Abstract
Abstract: :
Purpose:To determine if the recently established epithelial conjunctival human cell line (IOBA–NHC) is susceptible to HSV–1 infection and to evaluate the induction of NF–kB nuclear translocation in infected IOBA–NHC cells stimulated or not with TNF–alpha. Methods:IOBA–NHC cell monolayers grown in converslips inside 24–well plates were infected with HSV–1 Cgal (m.o.i. = 0.2), which contains the Escherichia coli lacZ reporter gene encoding ß –galactosidase under the control of a strong heterologous promoter from the human cytomegalovirus immediate–early (IE) regulatory region. After incubation for 1 h at 37 ºC, inocula were eliminated and cells were covered with MEM 1.5%. At 24 h post–infection (p.i.), supernatants were harvested for titration in Vero cells, and IOBA–NHC cells were stained in–situ for ß –gal. IOBA–NHC cells were infected with HSV–1 KOS strain at a m.o.i of 10 PFU/cell, and treated or not with 10 ng/ml of TNF–alpha. After 10 and 24 h p.i., cells were processed for a double immunofluorescence staining (IFI) using monoclonal anti–glycoprotein D (mgD) of HSV–1 and polyclonal anti NF–kB antibodies. Results:IOBA–NHC cells were effectively infected by both viral stocks. HSV–1 Cgal propagated well since "blue" cells clustered in characteristic herpesvirus foci were observed, yielding a viral titer of 104 PFU/ml at 24 h p.i., in Vero cells. Infected IOBA–NHC cells treated or not with TNF–alpha along 10 h. p.i. evidenced NF–kB translocation to the nucleus by means of an IFI assay. Similar results were obtained at 24 h.p.i., though TNF–alpha treated infected cells exhibited a stronger cytopathic effect than untreated cells. Conclusions:1.– HSV–1 is able to multiplicate in a human conjunctival cell line (IOBA–NHC). 2.– HSV–1 induces NF–kB nuclear translocation in IOBA–NHC cells at 10 and 24 h.p.i.. 3.– The addition of TNF–alpha enhances HSV–1 cytopathic effect in IOBA–NHC cells at 24 h.p.i..
Keywords: conjunctiva • herpes simplex virus • transcription factors