Abstract
Studies suggest certain stimuli like apoptosis behind the aberrant expression of circulating specific auto-antibodies in Type-1 diabetic patients. In the present study, we investigated whether Streptozotocin (a diabetogenic compound) induced apoptosis in Rat RINm5f Insulinoma cells can expose immunogenic cryptic antigens. A time course treatment of RINm5f cells with 4 mM Streptozotocin revealed 6hrs as minimal time period to induce apoptosis without much effect on viability.Subsequent immunization of mice with the untreated and Streptozotocin treated RINm5f cells revealed highly reactive sera from 6hr STZ treated cells. Hybridoma technique showed a highly reactive clone named sup160which secretedIgG1 type monoclonal antibody.Parallely, we show that 6hr STZ-RINm5f challenged mice displayed both humoral and cellular immune response, as shown by increased presence of IgG1 / IgG2a subclass of antibodies and overexpression of IFNγ, IL-4 and TNFα in splenocytes. FACS and confocal imaging further established the reactivity of sup160 antibody with RINm5f cell surface antigen. The antigen was identified to be cytokeratin18 protein as detected by 2D gel electrophoresis and mass spectrometry. In conclusion, we have successfully established a novel xenogenic mice model system for identification of new auto-antigen in Type-1 diabetes, although further studies are warranted in human subjects.
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