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C. Ramachandran, Y. Guo, S. Roy, C.K. Sen, S.P. Srinivas; TLR Expression and LPS Induced MLC Phosphorylation in Bovine Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1617.
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The aim of this study is twofold 1) to determine the expression of Toll–Like–Receptor’s (TLR’s) 1–10 in bovine corneal epithelial cells, and evaluate the effect of LPS (Lipopolysaccharide) on MLC phosphorylation. Of specific interest is the TLR–4 which identifies the bacterial endotoxin LPS, a major virulence factor of Pseudomonas aeruginosa, a common cause of bacterial infections in Contact Lens wearers.
The basal level expression of TLR genes 1–10 was studied in vitro at the mRNA level using QRT–PCR on normal bovine corneal epithelial cells (3rd passage). RNA was extracted from these cells using the RNEasy kit from Stratagene and 5 microgram of the total RNA was subjected to RT–PCR in order to obtain single stranded cDNA. The expression of these genes in the corneal epithelium was studied by using specific primer pair sequences. The PCR product was run on a 2% agarose gel for confirmation of the specificity of the gene product. After treatment with LPS for 24hrs at a concentration of 300ng/ml, MLC phosphorylation was quantified using urea–glycerol electrophoresis followed by Western blotting.
TLR’s 1,2,3,4 and 6 were expressed in abundance in the normal bovine corneal epithelial cells. Bands of the expected product size were evident from agarose gel electrophoresis confirming the specificity of these products. Western blot analysis revealed significant MLC phophorylation in response to LPS treatment.
MLC phosphorylation induces contractility of actin cytoskeleton leading to a loss of barrier intergrity. This, study thus suggests that the TLR’s and in specific TLR4 present on the bovine corneal epithelial cells may signal the loss of the barrier function by inducing MLC phosphorylation. This is consistent with the report by Yi et al (2000) who have shown that LPS induces a loss in the barrier integrity in human/rat corneal epithelial cells.
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