A fundamental question about the pathogenesis of spontaneous autoimmune diabetes is whether there are primary autoantigens. For type 1 diabetes it is clear that multiple islet molecules are the target of autoimmunity in man and animal models. It is not clear whether any of the target molecules are essential for the destruction of islet beta cells. Here we show that the proinsulin/insulin molecules have a sequence that is a primary target of the autoimmunity that causes diabetes of the non-obese diabetic (NOD) mouse. We created insulin 1 and insulin 2 gene knockouts combined with a mutated proinsulin transgene (in which residue 16 on the B chain was changed to alanine) in NOD mice. This mutation abrogated the T-cell stimulation of a series of the major insulin autoreactive NOD T-cell clones. Female mice with only the altered insulin did not develop insulin autoantibodies, insulitis or autoimmune diabetes, in contrast with mice containing at least one copy of the native insulin gene. We suggest that proinsulin is a primary autoantigen of the NOD mouse, and speculate that organ-restricted autoimmune disorders with marked major histocompatibility complex (MHC) restriction of disease are likely to have specific primary autoantigens.

T cells are known to play an important role in beta cell destruction in the nonobese diabetic (NOD) mouse model of Type I diabetes and islet-specific T cell clones have been demonstrated to be capable of adoptive transfer of diabetes. One important issue involves the identity of beta cell antigens that are recognized by nominally islet cell-specific T cell clones. We have previously reported that insulin-specific T cells are a predominant component of islet-specific T cells isolated from islet infiltrates of pre-diabetic NOD mice. In this report we examine six independently derived insulin-specific T cell clones established from islet infiltrates of pre-diabetic NOD mice in detail. All six clones were found to be specific to a region of the insulin molecule defined by a synthetic peptide encompassing residues 9-23 of the B chain. Despite this restricted specificity, each member of this panel exhibited a distinct receptor specificity defined either by V beta usage or antigen fine specificity. Five clones produced interferon (IFN)-gamma but not interleukin (IL)-4, placing them in the T helper type 1 (TH1)-like category whereas one clone produced both IL-4 and IFN-gamma, a characteristic of TH0 cells. All six clones were capable of either acceleration of diabetes in young NOD mice or adoptive transfer to NODscid mice. Taken together, these results suggest that spontaneously arising insulin-specific T cells participate in beta cell destruction during development of diabetes in NOD mice.

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.It is now well established that insulin-dependent diabetes mellitus (IDDM) is an autoimmune disorder in which the insulin-producing beta cells are specifically destroyed. Although the available data indicate that T cells are the predominant mediators of beta cell destruction (1-4), overt diabetes is often preceded by the appearance of circulating antibodies specific to a number of beta cell products, among which is insulin (5). Insulin autoantibodies (IAAs) have been found in many new onset diabetic subjects prior to the administration of exogenous insulin (5) and have been found to precede the onset of diabetes by many years in certain individuals. The nonobese diabetic (NOD) mouse, which develops IDDM with many similarities to the human disease, is considered to be a good model of type I diabetes (6, 7), and IAAs have also been found to be present in NOD mice prior to the onset of diabetes (8-10). The presence of IAAs implies that there is also a T-cell response to this antigen, and although NOD mice can respond to insulin when immunized with insulin (11), attempts to demonstrate spontaneous T-cell responses to insulin in NOD mice have failed (12). In contrast, results from our laboratory indicate that T-cell responses to insulin arise spontaneously in NOD mice but that insulinspecific T cells are sequestered within the islet infiltrates characteristic of the disease process in these mice (13). More detailed investigations have revealed that insulin-specific T The publicatio...

Numerous investigation have demonstrated that T cells are involved in destruction of beta cells in the NOD mouse, a widely studied model of type I diabetes. In this report we describe a series of islet-specific T cell lines established from islet-infiltrating lymphocytes obtained from individual pre-diabetic NOD mice as well as a large panel of clones derived from these lines. Proliferation assays indicated that these nominally islet-specific lines responded vigorously to porcine insulin. Furthermore, of 40 islet-specific clones derived from lines established from 12-week-old mice, 22 (55%) responded to insulin. A similar analysis of islet-specific clones established from 7-week-old mice indicated that 2 of 14 (14%) were insulin specific. These findings demonstrate that insulin-specific T cells can comprise a major portion of the spontaneously arising T cell response to islets in NOD mice.

The Ia+, H-2d BALB/c lymphoma cell line L10.A 2J was fused to T cell-depleted spleen cells from mouse strains bearing other H-2 haplotypes. A portion of the selected hybrids expressed Ia antigens of the normal spleen cell partner in the fusion as evidenced by their presentation of various antigens to a set of antigen-specific I-region-restricted T cell hybridomas. Three cloned hybrids were studied in detail. Antigen presentation was shown to be inhibited specifically by monoclonal anti-Ia antibodies. Both I-A and I-E molecules were expressed, including in the one case examined hybrid I-A and I-E molecules between the H-2d and H-2b haplotypes. These Ia+ B cell hybridomas provide a useful set of tools for studying the role of I-region-encoded molecules in antigen presentation to T cells.

Insulin-dependent diabetes mellitus (IDDM) is the result of a genetically associated autoimmune mediated process in which the insulin-producing pancreatic beta-cells are thought to be destroyed by autoreactive T cells [1,2]. The disease process is accompanied by autoreactive T cells and autoantibodies to various islet antigens. Many IDDM patients develop humoral immune responses to insulin [3,4], islet cells [5], tyrosine phosphatase homologue islet cell antibody ((ICA)512/islet cell antigen (IA)-2, 37 K) [6-9] and glutamic acid decarboxylase (GAD)65 [10][11][12]. Even though autoantibodies may not be directly involved in the destructive process, 60 % of new-onset diabetic patients are positive for insulin autoantibodies (IAA) compared to only 0.5 % of healthy control subjects [2]. The involvement of T cells in the pathogenesis has been implicated by insulitis [13], the recurrence of insulitis and diabetes after pancreas transplantation [14] and the transfer of diabetes with non-T-cell-depleted bone marrow transplants

No abstract

Drug-induced liver injury (DILI) is a major safety concern in drug development. Its prediction and prevention have been hindered by limited knowledge of the underlying mechanisms, in part the result of a lack of animal models. We developed a mouse model of halothane-induced liver injury and characterized the mechanisms accounting for tissue damage. Female and male Balb/c, DBA/1, and C57BL/6J mice were injected intraperitoneally with halothane. Serum levels of alanine aminotransferase and histology were evaluated to determine liver injury. Balb/c mice were found to be the most susceptible strain, followed by DBA/1, with no significant hepatotoxicity observed in C57BL/6J mice. D rug-induced liver injury (DILI) represents a major health problem that challenges not only health care professionals but also the pharmaceutical industry and drug-regulatory agencies. In the United States, DILI accounts for as many as 25% of liver failure cases in intensive care units. 1 Due to its association with significant patient morbidity and mortality, DILI is currently the most common cause for the withdrawal of drugs from the pharmaceutical market. 2 The key to predicting and preventing DILI is a thorough understanding of the underlying mechanisms. Prospective human clinical trials would be impossible given the unpredictable nature and low incidence of DILI. Retrospective studies may be conducted; however, they will not yield mechanistic information on the development of DILI. In vitro experiments may be used as a means to understand isolated events in DILI development, but these experiments are not likely to provide insights into the complex molecular and cellular mechanisms of DILI. The best approach for these purposes is to perform animal studies.At present, the only widely studied model of DILI is the acetaminophen (APAP)-induced liver injury model, particularly in mice. Although important information on the mechanism(s) of drug-induced acute inflammatory injury has been obtained using this model, 3-10 APAP hepatotoxicity in mice or humans does not encompass the complete clinical spectrum nor all possible mechanistic