Journal: PLoS ONE

Article Title: Infection of Female Primary Lower Genital Tract Epithelial Cells after Natural Pseudotyping of HIV-1: Possible Implications for Sexual Transmission of HIV-1

doi: 10.1371/journal.pone.0101367

Figure Lengend Snippet: Immunofluorescence analysis of HIV-1 infection in VK-2. VK-2 cells were infected with progeny virus from primary CD4+ T cells or CEMX174 and were stained for HIV-1 Gag expression 5 days post-infection. The input virus used to infect producer T cells are as indicated. An enlarged view of epithelial cells exposed to progeny virus from HIV-1/XMRV co-infected CEMX174 cells is shown (second panel). HIV-1 Gag expression is indicated by green fluorescence (FITC); green fluorescence merged to the corresponding bright field image is shown in the bottom panels. B. Target epithelial cells were exposed to progeny viruses in the presence or absence of AZT as indicated on the panels. Data shown are representative of six independent experiments. Bar = 10 µm. C and D , HIV-1 and XMRV viral RNA in the supernatants of the same infected VK-2 cells as in (A) and (B) were quantified by qRT-PCR. The viruses used to infect producer cells are shown on the X axis. The data shown represent the mean ± standard deviation from three independent experiments.

Article Snippet: Cultures of human primary vaginal epithelial cells (Celprogen, CA) and human primary cervical epithelial cells (Cell Application, CA) were also purchased and maintained in medium provided by the cell vendor following vendor protocols.

Techniques: Immunofluorescence, Infection, Staining, Expressing, Fluorescence, Quantitative RT-PCR, Standard Deviation

Journal: PLoS ONE

Article Title: Infection of Female Primary Lower Genital Tract Epithelial Cells after Natural Pseudotyping of HIV-1: Possible Implications for Sexual Transmission of HIV-1

doi: 10.1371/journal.pone.0101367

Figure Lengend Snippet: ( A, B ) Visualization of HIV-1 infection in primary endocervical epithelial cells by dual immunofluorescence staining with FITC-anti-HIV-1 Gag and anti-CK19 MAbs. Primary endocervical epithelial cells were exposed to progeny viruses from infected CEMX174 cells and immunofluorescence staining was performed 5 days post-infection. Epithelial cells exposed to virus in presence of AZT or anti-MLV polyclonal sera diluted 1∶300 are shown as indicated ( B , left two panels). HIV-1 Gag fluorescence is shown as green and CK19 as red. The corresponding bright field images are shown on the right ( A ) or at the bottom ( B ). HIV-1 Gag staining merged with CK19 staining and enlarged views of HIV-1 infected cells are shown in (A). C and D: XMRV RNA ( C ) and HIV-1 RNA ( D ) in supernatants from the same infected primary endocervical epithelial cells from ( A ) and (B) above were quantified by qRT-PCR. The input virus used to infect producer CEMX174 cells and the treatments are indicated on the X-axis of the graphs (pAb 1∶300 = anti-MLV polyclonal sera at dilution 1∶300; Control serum = normal goat serum diluted 1∶300; No serum = culture medium control). **, p<0.001, control serum vs indicated treatments. The data shown represent the mean ± standard deviation from three independent experiments. a , HIV-1 and XMRV from CEMX174 cells infected with each virus alone were quantified and mixed at a ratio equal to the ratio of the two viruses in the progeny virus from HIV-1 and XMRV co-infected CEMX174 cells. The virus mixture was then inoculated onto primary endocervical epithelial cells and immunostaining and qPCR was performed exactly as described for progeny virus from co-infected CEMx174 cells.

Article Snippet: Cultures of human primary vaginal epithelial cells (Celprogen, CA) and human primary cervical epithelial cells (Cell Application, CA) were also purchased and maintained in medium provided by the cell vendor following vendor protocols.

Techniques: Infection, Immunofluorescence, Staining, Fluorescence, Quantitative RT-PCR, Standard Deviation, Immunostaining

Journal: PLoS ONE

Article Title: Infection of Female Primary Lower Genital Tract Epithelial Cells after Natural Pseudotyping of HIV-1: Possible Implications for Sexual Transmission of HIV-1

doi: 10.1371/journal.pone.0101367

Figure Lengend Snippet: ( A, B ) Dual immunofluorescence staining with FITC-anti-HIV-1 Gag and anti-CK19 MAbs was performed on primary vaginal squamous epithelial cells which were exposed to progeny virus from infected CEMX174 cells. HIV-1 Gag is shown as green and CK19 is shown as red. Corresponding bright field images are also shown as indicated. The input viruses used to infect producer CEMX174 cells is indicated on the panels. Epithelial cells exposed to progeny virus in the presence of AZT or anti-MLV polyclonal sera diluted 1∶300 are shown in the left two columns in B .

Article Snippet: Cultures of human primary vaginal epithelial cells (Celprogen, CA) and human primary cervical epithelial cells (Cell Application, CA) were also purchased and maintained in medium provided by the cell vendor following vendor protocols.

Techniques: Immunofluorescence, Staining, Infection

Journal: PLoS ONE

Article Title: Infection of Female Primary Lower Genital Tract Epithelial Cells after Natural Pseudotyping of HIV-1: Possible Implications for Sexual Transmission of HIV-1

doi: 10.1371/journal.pone.0101367

Figure Lengend Snippet: Primary endocervical epithelial cells were co-cultured for two days with an equal number of mitomycin C-treated CEMX174 cells infected with HIV-1 alone, XMRV alone or co-infected with both viruses. After washing to remove non-adherent cells, dual immunofluorescence staining with FITC-anti-Gag and anti-CK19 MAbs was performed on the adherent primary endocervical epithelial cells on day 5. Merged images confirmed that HIV-1 infected cells were epithelial cells. Primary endocervical epithelial cells were co-cultured with (A) HIV-1/XMRV co-infected CEMX174; (B) same as (A) in presence of AZT; (C) HIV-1 infected CEMx174; (D) XMRV infected CEMx174; (E) mock infected CEMx174.

Article Snippet: Cultures of human primary vaginal epithelial cells (Celprogen, CA) and human primary cervical epithelial cells (Cell Application, CA) were also purchased and maintained in medium provided by the cell vendor following vendor protocols.

Techniques: Cell Culture, Infection, Immunofluorescence, Staining