| Summary: | Endogenous posterior uveitis (EPU) is currently treated with immunosuppressive drugs, the uses of which are limited by systemic side effects and the development of refractoriness towards the treatment. EPU is thought to be the result of an autoimmune response towards sequestered antigens in the retina. In animal models disease can be prevented by inducing tolerance to an initiating antigen. Knowing the role each antigen plays in human disease will be pivotal in developing a strategic approach to tolerance therapy in humans. There is an urgent need for the production of human sequence autoantigens to further our understanding of the diseases and develop novel therapies. In this project we produced the putative candidate autoantigens S-antigen and recoverin and used these to investigate the cellular and humoral immune responses in EPU. Recoverin was expressed in E. coli BL21 (DE3) using the expression vector pET14b. The cDNA was obtained from RT-PCR of healthy lymphocytes. The recombinant recoverin was purified with nickel chelation chromatography then reverse HPLC. The purified recoverin was used to develop an ELISA. S-antigen was expressed to CHO cells transfected with the expression vector pcDNA3.1zeo into which human S-antigen cDNA had been cloned. The S-antigen was purified, but not to the degree that it could be used for human cellular assays. Therefore, cellular assays investigating proliferation, and IL-10 and TGF-b secretion were carried out using an S-antigen synthetic peptide panel. We demonstrated that certain peptides could stimulate PBMC to produce IL-10 without proliferating, raising the possibility that certain epitopes may induce regulatory rather than pathogenic responses.
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