Functional status of the immune system after chronic administration of 2'-deoxycoformycin in the BB rat

Insulin-dependent diabetes mellitus (IDDM) is caused by autoimmune destruction of pancreatic beta cells with the primary mechanism being cell mediated. The BB rat develops insulitis and IDDM with many features analogous to the disease in man. In previous studies we reported that weekly administration of 2'-deoxycoformycin (dCF) for four months reduces significantly the incidence of IDDM in the BB rat by 70%, and that the animals remain free of diabetes for a minimum of two months after drug withdrawal. Since the diabetes-prone BB rat is lymphopenic, with a reduction of both CD4 and CD8 cells, the continuous failure of dCF treated animals to develop diabetes may have been due to generalized immunosuppression. To test this possibility, the ability of dCF treated diabetes-free BB rats to mount an immune response after challenge with Ovalbumin was examined five months after drug withdrawal. The results showed that the post-immunization levels of total IgG and specific IgG in these animals did not differ from those observed in non-dCF treated controls nor those of control diabetes-resistant non-lymphopenic BB rats. Moreover, FACS analysis indicated no change in the percentages of total numbers of CD4+ or CD8+ cells between the two groups of animals. Histological assessment of the pancreata of the post-dCF treated animals showed varying degrees of mononuclear cell infiltrates in the islets. These data demonstrate that treatment by dCF is not permanent, and may require intermittent or continuous administration to prevent development of diabetes. Further studies are needed to determine the mechanism of action of dCF in this model of IDDM.     

Effect of alpha-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.     

Autoimmunity-prone BB rats lack functional cytotoxic T cells

BB rats are prone to develop an autoimmune form of insulin-dependent diabetes mellitus (IDDM) and thyroiditis. Development of autoimmunity is thymus dependent. Previous studies have shown that BB rats lack a population of T cells bearing the RT6 antigen and have very low numbers of suppressor/cytotoxic T cells. In this study, we confirm that BB rats have decreased numbers of phenotypic T suppressor/cytotoxic (Ts/c) cells (OX19+, OX8+ cells) in their lymphoid organs. Moreover, we find that the phenotypic Ts/c cells of BB rats lack apparent cytotoxic activity. These T cells fail to kill allogeneic target cells in a cell-mediated lympholysis assay and fail to generate lectin-dependent cytotoxicity. The addition of interleukin 2, gamma-interferon, and other lymphokines to cultures of BB T cells does not induce functional cytotoxic T lymphocytes. We find that the activated T cells of newly diabetic rats are incapable of killing major-histocompatibility-complex-matched islet cells, despite the ability of these cells to cause IDDM in passive transfer experiments. We conclude that autoimmune disease occurs in BB rats in the absence of functional cytotoxic T cells.     

Virus-induced diabetes in a transgenic model: role of cross-reacting viruses and quantitation of effector T cells needed to cause disease

Virus-specific cytotoxic T lymphocytes (CTL) at frequencies of >1/1, 000 are sufficient to cause insulin-dependent diabetes mellitus (IDDM) in transgenic mice whose pancreatic beta cells express as "self" antigen a protein from a virus later used to initiate infection. The inability to generate sufficient effector CTL for other cross-reacting viruses that fail to cause IDDM could be mapped to point mutations in the CTL epitope or its COO(-) flanking region. These data indicate that IDDM and likely other autoimmune diseases are caused by a quantifiable number of T cells, that neither standard epidemiologic markers nor molecular analysis with nucleic acid probes reliably distinguishes between viruses that do or do not cause diabetes, and that a single-amino-acid change flanking a CTL epitope can interfere with antigen presentation and development of autoimmune disease in vivo.     

Combination therapy with an antioxidant and a corticosteroid prevents autoimmune diabetes in NOD mice.

Oxygen free radicals have been implicated as mediators of pancreatic islet beta cell damage in autoimmune, insulin-dependent diabetes mellitus (IDDM). In this study, we show that the antioxidant, probucol, produced only a small decrease in diabetes incidence in nonobese diabetic (NOD) mice, an animal model for human IDDM. However, combination of probucol with the antiinflammatory corticosteroid, deflazacort, produced an early synergistic effect, delaying diabetes onset by 3 weeks, and a later additive effect, decreasing diabetes incidence from 68% (17 of 25 mice) to 23% (6 of 26 mice, p < 0.005). Protection against diabetes by the combination of probucol and deflazacort was associated with a significant decrease in pancreatic islet infiltration by macrophages/lymphocytes (insulitis) and prevention of islet beta cell loss.     

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

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

Feeding a Protective Hydrolysed Casein Diet to Young Diabetes-prone BB Rats Affects Oxidation of L[U?C14] 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.     

BioBreeding/Worcester (BB/Wor) rats are deficient in the generation of functional cytotoxic T cells

The BioBreeding/Worcester (BB/Wor) rat provides a good model of spontaneous autoimmune diabetes. There are several sublines of the BB/Wor rat. The diabetes prone (DP) sublines develop diabetes at a frequency of 50 to 80% from 60 to 120 days of age. The DP rats are lymphopenic, have a severe deficit in phenotypic OX 19+ OX 8+ cytotoxic T cells (Tc), and lack RT 6.1 T cells. These rats have a relative increase in OX 19- OX 8+ natural killer (NK) cells and in NK activity as compared with the diabetes resistant (DR) sublines. The DR sublines have a normal complement of phenotypic Tc and RT 6.1 T cells, fewer NK cells, and lower NK activity than the DP rat. The ability to elicit functional Tc in the BB/Wor rat has not been well studied. In these experiments, by using a model of lymphocytic choriomeningitis virus (LCMV) infection in DP and DR rats, we have studied the functional activity of Tc in these lines. Seven days after infection with LCMV, DR rats develop lymphocytes which are cytotoxic for LCMV-infected syngeneic fibroblasts. These cytotoxic lymphocytes are phenotypic Tc (OX 19+ OX 8+), and do not kill Pichinde virus-infected syngeneic fibroblasts or LCMV-infected allogeneic fibroblasts. This cytotoxic activity is accompanied by an increase in phenotypic Tc from 17 to 33%. DP rats produced neither functional nor phenotypic Tc. These studies confirm that NK cells are the predominant cytotoxic lymphocyte in the BB/Wor rat and suggest that these rats may not utilize a Tc mechanism in islet destruction or another immunologic process such as graft rejection.     

Deficiencies in gut NK cell number and function precede diabetes onset in BB rats

Defects in the intestinal immune system may contribute to the pathogenesis of autoimmune diseases. Intraepithelial lymphocytes represent a substantial fraction of gut-associated lymphocytes, but their function in mucosal immunity is unclear. A newly described population of NK cells that spontaneously secrete IL-4 and IFN-gamma is present in the intraepithelial lymphocyte compartment of the rat. We hypothesized that defects in the number or function of these cells would be present in rats susceptible to autoimmunity. We report that the number of NKR-P1A(+)CD3(-) intraepithelial NK (IENK) cells is deficient before onset of spontaneous autoimmune diabetes in diabetes-prone BB (BBDP) rats. The absolute number of recoverable IENK cells was only approximately 8% of that observed in WF rats. Bone marrow transplantation from histocompatible WF donors reversed the IENK cell deficiency (and prevented diabetes) in these animals, suggesting a hemopoietic origin for their IENK cell defect. Analysis of diabetes-resistant BB rats, which develop autoimmune diabetes only after perturbation of the immune system, revealed IENK cell numbers intermediate between that of BBDP and WF rats. IENK cells were selectively depleted during treatment to induce diabetes. Prediabetic BBDP and diabetes-resistant BB animals also exhibited defective IENK cell function, including decreased NK cell cytotoxicity and reduced secretion of IL-4 and IFN-gamma. IENK functional defects were also observed in LEW and BN rats, which are susceptible to induced autoimmunity, but not in WF, DA, or F344 rats, which are resistant. Defects in IENK cell number and function may contribute to the pathogenesis of autoimmune diseases including type 1 diabetes.     

Infection of peripancreatic lymph nodes but not islets precedes Kilham rat virus-induced diabetes in BB/Wor rats.

A parvovirus serologically identified as Kilham rat virus (KRV) reproducibly induces acute type I diabetes in diabetes-resistant BB/Wor rats. The tissue tropism of KRV was investigated by in situ hybridization with a digoxigenin-labelled plasmid DNA probe containing approximately 1.6 kb of the genome of the UMass isolate of KRV. Partial sequencing of the KRV probe revealed high levels of homology to the sequence of minute virus of mice (89%) and to the sequence of H1 (99%), a parvovirus capable of infecting rats and humans. Of the 444 bases sequenced, 440 were shared by H1. KRV mRNA and DNA were readily detected in lymphoid tissues 5 days postinfection but were seldom seen in the pancreas. High levels of viral nucleic acids were observed in the thymus, spleen, and peripancreatic and cervical lymph nodes. The low levels of infection observed in the pancreas involved essentially only endothelial and interstitial cells. Beta cells of the pancreas were not infected with KRV. These findings suggest that widespread infection of peripancreatic and other lymphoid tissues but not pancreatic beta cells by KRV triggers autoimmune diabetes by perturbing the immune system of genetically predisposed BB/Wor rats.     

Cellular and molecular mechanism for Kilham rat virus-induced autoimmune diabetes in DR-BB rats

Kilham rat virus (KRV) causes autoimmune diabetes in diabetes-resistant BioBreeding (DR-BB) rats; however, the mechanism by which KRV induces autoimmune diabetes without the direct infection of beta cells is not well understood. We first asked whether molecular mimicry, such as a common epitope between a KRV-specific peptide and a beta cell autoantigen, is involved in the initiation of KRV-induced autoimmune diabetes in DR-BB rats. We found that KRV peptide-specific T cells generated in DR-BB rats infected with recombinant vaccinia virus expressing KRV-specific structural and nonstructural proteins could not induce diabetes, indicating that molecular mimicry is not the mechanism by which KRV induces autoimmune diabetes. Alternatively, we asked whether KRV infection of DR-BB rats could disrupt the finely tuned immune balance and activate autoreactive T cells that are cytotoxic to beta cells, resulting in T cell-mediated autoimmune diabetes. We found that both Th1-like CD45RC+CD4+ and cytotoxic CD8+ T cells were up-regulated, whereas Th2-like CD45RC-CD4+ T cells were down-regulated, and that isolated and activated CD45RC+CD4+ and CD8+ T cells from KRV-infected DR-BB rats induced autoimmune diabetes in young diabetes-prone BioBreeding (DP-BB) rats. We conclude that KRV-induced autoimmune diabetes in DR-BB rats is not due to molecular mimicry, but is due to a breakdown of the finely tuned immune balance of Th1-like CD45RC+CD4+ and Th2-like CD45RC-CD4+ T cells, resulting in the selective activation of beta cell-cytotoxic effector T cells.     

Congenic diabetes-prone BB.Sa and BB.Xs rats differ from their progenitor strain BB/OK in frequency and severity of insulin-dependent diabetes mellitus.

Two newly established congenic diabetes-prone BB rat strains designated BB.Sa and BB.Xs carrying a region of chromosome 1 (Sa-Lsn-Secr-Igf2-Tnt, 16 cM) and a region of chromosome X (DXMgh3-Mycs/Pfkb1-Ar, 36 cM) of the SHR rats, respectively, were studied to determine whether the transferred chromosomal regions influence diabetes frequency, age at onset, and clinical picture. Therefore, 4 complete litters of BB/OK (n = 43), BB.Sa (n = 45), and BB.Xs (n = 41) were observed for diabetes occurrence up to the age of 30 weeks. From these litters 6 diabetic males of each strain manifesting in an interval of 1 week were chosen to study body weight, blood glucose, insulin requirement to survive, and several diabetes-related serum constituents at onset of diabetes and after a diabetes duration of 150 days. The diabetes frequency was significantly lower in BB.Xs than in rats of the parental strain BB/OK, whereas comparable frequencies were found between BB/OK and BB.Sa rats. Obvious differences were observed 150 days after diabetes onset between BB/OK and both BB.Sa and BB.Xs rats. BB/OK rats were significantly heavier and needed significantly more insulin/100 g body weight than BB.Sa and BB.Xs rats. Comparisons of the serum constituents as lipids, proteins, and minerals revealed significant differences between diabetic BB/OK rats and their diabetic congenic derivatives in several traits studied at onset and after 150 days of insulin treatment. These results not only show the power of congenic lines in diabetes research, but indicate for the first time that there are genetic factors on chromosomes 1 and X influencing frequency and severity of diabetes in the BB/OK rat.     

Infections that induce autoimmune diabetes in BBDR rats modulate CD4+CD25+ T cell populations

Viruses are believed to contribute to the pathogenesis of autoimmune type 1A diabetes in humans. This pathogenic process can be modeled in the BBDR rat, which develops pancreatic insulitis and type 1A-like diabetes after infection with Kilham's rat virus (RV). The mechanism is unknown, but does not involve infection of the pancreatic islets. We first documented that RV infection of BBDR rats induces diabetes, whereas infection with its close homologue H-1 does not. Both viruses induced similar humoral and cellular immune responses in the host, but only RV also caused a decrease in splenic CD4(+)CD25(+) T cells in both BBDR rats and normal WF rats. Surprisingly, RV infection increased CD4(+)CD25(+) T cells in pancreatic lymph nodes of BBDR but not WF rats. This increase appeared to be due to the accumulation of nonproliferating CD4(+)CD25(+) T cells. The results imply that the reduction in splenic CD4(+)CD25(+) cells observed in RV-infected animals is virus specific, whereas the increase in pancreatic lymph node CD4(+)CD25(+) cells is both virus and rat strain specific. The data suggest that RV but not H-1 infection alters T cell regulation in BBDR rats and permits the expression of autoimmune diabetes. More generally, the results suggest a mechanism that could link an underlying genetic predisposition to environmental perturbation and transform a "regulated predisposition" into autoimmune diabetes, namely, failure to maintain regulatory CD4(+)CD25(+) T cell function.     

Molecular characterization of MHC class II antigens (β 1 domain) in the BB diabetes-prone and -resistant rat

The BB or BB/Worcester (BB/W) rat is widely recognized as a model for human insulin-dependent diabetes mellitus (IDDM). Of at least three genes implicated in genetic susceptibility to IDDM in this strain, one is clearly linked to the major histocompatibility complex (MHC). In an attempt to define the diabetogenic gene(s) linked to the MHC of the BB rat, cDNA clones encoding the class II MHC gene products of the BB diabetes-prone and diabetes-resistant sublines have been isolated and sequenced. For comparison, the ₁ domain of class II genes of the Lewis rat (RTl^L) were sequenced. Analysis of the sequence data reveals that the first domain of RT1.D and RT1.B chain of the BB rat are different from other rat or mouse class 11 sequences. However, these sequences were identical in both the BB diabetes-prone and BB diabetes-resistant sublines. The significance of these findings is discussed in relation to MHC class II sequence data in IDDM patients and in the nonobese diabetic (NOD) mouse strain.     

Adenoviral insulin gene therapy prolongs survival of IDDM model BB rats by improving hyperlipidemia.

Diabetes is frequently associated with hyperlipidemia, which results in atherogenic complications. Insulin-dependent diabetes mellitus (IDDM) model BB/Wor//Tky (BB) rats exhibit both hyperglycemia and hyperlipidemia and die within 3 weeks after the onset of diabetes unless insulin therapy is given. We performed insulin gene therapy in BB rats with adenovirus vectors through the tail vein. After infusion, plasma triglyceride levels dropped quickly and maintained low levels for 1 week, whereas blood glucose levels showed a slight decrease. The survival period of diabetic BB rats was prolonged to up to 75 days by infusing insulin gene-expressing adenoviral vectors. We suggest that the control of hyperlipidemia can be a life-saving measure when combined with hyperglycemia control in the treatment of diabetes.     

Coxsackieviruses and diabetes

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease whose etiology is complex. Both genetic susceptibility, which is polygenic, and environmental factors, including virus infections, appear to be involved in the development of IDDM. In this review, we have tried to balance the discussion of diabetes by examining both immunological and virological perspectives. Several mouse models, including viral and non-viral models, have been used to study diabetes. For this review, we include lessons gleaned from the non-obese diabetic (NOD) mouse and from mouse models of coxsackievirus- and encephalomyocarditis-virus-induced diabetes. Finally, we present a multi-stage model in which several viral infections, including the coxsackieviruses, are postulated to play a role in the autoimmune destruction of pancreatic beta cells.     

Induction of diabetes by PolyI:C and anti-RT6.1 antibody treatment in DR-BB rats.

To determine whether environmental factors could affect the incidence of diabetes in RT6.1+ lymphocytes-depleted diabetes resistant (DR) BB rats, we tested polyinosinic-polycytidylic acid (Poly I:C), as an immune activator, in conjunction with anti-RT6.1 antibody in DR-BB rats which were bred in a specific pathogen free (SPF) condition. Diabetes was induced by the combined administration of poly I:C and anti-RT6.1 antibody. The use of poly I:C or anti-RT6.1 antibody alone did not cause diabetes. These results suggest that RT6.1+ T lymphocytes regulate autoimmune diabetes and that non-specific immune activation caused by environmental factors plays a key role in inducing diabetes in DR-BB rats.     

Spontaneous diabetes mellitus syndrome in the rat. II. T lymphopenia and its association with clinical disease and pancreatic lymphocytic infiltration

We have studied the occurrence of two phenotypic components (pancreatic lymphocytic infiltration [PLI] of the pancreas and T lymphocytopenia) of the spontaneous insulin-dependent diabetic syndrome (IDDM) in the progeny of hybrids obtained by crossing BB diabetic rats with rats of inbred strains differing from the BB rat at the major histocompatibility complex, RT1. Both PLI and T lymphopenia were seen in animals with all three possible genotypes in both (BUF x BB) and (LEW x BB) lines. PLI was seen in all IDDM animals. T lymphopenia was strongly associated with overt IDDM in both lines (chi 2 = 22.28, p = 0.00002 and chi 2 = 19.28, p less than 0.00001). In addition, T lymphopenia was associated with PLI with and without IDDM in both lines (chi 2 = 8.32, p = 0.0039 an chi 2 = 3.95, p = 0.0467). Not all animals exhibiting PLI without overt IDDM had depressed T cells. Not all animals with T lymphopenia had PLI with or without IDDM. In both lines, the overt IDDM occurred only in animals with at least one RT1 u haplotype derived from the BB rat, confirming our previously reported association of IDDM and RT1. We interpret this evidence to suggest that the overt IDDM syndrome requires one MHC-linked gene and at least two non-MHC-linked genes, which determine susceptibility to PLI and to circulating T lymphocyte depression.