Antigen-specificity of antiretinal antibodies in patients with noninfectious uveitis.

OBJECTIVE: Antiretinal antibodies (ARAs) have previously been described in noninfectious uveitis. However, the antigen specificity of these ARAs has not been investigated. The purpose of this study was to identify antigen-specific ARAs in noninfectious uveitis. METHODS: A total of 18 patients with noninfectious uveitis were enrolled. Surface plasmon resonance was used to measure binding responses of patient and control sera against several uveitogenic proteins: recoverin, S-antigen, interphotoreceptor retinoid binding (IRBP), retinal-pigment-epithelium-specific 65-kDa protein (RPE65), tyrosinase-related protein 1 (TRYP1), and tyrosinase-related protein 2 (TRYP2). RESULTS: The frequency of ARA positivity against S-antigen, IRBP, RPE65, TYRP1, and TYRP2 in patients with uveitis did not differ significantly from that of normal controls. However, ARA positivity for recoverin was more frequently observed in patients with uveitis (p = 0.002). A total of 10 patients in the uveitis cohort had birdshot chorioretinopathy, and all 10 were positive for anti-recoverin ARAs. CONCLUSIONS: Patients with noninfectious uveitis have increased frequency of ARA positivity against recoverin. This ARA deserves further investigations as a potential biomarker and pathogenic agent in noninfectious uveitis, especially in birdshot chorioretinopathy.

Thinning of the RPE and choroid associated with T lymphocyte recruitment in aged and light-challenged mice.

PURPOSE: Thinning of the RPE and the underlying vascular layer, the choroid, is observed with age in many human eye disorders. The reasons for this thinning are ill-defined. Here, we highlight the possible role of T lymphocyte recruitment in choroidoretinal thinning in aged and light-challenged mice. METHODS: In age and light challenge models, we measured chemokine concentrations using enzyme-linked immunosorbent assay and used flow cytometry to characterize lymphocyte populations. We quantified thinning in eye immunosections and RPE65 expression using quantitative PCR. RESULTS: Age and light challenge led to increased levels of the lymphotactic protein CXCL10 alone (aging) or in conjunction with CXCL9 (light challenge). Increased numbers of CD3+ T lymphocytes, most of them CD8+ cytotoxic T lymphocytes, were also observed in the choroid and retina of old mice and following light challenge. Influx of T lymphocytes was associated with RPE and choroidal thinning and diminished expression of RPE65 mRNA, an essential enzyme of the visual cycle. CONCLUSIONS: The observations from this study suggest that cytotoxic CD8(+) T lymphocytes might participate in choroidal and RPE degeneration and that modulation of T lymphocyte recruitment might be a novel strategy to reduce choroidoretinal dysfunctions observed with age and following photo-oxidative stress.

Identification of key residues determining isomerohydrolase activity of human RPE65.

RPE65 is the retinoid isomerohydrolase that converts all-trans-retinyl ester to 11-cis-retinol, a key reaction in the retinoid visual cycle. We have previously reported that cone-dominant chicken RPE65 (cRPE65) shares 90% sequence identity with human RPE65 (hRPE65) but exhibits substantially higher isomerohydrolase activity than that of bovine RPE65 or hRPE65. In this study, we sought to identify key residues responsible for the higher enzymatic activity of cRPE65. Based on the amino acid sequence comparison of mammalian and other lower vertebrates' RPE65, including cone-dominant chicken, 8 residues of hRPE65 were separately replaced by their counterparts of cRPE65 using site-directed mutagenesis. The enzymatic activities of cRPE65, hRPE65, and its mutants were measured by in vitro isomerohydrolase activity assay, and the retinoid products were analyzed by HPLC. Among the mutants analyzed, two single point mutants, N170K and K297G, and a double mutant, N170K/K297G, of hRPE65 exhibited significantly higher catalytic activity than WT hRPE65. Further, when an amino-terminal fragment (Met(1)-Arg(33)) of the N170K/K297G double mutant of hRPE65 was replaced with the corresponding cRPE65 fragment, the isomerohydrolase activity was further increased to a level similar to that of cRPE65. This finding contributes to the understanding of the structural basis for isomerohydrolase activity. This highly efficient human isomerohydrolase mutant can be used to improve the efficacy of RPE65 gene therapy for retinal degeneration caused by RPE65 mutations.