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H. Alizadeh, H.-C. Li, S. Neelam, J. Y. Niederkorn; Modulation of Corneal Epithelium and Stromal Matrix Metalloproteinase by the Mannose-Induced Acanthamoeba Cytolytic Protein. Invest. Ophthalmol. Vis. Sci. 2008;49(13):502.
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The involvement of the mannose-induced Acanthamoeba cytopathic protein (MIP-133) in tissue injury and activation of metalloproteinase of corneal and stromal cells was examined in vitro.
Activation of MMP-1, MMP-2, MMP-3, MMP-9, and MMP-14 induced by MIP-133 on human corneal epithelial and stromal cell cultures was examined by reverse transcriptase polymerase chain reaction (RT-PCR), zymography, immunoblot analysis and ELISA.
MMP-1, MMP-2, MMP-3, MMP-9 and MMP-14 mRNA were expressed in both cultured human corneal epithelial and stromal cells. When the epithelial cells were exposed to MIP-133 protein, the mRNA expression for all MMPs tested was significantly down-regulated 4 and 8 hours after treatment. In contrast, the expression of MMP-2 and MMP-3 was significantly up-regulated in the stromal cells 1 and 4 hours after MIP-133 stimulation.Gelatin zymography of the medium obtained after incubation of corneal epithelial cells with MIP-133 for 6 hr revealed two major bands of 72 and 62 kDa corresponding to pro MMP-2 and active MMP-2 respectively. When corneal stromal cells were treated for 24 hr with MIP-133, activated forms of MMP-2 and MMP-3 were detected by ELISA. To verify the possibility of homology between MMPs and A. castellanii proteases, the mRNA from A. castellanii was prepared and analyzed for the expression of MMP genes by PT-PCR. The results showed that A. castellanii did not express mRNA for MMP-1, MMP-2, MMP-3, or MMP-9. Thus, A castellanii mRNA does not cross react with human MMPs. Furthermore, ELISA was used to determine the cross reactivity of MMP antibodies with the MIP-133 protein. Monoclonal antibodies against MMPs did not cross react with either the MIP-133 protein or BSA (negative control antigen).
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