Coupling of oral human or porcine insulin to the B subunit of cholera toxin (CTB) overcomes critical antigenic differences for prevention of type I diabetes.

Our earlier investigations have demonstrated a critical difference in the efficacy of orally administered porcine compared to human or mouse insulin (no effect) in preventing type I diabetes in two distinct experimental models. Based on these findings one has to assume that certain insulins might not be suitable for the induction of oral 'tolerance'/bystander suppression, which might be one cause for recent failures in human oral antigen trials. Here we demonstrate that coupling to the non-toxic subunit of cholera toxin (CTB) can abolish these differences in efficacy between human and porcine insulin. As expected, an added benefit was the much smaller oral antigen dose required to induce CD4+ insulin-B specific regulatory cells that bystander-suppress autoaggressive responses. Mechanistically we found that uptake or transport of insulin-CTB conjugates in the gut occurs at least partially via binding to GM-1, which would explain the enhanced clinical efficacy. Both B chains bound well to major histocompatibility complex (MHC) class II, indicating comparable immunological potential once uptake and processing has occurred. Thus, our findings delineate a pathway to overcome issues in oral antigen choice for prevention of type I diabetes.

The cholera toxin B subunit is a mucosal adjuvant for oral tolerance induction in type 1 diabetes.

When conjugated to various proteins, the nontoxic B-chain of cholera toxin (CTB) significantly increases the ability of these proteins to induce immunological tolerance after oral administration. Here, we investigated if a nonconjugated form of CTB enhances the induction of immune tolerance after oral insulin administration. Induction of immunological tolerance was studied after oral administration of insulin preparations in three mouse models; an insulin/ovalbumin coimmunization model, a model of virus-induced diabetes in transgenic RIP-LCMV-NP mice and in nonobese diabetic (NOD) mice serving as a model of spontaneous diabetes. In the immunization model, we demonstrate that mixing with CTB increases the tolerogenic potential of insulin, approximately 10 fold. Titration of the CTB concentration in this system revealed that an insulin : CTB ratio of 100 : 1 was optimal for the induction of bystander suppression. Further studies revealed that this insulin : CTB ratio also was optimal for the prevention of diabetes in a virus-induced, transgenic diabetes model. In addition, the administration of this optimal insulin-CTB preparation significantly prevented the onset of diabetes in old NOD mice with established islet infiltration. The data presented here demonstrate that CTB, even in its unconjugated form, functions as a mucosal adjuvant, increasing the specific tolerogenic effect of oral insulin.

Low prevalence of GAD and IA2 antibodies in schoolchildren from a village in the southwestern section of the Netherlands.

Glutamic acid decarboxylase (GAD) and insulinoma antigen 2 (IA2) antibodies are increasingly used as a tool to predict type I diabetes in children and as a differential diagnostic tool to distinguish type I and type II diabetes in adults. However, the background frequency of these antibodies in the general population has not been extensively studied and may differ between countries. The current study aims to establish the frequency of GAD and IA2 antibodies in an unselected population of schoolchildren and confirm the previously reported low prevalence of islet cell antibodies (ICA) in the general Dutch population. The study population consisted of 1403 unselected schoolchildren. All children were tested for GAD antibodies, and 1085 children were analyzed for IA2 antibodies by radiobinding assay. Development of diabetes was recorded during a 7-year follow-up. Five children (0.4%) were positive for GAD antibodies, one child (0.1%) was positive for IA2 antibodies. Two children developed diabetes during follow-up, one was positive for GAD antibodies only, the second was positive for both GAD and IA2 antibodies. The frequency of GAD and IA2 antibodies in the southwestern part of The Netherlands is low. This observation is in concordance with earlier studies on ICA in Dutch schoolchildren. For future diabetes prediction and intervention trials it is important to establish the background frequencies and predictive power of antibody screening in different populations.

Neither B lymphocytes nor antibodies directed against self antigens of the islets of Langerhans are required for development of virus-induced autoimmune diabetes.

We evaluated the role of the humoral arm of the immune response in causing or contributing to virus-induced diabetes. Transgenic mice expressing the nucleoprotein (NP) or glycoprotein (GP) of the lymphocytic choriomeningitis virus (LCMV) under control of the rat insulin promoter (RIP) in pancreatic beta cells (RIP-LCMV) and RIP-LCMV mice with genetic dysfunction of B cells (RIP-LCMV x microMT/microMT) were compared for development of diabetes after challenge with LCMV. After inoculation with LCMV, B and T lymphocytes and macrophages infiltrated into pancreatic islets in RIP-LCMV mice, and over 50% of these mice generated Abs against host insulin or glutamate decarboxylase. However, neither B cells nor the autoantibodies played a direct role in the initiation, kinetics, or severity of the virus-induced diabetes as judged by comparing disease in RIP-LCMV mice to littermates whose functional B cells were genetically eliminated. Furthermore, the quality and quantity of T lymphocyte and macrophage infiltration was similar in the B cell-deficient and non-B cell-deficient RIP-LCMV mice. Although the development of autoantibodies to islet Ags had no direct influence on the pathogenesis of insulin-dependent (type 1) diabetes mellitus, it served as a prediabetes marker, as such autoantibodies were often elevated before the onset of disease. Hence, the RIP-LCMV model is not only useful for understanding the pathogenetic mechanisms of how islets are destroyed and spared but also for evaluating therapeutic strategies before onset of clinical diabetes.

The antiviral 2',5'-oligoadenylate synthetase is persistently activated in type 1 diabetes.

Type 1 diabetes results from autoimmune destruction of the pancreatic beta-cells. Although viruses have been implicated as etiologic factors, specific pathogenic mechanisms have not been identified. Recently, increased attention has focused on the role of the innate antiviral defense system in directing adaptive immune responses. In this context, the pathogenesis of type 1 diabetes may involve an aberrant response to endogenous or exogenous viruses or their products. The family of 2',5' oligoadenylate synthetases (2', 5' AS) are IFN-alpha-inducible, RNA-dependent effector molecules in the antiviral defense system. We show that lymphocytic 2',5' AS activity is significantly increased in type 1 diabetes, both in recent-onset and in long-standing type 1 diabetes, and in diabetic twins from monozygotic twin pairs. The activity of 2',5' AS was not elevated in patients with type 2 diabetes or multiple sclerosis thus excluding hyperglycemia or autoimmunity per se as inducing upregulation of enzyme activity. In recent-onset diabetic patients, lymphocyte levels of protein kinase p68 and MxA, two other IFN-alpha-inducible antiviral proteins, were similar to control levels. These data suggest that the increased 2',5' AS activity may reflect an aberrant response to viruses or RNA molecules originating from exogenous or endogenous sources.

Autoreactive CD4+ T cells protect from autoimmune diabetes via bystander suppression using the IL-4/Stat6 pathway.

Targeted immune regulation can be achieved by use of tissue-specific T cells and offers the potential for organ-specific suppression of destructive autoimmune processes. Here, we report the generation and characterization of insulin B chain-specific "autoreactive" CD4+ regulatory T cells that locally suppress diabetogenic T cell responses against an unrelated self-antigen (viral transgene) in a virus-induced model for type 1 diabetes. Interleukin 4 (IL-4) is essential for prevention of diabetes since regulatory T cells cannot be induced in the absence of IL-4 or stat6 (IL-4 signaling pathway). Our observations demonstrate that autoreactive regulatory T cells can suppress autoreactive destructive T cell activity of differential antigenic specificity locally in the pancreatic draining lymph node, probably via cytokine-mediated modulation of antigen-presenting cells.

Insulin in oral immune "tolerance": a one-amino acid change in the B chain makes the difference.

Oral administration of self-Ags can dampen or prevent autoimmune processes by induction of bystander suppression. Based on encouraging results from experiments in nonobese diabetic (NOD) mice, clinical trials have been initiated in type 1 diabetes using human insulin as an oral Ag. However, neither the precise antigenic requirements nor the mechanism of bystander suppression are currently understood in detail. Here we report that 1) a 1-aa difference in position 30 of the insulin B chain abrogated the ability of insulin to confer protection in both NOD as well as a virus-induced transgenic mouse model for type 1 diabetes. In the latter model transgenic mice express the nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) under the control of the rat insulin promotor (RIP) in the pancreatic beta cells and develop diabetes only following LCMV infection; and 2) protection could be transferred with insulin B chain-restimulated but not LCMV-restimulated splenocytes from RIP-NP transgenic mice, demonstrating that the mechanism of diabetes prevention in the RIP-NP model is mediated by insulin B chain-specific, IL-4-producing regulatory cells acting as bystander suppressors.

GAD65-Reactive T cells in a non-diabetic stiff-man syndrome patient.

GAD65 (glutamic acid decarboxylase) is an important autoantigen in both type 1 (insulin-dependent) diabetes mellitus (IDDM) and the neurological autoimmune disease stiff-man syndrome (SMS), and is expressed in pancreatic islets as well as the nervous system. Still, only 30% of SMS patients also have type 1 diabetes. To study regulation of T cell responsiveness to GAD65, we investigated a non-diabetic SMS patient with HLA-DR3/7 (predisposing to type 1 diabetes) and high levels of type 1 diabetes-associated autoantibodies against GAD65 and islet cells, and compared the results with those of her diabetic son and two other SMS patients. T cell responses to GAD65 were repeatedly absent in primary stimulation, whereas IA-2, islet antigen and tetanus toxoid induced significant T cell proliferation. However, after in vitro restimulation, GAD65 reactive T cell lines and clones were obtained that were HLA-DR3 restricted, and cross-reactive with a homogenate of purified human pancreatic islets. These T cells produced the immunoregulatory cytokine IL-10 in combination with IFN-gamma and IL-4 (Th0). The dominant T cell epitope was mapped to the central region of GAD65. Although no primary response to whole GAD65 was detectable, the naturally processed GAD65 peptide epitope was recognized vigorously in the primary stimulation assay. The lack of detectable primary T cell responses to GAD65, together with the GAD65-specific cytokine production of restimulated T cells, suggest that GAD65-specific cellular autoimmunity in this patient is suppressed and may be related to the absence of diabetes despite humoral autoreactivity and genetic predisposition.

Progression to type 1 diabetes is associated with a change in the immunoglobulin isotype profile of autoantibodies to glutamic acid decarboxylase (GAD65). Childhood Diabetes in Finland Study Group.

To investigate whether type 1 diabetes in man is associated with a preferential Th1/Th2 response, and whether autoantibodies to one of the main autoantigens would reflect such a response, we characterized the immunoglobulin isotype profile to the 65-kDa isoform of glutamic acid decarboxylase (GAD65) in siblings to IDDM patients. Samples obtained from affected subjects before and at clinical onset of IDDM, from unaffected individuals at high risk and at low risk and from healthy controls were studied. The immunoglobulin isotype profile in the siblings at low risk reflected a more immature, i.e., IgM and Th2 like, i.e., IgE response compared to the progressors and siblings at high risk, with significantly higher median levels of IgM and IgE. The rank order of anti-GAD65 immunoglobulin isotypes was similar in the siblings before and at clinical onset of IDDM, IgG1 > IgG4 > IgM > IgE > IgA > IgG3 > IgG2, but markedly different in the individuals at low risk, IgG1 > IgM > IgE > IgG4 > IgG3 > IgA > IgG2. Based on these observations, we suggest that progression to clinical onset of IDDM is associated with a maturation and a decrease in the Th2 immune response against GAD65; findings which could have implications for future intervention and prediction strategies.

Diagnostic role of antibodies to glutamic acid decarboxylase in latent autoimmune diabetes mellitus in adults.

OBJECTIVE: To investigate the diagnostic role of antibodies to glutamic acid decarboxylase (GAD65-Ab) in latent autoimmune diabetes of adults (LADA) and the frequency of GAD-Ab in Chinese patients initially diagnosed as non-insulin-dependent diabetes mellitus (NIDDM). METHODS: Forty-five control subjects and 195 consecutive inpatients initially classified as NIDDM with > or = 35 years of age at onset and nonketotic history for > 6 months after diagnosis, were recruited. In vitro transcripted and translated recombinant human 35S-GAD65 was used in radioligand assay of GAD-Ab. RESULTS: The overall prevalence of GAD65-Ab was 14.8% (29/195) in NIDDM patients and 2.2% (1/45) in control subjects, respectively. Of the 29 GAD65-Ab positive patients, 17 (58.6%) were insulin-deficient while 12 (41.4%) were non-insulin-deficient. The prevalence of GAD65-Ab in NIDDM group with age of < 40 years at diabetes onset, ketotic history, body mass index (BMI) < 21 kg/m2, were significantly higher than that of corresponding control diabetic subgroups (2.5, 4.1 and 3.2 times, respectively). The sex, duration, symptoms of polyphagia, polydipsia, polyuria and weight loss at onset of the disease were not related to the prevalence of GAD65-Ab positivity. CONCLUSIONS: In China, patients initially diagnosed as NIDDM may in many cases suffer from LADA. Testing by GAD65-Ab may be of assistance to identifying LADA at the earliest stage of disease.

Immunization of diabetes-prone or non-diabetes-prone mice with GAD65 does not induce diabetes or islet cell pathology.

Glutamic acid decarboxylase autoimmunity was investigated by immunizing female BALB/c, C57B1/6, National Marine Research Institute (NMRI) and non-obese diabetic (NOD) mice once or twice with glumatic acid decarboxylase, GAD65, bovine serum albumin, or phosphate-buffered saline in incomplete Freunds adjuvant, or not treating. Mice immunized with GAD65, showed splinic T-cell reactivity to GAD 65 in vitro assessed by cytokine secretion. However untreated NOD mice did not. NOD mice showed a vigorous IFN-gamma response after one immunization, whereas NMRI mice showed a lower response. IL-4 and IL-10 were only detected after two immunizations with higher levels in BALB/c, NMRI and NOD mice, compared to C57B1/6 mice. High levels of GAD65 antibodies were detected in all mice immunized with GAD65, though lower levels were found in C57B1/6 mice. Histological analysis of pancreata revealed that no control mice, regardless of treatment, had mononuclear cell infiltration in the islets. In NOD mice, peri-insulitis was detected in all groups, but less so in GAD65 and bovine serum albumin (BSA) immunized animals. These data demonstrate that NOD mice respond more vigorously to immunization with GAD65 than non-diabetic mice strains. Furthermore, immunization with GAD65 is not sufficient to provoke onset of diabetes in NOD mice or induce islet cell pathology in non-diabetes prone mice.

Regulation of GAD expression in rat pancreatic islets and brain by gamma-vinyl-GABA and glucose.

Glutamic acid decarboxylase (GAD) is an important autoantigen in insulin-dependent diabetes mellitus (IDDM), but little is known about its regulation and function in islet cells. We investigated the effects of the GABA-transaminase inhibitor gamma-vinyl-GABA (GVG) on GAD expression in rat islets and brain in vitro and in vivo. In islets incubated in high glucose culture medium there was an increase in GAD activity, GAD65 and GAD67 protein levels compared to low-glucose conditions; however, even in high glucose, GVG still significantly suppressed GAD activity and GAD67 expression. Our observations suggest that glucose and GVG act on GAD in islets through different mechanisms. Quantitative immunohistochemistry of pancreatic sections from rats treated with GVG in vivo using novel monoclonal antibodies specific for GAD65 and GAD67, showed a decrease in GAD67 expression (p < 0.005) relative to untreated rats. The effects of GVG on rat pancreatic islets were very similar to those observed in brain of rats treated with GVG in vivo. In homogenates of cerebral tissue from GVG treated rats containing both membrane-bound and soluble protein GAD67 levels were significantly decreased while GAD65 levels were not significantly changed compared to untreated rats. In contrast, in homogenates of cerebral tissues containing only soluble cytosolic protein, GVG-treatment was also significantly found to decrease GAD65 levels. Taken together, these results suggest that GVG potentially could be of use to decrease GAD expression in islet cells and consequently to deviate/inhibit the autoimmune response against the beta cells seen in IDDM.

Molecular mimicry between non-self, modified self and self in autoimmunity.

Molecular mimicry, the presence of shared epitopes between foreign and self antigens is common both at the B and T cell level. That it only rarely leads to a cross-reactive autoimmune response is a testimony to the effectiveness of the immune system in regulating the immune response and avoiding harmful self-reactivity. However, despite this regulation, various auto-immune-mediated diseases have been associated with molecular mimicry. The concept of molecular mimicry between foreign and self antigen as well as between self and modified self epitopes is reviewed and the relevance of these phenomena for the initiation of autoimmune diseases is discussed.

Prediction of insulin-dependent diabetes mellitus in siblings of children with diabetes. A population-based study. The Childhood Diabetes in Finland Study Group.

An unselected population of 755 siblings of children with insulin-dependent diabetes mellitus (IDDM) was studied to evaluate the predictive characteristics of islet cell antibodies (ICA), antibodies to the IA-2 protein (IA-2A), antibodies to the 65-kD isoform of glutamic acid decarboxylase (GADA), insulin autoantibodies (IAA), and combinations of these markers. We also evaluated whether the histochemical ICA test could be replaced by the combined detection of other markers. 32 siblings progressed to IDDM within 7.7 yr of the initial sample taken at or close to the diagnosis of the index case (median follow-up, 9.1 yr). The positive predictive values of ICA, IA-2A, GADA, and IAA were 43, 55, 42, and 29%, and their sensitivities 81, 69, 69, and 25%, respectively. In contrast to the other three antibody specificities, GADA levels were not related to the risk for IDDM. The risk for IDDM in siblings with four, three, two, one, or no antibodies was 40, 70, 25, 2, and 0.8%, respectively. Combined screening for IA-2A and GADA identified 70% of all ICA-positive siblings, and all of the ICA-positive progressors were also positive for at least one of the three other markers. The sensitivity of the combined analysis of IA-2A and GADA was 81%, and the positive predictive value was 41%. In conclusion, combined screening for IA-2A and GADA may replace the ICA assay, giving comparable sensitivity, specificity, and positive predictive value. Accurate assessment of the risk for IDDM in siblings is complicated, as not even all those with four antibody specificities contract the disease, and some with only one or no antibodies initially will progress to IDDM.

Persistent GAD 65 antibodies in longstanding IDDM are not associated with residual beta-cell function, neuropathy or HLA-DR status.

Persistent humoral autoimmunity to the enzyme glutamic acid decarboxylase (GAD) has been described in a substantial proportion of patients with insulin-dependent diabetes mellitus (IDDM) of long duration. The source of the stimulus for this autoimmune reactivity is still unknown. Because the GAD 65 isoform is mainly expressed in pancreatic beta-cells and in the nervous system we investigated in the present study of the largest number of well characterized patients with longstanding IDDM (n = 105; median duration: 21 years; range: 10-46 years) the presence of autoantibodies to GAD 65 and their relationship to a residual C-peptide response or peripheral and autonomic neuropathy. Additionally we studied the HLA-DR status relative to GAD 65 antibodies in 86 out of the 105 individuals. One hundred healthy control subjects and 100 recent onset IDDM patients were also studied for GAD 65 antibodies. GAD 65 antibodies were detected in a radioligand-binding-assay with recombinant human GAD 65 and were present in 32% of the long-term diabetic patients, 82% of the recent onset IDDM patients and in 3% of the healthy control subjects. A preserved C-peptide response to i.v. glucagon (Hendriksen criteria) was observed in 23% of the long-term IDDM patients. Autonomic neuropathy and peripheral neuropathy was identified using criteria based on both symptoms and formal testing giving a frequency of 67% vs 79%. The HLA specific DR 4/X was observed in 47% and HLA-DR 3/X in 22% of the long-term IDDM patients. Patients who were heterozygous for DR3/DR4 were found in 23% of the cases. GAD 65 antibodies were significantly less frequent in the long-term IDDM patients compared to recent onset IDDM (p < 0.001), and diabetes duration showed a significant negative correlation with GAD 65 antibody index levels (r = 0.22, p < 0.01). Interestingly, GAD 65 antibodies were not significantly correlated either with residual beta-cell function or neuropathy and no particular HLA-DR status was associated with persistent GAD 65 antibodies. In conclusion neither residual beta-cell function nor diabetic neuropathy or a certain HLA-DR specificity are exclusively associated with persistent autoimmunity directed to GAD 65 in longstanding IDDM. The stimulus for the persistent humoral immune response and its significance for the disease process and its complications remain to be established.

Population based study of prevalence of islet cell autoantibodies in monozygotic and dizygotic Danish twin pairs with insulin dependent diabetes mellitus.

OBJECTIVE: To study the comparative importance of environment and genes in the development of islet cell autoimmunity associated with insulin dependent diabetes mellitus. DESIGN: Population based study of diabetic twins. SETTING: Danish population. SUBJECTS: 18 monozygotic and 36 dizygotic twin pairs with one or both partners having insulin dependent diabetes. MAIN OUTCOME MEASURES: Presence of islet cell antibodies, insulin autoantibodies, and autoantibodies to glutamic acid decarboxylase (GAD65) in serum samples from twin pairs 10 years (range 0-30 years) and 9.5 years (2-30 years) after onset of disease. RESULTS: In those with diabetes the prevalence of islet cell antibodies, insulin autoantibodies, and autoantibodies to glutamic acid decarboxylase in the 26 monozygotic twins was 38%, 85%, and 92%, respectively, and in the dizygotic twins was 57%, 70%, and 57%, respectively. In those without diabetes the proportions were 20%, 50%, and 40% in the 10 monozygotic twins and 26%, 49%, and 40% in the 35 dizygotic twins. CONCLUSION: There is no difference between the prevalence of islet cell autoantibodies in dizygotic and monozygotic twins without diabetes, suggesting that islet cell autoimmunity is environmentally rather than genetically determined. Furthermore, the prevalence of islet cell antibodies was higher in the non-diabetic twins than in other first degree relatives of patients with insulin dependent diabetes. This implies that the prenatal or early postnatal period during which twins are exposed to the same environment, in contrast with that experienced by first degree relatives, is of aetiological importance.

Treatment with GAD65 or BSA does not protect against diabetes in BB rats.

The M(r) 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 micrograms/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 hypothetically be explained by no or by an aberrant expression of GAD in BB-rat islet cells. However, immunohistochemistry of pancreata and immunoblotting 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.

GAD65 autoantibodies in women with gestational or insulin dependent diabetes mellitus diagnosed during pregnancy.

We have studied the presence of GAD65 autoantibodies in women with insulin-dependent diabetes mellitus (IDDM) (n = 28) or gestational diabetes (GDM) (n = 139) diagnosed during pregnancy and investigated the temporal relationship between these autoantibodies and the subsequent recurrence or development of IDDM. Among the GDM patients, 4.3% (6 of 139) developed true IDDM during a median follow-up period of 6.3 years (range 4.0-11.0). Of these, 2.2% (3 of 139) were positive for GAD65 autoantibodies at diagnosis of GDM compared to 0% (0 of 27) of healthy pregnant women. All 3 GAD65 autoantibody positive GDM patients subsequently developed IDDM after a median of 14 months (range 4-34). GAD65 autoantibodies were present in 50% (14 of 28) of sera from women with IDDM diagnosed during pregnancy. The non-insulin-requiring remission period was significantly shorter in GAD65 autoantibody positive patients (median 0.5 years [range 0-6.0 years]) than in GAD65 antibody negative patients (median 2.6 years; range 0-9.7 years; p < 0.05). The results suggest that screening for GAD65 autoantibodies in women with GDM or IDDM diagnosed during pregnancy may be useful for predicting the clinical course of the disease.

Oral insulin treatment suppresses virus-induced antigen-specific destruction of beta cells and prevents autoimmune diabetes in transgenic mice.

Oral administration of self-antigens has been proposed as a therapy to prevent and treat autoimmune diseases. Here we report that oral treatment with insulin prevents virus-induced insulin-dependent diabetes mellitus (IDDM) in a transgenic (tg) mouse model. Such mice express the viral nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) under control of the rat insulin promoter in their pancreatic beta cells and < 2% spontaneously develop diabetes. However, 2 mo after challenge with LCMV, IDDM occurs in > 95% of tg mice but not in controls. Oral treatment with 1 mg of insulin twice per week for 2 mo starting either 1 wk before or 10 d after initiating LCMV infection prevents IDDM in > 50% of the tg mice (observation time 8 mo). Thus, insulin therapy is effective in preventing progression to overt IDDM in prediabetic tg mice with ongoing islet infiltration. Oral administration of insulin does not affect the generation of LCMV-NP-specific anti-self cytotoxic T lymphocytes nor the infiltration of lymphocytes into the pancreas. However, less beta cells are destroyed in insulin-treated mice, upregulation of MHC class I and II molecules does not occur, and antiviral (self) cytotoxic T lymphocytes are not found in the islets, events present in tg mice developing IDDM. The majority of lymphocytes in the islets of insulin-treated tg mice without IDDM produces IL-4, IL-10, and TGF-beta. In contrast, lymphocytes from islets of tg mice developing IDDM mainly make gamma-IFN.