Host immune factors involved in bacterial ocular virulence were examined through the use of pneumolysin, a cytoplasmic protein and pneumococcal strain D39 utilizing human corneal epithelial cells (HCEC). Recombinant pneumolysin activity was determined by a hemolysis assay using the following concentrations: 50-1000 ng/mL. HCEC were grown to confluency and exposed to 103 to 107 colony forming units (CFUs) of mid-log phase Streptococcus pneumoniae D39 for 2, 4, 6, and 24 h. HCEC were exposed to pneumolysin at various concentrations: 200-1000 ng/mL. ELISA and human cytokine array was performed on the supernatant. Fluorescence studies using cytotoxicity assay were performed on the HCEC cells following infection to determine live versus dead cells. The hemolysis assay showed that a minimum of 100 ng/mL PLY will lyse the red blood cells. Cytokine array on the HCEC exposed supernatants showed the presence of IL-6 and IL-8. Four hour exposure of HCEC cells to a range of 103 to 107 CFU of D39 resulted in monolayer disruption and decreased viability as compared to cells exposed to the media alone. HCEC cells exposed to any of the concentrations of bacteria exhibited signs of cellular damage and death. Following 24 h exposure, there was an increased expression of IL-8 (p < 0.001) compared to media alone. IL-6, IL-8 and IFN-gamma levels were elicited at a minimal of 200ng PLY. In addition to changes in viability and morphology of cultured cells, we detected specific cytokines in the culture supernatants of HCEC following exposure to S. pneumoniae D39.
You have requested a machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Neither BioOne nor the owners and publishers of the content make, and they explicitly disclaim, any express or implied representations or warranties of any kind, including, without limitation, representations and warranties as to the functionality of the translation feature or the accuracy or completeness of the translations.
Translations are not retained in our system. Your use of this feature and the translations is subject to all use restrictions contained in the Terms and Conditions of Use of the BioOne website.
Vol. 85 • No. 4