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L.L. Wong, J. Chen, S. Sezate, M.M. Ramsey, W. Cao, J.F. McGinnis; In vitro Analysis of Antibody Mediated Cell Death . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1669.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Cancer Associated Retinopathy (CAR) is an autoimmune disease characterized by the presence of circulating antibodies against some retinal antigens, especially Recoverin (Rcvn), which results in the apoptotic death of rods and cones. One of the critical steps in this process is the entry of anti–Rcvn into cells, specifically Rcvn+ cells. In this study, we investigated the involvement of retinal proteins in the entry step and their identifications. Methods: Retinal cells were isolated from P1–2 rat pups and were grown in a defined medium for 10 to 14 days until confluent. Proteins were extracted from bovine retinas. We added anti–Rcvn (Ab110) in the presence or absence of retinal extract to the retinal cultures for 24 to 72 h. The amount of anti–Rcvn inside cells was analyzed by incubating cells with anti–rabbit IgG conjugated to Alexa–594. Apoptosis was monitored by staining cells with propidium iodide and anti–Annexin V. Flow cytometry, confocal and fluorescence microscopy were used to analyze these two processes. Results: After 24 h of incubation, a significant amount of anti–rabbit IgG was detected in retinal cells that were co–incubated with retinal extract and Ab110. Anti–rabbit signal was not observed inside cells at the same time interval in controls or samples incubated with Ab110 alone. By 72 h, we detected anti–rabbit IgG in samples containing Ab110 alone and in samples with retinal extract with Ab 110. However, the signal was significantly stronger in samples containing retinal extract with Ab 110. Conclusions: We think that one or more retinal proteins interact with anti–Rcvn and/or Rcvn to facilitate the entry of anti–Rcvn into cells resulting in increased death of Rcvn+ retinal neurons. We will identify the proteins that interact with the antigen/antibody complex because these antibody–entry facilitators may be potential targets for developing therapeutic agents for CAR or other autoimmune diseases.
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