The dynamic changes of zinc transporter 8 autoantibodies in Czech children from the onset of Type 1 diabetes mellitus.

AIMS: The prevalence of autoantibodies to zinc transporter 8 (ZnT8) in Czech children at the onset of Type 1 diabetes mellitus and dynamic changes in ZnT8 autoantibody levels during disease progression were studied. The value of ZnT8 autoantibody measurements in diagnosis of Type 1 diabetes was assessed. METHODS: Serum samples from 227 children with newly diagnosed Type 1 diabetes and from 101 control children without diabetes were analysed in a retrospective cross-sectional study. One hundred and seventy-one samples from 116 of the patients with diabetes were analysed in a follow-up study at (median) intervals of 1, 3, 5 and 10 years after onset of Type 1 diabetes. ZnT8 autoantibodies were measured using a bridging enzyme-linked immunosorbent assay, while antibodies to glutamic acid decarboxylase, insulinoma antigen 2 and insulin were measured by radioimmunoassays. RESULTS: ZnT8 autoantibodies were detected in 163/227 (72%) of children at Type 1 diabetes onset and in 1/101 (1%) of the control subjects. Sixteen out of 227 (7%) patients with Type 1 diabetes were antibody negative based on three antibodies (glutamic acid decarboxylase, insulinoma antigen 2 and insulin). This false-negative rate was reduced to 10/227 (4.4%) (P < 0.05) after inclusion of ZnT8 autoantibody measurements. Of the children, 142/227 (63%) were positive for at least three antibodies and the most common combination was insulinoma antigen 2, glutamic acid decarboxylase and ZnT8. ZnT8 autoantibody levels decreased over time after Type 1 diabetes onset and the presence and level of ZnT8 autoantibodies correlated with IA-2 autoantibodies. CONCLUSIONS: A ZnT8 autoantibody enzyme-linked immunosorbent assay showed 72% disease sensitivity and 99% specificity at Type 1 diabetes onset. Measurements of ZnT8 autoantibodies are important for Type 1 diabetes diagnosis and should be included in the panel of autoantibodies tested at the onset of Type 1 diabetes.

Autoantibodies and associated T-cell responses to determinants within the 831-860 region of the autoantigen IA-2 in Type 1 diabetes.

B-cells influence T-cell reactivity by facilitating antigen presentation, but the role of autoantibody-secreting B-cells in regulating T-cell responses in Type 1 diabetes is poorly defined. The aims of this study were to characterise epitopes on the IA-2 autoantigen for three monoclonal antibodies from diabetic patients by amino acid substitutions of selected residues of IA-2, establish contributions of these epitopes to binding of serum antibodies in Type 1 diabetes and relate B- and T-cell responses to overlapping determinants on IA-2. The monoclonal antibodies recognised overlapping epitopes, with residues within the 831-860 region of IA-2 contributing to binding; substitution of Glu836 inhibited binding of all three antibodies. Monoclonal antibody Fab fragments and substitution of residues within the 831-836 region blocked serum antibody binding to an IA-2 643-937 construct. IL-10-secreting T-cells responding to peptides within the 831-860 region were detected by cytokine-specific ELISPOT in diabetic patients and responses to 841-860 peptide were associated with antibodies to the region of IA-2 recognised by the monoclonal antibodies. The study identifies a region of IA-2 frequently recognised by antibodies in Type 1 diabetes and demonstrates that these responses are associated with T-cells secreting IL-10 in response to a neighbouring determinant.

Isolation and characterisation of a human monoclonal autoantibody to the islet cell autoantigen IA-2.

A hybridoma secreting a human monoclonal autoantibody to the islet cell autoantigen IA-2 was prepared from peripheral lymphocytes of a patient with type 1 diabetes and Graves' disease using EBV infection followed by fusion with a mouse/human hybrid cell line. The monoclonal antibody (M13) is an IgG1/kappa and in an immunofluorescence test M13 at 1 microg/mL showed islet cell antibody reactivity equivalent to 40 JDF units. M13 IgG bound (35)S-labelled IA-2 (26% at 100 microg/mL) and (125)I-labelled IA-2 (34% at 100 microg/mL) in an immunoprecipitation assay and reacted well with IA-2 in western blotting analysis. Amino acids 777-808 in the PTP domain of IA-2 were found to be important for M13 binding in an analysis using modified (35)S-labelled IA-2 proteins. M13 V region genes were from VH1-3, D3-22, JH4b, VKI DPK8/Vd+ and JK3 genes and showed a high replacement/silent mutation ratio for both the heavy (11.0) and the light (6.0) chain genes. Mouse monoclonal antibodies (mMAbs) reactive with at least three different epitopes within IA-2 aa 604-686 corresponding to the juxtamembrane domain were also obtained. F(ab')(2) or Fab from the mMAbs inhibited serum IA-2 autoantibody binding to IA-2 in 20/22 diabetic sera whereas M13 F(ab')(2) caused inhibition in only 6/22 sera. M13 is representative of some patient serum IA-2 autoantibodies and as such provides a useful tool to study autoimmune responses to IA-2.