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2005 OMIG, Abstract 6

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LIPID MEMBRANE RAFTS MEDIATE PSEUDOMONAS AERUGINOSA INTERNALIZATION IN CORNEAL EPITHELIUM FOLLOWING CTL WEAR IN VIVO AND IN VITRO. Cavanagh HD

PURPOSE: Comeal epithelium (CE) responds inversely to hypoxia induced by contact lens wear (CTLW) with increased binding and intemalization of Pseudomonas aeruginosa (PA). The purpose of this study was to determine if membrane lipid rafts (MLR) play a role in this process in vivo, in vitro and following CTLW.

METHODS: MLR formation was evaluated in vivo in rabbit CE with or without CTLW; immortalized human CE cells (hTERT) were used in vitro. MLR were detected by FITC-labelled (3-subunit cholera toxin known to bind MLR component GM1 with multiphoton confocal microscopy. Three pathogenic PA strains were tested; intemalization was assessed by gentamicin survival assay and confirmed by pretreatment of h-TERT cells with three cholesterol metabolism inhibitors. Cytotoxic effects of inhibitors were excluded by live-dead assays. Specific interactions of PA and MLR were confirmed by FACS analysis.

RESULTS: Normal rabbit CE does not exhibit MLR in vivo; these are induced by PMMA CTLW, but not by PA exposure alone to all three test strains. PA exposure after PMMA CTLW showed binding to MLR-forming cells in vivo followed by MLR aggregation and intemalization for all strains. A similar sequence of MLR formation and PA intemalization was seen in hTERT cells. Intemalization of all strains was blocked in a concentration dependent manner by cholesterol metabolism inhibitors (P < 0.01); cytotoxic effects were excluded by live-dead assays over concentrations studied. FACS analysis showed exposure of two PA test strains to cell lysate significantly increased fluorescence intensity, demonstrating binding of MLR-GMltoPA.

CONCLUSIONS: These findings demonstrate for the first time that CTLW- mediated PA intemalization involves MLR platforms. These findings offer a unique new strategy for prevention of CTL-related PA infection by blocking MLR formation.

Disclosure code: None

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