Diabetes-prone BB Research Articles

Prevention of Spontaneous Autoimmune Diabetes in Diabetes-Prone BB Rats by Prophylactic Treatment with Antirat Interferon- Antibody

Prevention of Spontaneous Autoimmune Diabetes in Diabetes-Prone BB Rats by Prophylactic Treatment with Antirat Interferon- Antibody

Treatment with GAD65 or BSA Does Not Protect Against Diabetes in BB Rats

The Mr 65,000 isoform of glutamic acid decarboxylase (GAD65) has been implicated as the initiating islet cell antigen in the pathogenesis of diabetes, primarily based on studies in non-obese diabetic (NOD) mice. To test the role of this islet cell autoantigen in the pathogenesis of spontaneously occurring diabetes in another animal model, purified recombinant human islet GAD65 was injected i.v. at 200 μg/animal into 18-day-old diabetes-prone BB rats. For controls, bovine serum albumin (BSA), which has also been implicated in the pathogenesis of diabetes, or buffer alone was injected into age matched BB rats. At 210 days of age there were no differences in diabetes incidence in the 3 groups, i.e. 73% (11 of 15) in the GAD65-treated, 81% (13 of 16) in the BSA-treated and 65% (11 of 17) in the buffer-treated animals, or in the median age at onset of disease, i.e. 79 days (range 65-111), 87 days (range 60–107) and 86 days (range 74–109), respectively. The lack of protection against diabetes following GAD65 treatment could hypothetical be explained by no or by an aberrant expression of GAD in BB-rat islet cells. However, immunohistochemistry of pancreata and immunoblot-ting analysis of isolated islets showed that the expression of GAD65 and GAD67 was similar in BB and Lewis rats.In conclusion, these data indicate that neither GAD65 nor BSA autoimmunity is important for the development of diabetes in BB rats, in contrast to the situation in NOD mice, and further emphasizes that extrapolation from only one animal model to autoimmune diabetes in general may not be appropriate.

Lack of Autoimmune Serological Reactions in Rodent Models of Insulin Dependent Diabetes Mellitus

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 β 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.

Interleukin-1 beta inhibits rat thyroid cell function in vivo and in vitro by an NO-independent mechanism and induces hypothyroidism and accelerated thyroiditis in diabetes-prone BB rats.

Interleukin-1 beta has been implicated as a pathogenic factor in the development of autoimmune thyroiditis. When given for 5 days to normal non-diabetes-prone Wistar Kyoto rats, it decreased plasma concentrations of total tri-iodothyronine and thyroxine and increased plasma TSH. These effects were not prevented by co-injection of nitroarginine methyl ester or aminoguanidine, inhibitors of NO synthases. Exposure to interleukin-1 beta dose-dependently reduced iodine uptake in FRTL-5 cells, but had no effect on thyroglobulin secretion. Nitrite was not detected in the FRTL-5 cell culture media after exposure to interleukin-1 beta. However, reverse transcription PCR analysis of mRNA isolated from interleukin-1 beta-exposed FRTL-5 cells revealed a transitory expression of the inducible NO synthase, which was markedly lower than inducible NO synthase induction in interleukin-1 beta-exposed isolated rat islets of Langerhans. Co-incubation with the NO synthase inhibitor NG-monomethylarginine did not ameliorate the effect of interleukin-1 beta on FRTL-5 cell iodine uptake. Furthermore, we demonstrate that daily injections of interleukin-1 beta for 13 weeks aggravated spontaneous thyroiditis and induced severe hypothyroidism in non-diabetic diabetes-prone BB rats. The data suggest that NO does not mediate interleukin-1 beta-induced inhibition of rat thyroid function in vivo or in vitro in FRTL-5 cells, and the induction of hypothyroidism by interleukin-1 beta in diabetes-prone BB rats is speculated to be due to exacerbation of recruitment and activation of intrathyroidal mononuclear cells.

Anti-endothelial cell autoantibodies in BB rats with spontaneous and induced IDDM.

Anti-endothelial cell (anti-EC) antibodies occur in several autoimmune diseases, including human IDDM, but the time course of their development and their importance in disease pathogenesis are unknown. To study such antibodies further, we investigated the BB rat model of autoimmunity. Diabetes-prone (DP) BB rats spontaneously develop autoimmune diabetes, whereas coisogenic diabetes-resistant (DR) BB rats are disease free but can be induced to become diabetic by the depletion of T-cells expressing the RT6 alloantigen. Anti-EC autoantibodies were readily detectable in both untreated DP-BB rats and RT6-depleted DR-BB rats before the onset of diabetes. Their concentration increased with time. The anti-EC antibodies in DP-BB rats were almost exclusively of the IgG2b subclass, whereas those in RT6-depleted DR-BB rats included both the IgG1/2a and the IgG2b subclasses. We also found that intravenous injections of purified immunoglobulins from RT6-depleted DR-BB rats induced abnormal pancreatic vascular leakage in mice. The preabsorption of immunoglobulins against cultured ECs abolished this activity. The pretreatment of mice with silica also abolished the ability of immunoglobulins of RT6-depleted DR-BB rats to induce pancreatic leakage, suggesting that monocytes are involved in the mechanism of anti-EC autoantibody-induced vascular leakage. We conclude that anti-EC autoantibodies are present in rat strains that are genetically predisposed to develop autoimmune diabetes. Their presence early in the disease process and their ability to induce pancreatic vascular leakage suggest that they may participate in diabetes pathogenesis.

Restoration of immune abnormalities in diabetic BB rats after pancreas transplantation. I. Macrochimerism of donor-graft-derived RT6+ T cells responsible for restoration of immune responsiveness and suppression of autoimmune reaction.

Diabetes-prone (DP) BB rats (RT1(u), RT6.1) spontaneously develop insulin-dependent diabetes mellitus (IDDM) and the disease manifestation resembles that in human IDDM. DP rats are immunodeficient with severe T lymphocytopenia due to the absence of T cells expressing the RT6 differential alloantigen, which have immunoregulatory functions. MHC- and non-MHC-compatible Wistar Furth (WF; RT1(u), RT6.2) pancreases were transplanted into DP rats. WF pancreas grafts were destroyed by IDDM recurrence (insulitis), but not by rejection, with a mean survival time of 65.3 +/- 21.7 days. To prevent the recurrence of IDDM in the grafts, monoclonal antibodies to intercellular adhesion molecule-1 and leukocyte function-associated antigen-1 were administered. WF pancreas grafts were indefinitely accepted (>108.0 +/- 26.8 days) in monoclonal antibody-treated DP recipients. The number of T cells was increased and cellular immune responses restored only in the DP rats that had accepted grafts. The increased number of T cells was due to the peripheral appearance of donor-type RT6.2+ T cells, which represented 34.3 +/- 7.0% of total splenic T cells. The cytotoxicity of splenic T cells to WF islet cells was suppressed in the presence of RT6+ T cells in vitro. These findings demonstrated that stable macrochimerism of donor-derived RT6+ T cells could restore the immune responses and prevent the recurrence of IDDM in the DP recipients.

Cytokine gene expression in islets and thyroids of BB rats. IFN-gamma and IL-12p40 mRNA increase with age in both diabetic and insulin-treated nondiabetic BB rats.

Inflammatory cytokines, particularly those produced by Th1 type lymphocytes, are hypothesized to play a major role in the pathogenesis of autoimmune diseases. The present studies investigated this hypothesis in the BB rat. Diabetes-prone (DP) BB rats develop spontaneous hyperglycemia and thyroiditis. Coisogenic diabetes-resistant (DR) BB rats do not develop either disorder spontaneously, but both diseases are induced by depletion of RT6+ T cells. Reverse transcriptase-PCR was used to measure mRNA encoding type 1 and type 2 cytokines. In both DP and RT6-depleted DR rats, IFN-gamma mRNA was present in islets before and during disease onset. IL-2 and IL-4 mRNAs were minimal or undetectable in infiltrated islets but present in activated peripheral T cells. IL-10 mRNA was present at low abundance in infiltrating T cells. These observations suggested a Th1 type inflammatory response, and consistent with this interpretation, we observed that mRNA encoding the p40 chain of IL-12 was also present before and during disease onset. Similar cytokine mRNA profiles were observed in the thyroids of RT6-depleted DR rats and in the islets of DP rats treated with prophylactic parenteral insulin to prevent diabetes. We conclude that IFN-gamma and IL-12 may play a major role in the expression of insulitis and thyroiditis in the BB rat, that Th1 lymphocytes may predominate over Th2 lymphocytes in these inflammatory lesions, and that prevention of diabetes by insulin is not associated with an alteration in the cytokine gene profile of islet infiltrating cells.

Abnormal thymocyte subset distribution and differential reduction of CD4+ and CD8+ T cell subsets during peripheral maturation in diabetes-prone BioBreeding rats.

Abstract In this study we quantified CD8+ and CD4+ T cells in T lymphocytopenic BB rats as compared with control rats at given stages along the maturational pathway from immature thymocytes to mature peripheral T cells. Our results show that BB rats exhibit abnormal thymocyte subset distribution. Numbers of mature TCRhigh/CD4-8+ thymocytes, and also their TCRhigh/CD4+8+ precursors were decreased, as were levels of CD8 expression on all thymocyte subsets investigated. By analogy with mouse thymocyte development, these findings suggest a decreased efficiency for positive selection of CD8 precursors in BB rats. Furthermore, as related to the number of available mature TCRhigh single positive thymocytes, numbers of CD4+ and CD8+ T cells most recently migrated from the thymus were severely decreased in BB blood, indicating either reduced thymic output or rapid cell death after migration. Subsequently, in peripheral blood and cervical lymph nodes, a 95% decrease of CD8+ and a 50 to 80% decrease of CD4+ T cells were demonstrated upon maturation from recent thymic migrants to mature peripheral T cells, leaving the BB rat with a severely reduced T cell population, consisting of CD4+ T cells and a minute population of CD8+ T cells. The vast majority of the latter was found to have an immature peripheral phenotype. Possible consequences of our findings for the generation of autoreactive CD8+ T cells are discussed.

Protection of nonobese diabetic mice from diabetes by intranasal or subcutaneous administration of insulin peptide B-(9-23).

The observation that overt type I diabetes is often preceded by the appearance of insulin autoantibodies and the reports that prophylactic administration of insulin to biobreeding diabetes-prone (BB-DP) rats, nonobese diabetic (NOD) mice, and human subjects results in protection from diabetes suggest that an immune response to insulin is involved in the process of beta cell destruction. We have recently reported that islet-infiltrating cells isolated from NOD mice are enriched for insulin-specific T cells, that insulin-specific T cell clones are capable of adoptive transfer of diabetes, and that epitopes present on residues 9-23 of the B chain appear to be dominant in this spontaneous response. In the experiments described in this report, the epitope specificity of 312 independently isolated insulin-specific T cell clones was determined and B-(9-23) was found to be dominant, with 93% of the clones exhibiting specificity toward this peptide and the remainder to an epitope on residues 7-21 of the A chain. On the basis of these observations, the effect of either subcutaneous or intranasal administration of B-(9-23) on the incidence of diabetes in NOD mice was determined. The results presented here indicate that both subcutaneous and intranasal administration of B-(9-23) resulted in a marked delay in the onset and a decrease in the incidence of diabetes relative to mice given the control peptide, tetanus toxin-(830-843). This protective effect is associated with reduced T-cell proliferative response to B-(9-23) in B-(9-23)-treated mice.

The role of NK cell activity in the pathogenesis of poly I:C accelerated and spontaneous diabetes in the diabetes prone BB rat

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.

Glimepiride-induced prevention of diabetes and autoimmune events in the BB rat: Revised

The purpose of this study was to determine whether Glimepiride, an oral sulfonylurea drug, prevents the onset of diabetes in diabetic prone BB rats. S750181, a sulfonylurea drug that has minimal in vivo glucose metabolic effects, was also tested. In addition, the shortest period of sulfonylurea treatment required for prevention was determined. Eighty rats were studied for all treatment periods with 40 receiving a daily oral gavage dosage of Glimepiride and 40 receiving a daily oral gavage dosage of vehicle solution. Diabetes onset was monitored by glycosuria and blood glucose levels. In study I, with a treatment period of 35–142 days of age, Glimepiride-treated rats showed a 32% incidence of diabetes, whereas control rats had a diabetes incidence of 55% (p < 0.04). In study II, with a treatment period of 60 –140 days of age, Glimepiride-treated rats showed a 29% incidence of diabetes compared to 54% in controls (p < 0.03). Further, comparing the time of diabetes onset between the Glimepiride and control groups showed that Glimepiride delays diabetes onset (p < 0.02). In study III, with a treatment period of 60 –100 days of age, Glimepiridetreated rats showed a 17% overall diabetes incidence at 170 days, whereas the controls were 43% (p < 0.01). In study IV, with a treatment period of 60 –140 days of age, S750181-treated rats showed a 38% diabetes incidence and the control group showed a 43% diabetes incidence. There was no significant delaying or prevention effect observed in the S750181 group. To determine if Glimepiride affected autoimmune events, the severity of islet inflammation was examined. In study I, islet histology from total and nondiabetic animals indicated that Glimepiride-treated rats had a lower severity of islet inflammation than that of the control rats (p=0.023). These studies show that a) Glimepiride has diabetes preventive effects, b) shorter treatment periods of only 40 days can be effective and c) Glimepiride decreases the severity of islet inflammation.

Thyroiditis in the BB rat is associated with lymphopenia but occurs independently of diabetes

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.

Tumor Necrosis Factor Mediates the Protective Effect of Freund's Adjuvant against Autoimmune Diabetes in BB Rats

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 (TNFα) production by macrophages of DP rats. In this study, we asked if CFA-induced prevention of diabetes in DP rats might be mediated by TNFα. Chronic intraperitoneal injection of TNFα, 20μg 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, 100μl 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 TNFα or CFA was associated with decreased insulitis and preservation of islet β-cells. By contrast, treatment of DP rats with CFA, plus antiserum to TNFα three times a week from age 26 to 110 days, obviated CFA-induced protection against β-cell destructive insulitis and diabetes. Thus, diabetes incidence was 75% (18 of 24) in DP rats treated with CFA plus anti-TNFα 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 TNFα may be a mediator of the protective effects of CFA against autoimmune diabetes development in BB rats.

High-Frequency, but Reduced Absolute Numbers of Recent Thymic Migrants among Peripheral Blood T Lymphocytes in Diabetes-Prone BB Rats

In rats, RT6 and CD45RC are expressed by mature peripheral T cells. The underrepresentation of T cells expressing these markers in the T lymphocytopenic BB rat might therefore be a reflection of a relatively immature T cell population. With the use of Thy-1 as a marker for recent thymic migrants, it was demonstrated that BB rats indeed have a phenotypically less mature T cell population than age-matched control rats. However, this could not account for the reduced percentages of RT6+ and CD45RC+ T cells, as these were also decreased among mature Thy-1- T cells of BB rats. Although relatively overrepresented, absolute numbers of Thy-1+ T cells were reduced in BB rats. Absolute numbers of mature Thy-1- T cells were also reduced in BB rats, but to a much larger degree than would proportionally be expected. Our findings taken together led us to conclude that both reduced thymic output and a defect in peripheral expansion are involved in the T lymphocytopenia of BB rats.

Nitric Oxide Synthesis and the Effect of Aminoguanidine and NG-monomethyl-L-Arginine on the Onset of Diabetes in the Spontaneously Diabetic BB Rat

Nitric oxide (NO) synthesis and the effect of aminoguanidine (AG) and NG-monomethyl-L-arginine (NMMA) (inhibitors of NO synthase) on the onset of diabetes were studied in the spontaneously diabetic BB rat. To measure in vivo NO production, 20 male 50-day-old diabetes-prone BB (BBdp) rats and age-matched non-diabetes-prone BB (BBn) rats were individually placed in metabolism cages. The animals had free access to a casein-based semipurified diet and deionized and double-distilled water. Urine excretion was collected every other day for 70 days, and urinary excretion of nitrate was measured as an index of in vivo NO synthesis. The urinary excretion of nitrate was enhanced by 150-200% in BBdp rats 4-6 days before the onset of diabetes, compared with aged-matched BBn rats. There was no difference in urinary excretion of nitrate between BBn rats and those BBdp rats that did not develop diabetes by the age of up to 120 days. To determine a role of NO in the development of spontaneous diabetes, 40-day-old male BBdp rats (30 rats per group) received daily subcutaneous injections of NMMA (acetate salt) (5 mg/kg body wt) or equal amounts of acetate (control) or oral administration of AG (0 or 3 g/l of drinking water) for 80 days. Both NMMA and AG delayed the onset of diabetes in BBdp rats by 13-15 days without altering the rate of incidence of diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytotoxicity Towards Neonatal Versus Adult BB Rat Pancreatic Islet Cells

Cell-mediated autoimmunity is believed to influence the development of diabetes in BB rats. It has been suggested that the autoimmune destruction is due to lack of tolerance induction in early neonatal life caused by delayed maturation of the pancreatic beta cells. The present experiment has been initiated to investigate if there is any difference in in vitro cytotoxicity, and therefore antigenicity, to islet cells from neonatal, young and adult diabetes prone BB rats, and to establish the possible developmental difference in these rats compared to non diabetes prone Wistar Furth rats. Islets from rats of different ages were isolated, dispersed and 51C-labeled. In vitro cytotoxicity mediated by mononuclear spleen cells from newly diabetic BB rats was measured by counting gamma-ray emission from the culture supernatant after 16 h coincubation. We found that full adult-like islet cell maturation in BB rats--as evidenced by sensitivity to cytotoxicity--is not seen before the age of 8 to 21 days after birth. In contrast adult-like cytotoxicity to neonatal islets cells from Wistar Furth rats is seen already at the age of 8 days. Thus delayed islet cell maturation is a fact observed in BB rats.

Adoptive transfer of diabetes to and from old normoglycaemic BB rats

Approximately 4% of diabetes-prone BB/ Mol rats escape overt diabetes which occurs in other rats between 56 and 130 days of age. The ability of preactivated spleen cells from older non-diabetic and from acutely diabetic rats to adoptively transfer diabetes into young diabetes-prone rats was compared, and it was found that they transferred disease with similar incidence and with overlapping onset times in the recipients. Old non-diabetic rats were themselves susceptible to diabetes adoptively transferred from acutely diabetic or from old non-diabetic donors. Lymphocytic insulitis and pancreatic insulin content in unmanipulated old non-diabetic rats were both intermediate between those seen in acutely diabetic and in diabetes-resistant rats. In vivo treatment with polyinosinic-polycytidylic acid induced diabetes with faster onset in old non-diabetic rats than in young diabetes-prone rats. Adoptive transfer of fresh, whole spleen cells from old non-diabetic rats did not protect young BB rats against spontaneous diabetes, while cells from diabetes-resistant rats did. Spleens from old non-diabetic rats contained significantly lower percentages of T cells than spleens from acutely diabetic rats but not lower than spleens from age-matched diabetic rats, suggesting that this reduction was age-related. Finally, spleens from both old non-diabetic and from acutely diabetic rats were negative for the regulatory RT6+ T-cell subset. It is concluded that quiescent beta-cell autoimmunity seen in a fraction of BB/ Mol rats can be reactivated upon non-antigen-specific immune stimulation.

Glipizide-Induced Prevention of Diabetes and Autoimmune Events in the BB Rat

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.

Changes in pancreatic glutathione peroxidase and superoxide dismutase activities in the prediabetic diabetes-prone BB rat.

Pancreatic superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) activities were measured during the development of diabetes in diabetes-prone BB rats (BBdp) prior to insulin dependence. The pancreata from seven to eight BBdp rats of each sex were examined at ages 5, 7, 10, and 18 weeks and compared with age-matched control BB rats (BBc). At Week 18, BBdp rats had moderate to high insulitis but normal levels of blood glucose and insulin. Pancreatic CuZnSOD activity in BBdp rats was two times higher than the activity seen in BBc rats at age 5-10 weeks but then declined to the same level as seen in BBc rats at 18 weeks of age. MnSOD activity increased over time in the BBdp rats but remained very low in BBc rats. These changes in CuZnSOD and MnSOD activity resulted in BBdp rats having twice the pancreatic total SOD activity compared with BBc rats (P < 0.0001). Total GSHPx activity was significantly reduced in the pancreata from both male and female BBdp rats compared with their respective controls (P < 0.01 and P < 0.0001, respectively). The lower total GSHPx activity was due to reduced selenium-dependent GSHPx (SeGSHPx) activity. Erythrocyte and plasma activity of these enzymes was not different between rats with or without insulitis, indicating that differences in enzyme activities were confined to the pancreas. Thus, changes in pancreatic antioxidant enzyme activities occur prior to the development of diabetes symptoms in BBdp rats and may be related to the destruction of the pancreatic B cells and ultimate development of diabetes.

Prevention of autoimmune type I diabetes in biobreeding (BB) rats by a newly established, autoreactive T cell line from acutely diabetic BB rats.

An autoreactive T cell line, BNY-2, was established from lymphocytes isolated from the islets of acutely diabetic BioBreeding (BB) rats after continuous stimulation of the isolated lymphocytes by irradiated lymph node cells. Flow cytometric analysis showed that BNY-2 cells were positive for CD4 and CD5 and were negative for CD8 and RT6, indicating that these cells were phenotypically Th cells rather than cytotoxic T cells. mAbs against rat MHC class II Ags (anti-RT1.D) blocked the proliferation response of BNY-2 cells, suggesting that these cells recognize the MHC class II Du molecule. When splenic T lymphocytes from diabetes-prone BB rats were stimulated by Con A in the presence of irradiated BNY-2 cells or irradiated lymph node cells, the BNY-2 cells had a significant suppressive effect on splenic T cell proliferation, whereas lymph node cells had no effect. When we injected diabetes-prone BB rats i.v. at 30 and 60 days of age with activated BNY-2 cells, the incidence of diabetes was significantly reduced compared with that seen in saline-injected control rats (diabetic incidences were 12 and 80%, respectively). On the basis of these observations, we conclude that autoreactive BNY-2 T cells, established from the pancreatic islets of acutely diabetic BB rats, can prevent the development of autoimmune diabetes in the diabetes-prone BB rat by an immunosuppressive effect.