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1.
FIG. 3.

FIG. 3. From: Respiratory Syncytial Virus Infection of Human Airway Epithelial Cells Is Polarized, Specific to Ciliated Cells, and without Obvious Cytopathology.

Polarity of rgRSV infection of WD HAE cultures. Shown are confocal fluorescent-optical-section photomicrographs of HAE cultures inoculated via either the apical (Ap) or basolateral (Bl) surfaces with rgRSV or AdVGFP. Twenty-four hours after infection, the cultures were fixed and immunostained with antibody specific for KS and detected by a secondary antibody conjugated to Texas Red. The KS-expressing apical surfaces of ciliated cells are shown in red, and virus-infected cells are shown in green. Original magnification, ×63.

Liqun Zhang, et al. J Virol. 2002 June;76(11):5654-5666.
2.
FIG. 2.

FIG. 2. From: Respiratory Syncytial Virus Infection of Human Airway Epithelial Cells Is Polarized, Specific to Ciliated Cells, and without Obvious Cytopathology.

Comparison of the abilities of rgRSV and AdVGFP to infect the apical (Ap) versus the basolateral (Bl) surfaces of WD HAE cultures. rgRSV (7 × 106 PFU; MOI, ∼20) or AdVGFP (108 PFU; MOI, ∼300) was applied to either the apical or basolateral surface of the cultures as detailed in Materials and Methods. Twenty-four hours later, the cultures were analyzed en face for GFP expression by fluorescence photomicroscopy. Original magnification, ×10.

Liqun Zhang, et al. J Virol. 2002 June;76(11):5654-5666.
3.
FIG. 6.

FIG. 6. From: Respiratory Syncytial Virus Infection of Human Airway Epithelial Cells Is Polarized, Specific to Ciliated Cells, and without Obvious Cytopathology.

Polarized release of rgRSV from the apical surfaces of WD HAE cultures. Virus shed from either the apical or basolateral surfaces of six independent cultures was collected at 24 h intervals as described in Materials and Methods. Titration of the collected samples on HEp-2 cells revealed significant shedding of rgRSV from the apical surface (diamonds), whereas within the limits of detection, no viral shedding was measured from the basolateral surface (below limits of detection). The values shown represent the mean ± standard deviation (n = 6).

Liqun Zhang, et al. J Virol. 2002 June;76(11):5654-5666.
4.
FIG. 7.

FIG. 7. From: Respiratory Syncytial Virus Infection of Human Airway Epithelial Cells Is Polarized, Specific to Ciliated Cells, and without Obvious Cytopathology.

Spread of rgRSV infection with time in WD HAE cultures. The apical surfaces of cultures were inoculated with a low titer of rgRSV (7 ×103 PFU) to achieve a submaximal number of cells expressing GFP at 24 h. Infection was then allowed to proceed over 4 days, and GFP expression was examined en face by fluorescence photomicroscopy on days 1 (A), 2 (B), 3 (C), and 4 (D) postinoculation. Note the counterclockwise circular spread of rgRSV infection by day 2 (B) and the increased number of rgRSV-infected cells by day 4. Original magnification, ×10.

Liqun Zhang, et al. J Virol. 2002 June;76(11):5654-5666.
5.
FIG. 1.

FIG. 1. From: Respiratory Syncytial Virus Infection of Human Airway Epithelial Cells Is Polarized, Specific to Ciliated Cells, and without Obvious Cytopathology.

Cell morphology and KS expression at the apical ciliated surfaces of WD HAE cell cultures. (A) Light micrograph of a cross section of a WD HAE culture grown at an ALI on a semipermeable membrane support for 4 weeks. Under these conditions, pseudostratified mucociliary epithelial cell morphology was generated. The cells were counterstained with hematoxylin and eosin. (B) Confocal fluorescent optical section of a live WD HAE culture exposed to an antibody specific for KS and detected with a secondary antibody conjugated to Texas Red. Note that KS serves as a marker for ciliated columnar epithelial cells at the apical surface of the culture and that the permeable support, a 10-μm-deep layer underlying the basal epithelial cells, displays non-KS-specific autofluorescence. Original magnification, ×100.

Liqun Zhang, et al. J Virol. 2002 June;76(11):5654-5666.
6.
FIG. 4.

FIG. 4. From: Respiratory Syncytial Virus Infection of Human Airway Epithelial Cells Is Polarized, Specific to Ciliated Cells, and without Obvious Cytopathology.

Effect of mechanical damage to the epithelium of WD HAE cultures on rgRSV and AdVGFP infection. WD HAE cultures were scratched with a pipette tip across the apical surface to expose the underlying basal cells along the injury path (white lines). The apical surfaces of injured cultures were immediately inoculated with rgRSV or AdVGFP as for Fig. ; 24 h later, the cultures were fixed and immunostained with antibody to KS, and the cultures were examined en face by fluorescence photomicroscopy. Note that in the left panel few cells expressed GFP within the injury path, while the undamaged regions colocalized with both GFP and KS expression, reflecting the presence of rgRSV infection and ciliated cells, respectively, in this region. In contrast, in the right panel the injury tract expressed GFP, reflecting AdVGFP infection, but did not colocalize with the undamaged tissue that expressed KS, reflecting the presence of intact ciliated cells. Original magnification, ×10.

Liqun Zhang, et al. J Virol. 2002 June;76(11):5654-5666.
7.
FIG. 8.

FIG. 8. From: Respiratory Syncytial Virus Infection of Human Airway Epithelial Cells Is Polarized, Specific to Ciliated Cells, and without Obvious Cytopathology.

Inhibition of initial rgRSV infection and spread in WD HAE cultures with an RSV-neutralizing monoclonal antibody or ribavirin. The apical surfaces of HAE cultures were inoculated with rgRSV (105 PFU; MOI, ∼0.3), and GFP expression was monitored en face by fluorescence photomicroscopy 1 (A) and 3 (B) days later. To assess the effects of potential RSV inhibitors on initial rgRSV infection, parallel cultures were treated prior to rgRSV inoculation with either 250 μg of the F-specific RSV-neutralizing monoclonal antibody Synagis/ml applied to the apical surface (C) or 100 μg of ribavirin/ml included in the basolateral medium (D). The cultures were then inoculated with rgRSV as described above, and GFP expression was assessed 1 day later by fluorescence photomicroscopy. To assess the effects of RSV inhibitors on viral spread, parallel cultures were inoculated as described above and then treated with Synagis 6 h postinoculation, and GFP expression was assessed on day 1 (E) and day 3 (F) postinoculation. Cultures treated with ribavirin 24 h postinoculation were assessed for GFP expression by fluorescence photomicroscopy on day 2 (G) and day 4 (H) postinoculation. Original magnification, ×10.

Liqun Zhang, et al. J Virol. 2002 June;76(11):5654-5666.
8.
FIG. 9.

FIG. 9. From: Respiratory Syncytial Virus Infection of Human Airway Epithelial Cells Is Polarized, Specific to Ciliated Cells, and without Obvious Cytopathology.

Lack of RSV-specific obvious cytopathology in WD HAE cells. (A) Confocal optical section of rgRSV-mediated GFP expression 36 days after apical inoculation of a WD HAE culture with rgRSV (7 × 106 PFU). GFP expression (green) was predominately in ciliated cells, as shown by colocalization with KS-specific antibodies (apical red signal). Note the lack of cell-cell fusion, i.e., syncytium formation. Original magnification, ×63. (B) No obvious cytopathology of different RSV isolates was observed after apical inoculation of WD HAE cultures. The apical surfaces of HAE cultures were inoculated with either rgRSV (106 PFU); GP1, an isogenic recombinant RSV that lacks GFP (106 PFU); Hep-4, a biologically derived wild-type RSV (106 PFU); or the Udorn strain of influenza A virus (106 PFU). The RSV- and influenza virus-inoculated cultures were incubated for 37 and 2 days, respectively. Histological cross sections counterstained with hematoxylin and eosin showed no gross histological differences in cell morphology for the RSV-inoculated cultures compared to cultures not inoculated with any virus. In contrast, cultures inoculated with influenza A virus underwent significant cytopathology 2 days postinoculation. Original magnification, ×63.

Liqun Zhang, et al. J Virol. 2002 June;76(11):5654-5666.
9.
FIG. 5.

FIG. 5. From: Respiratory Syncytial Virus Infection of Human Airway Epithelial Cells Is Polarized, Specific to Ciliated Cells, and without Obvious Cytopathology.

Susceptibility of HAE cultures to rgRSV infection as a function of the differentiation state of the culture. (A) Freshly plated cells were grown to confluence to represent a PD cell type and allowed to differentiate with time. On the indicated days following establishment of an ALI, replicate cultures were inoculated with rgRSV (7 × 106 PFU), and the percentage of GFP-positive cells was quantitated by fluorescence photomicroscopy 24 h later. Each datum point represents the mean of three independent measurements ± standard error of the mean. (B) Representative photomicrographs of the differentiation status of HAE cultures on day 2 (i), day 8 (ii), and day 14 (iii) after initiation of an ALI. Note the abundant ciliated cells on day 14. The cells were counterstained with hematoxylin and eosin. Also shown are en face fluorescence photomicrographs of corresponding cultures expressing GFP 24 h after inoculation with rgRSV on day 2 (iv), day 8 (v), and day 14 (vi). Original magnifications, ×100 (light) and ×10 (fluorescence).

Liqun Zhang, et al. J Virol. 2002 June;76(11):5654-5666.

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