April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Primary human corneal fibroblasts release specific matrix metalloproteinases in response to live Pseudomonas aeruginosa infection in-vitro
Author Affiliations & Notes
  • Ahmad Elsahn
    Molecular Microbiology, University of Southampton, Southampton, United Kingdom
    Eye Unit, University Hospital Southampton NHS Foundation Trust, Southampton, United Kingdom
  • Paula Rodas
    Molecular Microbiology, University of Southampton, Southampton, United Kingdom
    Medicine and Innovative Science (CIMIS), Facultad de Medicina, Andres Bello University, Santiago, Chile
  • Myron Christodoulides
    Molecular Microbiology, University of Southampton, Southampton, United Kingdom
  • Parwez Hossain
    Molecular Microbiology, University of Southampton, Southampton, United Kingdom
    Eye Unit, University Hospital Southampton NHS Foundation Trust, Southampton, United Kingdom
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 6275. doi:
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      Ahmad Elsahn, Paula Rodas, Myron Christodoulides, Parwez Hossain; Primary human corneal fibroblasts release specific matrix metalloproteinases in response to live Pseudomonas aeruginosa infection in-vitro. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6275.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To test the hypothesis that human corneal fibroblasts (CF) release specific matrix metalloproteinases (MMP) in respone to infection by live Pseudomonas aeruginosa bacteria.

Methods: Human CF were extracted from clinical samples, cultured to confluence in vitro and challenged with live Pseudomonas aeruginosa PA01 bacteria at an infecting dose of 107 cfu/ml as well as heat killed (HK) and freeze-thaw killed (FT) bacteria of the same dose, and lipopolysaccharides (LPS) at a dose of 100 ng/ml. Gentamicin was added to a group of cells after 3h of infection to kill all extra cellular bacteria and assess the role of internalized bacteria. Plain medium, bacteria alone (at a dose of 107 cfu/ml), and uninfected cells were used as controls. Supernatants were collected at 24h and analysed by gelatine zymography.

Results: Human CF challenged with HK bacteria, FT bacteria and LPS only produced the constitutive 65 kDa gelatinase (inactive MMP-2), similar to unchallenged cells. The same was true for CF treated with gentamicin after 3h of infection. Bacteria alone produced a 54 kDa gelatinase (alkaline protease). Human CF challenged with live bacteria released additional gelatinases of smaller sizes (25-37 kDa) that were not produced constitutively or in association with challenge with bacterial endotoxins or killed whole bacteria.

Conclusions: Human CF release specific MMPs in response to infection with live PA01 bacteria. This is probably related to exotoxins actively produced by live invading bacteria rather than passive bacterial endotoxins or structural components.

Keywords: 480 cornea: basic science • 594 microbial pathogenesis: experimental studies  
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