Type 1 diabetes is considered as Th1 cell mediated autoimmune disease and the suppression of Th1 cells or the activation of Th2 cells has been regarded as a plausible immunologic intervention for the prevention of type 1 diabetogenesis in a rodent model. CpG ODN is an immunostimulatory sequence primarily present in bacterial DNA, viral DNA and BCG. CpG ODN is conventionally classified as a Th1 cell activator, which has been clinically applied to cancer, allergy and infectious disease. Recently, there was a promising report of that CpG ODN administration suppressed the development of type 1 diabetes in NOD mice by inducing Th2 cell mediated cytokine. However, the antidiabetogenic effect of CpG ODN on NOD mice is controversial. Thus, two studies were serially undertaken with various kinds of CpG motif to find a more optimal sequence and administration method. In the first study, CpG ODN was vaccinated four times and pancreatic inflammation and the quantity of serum insulin subsequently evaluated. In the second study, the amounts of IFN gamma and IL-4 in sera were measured as representative cytokines of Th1 and Th2 cells, respectively. As a result, vaccination or continuous injection of CpG ODN failed to show a preventive effect on type 1 diabetogenesis in NOD mice. Structural differences of CpG ODN also had no affect on the result. CpG ODN also consistently showed affect on the pancreatic pathology. The productions of IFN gamma and IL-4 were detected only in the K and D type CpG ODN administration groups. Comparison of the two cytokines leads to the conclusion that CpG ODN generated a Th1-weighted response in both study groups. It was assumed that CpG ODN failed to produce Th2-weighted cytokine milieu, which can overcome the genetically determined phenotype of NOD mice. Given these results, it was concluded that the immunotherapeutic application of CpG ODN on Type 1 diabetes had clear limitations.
Animals ; DNA/*pharmacology ; Diabetes Mellitus, Type 1/*immunology/*prevention & control ; Female ; Mice ; Mice, Inbred NOD ; Th1 Cells/*immunology

OBJECTIVE: To investigate the effects of subcutaneous administration of insulin on insulitis,beta cell apoptosis and diabetes in non-obese diabetic (NOD) mice, and to explore the mechanism of immune tolerance induced by insulin. METHODS: Sixty female NOD mice were randomly divided into insulin group (n=32) and phosphate buffered saline (PBS) group (PBS group, n=28). Insulin was subcutaneously injected with humulin N (60 microL, 6U)+IFA (60 microL) at 4, 12, 20, and 28 weeks respectively, while the PBS group received PBS (60 microL) + IFA (60 microL). Insulitis and beta cell apoptosis of islets were observed at 12 weeks. IL-4 and IFN-gamma in the sera were measured by enzyme linked immunosorbent assay (ELISA). The expression levels of I-Abeta(g7), IL-4, IFN-gamma, IL-1beta, and Fas mRNA of islets were measured by reverse transcription-polymerase chain reaction (RT-PCR) at 12 weeks. RESULTS: The incidences in the insulin group were significantly lower than those in the PBS group (21.4% vs 71.4% at 30 weeks, 28.6% vs 85.7% at 52 weeks, P<0.05). The insulitis scores in the insulin group were lower than those in the PBS group, but there was no statistical significance. Fas expression on islets and apoptotic beta cell rates in the insulin group were lower than those in the PBS group (P<0.05). In the insulin group, serum IL-4 levels were higher, but IFN-gamma levels were lower than those in the PBS group (P<0.05). The levels of I-Abeta g7, IFN-gamma, IL-1beta and Fas mRNA transcription in islets were lower in insulin group, but IL-4 mRNA levels were higher than those in the PBS group (P<0.05). CONCLUSION The specific autoantigen insulin may induce immune tolerance and prevent diabetes in NOD mice, but it can't block the progression of insulitis. Subcutaneous administration of insulin can induce the regulatory T cells, and make Th1 to Th2 cytokine shifts in system and islets, thus preventing the Fas-mediated beta-cell apoptosis and diabetes.
Animals ; Apoptosis ; drug effects ; Diabetes Mellitus, Experimental ; prevention & control ; Diabetes Mellitus, Type 1 ; prevention & control ; Female ; Injections, Subcutaneous ; Insulin ; administration & dosage ; pharmacology ; Islets of Langerhans ; immunology ; pathology ; Mice ; Mice, Inbred NOD ; Pancreatitis ; prevention & control ; Random Allocation

Type 1 diabetes (T1D), the most prevalent human autoimmune disease, occurs in genetically susceptible individuals. Regulatory T cells (Tregs) are defective in T1D setting. Therefore, efforts to repair or restore Tregs in T1D may prevent or reverse this autoimmune disease. Here, we studied the potential role of rgp96 in preventing T1D, using non-obese diabetic (NOD) mice as an animal model. High-dose rgp96 immunization elicited efficient protection of mice against T1D, as evidenced by stable blood glucose, decreased disease incidence. Significantly increased CD4⁺ CD25⁺ Foxp3⁺ Tregs were observed in immunized mice. In vitro co-culture experiments demonstrated that rgp96 stimulation enhanced Treg proliferation and suppressive function by up-regulation of Foxp3 and IL-10. Our work shows that activation of Tregs by high-dose rgp96 immunization protects against T1D via inducing regulatory T cells and provides preventive and therapeutic potential for the development of an rgp96-based vaccine against T1D.
Animals ; Antigens, Neoplasm ; administration & dosage ; immunology ; Coculture Techniques ; Diabetes Mellitus, Type 1 ; prevention & control ; therapy ; Forkhead Transcription Factors ; Heat-Shock Proteins ; administration & dosage ; immunology ; Interleukin-10 ; immunology ; Mice ; Mice, Inbred NOD ; T-Lymphocytes, Regulatory ; immunology ; Up-Regulation ; Vaccination

To improve the efficacy of peptide P277 in preventing autoimmune diabetes, heat shock protein 65 kD (HSP65) of Mycobacterium tuberculosis var. bovis was fused with linear polypeptide epitope of P277 and expressed as soluble protein in Escherichia coli. The fusion protein HSP65-P277 was purified by anion exchange column chromatography and then used to immunize prediabetic NOD mice with three ip inoculations in absence of adjuvants. Serum samples from the immunized mice were collected monthly and the concentration of blood glucose was measured. The study showed that administration of HSP65-P277 to NOD mice could prevent the development of diabetes more efficiently than the peptide P277 itself or HSP65. Fused to heat shock protein 65 of Mycobacterium tuberculosis could improve the efficacy of diabetes prevention of P277 in nonobese diabetic mice. The results suggest the fusion protein of HSP65-P277 would be useful for treating insulin-dependent diabetes mellitus.
Animals ; Bacterial Proteins ; biosynthesis ; genetics ; immunology ; Chaperonin 60 ; Chaperonins ; biosynthesis ; genetics ; immunology ; Diabetes Mellitus, Type 1 ; prevention & control ; Escherichia coli ; genetics ; metabolism ; Female ; Heat-Shock Proteins ; genetics ; immunology ; Immunization ; Mice ; Mice, Inbred NOD ; Mycobacterium bovis ; Peptide Fragments ; biosynthesis ; genetics ; immunology ; Random Allocation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Vaccines, Synthetic ; genetics ; immunology