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OBJECTIVE -Mucosal administration of insulin retards development of autoimmune diabetes in the nonobese diabetic mouse model. We conducted a double-blind crossover study in humans at risk for type 1 diabetes to determine if intranasal insulin was safe, in particular did not accelerate ␤-cell destruction, and could induce immune effects consistent with mucosal tolerance. RESEARCH DESIGN AND METHODS-A total of 38 individuals, median age 10.8 years, with antibodies to one or more pancreatic islet antigens (insulin, GAD65, or tyrosine phosphatase-like insulinoma antigen 2) were randomized to treatment with intranasal insulin (1.6 mg) or a carrier solution, daily for 10 days and then 2 days a week for 6 months, before crossover. The primary outcome was ␤-cell function measured as first-phase insulin response (FPIR) to intravenous glucose at 0, 6, and 12 months and then yearly; the secondary outcome was immunity to islet antigens, measured monthly for 12 months.RESULTS -No local or systemic adverse effects were observed. Diabetes developed in 12 participants with negligible ␤-cell function at entry after a median of 1.1 year. Of the remaining 26, the majority had antibodies to two or three islet antigens and FPIR greater than the first percentile at entry, as well as ␤-cell function that generally remained stable over a median follow-up of 3.0 years. Intranasal insulin was associated with an increase in antibody and a decrease in T-cell responses to insulin. CONCLUSIONS -Results from this pilot study suggest that intranasal insulin does not accelerate loss of ␤-cell function in individuals at risk for type 1 diabetes and induces immune changes consistent with mucosal tolerance to insulin. These findings justify a formal trial to determine if intranasal insulin is immunotherapeutic and retards progression to clinical diabetes. Diabetes Care 27:2348 -2355, 2004T ype 1 diabetes is an autoimmune disease in which T-cells mediate destruction of insulin-secreting ␤-cells in the pancreatic islets. Asymptomatic individuals with preclinical type 1 diabetes can be identified by the presence of circulating antibodies (Abs) to insulin, GAD 65-kDa isoform, and tyrosine phosphatase-like insulinoma antigen 2 (IA2) (1-3). Insulin is the only self-antigen specific for ␤-cells, and several lines of evidence indicate that it may play a major role in driving autoimmune ␤-cell destruction (4 -7). In experimental rodent models, administration of self-antigens to mucosa-associated lymphoid tissues can induce immune tolerance and prevent autoimmune disease (8,9). In the nonobese diabetic (NOD) mouse, a spontaneous model of autoimmune type 1 diabetes, oral (10) or naso-respiratory (11) insulin induces regulatory, diabetes-protective Tcells. After naso-respiratory insulin, there was a decrease in T-cell and an increase in Ab responses to insulin (11), conforming to the shift from T-helper (Th)-1 cellular immunity to Th2 humoral immunity that is associated with protection against diabetes in this rodent model (12). In human volunteers, oral (13)...

Autoimmune destruction of pancreatic beta cells in type I, insulin-dependent diabetes mellitus (IDDM) results in the loss of endogenous insulin secretion, which is incompletely replaced by exogenous insulin administration. The functional restoration provided by allogeneic beta-cell transplantation is limited by adverse effects of immunosuppression. To pursue an insulin replacement therapy based on autologous, engineered human non-beta cells, we generated a retroviral vector encoding a genetically modified human proinsulin, cleavable to insulin in non-beta cells, and a human nonfunctional cell surface marker. Here we report that this vector efficiently transduced primary human cells, inducing the synthesis of a modified proinsulin that was processed and released as mature insulin. This retrovirally derived insulin displayed in vitro biological activity, specifically binding to and phosphorylation of the insulin receptor, comparable to human insulin. In vivo, the transplantation of insulin-producing fibroblasts reverted hyperglycemia in a murine model of diabetes, whereas proinsulin-producing cells were ineffective. These results support the possibility of developing insulin production machinery in human non-beta cells for gene therapy of IDDM.

SUMMARYType 1 diabetes is associated with autoimmunity to insulin. Genetic susceptibility to type 1 diabetes is polygenic and includes the INS VNTR-IDDM2 locus which may regulate the expression of insulin in pancreas and thymus. In order to determine whether insulin autoimmunity could be attributed to a genetic susceptibility conferred by the INS VNTR-IDDM2 locus, peripheral blood T cell proliferation to human insulin and insulin autoantibodies (IAA) was measured in patients with new onset type 1 diabetes and control subjects. IAA were detected in 21 of 53 patients and in none of 25 control subjects, while T cell responses were low (stimulation index range 0·4-7·2) and similar in both groups. Both antibody and T cell responses were higher in younger subjects and IAA were more prevalent in patients with the HLA-DR4 allele. No relationship was observed between humoral and cellular responses to insulin. No association was found between the INS VNTR-IDDM2-susceptible allele and insulin autoimmunity. Increased T cell responses and IAA were found in patients with either the diabetes-susceptible or the diabetes-protective INS VNTR-IDDM2 locus genotypes, and increased T cell responses were also found in control subjects with either susceptible or protective INS VNTR-IDDM2 locus genotypes. This study confirms that primary T cell proliferative responses to insulin are low and detectable also in control subjects. The detection of T cell proliferation and autoantibodies to insulin in subjects with and without the protective INS VNTR-IDDM2 locus genotypes does not support the hypothesis of an allele-specific capacity for tolerance induction which could determine a susceptibility to develop autoimmunity against the insulin protein and subsequently diabetes.