Insulin-dependent diabetes is associated with a specific loss of the pancreatic B-cells and with the presence of circulating islet cell antibodies at the time of diagnosis. Islet cell antibodies, found to react with antigenic determinants in the B-cell plasma membrane, seem capable of mediating complement-dependent cytotoxicity but also of inhibiting the B-cell function. The present project will identify and characterize islet cell antibodies and determine their biochemical and cellular specificities. The possibility that islet cell surface antibodies may be of pathogenetic importance will be tested in in vitro perifusion experiments of single cell suspensions of pancreatic islet cells and in in vivo passive transfer experiments from man to mouse of immunoglobulin from diabetic children. Quantitative methods to determine both cell surface and cytoplasmic islet cell antibodies will be used in analyzing plasma samples collected prospectively from newly diagnosed insulin-dependent diabetic patients and their first-degree relatives and correlated to the presence of islet cell cytotoxic antibodies. The reactivity of islet cell and lymphocyte antibodies in spontaneously diabetic BB Wistar rats, with endocrine islet cells and lymphocytes will be approached by cell sorting and in quantitative radioligand assays. The present project also aim at identifying antigenic determinants recognized by islet cell antibodies. Pancreatic islet cells or tumor B-cells will be labelled with radioactive amino acids and detergent solubilized proteins subjected to immunoprecipitation followed by gel electrophoretic analysis. A hypothetical B-cell specific glycoprotein (Mr 40000) and a Mr 64000 human islet cell protein detected by diabetic sera will be characterized and attempts made to establish their subcellular localization. Further characterization will be approached by radiosequence analysis to obtain a partial amino acid sequence. Long-term objectives include the use of recombinant DNA techniques to isolate cloned cDNA sequences for the antigens. A successful isolation of cloned genes will permit nucleotide sequene determination to allow prediction of amino acid sequences. The long-term goal is to identify and characterize islet cell membrane antigens which may be important in an immunopathological disease process leading to insulin-dependent diabetes.
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