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S.V. Maharajan, R.S. McIntosh, J. McElveen, A. Browning, H.S. Dua; Cytokine Expression by Corneal Epithelial Cells Grown on Amniotic Membrane as Compared to Falcon (collagen coated) and Nunc (non-coated) Plates . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1384.
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Purpose: It has been shown clinically that the use of amniotic membrane for ocular resurfacing reduces inflammation. We quantitatively assessed the mRNA expression of pro-inflammatory and anti-inflammatory cytokines by corneal epithelial cells, in culture, on amniotic membrane as compared to Falcon and Nunc plates. We also analysed the culture supernatants for the same. Methods: Eye bank preserved corneo scleral rims (n =14) were used to expand sheets of epithelial cells simultaneously on the basement membrane side of amniotic membrane, collagen coated (Falcon) and non-coated (Nunc) plates. At 3 weeks, when confluence was achieved, the supernatants were collected and RNA was isolated from the cells. cDNA was prepared from the RNA and analysed using PCR amplification for messenger RNA for various proinflammatory cytokines, including IL-1α, IL-1ß, IL-4, IL-6, IL-8, IL-10 and TNFα. Quantification of the PCR products was carried out on an ABI310 genetic analyser and compared to known quantities of internal mimics. Cytokines in the supernatants were analysed by cytometric bead array (CBA) (BD Biosciences, human inflammatory and human TH1/TH2 CBA) using flow cytometry. Results: Expression of mRNA was seen for all the tested cytokines by PCR in varying quantities on all the 3 surfaces. There was a trend for the pro-inflammatory cytokines IL1 alpha, IL1 beta, IL8 to be less in amnion compared to Falcon and Nunc. The anti-inflammatory cytokine IL4 was found to be higher in amnion as compared to Nunc. Statistical significance however, could not be shown. The CBA on the supernatants showed presence of IL6 and IL8. Parametric ttest showed statistically significantly low IL8 (p< 0.03) in supernatants from amnion as compared to Nunc. Conclusions: The results seem to compare with the clinical observation that the use of the amniotic membrane helps to decrease the inflammatory response
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