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The BB rat is a model of spontaneous autoimmune diabetes. To characterize quantitatively all known immune cell subsets involved in disease pathogenesis, FACS analysis of spleen cells was performed in diabetes-prone (DP) and acutely diabetic (D) BB rats and compared with diabetes-resistant (DR) BB and normal Wistar-Furth (WF) strains. We observed increased percentages of splenic NK cells in DP and D animals compared with DR rats using an NK-specific monoclonal antibody. We found increased proportions of splenic macrophages in the T-lymphopenic DP and D rats and low macrophage contents in DR spleens compared with WF spleens. We observed that percentages of the CD4-CD8- T cell receptor alpha/beta+ (double-negative) T cell subset were strikingly increased in the lymphopenic DP and D animals, compared with DR animals. We observed increased percentages of activated splenic CD5+ T cells expressing the IL-2 receptor and MHC class II antigen in DP and D rats compared with DR animals. Our studies suggest that (a) splenic NK cells and macrophages quantitatively appear to be involved in the pathogenesis of diabetes; (b) double-negative T cells escape from the T cell depletion process; (c) a marked increase of activated splenic T cells suggests diabetes is associated with general T cell activation processes; and (d) an altered balance among the different immune cell subsets may in part explain the pathogenesis of diabetes, since marked relative changes are observed when comparing the DR strain to the DP strain in both the prediabetic and diabetic stages.

Insulin-dependent diabetes is a chronic autoimmune disease probably mediated by T cells. We examined the alpha chain of the T-cell antigen receptor in two models of this illness (man and BB rat) to determine any association with autoimmune diabetes. We conducted a population study in man, using a human alpha chain probe, pGA-5, and restriction enzyme Bgl 11. Two allelic forms and three RFLP patterns, 2.8 and 3.0 kb homozygous and 2.8/3.0 heterozygous, were detected. There was no difference in the frequency of these RFLPs among the 50 Type I diabetic patients and 48 controls tested. BB rats develop a spontaneous T-cell mediated autoimmune diabetes. The diabetes has been linked in several breeding studies to an undetermined autosomal recessive gene causing T-cell lymphopenia. We were able to differentiate the T-cell antigen receptor alpha chain of the diabetic BB and control BBN rats using the restriction enzyme EcoR1 and a murine alpha chain probe, TT11. The BB rat had a haplotype characterized by the presence of 4.7 and 5.8 kb bands, and the absence of 1.4, 2.2, 2.6, 3.6, 3.9, 4.1, and 6.1 kb bands. In a breeding study with BB and BBN rats, diabetic animals of the F2 generation demonstrated no linkage with the BBs' alpha chain, nor was lymphopenia linked to the alpha chain of the BB rat. These results suggest that autoimmune diabetes is not linked to the T-cell antigen receptor alpha chain in the BB rat, nor is it associated with alpha chain constant region polymorphisms in Type I diabetes in man.

Type 1 Diabetes (T1D) is an autoimmune disease characterized by the pancreatic infiltration of immune cells resulting in T cell-mediated destruction of the insulin-producing beta cells. The successes of the Non-Obese Diabetic (NOD) mouse model have come in multiple forms including identifying key genetic and environmental risk factors e.g. Idd loci and effects of microorganisms including the gut microbiota, respectively, and how they may contribute to disease susceptibility and pathogenesis. Furthermore, the NOD model also provides insights into the roles of the innate immune cells as well as the B cells in contributing to the T cell-mediated disease. Unlike many autoimmune disease models, the NOD mouse develops spontaneous disease and has many similarities to human T1D. Through exploiting these similarities many targets have been identified for immune-intervention strategies. Although many of these immunotherapies did not have a significant impact on human T1D, they have been shown to be effective in the NOD mouse in early stage disease, which is not equivalent to trials in newly-diagnosed patients with diabetes. However, the continued development of humanized NOD mice would enable further clinical developments, bringing T1D research to a new translational level. Therefore, it is the aim of this review to discuss the importance of the NOD model in identifying the roles of the innate immune system and the interaction with the gut microbiota in modifying diabetes susceptibility. In addition, the role of the B cells will also be discussed with new insights gained through B cell depletion experiments and the impact on translational developments. Finally, this review will also discuss the future of the NOD mouse and the development of humanized NOD mice, providing novel insights into human T1D., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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.

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 alpha-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 alpha-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.

Cardiotrophin-1 (CT-1) produces longitudinal elongation of neonatal cardiomyocytes, but its effects in adult cardiomyocytes are not known. Recent observations indicate that CT-1 may be involved in pressure overload left ventricular hypertrophy (LVH). We investigated whether the hypertrophic effects of CT-1 are different in cardiomyocytes isolated from adult normotensive and spontaneously hypertensive rats (SHR). Hypertrophy was evaluated by planimetry and confocal microscopy, contractile proteins were quantified by Western blotting and real-time RT-PCR, and intracellular pathways were analyzed with specific chemical inhibitors. CT-1 increased c-fos and ANP expression (p<0.01) and cell area (p<0.01) in cardiomyocytes from both rat strains. In Wistar cells, CT-1 augmented cell length (p<0.01) but did not modify either the transverse diameter or cell depth. In SHR cells, CT-1 increased cell length (p<0.05), cell width (p<0.01) and cell depth, augmented the expression of myosin light chain-2v (MLC-2v) and skeletal alpha-actin (p<0.01) and enhanced MLC-2v phosphorylation (p<0.01). The blockade of gp130 or LIFR abolished CT-1-induced growth in the two cell types. All distinct effects observed in cardiomyocytes from SHR were mediated by STAT3. Baseline angiotensinogen expression was higher in SHR cells, and CT-1 induced a 1.7-fold and 3.2-fold increase of angiotensinogen mRNA in cardiomyocytes from Wistar rats and SHR respectively. In addition, AT1 blockade inhibited the specific effects of CT-1 in SHR cells. Finally, ex vivo determinations revealed that adult SHR exhibited enhanced myocardial CT-1 (mRNA and protein, p<0.01), increased cell width (p<0.01) and concentric LVH compared with pre-hypertensive SHR. These findings reveal a specific cell-broadening effect of CT-1 in cardiomyocytes from adult SHR and suggest that the hypertensive phenotype of these cells may influence the hypertrophic effects of CT-1, probably by means of an exaggerated induction of angiotensinogen expression. We suggest that CT-1 might facilitate LVH in genetic hypertension through a cross-talk with the renin-angiotensin system.

Lymphopenia is due to a frameshift mutation in Gimap5 on rat chromosome 4 and is linked to type 1 diabetes in the diabetes prone (DP) BB rat. The hypothesis that bone marrow derived cells confer the lymphopenia phenotype was tested by reciprocal bone marrow transplantation in 40-day-old lethally irradiated diabetes resistant (DR) congenic DR.lyp/lyp (lymphopenia and diabetes) and DR.+/+ (no lymphopenia and no diabetes) rats. In two independent series of transplants, all DR.lyp/lyp rats (n=5 and 4) receiving DR.lyp/lyp bone marrow retained lymphopenia and developed insulitis (5/5 and 4/4) as well as diabetes in some (2/5 and 3/4). Both DR.+/+ and DR.lyp/lyp rats receiving DR.+/+ bone marrow cells as well as DR.+/+ rats receiving DR.lyp/lyp bone marrow cells showed no lymphopenia or diabetes. In accordance with earlier studies in non-congenic BB rats, the DR.+/+ rats receiving DR.lyp/lyp bone marrow cells recapitulated an intermediary phenotype rather than the +/+ or lyp/lyp phenotypes. Our data demonstrate that BBDP rat lymphopenia and diabetes are transferred by bone marrow transplantation to syngeneic DR.lyp/lyp but not DR.+/+ recipients. The intermediary recapitulation of DR.lyp/lyp T cells in recipient DR.+/-/+/- rats suggests that radiation resistant +/-/+/- T cells, the Gimap5 mutation in bone marrow cells, or both may not support the development of lymphopenia.

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 (42.5-93.6Mb; including the lyp gene, but also iddm4) display an in vitro DC differentiation different from normal F344 rats. Here we show that the 42.5-93.6Mb 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.

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.

Spontaneous diabetes in B(io)B(reeding) rats is complex, polygenic, and recessively inherited. Several crossing studies have demonstrated that beside the class II genes of the major histocompatibility complex (MHC, Iddm1) additional non-MHC genes are involved in diabetes development. One of them, Iddm4, was initially mapped on chromosome 6q32. To study the physiologic importance of Iddm4 a congenic BB.SHR rat strain (BB.6S) was established. The BB.6S is characterised by a drastic reduction of diabetes frequency (86 vs. 14%) indicating existence of diabetes protective genes of SHR on the exchanged chromosomal segment. One of the possible diabetes susceptibility candidate genes located within this exchanged region is the multifunctional transcription factor Yin yang 1 (Yy1). Yy1 was therefore sequenced in BB/OK and SHR rats. No genetic variation in exons between BB/OK and SHR was found. However, three single nucleotide polymorphisms (SNPs) were detected in intron 4. To determine the "wild type" allele, intron 4 of several diabetes-resistant inbred rat strains (DA, LEW, BN, and WOKW) and wild rats was sequenced. In addition, a congenic BB/OK strain was established by introgressing the same segment of chromosome 6 (D6Rat184-D6Rat3) of wild rats onto BB/OK background (BB.6W). The sequence analysis showed the SNP pattern of SHR (A/C/C) in all inbred rat strains studied whereas both unrelated wild rats showed the pattern of BB/OK rats (T/G/A). The congenic BB.6W rats developed diabetes in the same extent than BB/OK rats. This finding may support the assumption that the SNP pattern of BB/OK and wild rats favours and that of SHR suppresses diabetes development. Because of strong synteny between rat chromosome 6q32 and human 14q32, Yy1 may be also of interest in human type 1 diabetics showing significant linkage to markers on chromosome 14q32.

A transfer model for studying both the development and prevention of diabetes in rats is described in detail. Diabetes was induced in BBDR rats by combining RT6-depletion with PolyI:C treatment. Autoreactive cells were isolated from acutely diabetic donors, reactivated in vitro and transferred intravenously into young (<34-day-old) BBDP rats. Accelerated diabetes occurred 13+/-3 days or 18+/-4 days after transfer of reactivated splenocytes or purified T cells (42/43 or 26/27 recipients, respectively). Freshly isolated mesenteric and splenic leukocytes from adult, healthy BBDR rats prevented spontaneous diabetes in BBDP rats, but were not able to prevent the accelerated diabetes when co-transferred with the autoreactive cells. By contrast, diabetes was significantly delayed (P<0.001) when protective cells were transferred 4 days prior to the autoreactive cells (16+/-3 days). In vivo tracking studies of the two types of transferred cells suggest different homing patterns which may explain this finding. The data suggest that leukocytes from BBDR contain cells with the ability to regulate reactivated autoreactive T cells in an autoimmune environment. This in vivo model of recurrent diabetes can therefore be used to define which type of cells are most effective in suppressing established autoimmune destruction of beta-cells.

In the pathogenesis of type-1 diabetes insulin-producing beta-cells are destroyed by cellular autoimmune processes. The locality of beta-cell destruction is the inflamed pancreatic islet. During insulitis cytokines released from islet-infiltrating mononuclear cells affect beta-cells at several levels. We investigated whether cytokine-induced beta-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)-1beta alone or in combination with interferon (IFN)-gamma plus tumour necrosis factor (TNF)-alpha. 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-1beta treated islets cells were detectable, the cytokine combination caused damage of cell membranes. Independent of diabetes susceptibility IL-1beta treated islet beta-cells expressed a significantly increased amount of ICAM-1 on their surfaces which was not further increased by IFN-gamma+TNF-alpha. However, IL-1beta induced Fas expression was significantly enhanced only on beta-cells from diabetes-prone BB rats. From these results we suggest that IL-1beta mediates the major stimulus for ICAM-1 induction which is possibly a necessary but not sufficient step in the process of beta-cell destruction. Obviously, the additional enhancement of Fas expression on the surface of beta-cells is important for destruction. The combined action of all three cytokines induced the expression of Fas on the beta-cell surface independent of diabetes susceptibility, indicating that such a strong stimulus in vitro may induce processes different from the precise mechanisms of beta-cell destruction in vivo.

Diabetes in the biobreeding (BB) rat results from autoimmune destruction of pancreatic beta cells and thereby it is sharing many features with human type 1 diabetes. Independent crossing studies have demonstrated that diabetes in the BB rat is explained by at least three recessively acting genes termed Iddm1 (major histocompatibility complex), Iddm2 (lymphopenia), Iddm3 (unknown). About 50% of Iddm1 and Iddm2 homozygous first backcross hybrids (BC1) usually develop diabetes. However, 75% of these homozygotes become diabetic when using diabetic BB/HRI and diabetes-resistant BN/Mol rats. That prompted us to carry out a cross between BB/OK and BN/Crl rats in order to localise diabetogenic gene(s) of BB and/or BN rats. Fifty nine Iddm1 and Iddm2 homozygous [(BNxBB)F1xBB] BC1 hybrids (35 M, 24 F) were observed for diabetes occurrence up to an age of 30 weeks. All hybrids were used in a genome-wide scan carried out with 238 microsatellite markers covering about 92% of the genome. Significantly more Iddm1 and Iddm2 homozygous BC1 hybrids became diabetic (69 vs. 50%, p<0.003) with an age at onset of 91+/-31 days. Significant deviations from expected allele distribution between diabetic and non-diabetic BC1 hybrids were found at loci on chromosomes 1, 2, 3, 9, 10, 15, 16 and 19, with the strongest effect observed at locus D10Mgh2, where more heterozygous (91%) than homozygous diabetics (44%) were found. We conclude that BN rats possess more than one gene contributing to type 1 diabetes development.

Interleukin-2 (IL-2) is an important cytokine in the autoimmune process proceeding Type 1 diabetes. Our aim was to investigate, in two previously used animal models, the NOD mouse and the BB/W rat, the in vivo tissue distribution of radio-labeled IL-2. If the radio-labeled IL-2 accumulated significantly in the pancreas compared to surrounding organs it could allow imaging of lymphocyte infiltration of the islets of Langerhans by scintigraphic methods. IL-2 was labeled enzymatically with(125)Iodine. Radio-labeled IL-2 was injected iv in prediabetic NOD mice, diabetic NOD mice and Balb/c mice in the first animal model and in BB rats in the second model. Animals were sacrificed at different time points and the activity in different organs was measured. It was found that the mean activity in the pancreas in both diabetic and prediabetic NOD mice was significantly higher compared to pancreas from Balb/c mice (P< 0.001 and P=0.005, respectively). However, the mean activity in the pancreas was at the lower range of the surrounding organs in both animal models, thereby excluding the possibility of imaging the autoimmune process by scintigraphic methods. It is concluded that radio-labeled IL-2 did accumulate significantly in the pancreas of NOD mice compared to control mice but there is a need to develop new techniques in order to visualize the localized activity., (Copyright 2001 Academic Press.)

Feeding diabetes-prone BioBreeding (BBdp) rats a hydrolysed-casein (HC)-based semi-purified diet results in two-to-three-fold fewer diabetes cases compared with feeding cereal-based diets such as NIH-07 (NIH). We showed previously that young NIH-fed BBdp rats had decreased islet area at a time when classic insulitis was minimal. Rats fed an HC diet maintained near normal islet area followed 3-4 weeks later by a deviation of the pancreas cytokine pattern from Th1 to Th2/Th3. This finding raised the possibility that BBdp rats were more susceptible to diet-induced changes in islet homeostasis. To investigate this possibility further, BBdp rats were fed an NIH or HC diet from days 23 to 45. Bouin's fixed sections of pancreas were stained with H & E or antibodies for insulin and glucagon. Cell proliferation nuclear antigen (PCNA) was used as a marker of cell proliferation and cells were stained for putative markers of islet neogenesis, cytokeratin 20 (CK20) and Bcl-2. Apoptotic bodies were recognized by morphological features and by TUNEL-positive staining. BBdp rats fed an HC diet had a significantly higher beta-cell fraction than rats fed NIH, whereas alpha-cell fraction and beta-cell size were not affected by diet or rat type. Apoptotic bodies of beta-cells were rare and unaffected by diet. The number of PCNA(+)beta-cells was not affected by diet. CK20 expression was localized in the ductular system and at the periphery of islets in rats aged 7 and 45 days. There were more CK20(+)islets in BBdp rats fed NIH than in those fed HC but the CK20 area fraction was unaffected by diet. Bcl-2 expression was scattered among ducts and central acinar cells. The number of extra-islet insulin(+)and glucagon(+)clusters (

Dendritic cells (DCs) comprise a small population of cells in the normal thyroid. These excellent antigen-presenting cells (APCs) are thought to be involved in the initiation of thyroid autoimmune reactions. However it is not known whether the APCs involved in this process are indeed DCs, or thyrocytes. Our aims were as follows: (1) to isolate DCs from the thyroid of normal Wistar rats and BB-DP rats prior to the development of lymphocytic thyroiditis; (2) to determine the T-cell stimulatory capability of such isolated thyroid DCs and to compare this capability to that of BB-DP thyrocytes and splenic DCs; and (3) to investigate the phenotype of isolated thyroid DCs and to compare it to that of splenic DCs; and (4) to investigate the capability of such thyroid DCs to regulate thyrocyte growth and function, and to compare it to our earlier reports demonstrating such capability with splenic DCs. Leukokcytic cell fractions were isolated from the thyroids of BB-DP and control Wistar rats of 7-20 weeks of age. The isolation steps included gentle enzymatic tissue disruption, the collection of non-plastic adherent cells and density gradient centrifugation of these cells to yield a low and a high density non-adherent fraction. The low density cell (LDC) fraction was composed of 50-75% leukocytes in both strains. These leukocytes were almost exclusively ED1+ monocytes or MHC-class II+ DC. The high density cell (HDC) fractions of both strains were composed of about 70% MHC-class II-negative thyrocytes and 30% ED1+ monocytes. The thyroid LDCs of both strains had an APC capability in syngeneic(syn)-MLR comparable to that of splenic DCs. However, the HDCs were extremely poor in syngeneic T cell stimulation. There was a difference in composition between the Wistar and the BB-DP LDC fractions: The Wistar LDCs were composed of 30-35% ED1+ monocytes and 15-20% typical MHC-II+ DCs, while BB-DP LDC fractions contained more ED1+ monocytes (about 70%), but fewer DCs (5-10%). In comparison to splenic DCs, thyroid DCs had a low CD80 and CD86 expression in both strains (i.e., an 'immature' phenotype). The LDCs of both animal strains were shown to decrease both basal and TSH-stimulated thyrocyte proliferation and T(3)release by about half. This report shows that a cell fraction enriched for monocytes and DCs can be isolated from the thyroids of both Wistar and BB-DP rats. The cells in this fraction were as capable as splenic DCs to act as T cell stimulators in syn-MLR. Since the thyroid HDCs (predominantly thyrocytes) were very poor at such T cell stimulation, thyroid monocytes and DCs (and not thyrocytes) are the prime candidates to act as immune accessory cells in the initiation of thyroid autoimmunity in the rat. Wistar thyroid LDCs differed in phenotype from BB-DP LDCs. The latter contained a lower percentage of DCs and a higher percentage of their precursors, the monocytes. Interestingly, a defect in the transition of monocytes to DCs has been described in another animal model of autoimmune thyroiditis/insulitis (the NOD mouse), as well as in thyroiditis and diabetic patients., (Copyright 2000 Academic Press.)

The Lyp locus controls diabetes development in rats. The diabetogenic allele in diabetes-prone BB rats is responsible for T cell lymphopenia characterized by the absence of regulatory T cells. We present refined genetic and radiation hybrid maps of the Lyp region on rat chromosome 4, a single 800-kb rat yeast artificial chromosome and a rat P1-derived artificial chromosome (PAC) contig corresponding to approximately 550 kb, both encompassing the entire candidate region. The contig, consisting of 48 PACs, gives 3- to 12-fold coverage. Genetic, radiation hybrid, and physical data were all in agreement and supported the same marker order. Nine genes and ESTs were identified in the contig in addition to a rat EST from the University of Iowa rat EST database-all possible candidate genes for Lyp. Alignment of our rat PAC contig with sequenced human PAC/BAC contigs confirms the position within the region of 3 of the 10 candidates and identifies an additional 8 genes/ESTs as candidates. These data will facilitate identification of Lyp., (Copyright 2000 Academic Press.)

The rat diabetes susceptibility gene, Lyp or Lymphopenia, has been localized to RNO4. Proximal to Lyp are the genes caspase-2 (Casp2) and pancreatic trypsin 1 (Prss1), while neuropeptide Y (Npy) is the closest distally positioned gene. In human, the three genes are syntenic on HSA7, but they are not on a conserved segment: CASP2 and PRSS1 are localized to 7q35, while NPY is localized to 7p15.1. This raises the question whether the human homologue of Lyp is linked to CASP2/PRSS1 or to NPY. We present a comparative map of the Lyp region in rat and human, assigning the gene to a 1.3-Mb segment between RNY3 and ABP1 at 7q35., (Copyright 2000 Academic Press.)

The effects of substituting a plant-based control diabetogenic diet (NIH diet) by a protective hydrolyzed casein diet (HC diet) upon selected metabolic and functional variables were recently investigated in Peyer's patch cells, splenocytes, mesenteric lymph node cells, and pancreatic islets from either control (BBc) or diabetes-prone (BBdp) BB rats. In the present work, the plasma d-glucose and insulin concentrations, the protein and insulin content of pancreatic islets, the metabolism of d-glucose, and its insulinotropic action in islets first cultured for 24 h in the absence or presence of IL-1beta, the production of IFN-gamma and IL-10 by mesenteric lymph node cells cultured for 48 h in the absence or presence of concanavalin A, the mitogenic activity of Peyer's patch cells and pancreatic lymph node cells in the absence or presence of the same lectin, and the biosynthetic activity of Peyer's patch cells were measured in the BBc and BBdp rats fed either the NIH or the HC diet. Two major novel findings emerged from this study. First, in immune cells, diet HC increased to a greater extent the responsiveness to concanavalin A of certain metabolic and functional variables in BBdp rats than in BBc rats. Second, pancreatic islet cells of BBdp rats were less sensitive to IL-1beta than those of BBc rats and this difference was further accentuated when the animals were fed the HC rather than the NIH diet. These findings afford further support to the view that, in BB rats, changes in the biological behavior of Peyer's patch cells, mesenteric and pancreatic lymph node cells, and pancreatic islet cells participate in the pathogenesis of insulin-dependent diabetes mellitus and its prevention by a suitable dietary manipulation., (Copyright 1999 Academic Press.)

It has been previously suggested that alterations in sodium homeostasis, leading to calcium overload may play a part in the mediation of cardiac ischemic injury. It has been demonstrated that the Na+-H+ exchanger plays an important role with regard to the regulation of intracellular sodium during ischemia and reperfusion and that inhibition of the Na+-H+ exchanger during ischemia protects hearts from ischemic injury. Studies using chemically-induced diabetic animals have suggested that the cardiac Na+-H+ exchanger in the diabetic heart is impaired and is responsible for limiting the increase in sodium during ischemia. The extent to which the Na+-H+ exchanger contributes to increases in intracellular sodium during ischemia in diabetic hearts is unclear as direct measurements of exchanger activity have not been made in genetically diabetic hearts. Therefore, this paper aims to address the following issues: (a) is the Na+-H+ exchanger impaired in a genetically diabetic rat heart: (b) does this impairment result in lower [Na]i or [Ca]i during ischemia; and (c) does Na+-H+ exchanger inhibition limit injury and functional impairment in diabetic hearts during ischemia and reperfusion? These issues were examined by inhibiting the Na+-H+ exchanger with ethylisopropylamiloride (EIPA) in isolated perfused hearts from both genetically diabetic (BB/W) and non-diabetic rats. Levels of intracellular sodium, intracellular calcium, intracellular pH and high energy phosphates (using 23Na,19F, 31P NMR spectroscopies, respectively) during global ischemia and reperfusion were also measured. The impact of diabetes on Na+-H+ exchanger activity was assessed by measuring pH recovery of these hearts after an acid load. Creatine kinase release during reperfusion was used as a measure of ischemic injury. This study demonstrated that the Na+-H+ exchanger is impaired in diabetic hearts. Despite this impaired activity, inhibition of Na+-H+ exchanger protected diabetic hearts from ischemic injury and was associated with attenuation of the rise in sodium and calcium, and limitation of acidosis and preservation of ATP during ischemia. The data presented here favor the use of Na+-H+ exchanger inhibitors to protect ischemic myocardium in diabetics. Also, the data provides possible mechanisms for the altered susceptibility of diabetic hearts to ischemic injury., (Copyright 1999 Academic Press.)

To study the relative roles of CD4(+)and CD8(+)T cells and their cytokine products in autoimmune diabetes development, we selectively depleted CD4(+)and CD8(+)T cells in autoimmune diabetes-prone (DP) biobreeding (BB) rats, by administrations of anti-CD2 and anti-CD8 monoclonal antibody (mAb) respectively. We then analysed cytokine mRNA expression, by PCR assay, in mononuclear leukocytes isolated from islets and spleens of control and mAb-treated DP-BB rats. Depletion of CD4(+)T cells (by anti-CD2 mAb) in blood, spleen and islets prevented diabetes development in DP-BB rats, and depletion of CD8(+)T cells (by anti-CD8 mAb) delayed and significantly decreased diabetes incidence. Depletion of either CD4(+)or CD8(+)T cells completely prevented IFN-gamma mRNA upregulation in islets of DP-BB rats above the low level expressed in islets of diabetes-resistant (DR) BB rats. Also, IL-10 mRNA levels in islets of DP-BB rats were significantly decreased by depletion of either CD4(+)or CD8(+)T cells, whereas the effects of the anti-T cell mAb on mRNA levels of other cytokines in islets (IL-2, IL-4, IL-12p40, and TNF-alpha) were discordant. In contrast, both mAb treatments significantly upregulated IL-4 and TNF-alpha mRNA levels in spleens of DP-BB rats. These results demonstrate that islet infiltration by both CD4(+)and CD8(+)T cells is required for IFN-gamma and IL-10 production in islets and beta-cell destruction. Depletion of either CD4(+)or CD8(+)T cells may prevent beta-cell destruction by decreasing IFN-gamma and IL-10 production in islets and increasing IL-4 and TNF-alpha production systemically., (Copyright 1999 Academic Press.)

Poly I:C, an inducer of IFN-alpha and other cytokines, has been used to study the development of diabetes in both the BioBreeding (BB) diabetes prone rat and non-obese diabetic (NOD) mouse animal models of insulin-dependent diabetes mellitus (IDDM). Surprisingly, poly I:C accelerates the disease in the BB rat while inhibiting it in the NOD mouse. Since cytokines can have dose related opposing effects on immune responses, we hypothesized that the paradoxical effect of polyinosinic polycytidylic acid (poly I:C) on diabetes in the two animal models is dose related. Accordingly, we compared the incidence of diabetes and degree of insulitis in diabetes prone BB rats administered saline and poly I:C at doses (0.05 microg/g body weight and 0.1 microg/g body weight) up to 100-fold lower than doses (poly-5 microg/g) previously found to accelerate diabetes. In addition, the non-specific suppressor activity of mononuclear splenocytes from BB rats administered low dose (poly-0.05 microg/g body weight), high dose (poly-5 microg/g body weight), and saline were compared. The development of diabetes was inhibited in rats treated with each dose of poly I:C. The degree of insulitis in poly-I:C treated animals was also less severe. The total white blood cell count and proportion of RT6+ T-cells and each T-cell subset were unaltered by poly I:C. When compared to splenocytes of control animals, splenocytes from poly I:C (0.05 microg/g body weight) treated rats suppressed responder cell proliferation to concanavalin A and alloantigen. However, spleen cells from high dose poly-I:C did not suppress responder cell proliferation to alloantigen. In adoptive transfer studies, the administration of spleen cells from poly-0.05 treated rats decreased the development of diabetes in recipient BB rats. In vitro studies also demonstrated that poly-I:C inhibits the proliferative response of BB rat spleen cells to concanavalin A. The administration of poly-0.05, but not poly-5.0, decreased TNF-alpha mRNA and IL-10 mRNA content in spleen cells. We conclude that poly I:C, at a dose 100 times lower than that required to accelerate diabetes prevents the development of diabetes in BB rates by interfering with the development of insulitis. The induction of suppressor cell activity induced by low dose poly-I:C in vivo and the inhibition of T-cell responses by poly-I:C in vitro suggests that the diabetes sparing activity of poly I:C is mediated by augmented immunoregulatory cell activity. Further studies with poly I:C may be important in increasing our understanding of the pathogenesis of IDDM and provide a means to prevent it., (Copyright 1998 Academic Press)

Antibodies to ICA512/IA-2 are a well established marker of human IDDM and can be detected prior to and soon after the onset of insulin dependency. The non-obese diabetic (NOD) mouse and the diabetes-prone BB rat develop spontaneous diabetes as a consequence of T-cell mediated autoimmune destruction of islet beta-cells, but the occurrence of autoantibodies is controversial. We tested sera from NOD mice and BB-rats for anti-ICA512 by a radioimmunoprecipitation assay (RIP). In sequential serum samples from 20 NOD mice, of which 15 developed diabetes, low levels of anti-ICA512 were demonstrable. Anti-ICA512 appeared close to the onset of hyperglycaemia and was usually transient. Non-diabetic NOD mice also produced anti-ICA512, but at a later age and at lower levels than the diabetic NOD mice. In a cross-sectional analysis of sera from BB rats, low levels of anti-ICA512 were present in 11/20 (55%) of non-diabetic-diabetes prone (DP) BB rats, 0/4 (0%) of diabetic DP BB rats, and 1/6 (17%) of diabetes-resistant BB rats. Anti-ICA512 was not detected in rats of other strains, including three Sprague-Dawley rats with streptozotocin-induced diabetes. In both NOD mice and BB rats the anti-ICA512 reactivity was directed to the cytoplasmic domain of the protein. The transient appearance of anti-ICA512 close to the onset of diabetes in NOD mice and the loss of these antibodies after diabetes onset is consistent with the occurrence of anti-ICA512 in human IDDM. Thus in both human IDDM and rodent models, anti-ICA512 is a marker of the impending onset of diabetes and disappears after diabetes onset.

The goal of this study was to determine whether zinc supplementation in the diet of diabetes-prone BB Wistar rats will delay or prevent the onset of overt diabetes. Male Wistar BB rats were fed diets containing either 1000 ppm (HZ), 50 ppm (NZ), or 1 ppm zinc (LZ) starting at 30 days of age. Non-diabetes-prone rats were fed NZ and designated as controls (NORM). Beginning at 60 days, the rats were checked for glycosuria and, if positive, were given an i.p. glucose tolerance test (IPGTT). All remaining animals underwent an IPGTT at 100 days and were sacrificed. At 90 days of age HZ rats had a lower incidence of diabetes (19%) than NZ (53%) or LZ (44%) animals (P < 0.015). By age 100 days, for the HZ group, there was a 60% reduction in the number of expected overt diabetic rats. HZ animals also had higher concentrations of both pancreatic and serum insulin and exhibited lower serum glucose and triglycerides. Immunohistochemistry of HZ rats was clearly different from NZ rats and showed evidence of nearly normal pancreatic endocrine activity. Data indicate that dietary treatment of diabetes-prone BB Wistar rats with zinc appears to be an effective approach for delaying or preventing the onset of diabetes in genetically predisposed rodents. This finding may suggest further experimental studies regarding dietary means for preservation of pancreatic function.

The effect of dietary n-3 or n-6 polyunsaturated fatty acids on the development of autoimmune insulitis was analysed in diabetes-prone BB rats. Litter-matched groups of rats received a standard open formula NIH-07 (National Institutes of Health, NIH) diet enriched with 10% fish oil, 10% flaxseed oil or with 10% palm oil plus 2% cholesterol during the period of insulitis onset (50-70 days of age). Analysis of cytokine gene expression in pancreatic RNA revealed an increase of IFN-gamma and a decrease of IL-10 mRNA with onset of insulitis. When compared to unsupplemented NIH, none of the three fat-enriched diets depressed the rise of IFN-gamma gene expression or the influx of leukocytes into islets. However, all of the fat-enriched diets led to significantly higher IL-10 mRNA levels. Although a specific anti-inflammatory effect of fish oil was not seen in the pancreas, a clear shift of the Th1/Th2 cytokine mRNA ratio towards Th2 was seen in the gut-associated immune system. We conclude that diets high in fat support IL-10 without suppressing IFN-gamma gene expression in islet inflammation. A special anti-inflammatory effect of fish oil was not seen in pancreatic lesions of BB rats, although there was strong modulation of the IFN-gamma/IL-10 mRNA ratio in the gut associated immune system., (Copyright 1998 Academic Press Limited.)

Diabetes-prone DP-BB rats spontaneously develop insulin-dependent diabetes mellitus resembling type 1 diabetes mellitus in man. Expression of T cell lymphopenia and presence of at least one class II major histocompatibility complex (MHC) RT1u haplotype are required for development of diabetes. Diabetes segregation was studied in lymphopenic backcross (BC) offspring from a cross between male DP-BB/HRI and female BN/Mol rats. Diabetes occurred in 75% of BC rats with genotype RT1u/u and in 18% of those being RT1n/u in genotype. The latter developed diabetes significantly later than MHC homozygotes and parental DP-BBs. Our data further point to the existence of additional genes of minor importance for development of IDDM. One of these seemed to be positioned on the X chromosome. The recently published linkage to chromosome 18 could not be confirmed however. Finally, the BN-derived non-albino allele of the C gene was associated with higher diabetes incidence. This points to the existence of minor susceptibility genes in other strains of rats.

Spontaneous insulitis with insulin-dependent diabetes mellitus (IDDM) in rodent models, the BB rat and NOD mouse, has clarified the pathogenesis of and guided decisions on interventional therapy for human IDDM. However, the occurrence in such models of a standard marker of human IDDM, autoantibodies to beta islet cell constituents, has been controversial. Hence we assessed diabetes-prone rodents for the frequencies of raised levels of auto-antibodies to glutamic acid decarboxylase GAD (anti-GAD), insulin and heat shock protein 65 (HSP-65) in relation to levels in non-diabetes-prone animals and levels in human diabetic sera. Assays were performed sequentially at various ages of life. The immunoassays used for anti-GAD and anti-insulin were those validated for sensitivity and specificity for detection of the corresponding autoantibodies in human IDDM sera at international workshops. Positive controls included human IDDM sera with reactivity with GAD or insulin and, for mouse anti-GAD, the highly reactive monoclonal antibody, GAD-6. The results were that levels of autoantibodies in diabetes-prone BB rats or NOD mice to the "IDDM-relevant' autoantigens in our panel did not exceed levels in control rats or mice, and were much lower than levels in humans with IDDM. We conclude that the BB rat and NOD mouse represent a model, but not a facsimile, of human IDDM and that therapeutic successes in such models should be interpreted with caution in relation to interventional therapy for human IDDM.

Th1 cytokines are thought to play a key role in islet inflammation and destruction in insulin-dependent diabetes mellitus (IDDM). We studied this hypothesis in the diabetes-prone (DP)-BB and the diabetes-resistant (DR)-BB rats that are used as a model of human IDDM. The DP-BB rat develops spontaneous autoimmune diabetes at the age of 11-14 weeks. In the DR-BB rat, diabetes is inducible by depletion of RT6+ lymphocytes and coadministration of polyinosinic:polycytidylic acid (Poly I:C). We used reverse transcriptase-polymerase chain reaction (RT-PCR) and semi-quantitative PCR techniques to examine mRNA expression of Th1 and Th2 cytokines in inflamed islets and thyroids from DP-BB and DR-BB rats. We observed that in DP-BB and in treated DR-BB rats, the levels of TCR beta, IFN-gamma and IL-12p40 mRNA increase with disease progression. In contrast, expression of message for IL-2 and IL-4 is minimal to undetectable in DP-BB and RT6-depleted DR-BB animals at any age. Message for IL-10 is detectable in DP and DR islets; however, its level of expression does not change with disease progression. A similar cytokine mRNA profile is observed in inflamed thyroids from acutely diabetic RT6-depleted DR-BB rats. Incubation of 10 wk old DP islets for 48 h in the presence of anti-CD3 antibody, followed by an incubation with rIL-2 for an additional 5-7 days, results in an expansion of T lymphocytes, and these cells express high levels of IFN-gamma and IL-10 mRNA. Our results suggest that autoimmunity in DP-BB and DR-BB rats is mediated by Th1 lymphocytes and that IFN-gamma and IL-12 are likely to play a key role in islet and thyroid inflammation and destruction in IDDM.

Pancreatic islets obtained from the congenic LEW.1BB/OK rat strain (MHC identical, different in genetic background from BB/OK rats) were grated into diabetic BB/OK rats. The recipients were treated for 10 d with 1.0 mg/kg bw anti-IL2 receptor monoclonal antibody (ART-18) in combination with 1.5 mg/kg bw cyclosporin A, which resulted in indefinite graft survival in the majority of animals. The successfully treated recipients (normoglycaemia for > 120 days) relapsed immediately into hyperglycaemia after graft removal. A second donor-identical graft was accepted without any further immunotherapy, whereas MHC-different islet grafts (obtained from LEW.1A or DA rats) were rejected, demonstrating the induction of donor-specific tolerance. Splenocytes or thoracic duct lymphocytes (TDL) obtained from successfully treated recipients were transfused into naive diabetic BB/OK rats grafted with LEW.1BB/OK islets. Independent of the origin of the transfused cells, 64% of recipients maintained normoglycaemia for more than 120 days. To characterize the cell population(s) responsible for transfer of tolerance, B-lymphocytes were removed from the TDLs using the monoclonal antibody OX33 and magnetic beads. When OX33-depleted TDLs were transfused into naive diabetic BB/OK with a LEW.1BB/OK islet graft, all recipients maintained normoglycaemia. The OX33-depleted TDLs consisted only of CD4+ T-lymphocytes, which were either negative for CD45RC or coexpressed the CD45RC at low levels. We conclude that the cell-dependent tolerance transfer is mediated by a TH2-like suppressor cell.

To study the ability of interleukin 1-beta (IL-1) to induce apoptosis in the endocrine pancreas, rat islets of Langerhans obtained from 14-day-old BB.1A rats were exposed to 25 U/ml IL-1 for 40 h. In order to prove the role of nitric oxide (NO) in this process islets were exposed either to 1 mmol/l N-nitro-L-arginine methylester (NAME) or to 25 mmol/l nicotinamide (NA) or to a combination of NA or NAME with IL-1. In dispersed cells oligonucleosomes, resulting from cleavage of nuclear DNA due to apoptosis, were identified by enzymatic labelling the free 3'-OH-DNA ends with fluorescein-dUTP and quantified by means of flow cytometry. After exposure to IL-1, the islets were characterized by elevated basal (in response to 2 mmol/l glucose) insulin release while glucose-stimulated (20 mmol/l glucose) insulin secretion was nearly completely abolished. In contrast, glucose-stimulated insulin secretion was well preserved in NAME-exposed islets, but was markedly inhibited after NA treatment. Accordingly, only the IL-1-induced inhibition of glucose-stimulated insulin secretion was significantly restored in the presence of NAME but was reinforced by NA. IL-1 exposure resulted in a significant increase in the percentage of apoptotic cells (untreated controls 3.8 +/- 0.5% IL-1 18.8 +/- 1.8%, P < 0.01). This effect was significantly reduced in the presence of NA and NAME. Nitrite production which was assayed as an equivalent of NO generation of islets was highest under the influence of IL-1 (16.48 +/- 1.40 versus 2.89 +/- 0.37 pmol/islet for control islets) which was correlated with the percentage of apoptotic cells. IL-1-stimulated nitrite production was reduced to 9.25 +/- 0.48 and 3.41 +/- 0.36 pmol/islet in the presence of NA or NAME, respectively. The results demonstrate the potency of IL-1 to induce apoptosis in rat islets. Since inhibition of NO production was always paralleled by a reduced ability of IL-1 to induce programmed cell death, this radical appears to be involved in this process. Remarkably, the near-complete prevention of NO generation as demonstrated under the influence of NAME was able to prevent the IL-1-induced deterioration of glucose-stimulated insulin secretion in parallel to the prevention of apoptosis-related appearance of DNA double-strand breaks. It is concluded that the elimination of damaged beta cells due to IL-1 exposure is partly achieved by induction of apoptosis.

The development of insulin dependent diabetes mellitus (IDDM) and diabetes in the diabetes prone (DP) BB rat animal model of IDDM is thought to be due to an autoimmune process. Natural killer (NK) cells have been implicated but not proven to play a pathogenetic role in BB rats due to the increased NK cell number and activity found in these animals. We have recently reported that poly I:C, an inducer of cytokines and a potent enhancer of NK cell function, accelerates the development of diabetes in DP BB rats and induces diabetes in diabetes resistant (DR) BB rats. Since we have further demonstrated that poly I:C administration to BB rats increases NK cell number and levels of inducers of NK cell activity, interferon-alpha and IL-6 which is described therein, we tested the hypothesis that NK cell activity plays an important role in poly I:C accelerated disease. The role of NK cells in poly I:C accelerated diabetes and spontaneous diabetes was examined by determining whether selective depletion of NK cells using a rat NK cell specific antibody (anti-NKR-P1 antibody) alters the development of diabetes. The treatment of BB rats with anti-NKR-P1 antibody resulted in a significantly lower mean NK cell activity of splenic mononuclear cells than that found in control animals. However, the development of diabetes and degree of insulitis was not significantly different between treatment groups. BB rats administered anti-NKR-P1 antibody with poly I:C had a lower mean splenocyte NK cell activity and lower mean NK cell number within the peripheral blood and inflamed islets than rats administered poly I:C alone. However, anti-NKR-P1 antibody administration did not alter the accelerated development of diabetes or the degree of insulitis in poly I:C treated animals. These data document that NK cells do not play a major role in the pathogenesis of poly I:C accelerated diabetes or spontaneous diabetes in the DP BB rat.

The spontaneously diabetic BB rat is an excellent and well studied model for human insulin-dependent diabetes (IDDM), sharing many important features with the human disease. Similarities include an equal frequency of IDDM in males and females, production of antibodies against pancreatic cell antigens, and an MHC disease association. In addition, the BB rat shares with human IDDM patients an increased frequency of autoantibodies against the parietal cells of the stomach and colloid cells of the thyroid gland. Here we investigate the genetic basis of thyroiditis in the BB rat. Based on crosses between BB, Lewis and Fischer rats, we show that two susceptibility factors for diabetes--the lymphopenia trait present in diabetes prone BB rats and the MHC--also appear to be risk factors for thyroiditis. However, the nature of the susceptibility was different for the two autoimmune diseases, with lymphopenia being absolutely required for diabetes although it only conferred increased risk for thyroiditis. Also, in contrast to IDDM, the MHC conferred dominant susceptibility to thyroiditis. Despite these shared risk factors, diabetes per se did not show significant correlation with thyroiditis.

In previous studies we reported that a single injection of complete Freund's adjuvant (CFA) in early life inhibits the development of autoimmune diabetes in BB diabetes-prone (DP) rats, and that CFA upregulates tumor necrosis factor-alpha (TNF alpha) production by macrophages of DP rats. In this study, we asked if CFA-induced prevention of diabetes in DP rats might be mediated by TNF alpha. Chronic intraperitoneal injection of TNF alpha, 20 micrograms daily from age 50 to 110 days, significantly decreased diabetes incidence in DP rats from 83% (15 of 18) to 37% (seven of 19) at age 110 days. Similarly, a single intraperitoneal injection of CFA, 100 microliters at age 26 days, significantly decreased diabetes incidence from 80% (16 of 20) to 50% (10 of 20) at age 110 days. Prevention by either TNF alpha or CFA was associated with decreased insulitis and preservation of islet beta-cells. By contrast, treatment of DP rats with CFA, plus antiserum to TNF alpha three times a week from age 26 to 110 days, obviated CFA-induced protection against beta-cell destructive insulitis and diabetes. Thus, diabetes incidence was 75% (18 of 24) in DP rats treated with CFA plus anti-TNF alpha antiserum, similar to that in untreated DP rats (80%), but only 44% (eight of 18) in DP rats treated with CFA plus control serum, similar to that in DP rats with CFA alone (50%). These results suggest that TNF alpha may be a mediator of the protective effects of CFA against autoimmune diabetes development in BB rats.

To determine whether glipizide, a sulfonylurea, can prevent diabetes in the diabetic-prone BB rat model, rats were studied from 35 to 240 days of age. Treated animals received oral glipizide (10 or 100 mg/kg/day) from 35 to 200 days of age, and control rats received oral placebo. From 80 to 135 days of age at both drug doses, glipizide decreased the incidence of diabetes, thus delaying disease onset (P < 0.02). At the higher dose of glipizide, a diabetes preventive effect was observed (P < 0.025). There were no significant differences in body weights between the treated and control groups. At 240 days, i.e. 40 days after stopping glipizide and placebo treatments, diabetes incidence remained stable in the two groups; thus the effect of glipizide persisted after discontinuation of the drug. Serum glucose and insulin levels measured at 90 and 200 days did not reveal differences between the glipizide treated and control groups. To determine whether the sulfonylurea affected autoimmune events, the prevalence and severity of islet inflammation were examined. In glipizide-treated BB rats at 240 days, only 44% of rats had islet inflammation compared to 86% in the control group (P < 0.01). At both 90 and 240 days the severity of islet inflammation was decreased in the glipizide treatment groups compared with the control groups (P < 0.01). These data indicate that glipizide (a) prevents diabetes in the diabetic-prone BB rat strain, (b) decreases the prevalence and severity of islet inflammation even after drug withdrawal and (c) may dampen autoimmune events leading to diabetes onset.

We describe the induction and prevention of autoimmune insulin dependent diabetes mellitus (IDDM), and its pathological substrate, insulitis, in congenitally athymic nude rats following injections of major histocompatibility complex (MHC) compatible lymph node T cells. The cells capable of adoptive transfer of autoimmunity were obtained from diabetes resistant (DR) BB rats that had been rendered hyperglycemic by in vivo depletion of the RT6+ regulatory T cell subset. We first established that our adoptive transfer assay system is cell dose- and time dependent and therefore amenable to quantitative analysis. It was also observed that both CD4+ and CD8+ T cells are required for efficient transfer of autoimmunity. The data indicate that, as in the NOD mouse, a synergistic interaction between CD4+ and CD8+ T cells is important for beta cell destruction. Finally, we demonstrated that the admixture of equal numbers of lymph node T cells, 60% of which were RT6+, from intact, non-diabetic DR rats prevented the adoptive transfer of IDDM mediated by diabetogenic T cells from RT6-depleted DR-BB rats. We conclude that an equilibrium between autoreactive and regulatory cells determines the expression of autoimmunity in the DR-BB rat and in the adoptive transfer of diabetes in quantitative analytical systems.

We previously reported that nonspecific immunomodulations with a streptococcal preparation (OK-432), an inducer of tumor necrosis factor (TNF), or with recombinant TNF prevented development of insulin-dependent diabetes mellitus (IDDM) in animal models (NOD mice and BB rats). Recently we have further reported that lymphotoxin (LT), a cytokine with functional and structural characteristics similar to those of TNF, also protected NOD mice from diabetes. In this study, we have extended our observation on the LT to BB rats. Male and female BB/Wor rats were treated intraperitoneally with recombinant human LT three times a week from 4 to 11 weeks of age. The cumulative incidence of diabetes by 14 weeks of age was 24/30 (80.0%) in nontreated control rats, whereas it was 10/26 (38.5% vs control, P < 0.01) and 4/29 (13.8% vs control, P < 0.0001) in the rats treated with 1 x 10(3) and 1 x 10(4) of LT, respectively. There was no significant difference in nonfasting blood glucose levels and body weights between nontreated control and LT-treated rats, which were nondiabetic. In the LT-treated rats, intensity of insulitis was significantly reduced in comparison with the nontreated rats. Concanavalin A-stimulated TNF/LT productivity of spleen cells was significantly lower in BB/Wor and BB/Sendai rats than in Wistar rats or other normal rat strains. On the other hand, there was no difference between BB/Sendai and Wistar rats in the in vivo TNF/LT productivity induced with LPS or with IFN-gamma plus LPS, and the TNF/LT productivity of these rats was lower on stimulation with LPS alone, but higher with IFN-gamma plus LPS than the other normal rats. These results indicate that treatment with LT, as well as TNF, modulated autoimmunity and prevented development of IDDM in BB/Wor rats which may be low producers of TNF/LT.

Previous studies have shown that dietary iodine enhances the severity and incidence of focal thyroiditis in autoimmune BB rats and OS chickens. However, which lymphoid cells are involved in the development of the iodine-induced focal thyroiditis and what the consequences are for the anticolloid antibody production have not been studied in detail. We therefore performed a study in which 3-week-old female BB rats were kept on either an enriched iodine diet (EID; iodine intake, 100 micrograms iodine/day) or a normal iodine diet (NID; iodine intake, 7 micrograms iodine/day) for a period of 18 weeks. The development of the focal thyroiditis was immunohistologically studied. Immunohistological data were compared to the thyroid hormone status and anti-colloid antibody production. Our data confirm that a high dietary iodine intake results in an accelerated development of the focal lymphoid cell infiltrates in the thyroid of the BB rat. After 12-18 weeks of an EID 50% of the BB rats developed these infiltrates. Our data additionally show that: (a) the process starts with increases in the number of infiltrating MHC class II-positive dendritic cells and a clustering of these cells with T cells, B cells, and some macrophages and (b) the focal infiltrates are highly organized and consist of central B cell follicle-like structures surrounded by rims and areas of T cells. The architecture of the focal thyroiditis is hence very similar to mucosa-associated lymphoid tissue and secondary lymphoid organs (spleen and lymph node). Only minor signs of thyrocyte destruction were observed. We therefore consider the term "thyroiditis" as inappropriate and prefer the term "thyroid-associated lymphoid tissue." Since the thyroiditis component was small, it is also not surprising that the BB rats on the EID remained euthyroid. The presence of the thyroid-associated lymphoid tissue in the BB rats was positively correlated to the presence of anti-colloid antibody in the serum of the BB rats. We speculate that the dietary iodine might have direct effects on cells of the immune system or on cells forming the microenvironment of lymphoid tissue (reticulum cells). A role for highly iodinated thyroglobulin in the accelerated development of thyroid-associated lymphoid tissue is also possible.

Immunomodulatory azaspirane compounds have immunosuppressive activity in animal models of autoimmune disease such as adjuvant-induced arthritis and experimental autoimmune encephalomyelitis. The mechanism of action of azaspiranes appears to be the induction of antigen non-specific (natural) suppressor cell activity. In this study, we tested the azaspirane, SK&F 106610 in an animal model of autoimmune (type 1) diabetes, the BB rat. Oral administration of SK&F 106610 (15 mg/kg/day) to diabetes-prone BB rats, from age 30 days, significantly decreased diabetes incidence at 100 days from 80% (24 of 30 control rats) to 32% (10 of 31 drug-treated rats, P < 0.001). Protection from diabetes by SK&F 106610 was accompanied by decreased lymphocytic infiltration of the pancreatic islets (insulitis). No changes occurred in splenic T cell, B cell or macrophage subsets, or in proliferative responses to the mitogens lipopolysaccharide and concanavalin A (Con-A). Cell mixing experiments in vitro, however, revealed increased antigen non-specific suppressor activity (suppression of splenic lymphoproliferative response to Con-A) in spleens of SK&F 106610-treated rats. The suppressor cell activity was enriched in a low density fraction of splenic cells relatively depleted of T cells, B cells, macrophages and natural killer cells. These results indicate that the azaspirane compound, SK&F 106610 can prevent insulitis and autoimmune diabetes in BB rats and that these actions may be related to the activation of non-specific (natural) suppressor cells.

The Bio-Breeding (BB) rat develops spontaneous insulin-dependent diabetes mellitus (IDDM) and provides a useful animal model to study this human autoimmune disease. Treatment of BB rats with tumor necrosis factor (TNF) has been reported to prevent the development of IDDM. This suggests that deficient TNF production may be involved in the immunopathogenesis of autoimmune diabetes. In this study, we evaluated TNF production in diabetes-resistant (DR) BB rats, diabetes-prone (DP) BB rats, and DP BB rats protected from diabetes by the immunoadjuvant, complete Freund's adjuvant (CFA). TNF production in short-term cultures of peritoneal macrophages from DP rats was significantly less than that from control DR rats, both in the basal state and after stimulation with either interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS) in vivo and in vitro. In contrast, TNF production by macrophages from CFA-injected DP rats (basal and IFN-gamma or LPS-stimulated) was equal to or greater than that by macrophages from DP rats and similar to TNF production by macrophages from CFA-injected DR rats. These results suggest that development of autoimmune diabetes in BB rats may be causally related to deficient macrophage production of TNF, and that upregulation of TNF production may protect against diabetes development.

Considerable indirect evidence implicates participation of natural killer cells (NK) in the pathogenesis of diabetes in BB rats. The most convincing evidence derives from studies showing that anti-CD8 antibody effectively prevents both primary disease onset and autoimmune damage to transplanted islets. However, anti-CD8 treatment depletes both NK and cytotoxic T cells (CTL) since both cell types express the CD8 marker. To study directly the role of NK in diabetic BB rats we used MCA 3.2.3, a monoclonal antibody which selectively depletes normal Lewis rats of NK cells but not CTL. A regimen of ip injected antibody achieved rapid reduction of NK cells in diabetic and nondiabetic BB rats by FACS analysis. NK cell activity remained low in rats treated weekly as evidenced by YAC tumor cell killing. We next studied the effect of NK depletion on disease incidence in diabetes-prone BB rats of which about one half are expected to develop diabetes. Onset and incidence of diabetes in 3.2.3-treated and control antibody-treated aged matched litter mates were equal. These studies suggest that NK cells are not necessary for autoimmune islet destruction in spontaneously diabetic BB rats and support a role for CTL in pathogenesis of the disease.

We have recently reported that chronic and systemic administration of tumor necrosis factor alpha (TNF) inhibits development of autoimmune diabetes in NOD mice and BB rats, animal models of insulin-dependent diabetes mellitus (IDDM). During these experiments, we unexpectedly found that in vivo production of TNF stimulated by a single injection of lipopolysaccharide was enhanced approximately 10 times in the long-term diabetic BB rats (P less than 0.0001), whose mean duration of diabetes with more than 16.8 mM (300 mg/dl) of nonfasting blood glucose level was 26.2 +/- 2.1 days, as compared to that in the rats of nondiabetes and in the rats at the onset of diabetes, whose mean duration of diabetes was 1.4 +/- 0.6 days. The long-term diabetic, but not short-term-diabetic, rats were also associated with increased levels of serum fructosamine/albumin (P less than 0.01) and triglyceride (P less than 0.01) and with a decreased level of serum albumin (P less than 0.01). The in vivo TNF productivity in the diabetic rats, including the short-term- and long-term-diabetic rats, was correlated positively with the level of fructosamine/albumin (P less than 0.05) and negatively with the level of serum albumin (P less than 0.05), but not with levels of blood glucose. None of these correlations were observed in nondiabetic rats. The increased LPS-induced serum TNF activity in the long-term diabetic state was observed not only in BB rats but also in NOD mice and GK rats, a model of non-IDDM, irrespective of sexes and ages, indicating that the enhancement of in vivo TNF production was a result of long-term diabetes. These findings indicate that some factor(s) associated with the long-term-diabetic state may prime macrophages in vivo to produce TNF. Further study is needed to reveal a mechanism of the enhanced TNF production and its possible relevance to various abnormalities associated with the chronic hyperglycemic state.

During diabetes in rats, the choroid of the eye shows increased permeability to albumin, basement membrane thickening, and decreased anionic charge sites on the abluminal surfaces of the choriocapillary microvessels. In other microvascular beds, permeability differences are correlated with differences in luminal membrane microdomains as indicated by the distribution of luminal membrane anionic charge. To see whether luminal surface charge distribution or other structural features of the choroidal microvasculature become altered during diabetes, we studied spontaneously diabetic and control rats using ultrastructural tracers and morphometric techniques. Rats were injected with horseradish peroxidase and perfused with aldehydes, and then retina-choroid tissue sections were incubated with cationized ferritin, reacted to visualize peroxidase, and prepared for electron microscopic study. The most striking alterations in the diabetic rats were vascular debris and migrating cells resembling vascular cells in the choriocapillaris stroma, suggesting an increase in capillary turnover. In addition, extracellular matrix material was increased, and peroxidase uptake and ferritin binding were low in some vessels of the diabetic rats compared with the controls. Variability was large in the diabetic animals, however, and other vessels remained apparently normal.

Daily injections of high dose human recombinant interleukin-1 beta (IL-1 beta) accelerated the onset of both insulin-dependent diabetes mellitus and lymphocytic thyroiditis in genetically prone BB rats. In diabetes-resistant BB rats, high dose IL-1 beta failed to induce diabetes. Additionally, the presence of neutralizing IL-1 beta antibodies in these rats strongly correlated with inhibition of lymphocytic thyroiditis. Since low dose IL-1 beta protects diabetes-prone rats from IDDM, we conclude that IL-1 beta is a potent modulator of autoimmune diabetes and thyroid disease in genetically susceptible rats.

The occurrence of diabetes mellitus was analysed in F2 hybrids bred from diabetic BB male rats and females of congenic rat strains carrying different major histocompatibility (RT1) haplotypes on the common LEW strain genetic background. Permissiveness for the disease in BB rats is probably determined by class II genes of the RT1 complex, which are also present in normal rats. Class I and class III genes, notably the Hsp70 genes in the class III region, do not appear to be involved. Among the diabetic F2 hybrids, about 90% are shown by DNA analysis to be homozygous for the class II genes of the permissive RT1 haplotype. The segregation patterns are in accord with the action of two independent recessive genes, one of them being RT1-linked.

The BB rat spontaneously develops an insulin-dependent diabetes mellitus (IDDM) that closely resembles this disease in man. The pathogenesis involves autoimmune destruction of pancreatic islet beta-cells. In the present study, a single intraperitoneal injection of complete Freund's adjuvant (CFA) in diabetes-prone (DP) BB/Wor rats between 9 and 28 days of age reduced the incidence of diabetes at 120 days from 89% to 10-28%, whereas injection of CFA after 40 days of age was ineffective. The CFA-injected, diabetes-free DP rats had normal levels of pancreatic insulin and little or no mononuclear leukocyte infiltration in the islets. These protective effects of CFA were not associated with any changes in peripheral blood leukocyte counts or monoclonal antibody-defined T cell, B cell, or macrophage subsets in the spleen of the DP rats. These results suggest that it is possible to prevent diabetes by adjuvant treatment in early life without general immunosuppression or sustained therapy.

Immunoregulatory polypeptides (cytokines) produced by mononuclear cells of the immune system can inhibit insulin secretion and may be cytotoxic to pancreatic islet beta cells. We used a modified 51Cr release cytotoxicity assay to define the interactive cytotoxic effects of interleukin-1 (IL-1), tumor necrosis factor (TNF), and interferon gamma (IFN-gamma) on rat islet cells in monolayer culture. We here demonstrate that (a) IFN-gamma, but not IL-1 or TNF, can sensitize islet cells to subsequent cytolysis by other cytokines (IL-1 or TNF); (b) the cytolytic effect of sequential addition of IFN-gamma, then IL-1 or TNF (approximately 35-40% 51Cr release) is similar to that produced by the concurrent addition of IFN-gamma plus IL-1 or TNF; (c) the priming effect of IFN-gamma persists for 3 to 6 or more days after its removal; and (d) islet cells preincubated with IFN-gamma are also more sensitive to cytolysis by splenic mononuclear cells from diabetes-prone BB/Wor rats. These findings suggest that IFN-gamma produced by activated T lymphocytes and monocytic cells infiltrating islets in Type 1 diabetes may play a direct and important role in sensitizing beta cells to damage by other cytokines (IL-1, TNF) and cytotoxic cells in the immune/inflammatory infiltrate.

Endothelial cell permeability and luminal surface anionic sites were studied in the retinal microvasculature of spontaneously diabetic rats. Horseradish peroxidase (HRP) was used as a tracer of pinocytotic transport, and cationized ferritin (CF) was used as a marker of luminal surface anionic sites. Diabetic and control rats were injected with HRP, and their retinas were fixed. Retinal tissue sections were then incubated in CF, reacted to visualize HRP, and prepared for quantitative electron microscopic analysis. In both control and diabetic rats treated with serotonin and histamine antagonists to prevent HRP-induced vascular changes, the endothelium formed a barrier to the tracer. Pinocytotic uptake was relatively low in most vessels. Reaction product was restricted to pinocytotic vesicles, tubular cisternae, and multivesticular bodies. HRP uptake appeared high in some of the deep capillaries of the diabetic retinas as compared with that of the controls, but the difference was not statistically significant. HRP-induced transendothelial permeability was observed in both control and diabetic rats when serotonin and histamine antagonist pretreatment was omitted. CF studies showed anionic sites in four luminal surface microdomains in control and diabetic endothelial cells. CF-binding, anionic sites were present on the plasma membrane, on all coated vesicles, on some uncoated vesicles, and on most diaphragms of uncoated vesicles. Plasma membrane binding was sparse and patchy in some diabetic vessels, especially in the deep vessels of rats that were not treated with the serotonin and histamine antagonists. However, statistical analysis showed similar numbers of plasma membrane binding sites in diabetic and control rats pretreated with serotonin and histamine antagonists. Our data suggest that the retinal microvasculature in diabetic rats remains normal in terms of permeability and luminal membrane anionic charge.

Like human insulin-dependent diabetes, autoimmune diabetes in BB rats and NOD mice is under control of Class II genes of the major histocompatibility complex. The mechanisms of expression of these genes is still unclear. No aberrant expression of Class II antigens was found in BB rats at the onset of diabetes. The putative role of inadequate Class II-linked suppressor control is suggested, however, by the observation that in vivo treatment with anti-Class II monoclonal antibody prevents the onset of diabetes in NOD mice, and that this protection can be transfer by CD4+ T cells.