Reiko Kotani

A possible association of Pro12Ala polymorphism in peroxisome proliferator–activated receptor γ2 gene with obesity in native Javanese in Indonesia

Peroxisome proliferators–activated receptor γ (PPAR γ) is a nuclear hormone receptor that serves as a master regulator of adipocytes‐specific genes contributing to adipocytes differentiation, susceptibility to obesity, and insulin sensitivity. The substitution of proline to alanine at codon 12 of the PPAR γ2 gene (Pro12Ala polymorphism) is most frequently identified and the associations with diabetes, obesity, and other clinical parameters have been reported and discussed in several ethnic groups. Among native Javanese ethnicity, however, there is no report about this polymorphism.

T lymphocyte response against pancreatic beta cell antigens in fulminant Type 1 diabetes

Aims/hypothesisFulminant Type 1 diabetes is a novel subtype of Type 1 diabetes that involves the abrupt onset of insulin-deficient hyperglycaemia. This subtype appears to be non-autoimmune because of the absence of diabetes-related autoantibodies in the serum, and of insulitis in pancreatic biopsy specimens. The pathogenesis of the disease is still unknown. In this study, we investigated whether T cell autoimmune responses are involved in fulminant Type 1 diabetes.MethodsCellular immune responses to beta cell autoantigens were studied by enzyme-linked immunospot (ELISPOT) assay in 13 fulminant Type 1 diabetic patients and 49 autoantibody-positive autoimmune Type 1 diabetic patients. Results were compared with those of 18 Type 2 diabetic patients, six secondary diabetic patients (diabetes due to chronic pancreatitis) and 35 healthy controls.ResultsNine of 13 (69.2%) GAD-reactive Th1 cells, and three of 12 (25%) insulin-B9-23-reactive Th1 cells were identified in fulminant Type 1 diabetic patients by ELISPOT, as in autoantibody-positive Type 1 diabetic patients. Four fulminant Type 1 diabetic patients possessed the highly diabetes-resistant allele DR2, three of whom had GAD-reactive Th1 cells in the periphery.Conclusions/interpretationPeripheral immune reaction was observed in 69.2% of fulminant Type 1 diabetic patients, indicating that autoreactive T cells might contribute, at least in part, to the development of fulminant Type 1 diabetes.

Detection of Autoreactive T Cells in Type 1 Diabetes Using Coded Autoantigens and an Immunoglobulin-Free Cytokine ELISPOT Assay: Report from the Fourth Immunology of Diabetes Society T Cell Workshop

Abstract: The “gold standard” for evaluation of immunoassays is blinded testing, using coded samples and antigens. Although assays for autoreactive T cells are no exception to this rule, it is nonetheless rarely applied in this context. To facilitate such investigations, we coded a panel of 10 peptides representing T cell epitopes reported to be of relevance to islet autoimmunity. These were deployed in a novel cytokine ELISPOT assay, in which the use of immunoglobulin‐free medium reduces background reactivity and thus potentially enhances the specificity and sensitivity of detection of autoreactive T cells. Significant IFN‐γ production against GAD65 (554‐575), insulin (B9‐23), and IA‐2 (709‐736) peptides were observed in type 1 diabetic patients, whereas no significant changes from background were detected in healthy controls. These results confirm the utility of the blinded performance of T cell assays as the most robust platform for assessing technologies to T cell autoreactivity.

Critical roles of CD30/CD30L interactions in murine autoimmune diabetes

CD30/CD30L is a member of tumour necrosis factor (TNF) receptor/TNF superfamily and has been implicated in immune‐regulation. A genetic study has also suggested a possible implication of CD30 in spontaneous autoimmune diabetes in NOD mice. In this study, we investigated the involvement of CD30/CD30L in the development of diabetes in NOD mice. Flow cytometric analysis showed that CD30 and CD30L were highly expressed on CD4+ or CD8+ T cells in the spleen and pancreatic lymph node of younger NOD mice. In addition, islet‐specific CD4+ or CD8+ T cell lines expressed CD30 and CD30L. Administration of a neutralizing anti‐CD30L monoclonal antibody (mAb) from 2 to 10 week of age completely suppressed the development of spontaneous diabetes in NOD mice. In addition, the treatment with anti‐CD30L mAb also inhibited the development of diabetes induced by adoptive transfer of spleen cells from diabetic NOD mice or islet‐specific CD4+ or CD8+ T cell lines into NOD‐SCID mice. Furthermore, anti‐CD30L mAb inhibited T cell proliferation in response to islet antigens. These results suggested that CD30/CD30L interaction plays important roles in both induction and effector phases of autoimmune diabetes in NOD mice.

T cell epitope mapping study with insulin overlapping peptides using ELISPOT assay in Japanese children and adolescents with type 1 diabetes

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease. Insulin seems to be a critical antigen recognized by autoreactive T cells. In this study, we performed T cell epitope mapping of insulin using serial overlapping peptides in Japanese patients with T1D. Serial overlapping insulin peptides comprising 23 peptides, which were each 15-amino acid long, were prepared based on insulin sequence. Cytokine secretion from peripheral T cells against these peptides was studied by enzyme-linked immunospot (ELISPOT) assay in 18 patients with recent-onset T1D and 12 patients with established T1D, and compared with 17 healthy control subjects. In ELISPOT assay, IFN-γ-secreting T cells, but not IL-4, against several insulin peptides were observed in 77.8% of patients with recent-onset T1D, 50.0% of patients with established T1D, and 0% of healthy control subjects. All epitopes recognized by T cells were identified in the B-chain of insulin. The most frequent epitope existed at the B10–24 region (9/18), followed by B1–15 and B11–25 regions (6/18, each), with B4–18, B9–23, and B12–26 identified in some patients. These data did not correlate with insulin autoantibodies or HLA-DRB1 of the patients. This is the first report of T cell epitope mapping using one amino acid serial overlapping peptides of insulin in T1D. ELISPOT assay revealed the frequent existence of insulin peptide-specific T cells in patients with recent-onset and established T1D. The T cell epitopes of insulin were similar but not identical in our cohort, which probably explains the difficulty encountered in prevention of human T1D by using insulin.

Prevention of recurrent but not spontaneous autoimmune diabetes by transplanted NOD islets adenovirally transduced with immunomodulating molecules.

Pancreatic islet transplantation has the potential to maintain normoglycemia in patients with established type 1 diabetes, thereby obviating the need for frequent insulin injections. Our previous study showed that recombinant IL-12p40-producing islets prevented the recurrence of NOD diabetes. First, to see which immunomodulating molecule-secreting islet grafts can most powerfully prevent diabetes development in NOD mice without immunosuppressant, NOD islets were transfected with one of the following adenoviral vectors: Ad.IL-12p40, Ad.TGF-beta, Ad.CTLA4-Ig, or Ad.TNF-alpha after which they were transplanted under the renal capsule of acutely diabetic NOD mice. The immunomodulating molecules produced by these adenovirus-transfected islets in vitro were 74+/-19ng, 50+/-4ng, 821+/-31ng, and 77+/-18ng/100 islets, respectively. Transplantation of IL-12p40, TNF-alpha, and CTLA4-Ig but not TGF-beta-secreting islets displayed enhanced survival and delayed diabetes recurrence in recent-onset diabetic recipients. IL-12p40-producing islet grafts most powerfully prevented recurrent diabetes in NOD mice. In addition, local production of TNF-alpha and CTLA4-Ig significantly prolonged islet graft survival. In second series of experiment, these manipulated islets were transplanted under the renal capsule of 10-week-old NOD recipients and were also transplanted subcutaneously into 2-week-old NOD recipients. Transplantation of these islets into 2- or 10-week-old pre-diabetic mice failed to protect them from developing diabetes; in fact, transplantation of Ad.TNF-alpha-transfected islets into 2-week-old mice actually accelerated diabetes onset. Taken together, this approach was ineffectual as a prophylactic protocol. In conclusion, this study showed comparisons of the immunomodulating effects of 4 different adenoviral vectors in the same transplantation model and local production of IL-12p40, TNF-alpha and CTLA4-Ig significantly prevented recurrent diabetes in NOD mice.

Autoreactive T Cell Response in CD25‐Negative Fraction of Peripheral Blood Mononuclear Cells in Established Type 1 Diabetes

CD4+CD25+ T cells (Tregs) play a critical role in maintaining dominant peripheral tolerance, and pathogenic autoreactive T cells may be frequent in the CD25‐negative fraction of peripheral blood mononuclear cells (PBMCs) from patients with autoimmune disease. We therefore investigated whether T cell autoimmune responses to recombinant GAD65 can be detected by the use of ELISPOT assay in the CD25‐negative fraction of PMBCs from Japanese type 1 diabetes (T1D) patients. The frequency of CD4+CD25+ T cells was not different among patients with newly developed T1D, established T1D, and healthy controls. The CD25 positive cell–depleted fraction was obtained by negative selection with antihuman CD25 magnetic beads, reducing the number of CD4+CD25+ T cells from 4–5% to less than 1%. In whole PBMC fraction, there was a significant elevation of IFN‐γ spots in PBMCs from recently diagnosed patients with T1D (P < 0.05), whereas the number of IFN‐γ spots from patients with established T1D was not significant. In the CD25‐negative fraction, unlike whole PBMCs, we observed the significant IFN‐γ spots to GAD65 in the fraction from patients with established T1D (P < 0.05), but not in those with recently diagnosed disease. The phenomena were not observed for IL‐4 spots. Our data suggest a possible role of Tregs maintaining dominant peripheral tolerance in T1D and application of further improved T cell assay detecting autoimmunity even in established T1D.