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L.L. Wong, S. Sezate, J. Chen, J.F. McGinnis; Development of a Heterologous in vitro System and an in vivo Rat Model to Study Antibody Mediated Cell Death . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4842.
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© ARVO (1962-2015); The Authors (2016-present)
Cancer Associated Retinopathy (CAR) is an autoimmune disease in which autoantibodies, recognizing retinal specific proteins, are found in the sera of patients. In some CAR patients, the autoantibody binds to Recoverin (Rcvrn), a retinal protein found in rod and cone photoreceptors, and a subset of cone bipolar cells. The circulating antibodies eventually cause retinal degeneration and subsequent blindness. In vitro studies using rodent retinal cell cultures have demonstrated that the apoptotic death of retinal cells is highly specific; only Rcvrn–positive cells are susceptible to the effect of anti–Rcvrn–IgG. We report here that we have established a heterologous in vitro model to investigate the mechanism of antibody mediated cell death and an in vivo rat model for evaluating potential therapeutic small molecules for CAR.
We used PCR cloning to generate a stable HEK 293 cell line expressing rat Rcvrn (RR 293). To assess the effect of anti–Rcvrn–IgG on RR 293 cells, we added anti–Rcvrn (Ab110, 100 µg/mL) to the culture medium at 60% confluence for 24h to 48h. The amount of anti–Rcvrn inside cells was analyzed by incubating cells with anti–rabbit IgG conjugated to PE or Alexa–594 after 24h. Apoptosis was monitored by staining cells with propidium iodide and Annexin V at 24 and 48h. Flow cytometry and confocal microscopy were used to analyze these two processes. For in vivo studies, rat eyes were injected with anti–Rcvrn–IgG and electroretinography (ERG) performed subsequently to evaluate visual function followed by morphometric analysis on histological sections. The ability of small molecules to prevent photoreceptor cell death is being investigated.
After approximately ten passages of RR 293 with G418 selection, the morphology and growth rate of these cells became indistinguishable from untransfected 293 cells. The effects of anti–Rcvrn–IgG on these cells will be presented. For in vivo studies, within three weeks of anti–Rcvrn–IgG injection, ERG’s were decreased by 30–50% compared to control eyes. The therapeutic effect of a small molecule using this in vivo CAR model will be presented.
Both models are extremely valuable. The heterologous in vitro system is very useful for testing the involvement of cell–type specific factors whereas the in vivo model evaluates the actual rescue of vision by potential therapeutic molecules.
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