DR-BB Rats Research Articles

GIMAP GTPase family genes: potential modifiers in autoimmune diabetes, asthma, and allergy.

GTPase of the immunity-associated protein (GIMAP) family members are differentially regulated during human Th cell differentiation and have been previously connected to immune-mediated disorders in animal studies. GIMAP4 is believed to contribute to the Th cell subtype-driven immunological balance via its role in T cell survival. GIMAP5 has a key role in BB-DR rat and NOD mouse lymphopenia. To elucidate GIMAP4 and GIMAP5 function and role in human immunity, we conducted a study combining genetic association in different immunological diseases and complementing functional analyses. Single nucleotide polymorphisms tagging the GIMAP haplotype variation were genotyped in Finnish type 1 diabetes (T1D) families and in a prospective Swedish asthma and allergic sensitization birth cohort. Initially, GIMAP5 rs6965571 was associated with risk for asthma and allergic sensitization (odds ratio [OR] 3.74, p = 0.00072, and OR 2.70, p = 0.0063, respectively) and protection from T1D (OR 0.64, p = 0.0058); GIMAP4 rs13222905 was associated with asthma (OR 1.28, p = 0.035) and allergic sensitization (OR 1.27, p = 0.0068). However, after false discovery rate correction for multiple testing, only the associations of GIMAP4 with allergic sensitization and GIMAP5 with asthma remained significant. In addition, transcription factor binding sites surrounding the associated loci were predicted. A gene-gene interaction in the T1D data were observed between the IL2RA rs2104286 and GIMAP4 rs9640279 (OR 1.52, p = 0.0064) and indicated between INS rs689 and GIMAP5 rs2286899. The follow-up functional analyses revealed lower IL-2RA expression upon GIMAP4 knockdown and an effect of GIMAP5 rs2286899 genotype on protein expression. Thus, the potential role of GIMAP4 and GIMAP5 as modifiers of immune-mediated diseases cannot be discarded.

Iddm30 Controls Pancreatic Expression of Ccl11 (Eotaxin) and the Th1/Th2 Balance within the Insulitic Lesions

The autoimmune diabetic syndrome of the BioBreeding diabetes-prone (BBDP) rat is a polygenic disease that resembles in many aspects human type 1 diabetes (T1D). A successful approach to gain insight into the mechanisms underlying genetic associations in autoimmune diseases has been to identify and map disease-related subphenotypes that are under simpler genetic control than the full-blown disease. In this study, we focused on the β cell overexpression of Ccl11 (Eotaxin), previously postulated to be diabetogenic in BBDR rats, a BBDP-related strain. We tested the hypothesis that this trait is genetically determined and contributes to the regulation of diabetes in BBDP rats. Similar to the BBDR strain, we observed a time-dependent, insulitis-independent pancreatic upregulation of Ccl11 in BBDP rats when compared with T1D-resistant ACI.1u.lyp animals. Through linkage analysis of a cross-intercross of these two parental strains, this trait was mapped to a region on chromosome 12 that overlaps Iddm30. Linkage results were confirmed by phenotypic assessment of a novel inbred BBDP.ACI-Iddm30 congenic line. As expected, the Iddm30 BBDP allele is associated with a significantly higher pancreatic expression of Ccl11; however, the same allele confers resistance to T1D. Analysis of islet-infiltrating T cells in Iddm30 congenic BBDP animals revealed that overexpression of pancreatic Ccl11, a prototypical Th2 chemokine, is associated with an enrichment in Th2 CD4+ T cells within the insulitic lesions. These results indicate that, in the BBDP rat, Iddm30 controls T1D susceptibility through both the regulation of Ccl11 expression in β cells and the subsequent Th1/Th2 balance within islet-infiltrating T lymphocytes.

Pathological endoplasmic reticulum stress mediated by the IRE1 pathway contributes to pre-insulitic beta cell apoptosis in a virus-induced rat model of type 1 diabetes

We hypothesised that pathological endoplasmic reticulum (ER) stress contributes to beta cell death during development of type 1 diabetes. In this study, we investigated the occurrence of beta cell ER stress and the signalling pathways involved during discrete stages of autoimmune diabetes progression. The virus-inducible BBDR rat model was used to systematically interrogate the three main ER stress signalling pathways (IRE1 [inositol-requiring protein-1], PERK [double-stranded RNA-dependent protein kinase (PKR)-like ER kinase] and ATF6 [activating transcription factor 6]) in pancreatic beta cells during type 1 diabetes development. ER stress and apoptotic markers were assessed by immunoblot analyses of isolated pancreatic islets and immunofluorescence staining of pancreas sections from control and virus-induced rats. Various time points were analysed: (1) early stages preceding the development of insulitis and (2) a late stage during onset and progression of insulitis, which precedes overt hyperglycaemia. The IRE1 pathway, including its downstream component X-box-binding protein 1, was specifically activated in pancreatic beta cells of virus-induced rats at early stages preceding the development of insulitis. Furthermore, ER stress-specific pro-apoptotic caspase 12 and effector caspase 3 were also activated at this stage. Activation of PERK and its downstream effector pro-apoptotic CHOP (CCAAT/-enhancer-binding-protein homologous protein), only occurred during late stages of diabetes induction concurrent with insulitis, whereas ATF6 activation in pancreatic beta cells was similar in control and virus-induced rats. Activation of the IRE1 pathway and ER stress-specific pro-apoptotic caspase 12, before the development of insulitis, are indicative of ER stress-mediated beta cell damage. The early occurrence of pathological ER stress and death in pancreatic beta cells may contribute to the initiation and/or progression of virus-induced autoimmune diabetes.

Temporal induction of immunoregulatory processes coincides with age-dependent resistance to viral-induced type 1 diabetes

The dilute plasma cytokine milieu associated with Type 1 diabetes (T1D), while difficult to measure directly, is sufficient to drive transcription in a bioassay that uses healthy leukocytes as reporters. Previously, we reported disease-associated, partially IL-1 dependent, transcriptional signatures in both T1D patients and the BioBreeding (BB) rat model. Here we examine temporal signatures in congenic BBDR.lyp/lyp rats that develop spontaneous T1D, and BBDR rats where T1D progresses only after immunological perturbation in young animals. After weaning, the BBDR temporal signature showed early coincident induction of transcription related to innate inflammation as well as IL-10- and TGF-β-mediated regulation. BBDR plasma cytokine levels mirrored the signatures showing early inflammation, followed by induction of a regulated state that correlated with failure of virus to induce T1D in older rats. In contrast, the BBDR.lyp/lyp temporal signature exhibited asynchronous dynamics, with delayed induction of inflammatory transcription and later, weaker induction of regulatory transcription, consistent with their deficiency in regulatory T cells. Through longitudinal analyses of plasma induced signatures in BB rats and a human T1D progressor, we have identified changes in immunoregulatory processes that attenuate a preexisting innate inflammatory state in BBDR rats, suggesting a mechanism underlying the decline in T1D susceptibility with age.

Variations in Rodent Models of Type 1 Diabetes: Islet Morphology

Type 1 diabetes (T1D) is characterized by hyperglycemia due to lost or damaged islet insulin-producing β-cells. Rodent models of T1D result in hyperglycemia, but with different forms of islet deterioration. This study focused on 1 toxin-induced and 2 autoimmune rodent models of T1D: BioBreeding Diabetes Resistant rats, nonobese diabetic mice, and Dark Agouti rats treated with streptozotocin. Immunochemistry was used to evaluate the insulin levels in the β-cells, cell composition, and insulitis. T1D caused complete or significant loss of β-cells in all animal models, while increasing numbers of α-cells. Lymphocytic infiltration was noted in and around islets early in the progression of autoimmune diabetes. The loss of lymphocytic infiltration coincided with the absence of β-cells. In all models, the remaining α- and δ-cells regrouped by relocating to the islet center. The resulting islets were smaller in size and irregularly shaped. Insulin injections subsequent to induction of toxin-induced diabetes significantly preserved β-cells and islet morphology. Diabetes in animal models is anatomically heterogeneous and involves important changes in numbers and location of the remaining α- and δ-cells. Comparisons with human pancreatic sections from healthy and diabetic donors showed similar morphological changes to the diabetic BBDR rat model.

769 Is There Somatic Hypersensitivity and Central Sensitization in Functional Dyspepsia (FD)?

Results: Compared to BBDR rats (n=5), LMMP preparations from normoglycemic BBDP rats (n=8) had higher MPO levels (0.0±0.0 vs. 5.4±2.2 U/mg, p<0.01), lower GRP78 mRNA expression (1.0±0.1 vs. 0.5±0.1, p<0.01) and lower PERK mRNA expression (1.0±0.1 vs. 0.4±0.2, p<0.05). A significant linear correlation was obserbed between mucosal and LMMP expression of GRP78 mRNA (p<0.01, r = 0.72; Figure1). In LMMP, a significant linear correlation was also observed between MPO levels and GRP78 mRNA expression (p<0.05, r = -0.65). Compared to BBDR rats, BBDP rats had lower mucosal GRP78 mRNA expression (1.0±0.1 vs. 0.4±0.1, p<0.001), lower IREα mRNA expression (1.0±0.2 vs. 0.5±0.1, p= 0.07), lower IREβ mRNA expression (1.0±0.2 vs. 0.3±0.2, p<0.01) and lower PERK mRNA expression (1.0±0.2 vs. 0.3±0.1, p<0.01). Conclusion: ER stress response was impaired in the inflamed intestine in BBDP-rats, both in mucosa and LMMP. Impaired ER stress response may contribute to the pathogenesis of increased permeability and intestinal dysmotility.

Imaging the pancreatic vasculature in diabetes models

Vascular parameters, such as vascular volume, flow, and permeability, are important disease biomarkers for both type 1 and type 2 diabetes. Therefore, it is essential to develop approaches to monitor the changes in pancreatic microvasculature non-invasively. Here, we describe the application of the long-circulating, paramagnetic T1 contrast agent, protected Graft Copolymer bearing covalently linked gadolinium diethylenetriaminepentaacetic acid residues and labelled with fluorescein (PGC-GdDTPA-F) for the non-invasive semi-quantitative evaluation of vascular changes in diabetic models using magnetic resonance imaging. We observed a significantly higher accumulation of protected graft copolymer bearing covalently linked gadolinium diethylenetriaminepentaacetic acid residues and labelled with fluorescein in the pancreata of BBDR rats induced to develop diabetes, as compared to non-diabetic controls at 1 h post-injection. No differences were seen in the blood pool, kidney, or muscle, indicating that the effect is specific to the diabetic pancreas. Fluorescence microscopy revealed a marked increase in contrast agent availability in the pancreas with the development of the pathology. Similar changes were noted in the homozygous Leprdb mouse model of type 2 diabetes. This effect appeared to result both from the increase of vascular volume and permeability. High-molecular weight paramagnetic blood volume contrast agents are valuable for the in vivo definition of pancreatic microvasculature dynamics by magnetic resonance imaging. The increase in vascular volume and permeability, associated with diabetic inflammation, can be monitored non-invasively and semi-quantitatively by magnetic resonance imaging in diabetic BBDR rats. This imaging strategy represents a valuable research tool for better understanding of the pathologic process.

Leptin treatment confers clinical benefit at multiple stages of virally induced type 1 diabetes in BB rats

The adipokine, leptin, regulates blood glucose and the insulin secretory function of beta cells, while also modulating immune cell function. We hypothesized that the dual effects of leptin may prevent or suppress the autoreactive destruction of beta cells in a virally induced rodent model of type 1 diabetes. Nearly 100% of weanling BBDR rats treated with the combination of an innate immune system activator, polyinosinic:polycytidylic acid (pIC), and Kilham rat virus (KRV) become diabetic within a predictable time frame. We utilized this model to test the efficacy of leptin in preventing diabetes onset, remitting new onset disease, and preventing autoimmune recurrence in diabetic rats transplanted with syngeneic islet grafts. High doses of leptin delivered via an adenovirus vector (AdLeptin) or alzet pump prevented diabetes in>90% of rats treated with pIC+KRV. The serum hyperleptinemia generated by this treatment was associated with decreased body weight, decreased non-fasting serum insulin levels, and lack of islet insulitis in leptin-treated rats. In new onset diabetics, hyperleptinemia prevented rapid weight loss and diabetic ketoacidosis, and temporarily restored euglycemia. Leptin treatment also prolonged the survival of syngeneic islets transplanted into diabetic BBDR rats. In diverse therapeutic settings, we found leptin treatment to have significant beneficial effects in modulating virally induced diabetes. These findings merit further evaluation of leptin as a potential adjunct therapeutic agent for treatment of human type 1 diabetes.

Differential effects of leptin receptor mutation on male and female BBDR.Gimap5−/Gimap5− spontaneously diabetic rats

Rodents homozygous for autosomal leptin receptor gene mutations not only become obese, insulin resistant, and hyperleptinemic but also develop a dysregulated immune system. Using marker-assisted breeding to introgress the Koletsky rat leptin receptor mutant (lepr-/lepr-), we developed a novel congenic BBDR.(lepr-/lepr-) rat line to study the development of obesity and type 2 diabetes (T2D) in the BioBreeding (BB) diabetes-resistant (DR) rat. While heterozygous lepr (-/+) or homozygous (+/+) BBDR rats remained lean and metabolically normal, at 3 wk of age all BBDR.(lepr-/lepr-) rats were obese without hyperglycemia. Between 45 and 70 days of age, male but not female obese rats developed T2D. We had previously developed congenic BBDR.(Gimap5-/Gimap5-) rats, which carry an autosomal frameshift mutation in the Gimap5 gene linked to lymphopenia and spontaneous development of type 1 diabetes (T1D) without sex differences. Because the autoimmune-mediated destruction of pancreatic islet beta-cells may be affected not only by obesity but also by the absence of leptin receptor signaling, we next generated BBDR.(lepr-/lepr-,Gimap5-/Gimap5-) double congenic rats carrying the mutation for Gimap5 and T1D as well as the Lepr mutation for obesity and T2D. The hyperleptinemia rescued end-stage islets in BBDR.(lepr-/lepr-,Gimap5-/Gimap5-) congenic rats and induced an increase in islet size in both sexes, while T1D development was delayed and reduced only in females. These results demonstrate that obesity and T2D induced by introgression of the Koletsky leptin receptor mutation in the BBDR rat result in islet expansion associated with protection from T1D in female but not male BBDR.(lepr-/lepr-,Gimap5-/Gimap5-) congenic rats. BBDR.(lepr-/lepr-,Gimap5-/Gimap5-) congenic rats should prove valuable to study interactions between lack of leptin receptor signaling, obesity, and sex-specific T2D and T1D.

Role of cytokines in the pathogenesis of autoimmune IDDM in KRV-infected DR-BB rats

Role of cytokines in the pathogenesis of autoimmune IDDM in KRV-infected DR-BB rats

DNA microarray analysis for the identification of innate immune pathways implicated in virus-induced autoimmune diabetes

We have recently demonstrated that upregulation of the innate immune system plays a key role in KRV-induced autoimmune diabetes in the BBDR rat, but the nature of this proinflammatory reaction has not yet been addressed. Using a DNA microarray approach, we identified 569 genes upregulated in pancreatic lymph nodes following virus infection. Among the most highly activated are IL-1 pathways, IFN-gamma-induced chemokines, and genes associated with interferon production and signaling. Ex vivo and in vitro studies indicate that KRV upregulates proinflammatory cytokines and chemokines in B lymphocytes and Flt-3L-induced plasmacytoid DCs (pDCs). Finally, in contrast to KRV, infection of BBDR rats with the non-diabetogenic KRV homologue H-1 parvovirus fails to induce a robust proinflammatory response in pancreatic lymph nodes. Our findings provide new insights into KRV-induced innate immune pathways that may play a role in early mechanisms leading to islet inflammation and diabetes.

Angiogenic sprouting from the aortic vascular wall is impaired in the BB rat model of autoimmune diabetes

Background Diabetes is associated with impaired neovascularization leading to reduced revascularization of ischemic tissue and impaired wound healing. Endothelial progenitor cells in diabetes were previously shown to be numerically reduced and functionally impaired. We hypothesize that diabetes also has a long-term effect on angiogenic cells residing in the vessel wall. To test this hypothesis, angiogenic sprout formation from ex vitro cultured aortic rings isolated from diabetic and non-diabetic BioBreeding (BB) rats was assessed. Methods Diabetes prone BB (BBDP) rats spontaneously develop autoimmune diabetes were suboptimally treated with insulin by subcutaneous implantation of slow-release insulin-pellets. Neonatally thymectomized BBDP rats, pre-diabetic BBDP rats and diabetes resistant BBDR rats served as non-diabetic controls. After follow-up thoracic aortas were harvested and cultured in vitro in Matrigel to induce sprout formation. Sprout length was quantified after 4, 7, 10 and 14 days of culture. The total number of sprout-derived cells was measured and in vitro proliferative capacity of sprout cells was quantified. Finally, expression of Flk-1, CD31 and smooth muscle α-actin on sprout cells was determined. Results Mean blood glucose levels in diabetics were significantly elevated compared with non-diabetics. Both long-term and short-term diabetes significantly reduced sprout formation ( p < 0.05 vs. non-diabetics). Reduced sprout length in diabetics was reflected by significantly reduced numbers of sprout cells that could be isolated ( p < 0.05 vs. non-diabetics). Isolated sprout cells from diabetics revealed significantly reduced proliferative capacity after in vitro culture ( p < 0.05 vs. non-diabetics). Immunofluorescent staining indicated an endothelial phenotype of both freshly isolated and in vitro cultured sprout cells as indicated by CD31 and Flk-1 expression and absence of smooth muscle α-actin expression. Conclusions Diabetes in BB rats impairs angiogenic sprouting from cells residing in the vascular wall, independent of effects on circulating cells or circulating angiogenic/anti-angiogenic factors. The angiogenic impairment of diabetic sprout cells is, to some extent, imprinted upon the cells.

Disordered Meiotic Regulation of Oocytes by Duration of Diabetes Mellitus in BBdp Rat

Diabetes mellitus (DM) disturbs normal functions from the level of cells to the level of organs. In this study, the authors explore the detrimental effects of type 1 diabetes on meiotic regulation depending on the duration of DM. In non-diabetes-prone BioBreeding (BBdr) control rats, most of the large follicles had germinal vesicle (GV)-intact oocytes. Conversely, a decrease of intact GV that was dependent on the duration of diabetic symptoms was observed; only 54% of the large follicles of diabetes-prone BB (BBdp) rats had GV-intact oocytes at 6 weeks after diabetes induction. Furthermore, some of the secondary follicles in BBdp rats also had germinal vesicle breakdown (GVB) oocytes. The nuclear status of the euglycemia BBdp rat was similar to those of the BBdr rat. In BBdp rats, the rate of meiotic progression to the metaphase II stage was significantly lower; however, the rate of segregated oocytes was significantly increased compared with controls during induction of in vitro maturation. The rate of segregated oocytes was not affected by the presence of the cumulus after chronic symptoms. These results indicate that chronic DM has a detrimental effect on meiotic regulation during folliculogenesis and results in a reduced number of competent oocytes. In addition, these data suggest that the follicle cells can resume supporting the meiotic regulation under euglycemia through insulin administration, independent of the duration of DM.

Low-density cells isolated from the rat thymus resemble branched cortical macrophages and have a reduced capability of rescuing double-positive thymocytes from apoptosis in the BB-DP rat

Biobreeding-diabetes prone (BB-DP) rats spontaneously develop organ-specific autoimmunity and are severely lymphopenic and particularly deficient in ART2(+) regulatory T cells. A special breed, the so-called BB-diabetic-resistant (DR) rats, are not lymphopenic and do not develop organ-specific autoimmunity. The genetic difference between both strains is the lymphopenia (lyp) gene. Intrathymic tolerance mechanisms are important to prevent autoimmunity, and next to thymus epithelial cells, thymus APC play a prominent part in this tolerance. We here embarked on a study to detect defects in thymus APC of the BB-DP rat and isolated thymus APC using a protocol based on the low-density and nonadherent character of the cells. We used BB-DP, BB-DR, wild-type F344, and F344 rats congenic for the lyp gene-containing region. The isolated thymus, nonadherent, low-density cells appeared to be predominantly ED2(+) branched cortical macrophages and not OX62(+) thymus medullary and cortico-medullary dendritic cells. Functionally, these ED2(+) macrophages were excellent stimulators of T cell proliferation, but it is more important that they rescued double-positive thymocytes from apoptosis. The isolated thymus ED2(+) macrophages of the BB-DP and the F344.lyp/lyp rat exhibited a reduced T cell stimulatory capacity as compared with such cells of nonlymphopenic rats. They had a strongly diminished capability of rescuing thymocytes from apoptosis (also of ART2(+) T cells) and showed a reduced Ian5 expression (as lyp/lyp thymocytes do). Our experiments strongly suggest that branched cortical macrophages play a role in positive selection of T cells in the thymus and point to defects in these cells in BB-DP rats.

Refinement of the Iddm4 Diabetes Susceptibility Locus Reveals TCRVβ4 as a Candidate Gene

Iddm4 is a dominant non-major histocompatibility complex (MHC) determinant of diabetes susceptibility in BBDR rats treated with poly I:C, plus depletion of regulatory T cells. In congenic MHC-identical normal WF rats, Iddm4(d) sensitively and specifically predicts induced diabetes. We report a new diabetes-susceptible subcongenic line that carries Iddm4 in a < 2.6 megabase interval. Candidate genes include the T cell receptor beta chain variable (TCRVbeta) family. We found that TCRVbeta4 in WF rats contains a stop codon, whereas 5/5 diabetes-susceptible rat strains express TCRVbeta4. We conclude that Iddm4-mediated diabetes resistance in rats may be due to a recessive protective mutation in TCRVbeta4.

Aberrancies in the differentiation and maturation of dendritic cells from bone-marrow precursors are linked to various genes on chromosome 4 and other chromosomes of the BB-DP rat

BB-Diabetes Prone (BB-DP) rats, a model for endocrine autoimmune diseases, are severely lymphopenic, especially lacking ART2 + regulatory T cells. BB-Diabetes Resistant (DR) rats are not lymphopenic and do not develop autoimmunity. BB-DP and BB-DR rats only differ at the lymphopenia ( lyp) gene ( iddm2) on chromosome 4. Since BB-DP rats also show aberrancies in the differentiation of dendritic cells (DC) from bone-marrow precursors, we tested the hypothesis that F344 rats congenic for a BB-DP chromosome 4 region (42.5–93.6 Mb; including the lyp gene, but also iddm4) display an in vitro DC differentiation different from normal F344 rats. Here we show that the 42.5–93.6 Mb BB-DP chromosome 4 region is linked to an increased DC precursor apoptosis, a low MHC class II expression, a reduced IL-10 production and a reduced T cell stimulatory capacity of DC. From our previous report on DC differentiation defects in BB rats (only differing in iddm2) and the present report, we deduce that the abnormal apoptosis and low MHC class II expression is linked to iddm2. The reduced T cell stimulatory capacity is linked to other genes on chromosome 4 (candidate gene: iddm4). The reduced IL-10 production has a complex linkage pattern.

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.

In Vivo Control of Diabetogenic T-Cells by Regulatory CD4+CD25+ T-Cells ExpressingFoxp3

To understand the ability of regulatory T-cells to control diabetes development in clinically relevant situations, we established a new model of accelerated diabetes in young DP-BB rats by transferring purified T-cells from DR-BB rats made acutely diabetic. Transfer of 3, 5, 10, or 23 million pure in vitro-activated T-cells accelerated diabetes onset in >90% of the recipients, with the degree of acceleration being dosage dependent. Cotransfer of unfractionated leukocytes from healthy donors prevented diabetes. Full protection was achieved when protective cells were transferred 3-4 days before diabetogenic cells, whereas transfer 2 days before conferred only partial protection. Protection resided in the CD4(+) fraction, as purified CD4(+) T-cells prevented the accelerated diabetes. When CD25(+) cells were depleted from these cells before they were transferred, their ability to prevent diabetes was impaired. In contrast, two million CD4(+)CD25(+) cells (expressing Foxp3) prevented the accelerated diabetes when transferred both before and simultaneously with the diabetogenic T-cells. In addition, 2 million CD4(+)CD25(+) T-cells prevented spontaneous diabetes, even when given to rats age 42 days, whereas 20 million CD4(+)CD25(-) cells (with low Foxp3 expression) were far less effective. We thus demonstrated that CD4(+)CD25(+) cells exhibit powerful regulatory potential in rat diabetes.

The Rat Diabetes Susceptibility LocusIddm4and at Least One Additional Gene Are Required for Autoimmune Diabetes Induced by Viral Infection

BBDR rats develop autoimmune diabetes only after challenge with environmental perturbants. These perturbants include polyinosinic:polycytidylic acid (poly I:C, a ligand of toll-like receptor 3), agents that deplete regulatory T-cell (Treg) populations, and a non-beta-cell cytopathic parvovirus (Kilham rat virus [KRV]). The dominant diabetes susceptibility locus Iddm4 is required for diabetes induced by treatment with poly I:C plus Treg depletion. Iddm4 is penetrant in congenic heterozygous rats on the resistant WF background and is 79% sensitive and 80% specific as a predictor of induced diabetes. Surprisingly, an analysis of 190 (BBDR x WF)F2 rats treated with KRV after brief exposure to poly I:C revealed that the BBDR-origin allele of Iddm4 is necessary but not entirely sufficient for diabetes expression. A genome scan identified a locus on chromosome 17, designated Iddm20, that is also required for susceptibility to diabetes after exposure to KRV and poly I:C (logarithm of odds score 3.7). These data suggest that the expression of autoimmune diabetes is a complex process that requires both major histocompatibility complex genes that confer susceptibility and additional genes such as Iddm4 and Iddm20 that operate only in the context of specific environmental perturbants, amplifying the immune response and the rate of disease progression.