX-200 decreased inflammatory cell infiltration into the pancreas equipotent to that seen using CsA. These studies demonstrate the potential therapeutic efficacy of dithiocarbamates to oppose the development of autoimmune insulin-dependent diabetes mellitus by limiting inflammatory cell activation/infiltration.

Bioactive GLP-1 in gut, receptor expression in pancreas, and insulin response to GLP-1 in diabetes-prone rats.

Glucagon-like peptide-1 (GLP-1) is the most insulinogenic of the glucagon-like peptides secreted mainly by L cells in the small and large intestine in response to the ingestion of nutrients. It binds to a specific GLP-1 receptor (GLP-1R) on beta-cells and can increase islet neogenesis and beta-cell mass. It is not clear whether the transmission of information from the gut to islet beta-cells by messengers such as GLP-1 is different in individuals who develop autoimmune diabetes. In the present study the expression of bioactive GLP-1 protein in the gut and its receptor in the pancreas was examined in diabetes-prone BioBreeding (BBdp) rats in the period before overt diabetes and in age-matched control, non-diabetes-prone BB (BBc) rats. An N-terminal directed antibody specific for the bioactive forms of GLP-1 (GLP-1(7-37) and GLP-1(7-36amide)) was used to mea-sure GLP-1 by radioimmunoassay in proximal, median, and distal gut. Pancreas GLP-1R area fraction, GLP-1R gene expression, and insulin content were analyzed, as were plasma GLP-1, glucose, and insulin. The concentration of GLP-1 protein in the jejunum and ileum of BBdp rats was lower than in BBc rats. Although these animals maintained normal blood glucose, there was impaired pancreatic endocrine function, characterized by low baseline insulin concentration in plasma and pancreas. GLP-1R mRNA expression was threefold less in islets isolated from BBdp rats, and GLP-1R+ islet area fraction in pancreas sections was decreased. When injected iv with GLP-1, BBdp rats displayed lower second-phase insulin response (and insulin/glucose ratios) compared with BBc rats. Thus, young BBdp rats displayed decreased concentrations of bioactive GLP-1 in jejunum and ileum, reduced GLP-1R in islets, and lower second-phase insulin response to iv GLP-1 than controls. The decrease in insulinogenic and islet beta-cell mass-promoting signal from GLP-1 in BBdp rats may contribute to impaired glucoregulation and ineffective maintenance of normal islet mass that shifts islet homeostasis in favor of development of diabetes.

T‐Cell Survival Regulator LKLF Is Not Involved in Inappropriate Apoptosis of Diabetes‐Prone BBDP Rat T Cells

Diabetes-prone BB (dpBB) rats develop autoimmune insulin-dependent diabetes mellitus (IDDM) at high frequency as a consequence of a defect in T cell development, caused by a mutation in a single gene locus on rat chromosome 4 (lyp) which has recently been identified as immune-associated nucleotide 4 (ian4). A phenotype similar to dpBB rat lymphopenia has recently been described in the mouse as the result of the targeted inactivation of the gene for the transcription factor LKLF (Lung Krüppel-like factor, KLF2) in the immune system. We cloned the LKLF gene of the rat and screened a panel of rat/hamster radiation hybrid cell lines to determine its chromosomal localization. We conclude that the LKLF gene is not defective in dpBB rats and that its expression is not compromised by the lyp mutation.

The diabetes prone BB rat model of IDDM shows duration of breastfeeding to influence Type 1 diabetes development later in life.

The diabetes prone BB rat model of IDDM shows duration of breastfeeding to influence Type 1 diabetes development later in life.

Long-term prophylactic insulin treatment can prevent spontaneous diabetes and thyroiditis development in the diabetes-prone bio-breeding rat, while short-term treatment is ineffective.

Prophylactic insulin treatment has been demonstrated to reduce diabetes development in the diabetes-prone bio-breeding (DP-BB) rat. These prophylactic insulin treatments were given from 50 to 150 days of age. However, several data indicate that the diabetogenic process in DP-BB rats starts well before day 50. DP-BB rats were given bovine insulin pellets from 21 to 60 days of age, from 21 to 100 days of age and from 60 to 100 days of age. At 160 days of age a glucose tolerance test was performed to establish beta-cell function and pancreata collected for histological analysis. Prophylactic insulin treatment from 21 to 100 days of age gave a 42% reduction of diabetes incidence. The other treatment protocols had no effect. Non-diabetic rats treated with insulin from day 21 to 100 showed normal glucose tolerance and no sign of insulitis at 160 days of age. Non-diabetic rats of the control group and the other treatment groups showed normal glucose tolerance, but a slight increase of insulitis. Interestingly, the 21-100 day treated rats showed reduced serum levels of anti-colloid antibodies as compared with the control group. These results show that short-term prophylactic insulin treatment cannot prevent diabetes and thyroiditis development in DP-BB rats. The prophylactic treatment must start well before 60 days of age and be prolonged into the phase when the rats normally become diabetic to reduce diabetes incidence. These findings imply that in the human situation prophylactic insulin treatment must be prolonged over the normal range of diabetes onset.

IL-1β, IFN-γ and TNF-α increase vulnerability of pancreatic beta cells to autoimmune destruction

In the pathogenesis of type-1 diabetes insulin-producing β-cells are destroyed by cellular autoimmune processes. The locality of β-cell destruction is the inflamed pancreatic islet. During insulitis cytokines released from islet-infiltrating mononuclear cells affect β-cells at several levels. We investigated whether cytokine-induced β-cell destruction is associated with changes in the expression of the surface receptors intercellular adhesion molecule (ICAM)-1 and Fas. Islets from diabetes-prone and congenic diabetes-resistant BB rats were exposed to interleukin (IL)-1β alone or in combination with interferon (IFN)-γ plus tumour necrosis factor (TNF)-α. Cytokines decreased islet insulin content, suppressed glucose stimulated insulin secretion and generated enhanced amounts of nitric oxide and DNA-strand breaks. While no membrane alterations of IL-1β treated islets cells were detectable, the cytokine combination caused damage of cell membranes. Independent of diabetes susceptibility IL-1β treated islet β-cells expressed a significantly increased amount of ICAM-1 on their surfaces which was not further increased by IFN-γ+TNF-α. However, IL-1β induced Fas expression was significantly enhanced only on β-cells from diabetes-prone BB rats. From these results we suggest that IL-1β mediates the major stimulus for ICAM-1 induction which is possibly a necessary but not sufficient step in the process of β-cell destruction. Obviously, the additional enhancement of Fas expression on the surface of β-cells is important for destruction. The combined action of all three cytokines induced the expression of Fas on the β-cell surface independent of diabetes susceptibility, indicating that such a strong stimulus in vitro may induce processes different from the precise mechanisms of β-cell destruction in vivo.

Role of the thymus in the development of tolerance and autoimmunity towards the neuroendocrine system.

The thymus is the unique lymphoid organ inside which a confrontation occurs throughout life between neuroendocrine self-antigens and a recently evolved system with original recombination machinery driving random generation of immune response diversity. Through transcription of neuroendocrine genes in the thymus stromal network and expression of cognate receptors by immature T cells, the neuroendocrine system regulates early T cell differentiation. In addition and more specifically, intrathymic presentation of neuroendocrine self-antigens by, or in close association with, major histocompatibility complex (MHC) proteins is responsible for the establishment of central immune self-tolerance of neuroendocrine principles. All members of the insulin gene (INS) family are expressed in the thymus stroma according to a precise hierarchy and cell topography: IGF2 (thymic epithelial cells) > IGF1 (thymic macrophages) >> INS (thymic medullary epithelial cells and/or dendritic cells). Given this hierarchical pattern in gene expression, the protein IGF-2 is more tolerated than INS. Igf2 transcription is defective in the thymus of bio-breeding (BB) rat, one animal model of type 1 diabetes (T1DM). This thymus-specific defect in Igf2 expression may explain both the absence of central tolerance to INS-secreting beta cells and the lymphopenia (including lack of regulatory RT6(+) T cells) in diabetes-prone BB rats. INS B:9-23 and the homologous sequence of IGF-2 compete for binding to DQ8, an MHC class II allele conferring major susceptibility to T1DM. In young DQ8(+) T1DM patients, INS B:9-23 presentation by DQ8 elicits a dominant IFN-gamma secretion by isolated PBMCs, whereas presentation of the IGF-2 self-antigen promotes a dominant regulatory interleukin-10 secretion. These data demonstrate that opposite immune responses are driven by MHC presentation of a self-antigen (here, IGF-2) and an autoantigen (INS, as "altered" self). The important tolerogenic properties of thymic self-antigens deserve now to be exploited for prevention and/or cure of devastating autoimmune diseases such as T1DM.

Necrosis Is the Predominant Type of Islet Cell Death During Development of Insulin-Dependent Diabetes Mellitus in BB Rats

Several reports propose that apoptosis of pancreatic beta cells may play a central role in the pathogenesis of both spontaneous and induced insulin-dependent diabetes mellitus (IDDM) in animal models. Whether apoptosis is a major cell death pathway during diabetes development, however, is highly controversial. The aim of this study was to examine the mode of islet cell death in prediabetic diabetes-prone (dp) BB rats, which spontaneously develop diabetes and serve as an animal model for human IDDM. In addition we investigated the cell death pathway of islet cells treated with the widely used diabetogenic compound streptozotocin or with nitric oxide (NO), which during IDDM development has been found to be present in inflamed islets in high concentrations because of the expression of inducible NO synthase. Islets of prediabetic BBdp rats were analyzed for DNA strand breaks and screened by electron microscopy. The mode of islet cell death in vitro after treatment with cytotoxic concentrations of streptozotocin or of NO was investigated using different methods including morphologic analysis by electron microscopy, detection of DNA strand breaks, poly(ADP-ribose) polymerase cleavage, and annexin V staining. Although cells with DNA stand breaks—often accepted as a proof for apoptosis—could be identified, we did not find apoptosis-specific features during islet cell death. Instead we observed massive necrosis as evidenced by disrupted plasma membranes and spilled-out cellular constituents in vitro as well as during disease manifestation in BBdp rats. These results may have serious consequences with regard to the treatment of humans to prevent the development of IDDM.