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David J McCanna, Sarah Oh, Junghee Seo, Lakshman N Subbaraman, Chantal Coles-Brennan, Zohra Fadli, Lyndon William Jones; Effect of Denatured Lysozyme on Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3511.
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© ARVO (1962-2015); The Authors (2016-present)
Protein denaturation on contact lenses (CL) has been hypothesized as one of the possible leading causes for unsuccessful CL wear probably due to the loss in proteins functions, change in the exposed chemical structure or to the fact that denatured proteins can be recognized as an antigen and therefore induces an immune response. The binding of lysozyme to some CL materials has been shown to result in a conformational change of the protein, leading to a loss in enzymatic activity. This investigation evaluated the effect of denatured lysozyme on human corneal epithelial cells (HCEC) by measuring cell viability and the release of inflammatory cytokines after HCEC exposure to heat-inactivated lysozyme
HCEC were cultured in multi-well plates and exposed to lysozyme that was denatured to various enzymatic activity levels. After a 16 hr exposure to the lysozyme (1.9mg/mL in media), cell viability was evaluated by staining the cells with the viability dyes calcein, ethidium homodimer-1 and annexin V and imaging the cells using a confocal microscope. The metabolic activity was also determined using the alamarBlue assay. The media from the cell wells were analyzed for cytokine levels using the human pro-inflammatory V-plex assay (IFN-γ, IL-10, IL-12 p70, IL-13, IL-1β, IL-2, IL-4, IL-6, IL-8 and TNF-α) from MesoScale Discovery.
The confocal images of stained HCEC exposed to various levels of denatured lysozyme did not show a change in the cell viability. However, a significant decrease in metabolic activity was observed following exposure to different levels denatured lysozyme when compared to native (100% active) lysozyme and the media controls (p<0.05). Exposure of HCEC to the denatured lysozyme also caused asignificant increase in the release of several inflammatory cytokines (IFN-γ, IL-2, IL-4, IL-6, IL-8, IL-12 and IL-13) from the HCEC (all p<0.05) depending on the level of denatured lysozyme in contact with the cells.
The results of this study for the first time show that denatured lysozyme can have a detrimental effect on HCEC. Both a reduction in metabolic activity and the release of inflammatory cytokines occurred after HCEC exposure to denatured lysozyme. Preventing proteins such as lysozyme from denaturing due to deposition on contact lenses may be desired to preserve corneal cell homeostasis essential for successful contact lens wear.
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