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T. Kezuka, Y. Usui, C. Nishiyama, T. Hattori, Y. Okunuki, N. Yamakawa, M. Takeuchi, H. Goto; Gene Therapy With Calcitonin Gene-Related Peptide Suppresses Murine Experimental Autoimmune Uveoretinitis. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2517.
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© ARVO (1962-2015); The Authors (2016-present)
Previously we reported that peritoneal exdate cells treated with calcitonin gene-related peptide (CGRP) suppress murine experimental autoimmune uveoretinitis (EAU) in an antigen-specific manner, even in the efferent phase, and IL-10 secreted from PEC might play an important role in the CGRP-mediated suppression of murine EAU. In this study, we investigate the role of the suppression in murine EAU by gene therapy with dendritic cells (DC) introduced CGRP.
Bone marrow cells derived from C57BL/6 mice were cultured with GM-CSF, and differentiated CD11+ immature DC were purified by autoMACS. Immature DC were further cultured with LPS as mature DC (BMDC). Coding region of CGRP cDNA was amplified by PCR using cDNA synthesized from total RNA of ARPE-19 cell line. Human CGRP cDNA fragment was inserted into pCR3.1-2FL plasmid by using site-directed mutagenesis. The resulting plasmid, pCR3.1-2FL-hCGRP, or control vector, pCR3.1-2FL (mock) as control group, was introduced into mouse BMDC by electroporation with Nucleofector II according to manufacturer's instruction. Then, BMDC with CGRP gene-transfer were cultured with human interphotoreceptor retinoid binding protein (IRBP) residues 1-20 (hIRBP-p). EAU was induced by immunization with hIRBP-p in mice. Gene therapy group mice were injected BMDC with CGRP gene-transfer intravenously. On day 19 after immunization, hIRBP-p-specific delayed hypersensitivity (DH) were measured. On day 21 after immunization, animals were sacrificed, and assessed the extent of EAU pathologically. The severity of EAU in each eye was scored on scale of 0 to 4.
Gene transfer efficiency was 70% cell viabilty and 50% transfection efficiency in suspension cells. EAU and antigen-specific DH was markedly suppressed by injection of DC with CGRP-gene-transfer compared to control group. Incidence of EAU on control group was 71% (5 of 7 EAU mice, average score 1.9), and CGRP gene therapy group was 14% (1 of 7 EAU mice, average score 0.3).
It was demonstrated that BMDC with CGRP gene-transfer play an immunosuppressive role in development of EAU.
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