Induction of experimental autoimmune uveoretinitis in Lewis rats with purified recombinant human retinal S‐antigen fusion protein

Abstract

Full-length human retinal cDNA for S antigen (S-ag) and for the alpha subunit of transducin (alpha-Td) were subcloned into a bacterial expression plasmid vector to generate recombinant fusion proteins with glutathione-S-transferase (GST). The recombinant GST-S-ag and rGST-alpha-Td fusion proteins were purified from bacterial extracts by continuous flow preparative gel electrophoresis under denaturing conditions, and were assessed for their ability to induce experimental autoimmune uveoretinitis (EAU). Immunization of Lewis rats with single doses of 10 micrograms-100 micrograms rGST-S-ag in Freund's complete adjuvant supplemented with Bordetella pertussis readily induced clinical signs of EAU. Immunization with GST alone did not induce EAU indicating that disease activity was ascribable to the S-ag residues in the fusion protein. Although the alpha-Td shares limited sequence homology with S-ag, the rGST-alpha-Td fusion protein was also not uveitogenic in Lewis rats. The clinical severity of EAU in Lewis rats sensitized with rGST-S-ag was found to be milder than that induced with native S-ag preparations purified from human retina. However, humoral antibody responses to sensitization with the recombinant S-ag fusion protein were of a higher magnitude than with native S-ag. The availability of recombinant preparations of human S-ag protein will be of value in studying its processing and presentation to T cells derived from patients with autoimmune retinal vasculitis.

Expression of autoimmune disease-related antigens by cells of the immune system

“…Electrophoresis of the PCR products (Fig. 2) shows expression of MBP, insulin (implicated as an auto-antigen in type I diabetes [Haskins and Wegmann, 1996]), cardiac myosin (CM; implicated in the pathogenesis of experimental autoimmune myocarditis [Wegmann et al, 1994]), and retinal S-antigen (one of the wellcharacterized self-antigens in autoimmune uveitis ([Roberts et al, 1992]) in thymocytes. In contrast, there was no PCR evidence for the expression of glutamic acid decarboxylase (GAD) 65 or thyroglobulin auto-antigens implicated in pathogenesis of autoimmune type I diabetes (Tisch et al, 1993) and thyroiditis (Bhatia et al, 1996), respectively.…”

Antigen-specific T cell recognition of affinity-purified and recombinant thyroid peroxidase in autoimmune thyroid disease

“…The recombinant preparations were purified from other bacterial proteins by 3 M urea extraction [13] followed by preparative gel electrophoresis employing an electroendosmotic preparative gel (ELFE, Genofit, Heidelberg, Germany) [14,15]. Before cell culture work SDS was removed by detergent exchange with 0 03% DOC.…”

“…For the preparation ofthe various rTPO fragments in E. coli, extraction of bacterial inclusion bodies by 3 Molar urea was found to enrich considerably the recombinant protein in the insoluble pellet [15], This is illustrated in the urea extraction studies of two comparatively sized rTPO preparations Rla4-Rlb and R2a, both of approximately 43 kD, Extraction of inclusion bodies with 3 Molar urea removes a large proportion of other E. coli proteins (Fig, 2a, lanes 2 and 3 and 6 and 7), leaving the rTPO proteins in the insoluble pellet (Fig, 2a, lanes 4 and 8), The final purity of the rTPO preparations following ELFE purification, as judged by SDS-PAGE, is shown in Fig, 2b,…”

Immunopathogenicity of thyrotropin receptor – ability to induce autoimmune thyroid disease in an experimental animal model

“…or thyroid function has also been recently examined (Seetharamaiah et al, 1995, abstract). lt is possible that ancillary adjuvants such as Bordetella pertussis, which in other experimental autoimmune diseases potentiates the immune insult (Roberts et al, 1992) may be an important additive to test. The TSE-1-R protein is extremely immunogenic when injected into animals in adjuvant since two injections gives rise to high titre serum and substantial T cell reactivity (Carayanniotis et al, 1995).…”