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Results: 7

1.
Fig. 7.

Fig. 7. From: Latent infection by ?herpesvirus stimulates profibrotic mediator release from multiple cell types.

Lung T cells in latently infected mice produce IFN-γ but not IL-4. Mice were infected with γHV-68 on days 0 and 28 dpi, and lungs were harvested and digested with collagenase and DNase. Isolated cells were stimulated with PMA and ionomycin for 6 h in the presence of GolgiStop protein transport inhibitor. Cells were then stained using fluorochrome-conjugated antibodies against the cell surface markers CD45, CD4, and CD8. Cells were then fixed, permeabilized, and stained with anti-IFN-γ and anti-IL-4. A: representative flow plots from CD4 or CD8 cells from both saline and virus-infected mice. B: summary showing the percentages of IFN-γ- and IL-4-producing cells (n = 5 mice per group). Q, quadrant.

Joshua S. Stoolman, et al. Am J Physiol Lung Cell Mol Physiol. 2011 February;300(2):L274-L285.
2.
Fig. 1.

Fig. 1. From: Latent infection by ?herpesvirus stimulates profibrotic mediator release from multiple cell types.

Murine γherpesvirus-68 (γHV-68) infection can augment subsequent fibrotic responses or exacerbate established ones. A: mice were infected with γHV-68 or mock-infected intranasally on day 0. On day 15, mice received an intratracheal injection of either FITC or saline (Sal; control group). On day 36, a time point that represents day 36 postinfection and day 21 postfibrotic stimuli, the lungs were collected, and collagen deposition was determined by hydroxyproline assay, n = 4–5 per group. *P < 0.05. B: mice were injected with FITC intratracheally on day 0. On day 14, mice received γHV-68 or mock infection with saline intranasally. Lungs were harvested on days 28 and 35, and collagen deposition was measured by hydroxyproline. The values for the mock and FITC group were similar at both time points and thus were combined in the 1st column (n = 10). The other 2 groups represent n = 5. *P < 0.05. NS, not significant.

Joshua S. Stoolman, et al. Am J Physiol Lung Cell Mol Physiol. 2011 February;300(2):L274-L285.
3.
Fig. 3.

Fig. 3. From: Latent infection by ?herpesvirus stimulates profibrotic mediator release from multiple cell types.

Gene expression of markers of classic or alternative activation in AMs, 28 dpi. C57BL/6 mice were infected intranasally with saline or 5 × 104 PFU of γHV-68 on day 0. Twenty-eight dpi, AMs harvested from mock-infected mice (white bars) or virally infected mice (black bars) were cultured in serum-free media for 24 h before RNA isolation. Markers of classic [inducible nitric oxide synthase (iNOS)] and alternative (arginase-1, Ym1/2, and Fizz-1) macrophage activation as well as expression of heparin-binding EGF-like growth factor (HB-EGF) were assessed by real-time RT-PCR analysis of mRNA levels normalized to expression of the housekeeping gene, β-actin. All samples were run in triplicate. A single sample from the saline-treated group was normalized to 1 for each gene, and all other samples are presented relative to that 1. There was a 32-fold increase in iNOS gene expression and a 4-fold increase in HB-EGF gene expression in AMs isolated from γHV-68-infected mice (n = 5; **P < 0.01, ***P < 0.001 compared with mock-infected). Arginase-1, Ym1/2, and Fizz-1 mRNA levels did not change significantly in AMs from γHV-68-infected mice compared with uninfected controls (n = 6).

Joshua S. Stoolman, et al. Am J Physiol Lung Cell Mol Physiol. 2011 February;300(2):L274-L285.
4.
Fig. 4.

Fig. 4. From: Latent infection by ?herpesvirus stimulates profibrotic mediator release from multiple cell types.

Proinflammatory and profibrotic factor expression is higher in mesenchymal cells from virally infected mice 42 dpi. C57BL/6 mice were infected intranasally with saline or 5 × 104 PFU of γHV-68. Twenty-eight dpi, lung minces were prepared and cultured in complete media supplemented with cidofovir (10 μM) for 14 days. Cells were changed to serum-free media for 24 h before supernatant and RNA or DNA harvest. A: mesenchymal cell supernatants from mock-infected mice (white bars) or γHV-68-infected mice (black bars) were assayed by ELISA for protein concentration. Viral infection upregulates TGF-β1, CCL12, and TNF-α; however, IFN-γ, IL-12, IL-4, and IL-13 were not detectable in cell supernatants of either group (n = 8 mice per group; nd; *P < 0.05, ****P < 0.0001 compared with mock-infected). B: real-time RT-PCR demonstrates that the viral gene M3 is expressed in mesenchymal cells 42 dpi following the 14-day in vitro culture period in cidofovir, but gB was not detectable. There was no detectable expression of M3 or gB gene expression in mesenchymal cells from mock-infected mice (n = 8; nd). Expression levels are presented relative to the housekeeping gene β-actin, which is set to 1. C: copies of gB viral DNA were measured from DNA of infected cells, quantified against a standard curve, and normalized to levels of gB per 100 ng of DNA (n = 4).

Joshua S. Stoolman, et al. Am J Physiol Lung Cell Mol Physiol. 2011 February;300(2):L274-L285.
5.
Fig. 5.

Fig. 5. From: Latent infection by ?herpesvirus stimulates profibrotic mediator release from multiple cell types.

CD19-enriched cells from the spleen and lungs of γHV-68-infected mice produce more TGF-β1. Spleens harvested from mice 28 dpi with saline or γHV-68 were positively selected for CD19 expression on a magnetic column and cultured for 24 h in serum-free media before supernatants were collected and RNA or DNA was isolated. A: ELISAs demonstrated CD19-enriched splenic cell supernatants from γHV-68-infected mice (black bars) contained significantly higher levels of total TGF-β1 and significantly lower levels of IL-6 compared with supernatants from CD19-enriched cells from mock-infected controls (white bars). IL-10 was produced at similar levels by CD19-enriched cells from both groups. CCL2, CCL12, TNF-α, IFN-γ, IL-4, and IL-13 were not detectable in cell supernatants of either group (n = 12; nd; ***P < 0.001, ****P < 0.0001 compared with mock-infected). B: real-time RT-PCR detected higher levels of M3 viral gene expression than gB in CD19-enriched cells from spleens of γHV-68-infected mice. CD19-enriched cells from spleens of mock-infected mice had no detectable expression of M3 or gB (n = 3; *P < 0.05). C: copies of gB viral DNA were measured from DNA of infected spleen CD19+ cells, quantified against a standard curve, and normalized to total DNA levels (n = 6). D: CD19-enriched cells from collagenase digests of the lungs were cultured for 24 h in serum-free media before harvests of supernatants and RNA or DNA. CD19-enriched cell supernatants from the lungs of γHV-68-infected mice (black bar) contained higher levels of TGF-β1 than CD19-enriched cell supernatants from mock-infected controls (white bar) as measured by ELISA (n = 8–9; ****P < 0.0001 compared with mock-infected). E: real-time RT-PCR analysis demonstrated that viral genes gB and M3 are expressed in CD19-enriched cells from the lungs of γHV-68-infected mice. No viral mRNA transcripts were detected in CD19-enriched cells from the lungs of mock-infected mice (n = 2). F: copies of gB viral DNA were measured from DNA of infected CD19+ lung cells, normalized to total DNA levels, and quantified against a standard curve (n = 7).

Joshua S. Stoolman, et al. Am J Physiol Lung Cell Mol Physiol. 2011 February;300(2):L274-L285.
6.
Fig. 2.

Fig. 2. From: Latent infection by ?herpesvirus stimulates profibrotic mediator release from multiple cell types.

Proinflammatory and profibrotic factor expression is higher in alveolar macrophages (AMs) from virally infected mice 28 days postinfection (dpi). C57BL/6 mice were infected intranasally with saline or 5 × 104 plaque-forming units (PFU) of γHV-68 on day 0. Twenty-eight dpi, AMs were harvested and cultured in serum-free media for 24 h. A: AM supernatants from mock-infected mice (white bars) or γHV-68-infected mice (black bars) were assayed by ELISA for protein concentration. Viral infection upregulated transforming growth factor (TGF)-β1, CCL2, CCL12, TNF-α, and IFN-γ; however, IL-12, IL-4, and IL-13 were not detectable (nd) in cell supernatants of either group (n = at least 3 mice per group representative of 3 similar experiments; nd; ***P < 0.001, ****P < 0.0001 compared with mock-infected). Error bars represent the SE between cell culture wells. B: real-time RT-PCR demonstrated that viral genes glycoprotein B (gB) and M3 are expressed in AMs from mice 28 dpi. Viral mRNA transcripts were not detected in AMs from the mock-infected mice. Expression levels are presented relative to the housekeeping gene β-actin, which is set to 1 (n = 21 combined from 3 experiments). C: copies of gB viral DNA were measured from DNA of infected cells, quantified against a standard curve, and normalized to total DNA levels (n = 6). d28, day 28.

Joshua S. Stoolman, et al. Am J Physiol Lung Cell Mol Physiol. 2011 February;300(2):L274-L285.
7.
Fig. 6.

Fig. 6. From: Latent infection by ?herpesvirus stimulates profibrotic mediator release from multiple cell types.

CD19-negative cells from spleens of γHV-68-infected mice produce more TGF-β1, but T cells from the lungs of γHV-68-infected mice produce less TGF-β1. Spleens harvested from mice 28 dpi with saline or γHV-68 were negatively selected for CD19 expression on a magnetic column and cultured for 24 h in serum-free media at 5 × 105 cells per milliliter before supernatants were collected and RNA or DNA was isolated. This cell population contained T cells, monocytes, and dendritic cells. A: ELISAs on supernatants showed higher levels of total TGF-β1 and CCL2 in CD19-negative cell supernatants from spleens of virally infected mice (black bars) than mock-infected mice (white bars). There were no significant differences between CCL12, IL-6, or IL-10 levels expressed by cells of the 2 groups, and TNF-α, IFN-γ, and IL-13 were not detectable by cells of either group (n = 6–12; nd; *P < 0.05, ***P < 0.001 compared with mock-infected). B: real-time RT-PCR detected similar levels of gB and M3 viral gene expression in CD19-negative cells from spleens of γHV-68-infected mice. CD19-negative cells from spleens of mock-infected mice had no detectable expression of M3 or gB (n = 6). C: copies of gB viral DNA were measured from DNA of infected CD19-negative spleen cells, quantified against a standard curve, and normalized to total DNA (n = 4). D: pan T cells isolated from lung collagenase digests 28 dpi from saline or γHV-68-infected mice were cultured at 5 × 106 cells per milliliter and incubated in serum-free media for 24 h before supernatants and RNA were harvested. Total TGF-β1 levels in cell supernatants measured by ELISA tended to be lower in T cells isolated from lungs of virally infected mice (black bar) compared with mock-infected controls (white bar; n = at least 11 per group combined from 2 experiments), whereas levels of IL-10 were increased (n = 6 per group; P < 0.001). E: copies of gB viral DNA were measured from DNA of infected lung pan T cells, quantified against a standard curve, and normalized to total DNA levels (n = 4).

Joshua S. Stoolman, et al. Am J Physiol Lung Cell Mol Physiol. 2011 February;300(2):L274-L285.

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