BBdp Rats Research Articles

The RT6 system of the rat: developmental, molecular and functional aspects

RT6 is a developmentally regulated cell-surface membrane adenosine 5'-diphosphate-ribosyltransferase/nicotinamide adenine dinucleotide-glycohydrolase inserted within the membrane by a glycophosphatidylinositol anchor. In the rat it is restricted to mature T lymphocytes and a subpopulation of natural killer cells. With respect to the data now available, three aspects concerning the function of RT6 are discussed: first, the meaning of the marked polymorphisms; second, its enzymatic activity; third, its possible role concerning T-cell survival. The observation that the rat RT6 gene contains two transcription start sites suggests their different use by distinct subpopulations of T cells. The fact that the expression of RT6 is defective in lymphopenic diabetes prone (DP-BB) rats, although the RT6 gene is structurally not grossly altered in these animals, makes this rat strain a promising model to study the biological meaning of RT6. While it mostly is believed that the RT6 expression defect of the DP-BB rat is a consequence of the lymphopenia, the present paper discusses the possibility that the RT6 expression defect is causally involved in the lymphopenia, and that a normal expression of RT6 may protect the recent thymic emigrants from apoptosis.

Thymic expression of insulin‐related genes in an animal model of autoimmune type 1 diabetes

Background Insulin and multiple other autoantigens have been implicated in the pathogenesis of autoimmune type 1 diabetes, but the origin of immunological self-reactivity specifically oriented against insulin-secreting islet β-cells remains obscure. The primary objective of the present study was to investigate the hypothesis that a defect in thymic central T-cell self-tolerance of the insulin hormone family could contribute to the pathophysiology of type 1 diabetes. This hypothesis was investigated in a classic animal model of type 1 diabetes, the Bio-Breeding (BB) rat. Methods The expression of the mammalian insulin-related genes (Ins, Igf1 and Igf2) was analysed in the thymus of inbred Wistar Furth rats (WF), diabetes-resistant BB (BBDR) and diabetes-prone BB (BBDP) rats. Results RT-PCR analyses of total RNA from WF, BBDP and BBDR thymi revealed that Igf1 and Ins mRNAs are present in 15/15 thymi from 2-day-old, 5-day-old and 5-week-old WF, BBDR and BBDP rats. In contrast, a complete absence of Igf2 mRNA was observed in more than 80% of BBDP thymi. The absence of detectable Igf2 transcripts in the thymus of BBDP rats is tissue-specific, since Igf2 mRNAs were detected in all BBDP brains and livers examined. Using a specific immunoradiometric assay, the concentration of thymic IGF-2 protein was significantly lower in BBDP than in BBDR rats (p<0.01). Conclusions The present study suggests an association between the emergence of autoimmune diabetes and a defect in Igf2 expression in the thymus of BBDP rats. This tissue-specific defect in gene expression could contribute both to the lymphopenia of these rats (by impaired T-cell development) and the absence of central T-cell self-tolerance of the insulin hormone family (by defective negative selection of self-reactive T-cells). Copyright © 2001 John Wiley & Sons, Ltd.

Thymic expression of insulin-related genes in an animal model of autoimmune type 1 diabetes.

Insulin and multiple other autoantigens have been implicated in the pathogenesis of autoimmune type 1 diabetes, but the origin of immunological self-reactivity specifically oriented against insulin-secreting islet beta-cells remains obscure. The primary objective of the present study was to investigate the hypothesis that a defect in thymic central T-cell self-tolerance of the insulin hormone family could contribute to the pathophysiology of type 1 diabetes. This hypothesis was investigated in a classic animal model of type 1 diabetes, the Bio-Breeding (BB) rat. The expression of the mammalian insulin-related genes (Ins, Igf1 and Igf2) was analysed in the thymus of inbred Wistar Furth rats (WF), diabetes-resistant BB (BBDR) and diabetes-prone BB (BBDP) rats. RT-PCR analyses of total RNA from WF, BBDP and BBDR thymi revealed that Igf1 and Ins mRNAs are present in 15/15 thymi from 2-day-old, 5-day-old and 5-week-old WF, BBDR and BBDP rats. In contrast, a complete absence of Igf2 mRNA was observed in more than 80% of BBDP thymi. The absence of detectable Igf2 transcripts in the thymus of BBDP rats is tissue-specific, since Igf2 mRNAs were detected in all BBDP brains and livers examined. Using a specific immunoradiometric assay, the concentration of thymic IGF-2 protein was significantly lower in BBDP than in BBDR rats (p<0.01). The present study suggests an association between the emergence of autoimmune diabetes and a defect in Igf2 expression in the thymus of BBDP rats. This tissue-specific defect in gene expression could contribute both to the lymphopenia of these rats (by impaired T-cell development) and the absence of central T-cell self-tolerance of the insulin hormone family (by defective negative selection of self-reactive T-cells).

The identification of a novel T cell activation state controlled by a diabetogenic gene.

The cyclin-dependent kinase inhibitor p27(kip) regulates the cell cycle at the G(1)-S phase restriction point. S phase entry and cell cycle commitment in peripheral T cells requires p27(kip) degradation, normally initiated by the receipt of costimulatory signals such as those provided by B7.1 or IL-2. We have previously reported that T cells from BioBreeding (BB)-diabetes-prone (DP) rats exhibit decreased costimulatory requirements for activation and cell cycle entry. In the present study, we find that peripheral T cell subsets from BB-DP rats demonstrate activation-like characteristics, including significantly reduced levels of p27(kip) as well as increased levels of proliferating cell nuclear Ag (PCNA). Since our previous studies have established that expression of extracellular activation markers are relatively low in unmanipulated peripheral BB-DP T cells; this p27(low) PCNA(high) phenotype represents a novel activation state. Analyses of T cell subsets from congenic rats demonstrate that this phenotype segregates with the lyp diabetogenic locus and that the p27(low) PCNA(high) phenotype is T cell specific. This p27(low) PCNA(high) phenotype is not seen in medullary thymocytes, but appears abruptly in the recent thymic emigrant population, suggesting that the lyp locus does not act directly on cell cycle regulators but rather alters the interaction between T cells and the peripheral environment. These results provide a biochemical basis for costimulation-independent activation and suggest a mechanism whereby a diabetes susceptibility gene contributes to disease development.

Hydrolysed Casein Diet Protects BB Rats from Developing Diabetes by Promoting Islet Neogenesis

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 β-cell fraction than rats fed NIH, whereas α-cell fraction and β-cell size were not affected by diet or rat type. Apoptotic bodies of β-cells were rare and unaffected by diet. The number of PCNA+β-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 (<four cells) was significantly higher in animals fed the HC diet compared with those fed NIH. Most of the insulin+clusters were also homeodomain-containing transcription factor pancreas duodenum homeobox gene-1 (PDX-1) positive. Glucagon+/PDX-1+clusters were rarely found. These data are consistent with a shift in pancreas homeostasis that maintains islet cell mass by increased islet neogenesis, a process that was enhanced in animals fed a diabetes-retardant diet.

A Functional and Phenotypic Study on Immune Accessory Cells Isolated from the Thyroids of Wistar and Autoimmune-prone BB-DP Rats

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 T3release 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.

The immune responses to diabetes in BB rats supplemented with vitamin A

A substantial amount of evidence suggests that in type I diabetes, vitamin A and zinc status could be of concern because of their impaired metabolic availability. Because both vitamin A and zinc play important roles in the regulation of immune function, the present study was undertaken to examine the immune responses to vitamin A and zinc supplements in diabetic-prone Bio-Breed rats (BBdp), and if the supplements increase the incidence of diabetes. Weanling BBdp rats were fed a NIH-07 diet supplemented with vitamin A either alone or in combination with zinc up to 120 days of age. A greater percentage of rats developing diabetes was found in rats that had supplements of vitamin A and zinc (67%) than those on the basal diet (55%) or with vitamin A supplementation alone (50%). The B cells and macrophages were all markedly increased, whereas CD 4 + and CD 8 + T cells were decreased at the onset of diabetes. However, this immune status was not changed by vitamin A and zinc supplements. The plasma vitamin A levels were significantly decreased in the presence of diabetes and the vitamin A status did not improve when the rats were given vitamin A and zinc supplements. The Natural Killer cell cytotoxicity on a per-cell basis was significantly decreased in the presence of diabetes, irrespective of supplements with vitamin A and zinc. Overall, results indicated that vitamin A and immune status are both affected by type I diabetes; these effects, however, are not responsive to supplemental intakes of vitamin A either alone or in combination with zinc.

Dietary effects on insulin and nutrient metabolism in mesenteric lymph node cells, splenocytes, and pancreatic islets of BB rats

The present studies were performed to determine if a protective diet has different effects on the metabolic activity or function of islet cells, as well as the metabolic activity of mesenteric lymph node (MLN) cells and spleen cells, from BioBreeding (BB) rats. Diabetes-prone BB (BBdp) rats and control non–diabetes-prone BB (BBc) rats were fed for about 20 days either a mainly plant-based diabetogenic diet, NIH-07 (NIH), or a protective semipurified diet with hydrolyzed casein (HC) as the amino acid source. At 6 to 8 weeks of age, BBdp rats had high plasma D-glucose and low insulin concentrations, low insulin content, and low metabolic and secretory responses to D-glucose in isolated pancreatic islets. Islet metabolism, as measured by accumulation of 14C-acidic metabolites, amino acids, and the ratio of D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization was increased in control rats fed HC (P < .05); a similar trend in BBdp rats was not significant. Feeding the HC diet increased islet insulin content (P < .01) by 13% in BBdp and 23% in BBc rats; other metabolic and hormonal variables were unaffected. Compared with BBc rats, BBdp rats displayed higher rates of L-[U-14C]glutamine oxidation, D-[5-3H]glucose utilization, and D-[U-14C]glucose oxidation in MLN cells, but not in splenocytes. There was a dramatic decrease of L-[U-14C]glutamine oxidation in MLN cells from BBc and BBdp rats fed HC. Glycolysis was decreased in control rats. We conclude that the protection afforded by feeding BBdp rats a HC diet is associated with increased insulin in target β cells and downregulation of metabolic activity in gut-associated MLN cells. Metabolic activity in splenocytes, cells representative of the systemic immune system, was less affected. These data suggest that diet-induced metabolic changes occur in the islets and nearby cells of the gut immune system in the period before classic insulitis. Changes in the islets were smaller in comparison to the dramatic remodeling of nutrient catabolism in MLN cells. MLN downregulation may reflect baseline metabolic activity in the absence of diabetogenic (or other) food antigens and further highlights an important interaction between diabetogenic food antigens and the gut immune tissues.

The Metabolic Availability of Vitamin A Is Decreased at the Onset of Diabetes in BB Rats

Streptozotocin (STZ)-induced diabetic rats have been associated with an impaired metabolic availability of vitamin A (retinol). This study was undertaken to investigate whether Biobreeding (BB) rats, in which diabetes mellitus resembling human type I diabetes develops spontaneously, respond the same way at the onset of diabetes. Weaning diabetes-prone (BBdp) and normal (BBn) BB rats consumed NIH-07 nonpurified diet ad libitum until 120 d of age. Plasma and hepatic concentrations of retinol and its carriers, retinol-binding protein (RBP) and transthyretin (TTR) were lower in diabetic BB (BBd) rats than in BBn rats. In parallel with RBP, the abundance of mRNA was lower in the liver of BBd rats. Furthermore, the status of zinc, an important factor for the synthesis of RBP, was also disturbed in BBd rats, as indicated by lower circulatory levels and greater urinary excretion. To determine whether the biochemical evidence of vitamin A deficiency in BBd rats could be reversed, BBdp rats were fed a diet supplemented with vitamin A either alone or in combination with zinc. None of these treatments increased plasma vitamin A concentration. The hepatic abundance of RBP mRNA was significantly greater, whereas circulatory RBP concentrations were unaffected by vitamin A plus zinc supplementation. Overall, these results suggest that impaired metabolic availability of vitamin A, possibly caused by its decreased transport from hepatic stores, is another metabolic derangement associated with type I diabetes.

Islet Protein Expression Changes during Diabetes Development in Islet Syngrafts in BB-DP Rats and during Rejection of BB-DP Islet Allografts

Interleukin 1β (IL-1) is cytotoxic to rat pancreatic β-cells in vitro, and increased expression of IL-1 mRNA is found in the islets of Langerhans during development of diabetes in BB/ Wor/Mol-BB2 (BB-DP) rats and NOD mice. It has been proposed that IL-1 induces a race between protective and deleterious proteins in the β-cells during development of diabetes, and that heat shock proteins 70 and 90, and manganese superoxide dismutase, all inducible by IL-1 are potentially protective proteins. We have established a database of approximately 2000 neonatal rat-islet proteins by two-dimensional gel (2-D gel) electrophoresis of [35S|-methionine labelled neonatal Wistar Furth rat islets. In these IL-1 was shown to up- or down-regulate the islet-expression level of 99, and to induce de novo synthesis of 6 proteins. The identity of most of the IL-1 induced proteins is unknown and under study. In this study we wished to investigate if changes in protein expression induced in vitro by IL-1 stimulation of islets are also seen in vivo during spontaneous development of diabetes in BB-DP rats, and during islet allograft rejection. Two-hundred neonatal BB-DP rat islets were grafted under the kidney capsule of either 30-day-old BB-DP rats killed at onset of diabetes or of 30-day-old Wistar Kyoto (WK) rats, killed 12 days after grafting. Proteins in excised islet-grafts and in vitro IL-1 exposed isolated neonatal BB-DP rat islets were labelled with [35S|-methionine, and processed for 2-D gel electrophoresis. Fluorographs of the gels were analysed by computer. A total of 1815 proteins were found in 3 of 3 12.5% polyacrylamide gels. Interleukin-1 was found to change expression level of 82 of these proteins (22 up- and 60 down-regulated) in neonatal BB-DP rat islets in vitro. Of these 82 proteins 33 (4 up- and 29 down-regulated) also changed level of expression during disease occurrence in syngeneic islet grafts from diabetic BB-DP rats, and 29 (4 up- and 25 down-regulated) during rejection of BB-DP islets grafted to WK rats. Changes in the expression level of 14 (3 up- and 11 down-regulated) of the 82 proteins altered by IL-1 in vitro were only found in syngeneic islet grafts in diabetic BB-DP rats, and changes in the expression level of 8 (2 up- and 6 down-regulated) of these 82 proteins expression were only found in BB-DP islet allografts in WK recipients. Identification of these proteins may be important in understanding the mechanisms of islet destruction during development of insulin-dependent diabetes mellitus and during islet allograft rejection.

Feeding a protective hydrolysed casein diet to young diabetic-prone BB rats affects oxidation of L[U-14C]glutamine in islets and Peyer's patches, reduces abnormally high mitotic activity in mesenteric lymph nodes, enhances islet insulin and tends to normalize NO production.

The present studies were undertaken to examine concomitant diet-induced changes in pancreatic islets and cells of the gut immune system of diabetes-prone BB rats in the period before classic insulitis. Diabetes-prone (BBdp) and control non-diabetes prone (BBc) BB rats were fed for ~ 17 days either a mainly plant-based standard laboratory rodent diet associated with high diabetes frequency, NIH-07 (NIH) or a protective semipurified diet with hydrolyzed casein (HC) as the amino acid source. By about 7 weeks of age, NIH-fed BBdp rats had lower plasma insulin and insulin/glucose ratio, lower insulin content of isolated islets, lower basal levels of NO but higher responsiveness of NO production to IL-1β in cultured islets, and higher Con A response and biosynthetic activities in mesenteric lymphocytes than control rats fed the same diet. In control rats, the HC diet caused only minor changes in most variables, except for a decrease in oxidation of L-[U−C14]glutamine in Peyer's patch (PP) cells and an increase in protein biosynthesis in mesenteric lymphocytes. In BBdp rats, however, the HC diet increased plasma insulin concentration, islet insulin/ protein ratio, and tended to normalize the basal and IL-1β-stimulated NO production by cultured islets. The HC diet decreased oxidation of L-[U−C14]glutamine in BBdp pancreatic islets, whereas oxidation of L-[U−C14]glutamine in PP cells was increased, and the basal [Methyl-H3] thymidine incorporation in mesenteric lymphocytes was decreased. These findings are compatible with the view that alteration of nutrient catabolism in islet cells as well as key cells of the gut immune system, particularly changes in mitotic and biosynthetic activities in mesenteric lymphocytes, as well as basal and IL-1β stimulated NO production, participate in the sequence of events leading to autoimmune diabetes in BB rats. Thus, the protection afforded by feeding a hydrolysed casein-based diet derives from alterations in both the target islet tissue and key cells of the gut immune system in this animal model of type 1 diabetes.

Oxidative stress as a mechanism of diabetes in diabetic BB prone rats: effect of secoisolariciresinol diglucoside (SDG).

Secoisolariciresinol diglucoside (SDG) isolated from flaxseed has antioxidant activity and has been shown to prevent hypercholesterolemic atherosclerosis. An investigation was made of the effects of SDG on the development of diabetes in diabetic prone BioBreeding rats (BBdp rats), a model of human type I diabetes [insulin dependent diabetes mellitus (IDDM)] to determine if this type of diabetes is due to oxidative stress and if SDG can prevent the incidence of diabetes. The rats were divided into three groups: Group I, BioBreeding normal rats (BBn rats) (n = 10); group II, BBdp untreated (n = 11); and group III, BBdp treated with SDG 22 mg/kg body wt, orally) (n = 14). Oxidative stress was determined by measuring lipid peroxidation product malondialdehyde (MDA) an index of level of reactive oxygen species in blood and pancreas; and pancreatic chemiluminescence (Pancreatic-CL), a measure of antioxidant reserve. Incidence of diabetes was 72.7% in untreated and 21.4% in SDG-treated group as determined by glycosuria and hyperglycemia. SDG prevented the development of diabetes by approximately 71%. Development of diabetes was associated with an increase in serum and pancreatic MDA and a decrease in antioxidant reserve. Prevention in development of diabetes by SDG was associated with a decrease in serum and pancreatic-MDA and an increase in antioxidant reserve. These results suggest that IDDM is mediated through oxidative stress and that SDG prevents the development of diabetes.

Angiotensin converting-enzyme inhibitor treatment reduces glomerular p16INK4 and p27Kip1 expression in diabetic BBdp rats.

Renal hypertrophy occurs early in diabetes mellitus and precedes the development of glomerulosclerosis and tubulointerstitial fibrosis. We have previously shown that cultured mesangial cells exposed to high glucose are arrested in the G1-phase of the cell cycle and undergo cellular hypertrophy. High glucose-mediated induction of p27Kip1, an inhibitor of cyclin-dependent kinases, is essential in this process. Further investigations have also shown that p27Kip1 and p21Cip1, other cyclin-dependent kinase inhibitors, are up regulated in the kidneys of mice with Type I (insulin-dependent) as well as Type II (non-insulin-dependent) diabetes mellitus. Our study was undertaken to test a potential effect of short-term treatment with the angiotensin-converting enzyme inhibitor enalapril on the glomerular expression of the cyclin-dependent kinase inhibitors p16INK4, p21Cip1, and p27Kip1 in BBdp rats, an autoimmune model of Type I diabetes. We evaluated p16INK4, p21Cip1, and p27Kip1 protein expression in isolated glomeruli by western blots. We also assessed p27Kip1 positive glomerular cells by immunohistochemistry. Glomerular expression of all three cyclin-dependent kinase inhibitors were stimulated in BBdp rats compared with non-diabetic BBdr animals. Enalapril treatment for 3 weeks, started after the onset of diabetes, reduced the glomerular expression of p16INK4 and p27Kip1 but not of p21Cip1. Enalapril also prevented the increase in kidney weights observed in BBdp rats but had no effect on systolic blood pressure or glucose concentrations. Our data show that enalapril attenuates the glomerular expression of cyclin-dependent kinase inhibitors in diabetes and suggest a molecular mechanism of how angiotensin-converting enzyme inhibitors prevent renal hypertrophy in diabetes.

Effects of a Protective Hydrolyzed Casein Diet upon the Metabolic and Secretory Responses of Pancreatic Islets to IL-1β, Cytokine Production by Mesenteric Lymph Node Cells, Mitogenic and Biosynthetic Activities in Peyer's Patch Cells, and Mitogenic Activity in Pancreatic Lymph Node Cells from Control and Diabetes-Prone BB Rats

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-1β, the production of IFN-γ 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-1β 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.

Recapitulation of Normal and Abnormal BioBreeding Rat T Cell Development in Adult Thymus Organ Culture

Congenitally lymphopenic diabetes-prone (DP) BioBreeding (BB) rats develop spontaneous T cell-dependent autoimmunity. Coisogenic diabetes-resistant (DR) BB rats are not lymphopenic and are free of spontaneous autoimmune disease, but become diabetic in response to depletion of RT6+ T cells. The basis for the predisposition to autoimmunity in BB rats is unknown. Abnormal T cell development in DP-BB rats can be detected intrathymically, and thymocytes from DR-BB rats adoptively transfer diabetes. The mechanisms underlying these T cell developmental abnormalities are not known. To study these processes, we established adult thymus organ cultures (ATOC). We report that cultured DR- and DP-BB rat thymi generate mature CD4 and CD8 single-positive cells with up-regulated TCRs. DR-BB rat cultures also generate T cells that express RT6. In contrast, DP-BB rat cultures generate fewer CD4+, CD8+, and RT6+ T cells. Analysis of the cells obtained from ATOC suggested that the failure of cultured DP-BB rat thymi to generate T cells with a mature phenotype is due in part to an increased rate of apoptosis. Consistent with this inference, we observed that addition of the general caspase inhibitor Z-VAD-FMK substantially increases the number of both mature and immature T cells produced by DP-BB rat ATOC. We conclude that cultured DR-BB and DP-BB rat thymi, respectively, recapitulate the normal and abnormal T cell developmental kinetics and phenotypes observed in these animals in vivo. Such cultures should facilitate identification of the underlying pathological processes that lead to immune dysfunction and autoimmunity in BB rats.

A regulatory defect of constitutive no-synthase in islet endothelial cells correlates with probability of disease manifestation in BBdp rats.

Type I (insulin-dependent) diabetes mellitus is characterised by leucocyte infiltration of pancreatic islets and a progressive destruction of insulin-producing beta cells. As endothelial nitric oxide production is known to regulate adhesion molecule expression and leucocyte permeation, we examined the activity and expression of the constitutive nitric oxide synthase (ecNOS) of islet endothelial cells from prediabetic BBdp rats. Cultures of aortic endothelial cells and islet capillary endothelial cells were established from young normoglycaemic BBdp rats, Wistar rats and diabetes-resistant BBdr rats, all matched for age. Nitrite and citrulline production was measured in all culture supernatants as indicators for ecNOS activities. Expression of ecNOS mRNA was assessed by reverse transcription-polymerase chain reaction. In contrast to those of the aorta, the Wistar rat islet derived endothelial cells exhibited a strong positive correlation of ecNOS activity with the culture medium glucose concentration but none of the BB rat-derived islet endothelial cells showed a similar glucose-responsiveness. Furthermore, at physiological as well as at increased glucose concentrations islet endothelia from all BBdp rats exhibited a considerable decrease in ecNOS activity by a factor of 3 to 6, indicating a specific dysfunction which is also found for the inducible nitric oxide synthase activity after cytokine challenge but effects were less (2.5 to 3 times) dramatic. In contrast, aorta endothelia from all rats exhibited identical ecNOS activities and no glucose responsiveness. We also found a correlation between ecNOS activities and ecNOS-mRNA expression and can exclude the involvement of the inducible isoform. A reproducible and highly significant dysfunction of islet ecNOS expression and activity in young normoglycaemic BBdp rats, which strongly correlates with the probability for disease manifestation is shown.

Both CD4+and CD8+T cells are Required for IFN-γ Gene Expression in Pancreatic Islets and Autoimmune Diabetes Development in Biobreeding Rats

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-CD2mAb) in blood, spleen and islets prevented diabetes development in DP-BB rats, and depletion of CD8+T cells (by anti-CD8mAb) delayed and significantly decreased diabetes incidence. Depletion of either CD4+or CD8+T cells completely prevented IFN-γ mRNA upregulation in islets of DP-BB rats above the low level expressed in islets of diabetes-resistant (DR) BB rats. Also, IL-10mRNA 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-α) were discordant. In contrast, both mAb treatments significantly upregulated IL-4 and TNF-α 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-γ and IL-10 production in islets and β-cell destruction. Depletion of either CD4+or CD8+T cells may prevent β-cell destruction by decreasing IFN-γ and IL-10 production in islets and increasing IL-4 and TNF-α production systemically.

Effect of α-phenyl-N-tert-butylnitrone on diabetes and lipid peroxidation in BB rats

Oxygen free radicals have been shown to interfere with pancreatic islet beta cell function and integrity, and have been implicated in autoimmune type 1 diabetes. We hypothesized that the spontaneous autoimmune type 1 diabetes of the BB rat would be prevented by in vivo administration of a free-radical spin trap, alpha-phenyl-N-tert-butylnitrone (PBN). Twenty-eight diabetes-prone (BBdp) and 13 non-diabetes-prone (BBn) rats received PBN (10 mg/kg) subcutaneously twice daily, and 27 BBdp and 12 BBn rats received saline as controls. Rats were treated from age 47 +/- 6 days until diabetes onset or age 118 +/- 7 days. PBN caused no growth, biochemical, or hematological side effects. Sixteen control BBdp rats became diabetic (BBd, mean age 77 +/- 6 days) and six demonstrated impaired glucose tolerance (IGT rats). The incidence of diabetes and IGT was not different in PBN-treated BBdp rats. Saline-treated rats showed no differences in pancreatic malondialdehyde (MDA) contents of BBd, IGT rats, and the BBdp that did not develop diabetes, versus BBn rats (2.38 +/- 0.35 nmoL/g). Among rats receiving PBN, BBn had lower pancreatic MDA than BBd and IGT rats (1.38 +/- 0.15 vs. 1.88 +/- 0.15 and 2.02 +/- 0.24 nmoL/g, p < 0.05), but not than BBdp rats (1.78 +/- 0.12 nmoL/g, ns). BBn rats receiving PBN also had lower pancreatic MDA than the saline controls (p < 0.05). Thus, PBN is remarkably nontoxic and is able to decrease MDA in the absence of the autoimmune process, but does not prevent diabetes. A combination of PBN with other complementary antioxidant agents may hold better promise for disease prevention.

Signs of Immaturity of Splenic Dendritic Cells from the Autoimmune Prone Biobreeding Rat: Consequences for the In Vitro Expansion of Regulator and Effector T Cells

From the biobreeding-diabetic prone (BB-DP) rat, an animal model for endocrine autoimmunity, phenotype and function of splenic dendritic cells (DC) were studied. Furthermore, the suppressive effect of peritoneal macrophages (pMphi) from the BB-DP rat in the MLR was investigated. Lower numbers of splenic DC were isolated from BB-DP rats than from control Wistar rats. In the preautoimmune phase, DC of the BB-DP rat had a lower surface MHC class II expression (and in preliminary data, a lower CD80 expression), ingested more bacteria, and had a lower stimulatory potency in the syngeneic (syn)MLR as compared with control DC. During disease development, the MHC class II expression further decreased, and a low stimulatory activity became evident in the allogeneic (allo)MLR. With regard to the expansion of suppressor/regulatory T cells, a lower percentage of RT6+ T cells but higher percentages of CD45RClow T cells were induced by BB-DP DC in synMLR, but not in alloMLR. An increase in the CD4/CD8 T cell ratio was observed in both the syn- and alloMLR due to a relative weak expansion of CD8+ T cells with DC of the BB-DP rat. Resident pMphi isolated from BB-DP or Wistar rats were equally effective in suppressing the DC-driven synMLR. In conclusion, splenic DC from the BB-DP rat have a lower accessory cell function already at young age, before the development of disease, and expanded different subsets of effector/suppressor T cells in vitro as compared with those from Wistar rats. The dysfunction of DC from BB-DP rats is likely to be caused by their relative immaturity as indicated by their low class II and costimulatory molecule expression and relatively high phagocytic activity.

Effect of Diet on the Development of the Immune System in the BB Rat.

The incidence of autoimmune diabetes in the BB diabetic-prone (BBdp) rat can be substantially delayed or prevented by replacing cereal-based mixed ingredient (chow) rodent diets with semi-purified (SP) diets differing in protein sources. Replacing a chow diet with a casein-based SP diet early in life reduces the incidence of BB rat diabetes. Feeding soy-based SP diets results in lower diabetes rates than chow but higher than casein. The following study was conducted to determine the effect of early feeding of chow to casein and soy-based SP diets on the development of the immune system in the BB rat. BBdp and non-diabetes prone (BBn) rats were fed a defined chow (NIH) or one of a casein or soy SP diet, to the dams at least one week prior to birth and to the pups of 21-30 days of age. Pups were killed at 15 and 30 days of age and effects of early diet on several immune parameters were determined. At 30 days of age, all chow fed rats were heavier (p<0.05) than the rats of both SP diets. Although the BBdp rats in the chow and soy diet groups were significantly heavier (p<0.05) than BBn rats of the same diet groups, the body weights of casein-fed BBdp and BBn rats were similar. The BBdp animals had a lower (p<0.05) proportion of T cells than BBn rats at 30 days of age. However, the proportion of CD4 and CD8 cells did not differ significantly from BBn for the casein-fed rats. The BBdp animals had poor proliferative responses and their production of IL-2 was unchanged by diet, but the natural killer (NK) activity of the casein-fed rats was reduced (p<0.05) to a level not different from the BBn rats. Diet also altered the immune response of the BBn rats. Feeding a soy-based as compared to a casein-based diet reduced (p<0.05) T cell responses (proliferation and IL-2 production) but increased (p<0.05) parameters of the innate immune system (nitric oxide production and NK cell activity). The findings of this study have important implications in identifying an immune mechanism for dietary effects on the incidence of diabetes in the BB rat and are of potential interest in defining the role of dietary protein source on the development of the immune system.