C A Buchman1, N Fregien. 1. Department of Otolaryngology, University of Miami School of Medicine, Florida, USA. cbuchman@med.miami.edu
Abstract
HYPOTHESIS: Human-derived normal middle ear mucosal cells can be harvested and cultured and will support influenza A virus (INF A) infection. STUDY DESIGN: Protocols for the collection and in vitro culture of middle ear mucosal cells were developed and used to investigate the effects of INF A infection as it relates to the pathogenesis of otitis media. MATERIALS AND METHODS: Middle ear mucosa was harvested during surgeries that opened the normal middle ear. Middle ear mucosal cells were plated and grown in collagen-coated dishes. Cells were characterized before and after INF A exposure using phase-contrast and immunofluorescence microscopy as well as reverse transcriptase-polymerase chain reaction (RT-PCR) for cytokeratin 18 gene expression and INF A. RESULTS: Primary cultures of human middle ear epithelial cells were established. Prolonged growth of middle ear cells yielded a second cell type that failed to stain for cytokeratin on immunofluorescence but continued to produce positive RT-PCR results on cytokeratin 18 analysis. After INF A exposure, cytological changes and immunofluorescence staining showed cellular infection. RT-PCR analysis using INF A-specific primers showed positive results for up to 72 hours after viral exposure. CONCLUSIONS: Primary cultures of human middle ear mucosal cells have been established. Two distinctly different cell culture systems have been developed: 1) middle ear epithelial cells and 2) either dedifferentiated epithelial cells or fibroblasts. Exposure of both cell types to INF A demonstrates that each can support cellular infection and viral replication. These models should be useful for studies of the pathogenesis of virus-mediated otitis media.
HYPOTHESIS: Human-derived normal middle ear mucosal cells can be harvested and cultured and will support influenza A virus (INF A) infection. STUDY DESIGN: Protocols for the collection and in vitro culture of middle ear mucosal cells were developed and used to investigate the effects of INF A infection as it relates to the pathogenesis of otitis media. MATERIALS AND METHODS: Middle ear mucosa was harvested during surgeries that opened the normal middle ear. Middle ear mucosal cells were plated and grown in collagen-coated dishes. Cells were characterized before and after INF A exposure using phase-contrast and immunofluorescence microscopy as well as reverse transcriptase-polymerase chain reaction (RT-PCR) for cytokeratin 18 gene expression and INF A. RESULTS: Primary cultures of human middle ear epithelial cells were established. Prolonged growth of middle ear cells yielded a second cell type that failed to stain for cytokeratin on immunofluorescence but continued to produce positive RT-PCR results on cytokeratin 18 analysis. After INF A exposure, cytological changes and immunofluorescence staining showed cellular infection. RT-PCR analysis using INF A-specific primers showed positive results for up to 72 hours after viral exposure. CONCLUSIONS: Primary cultures of human middle ear mucosal cells have been established. Two distinctly different cell culture systems have been developed: 1) middle ear epithelial cells and 2) either dedifferentiated epithelial cells or fibroblasts. Exposure of both cell types to INF A demonstrates that each can support cellular infection and viral replication. These models should be useful for studies of the pathogenesis of virus-mediated otitis media.
Authors: Kirsty R Short; Dimitri A Diavatopoulos; Ruth Thornton; John Pedersen; Richard A Strugnell; Andrew K Wise; Patrick C Reading; Odilia L Wijburg Journal: J Infect Dis Date: 2011-09-19 Impact factor: 5.226