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F. Giansanti, L. Papucci, L. Vannozzi, S. Capaccioli, E. Rapizzi, E. Witort, U. Menchini; Safety of Infliximab on Retinal Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5623.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the cytotoxic properties of infliximab on different ocular cells.
Cell culture: ARPE-19 (RPE) cells were maintained in a mixture of Dulbecco’s Modified Eagle’s Medium and Ham’s F12 with 2,5 mM L-glutamine and supplemented with 10% fetal bovine serum in humidified atmosphere of 5% CO2 at 37°C. RGC-5 cells were maintained in Dulbecco’s Modified Eagle’s Medium with 2,5 mM L-glutamine and supplemented with 10% fetal bovine serum in humidified atmosphere of 5% CO2 at 37°C. This cell line was kindly provided by Prof. Neeraj Agarwal, University of North Texas Health Sciences Center - Texas, USA. Treatments: Infliximab (0.1-0.3-1-3-6 mg/ml) diluted in culture medium was added to each cells maintaned in 6-well cell culture plates. Cells were harvested and assayed for apoptosis induction 24-48-72 hours after treatment. Western Blot analysis of Caspase 3 activity:The assays were performed according to standard conditions. The cells were lysed and proteins extracted in Ripa buffer. The protein lysates were obtained by centrifugation and then were analyzed by 12% SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose membrane in a Trans-blot apparatus at 100 V for 90 min. The blots were probed with mouse monoclonal anti-Caspase-3 antibody. A mouse monoclonal anti-alpha-Tubulin antibody was used as a protein loading control.
No apoptotic effect of infliximab at any of the concentrations tested was observed in RPE and RGC-5 cells. In fact, the pro-caspase 3 level were not affected by any of the infliximab concentration tested at any of the time points analysed.
The experimental findings support the safety of the infliximab at the concentrations and in the conditions tested.
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