42 research outputs found

    Selective expansion of cDC and pDC in the lung and the lung draining lymph nodes by Flt3L and 120G8 treatment.

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    <p>After Flt3L and 120G8 treatment, lung and lung lymph node cell suspensions from control, Flt3L treated (Flt3L) and pDC depleted Flt3L treated (Flt3L+120G8) mice were analyzed by flow cytometry for the presence of pDC and cDC (mDC). Numbers indicate the percentage of total cells in the gate shown. Shown are the representative flow plots of 3 experiments (A). The mean±SE from all of the mice in 3 experiments demonstrates that only pDC have been depleted in the lung and lymph node (LN) with the 120G8 (B).</p

    Selective expansion of cDC and pDC affects CD8+ T cell responses but not CD4+ T cell responses in the lung after RSV infection.

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    <p>Six days after infection, single cell suspensions of lungs from control (Cont), RSV-infected (RSV), Flt3L treated and RSV-infected (Flt3L+RSV) and Flt3L treated, pDC depleted, RSV infected (Flt3L+120G8+RSV) mice were analyzed by flow cytometry for the presence or expression of (A) CD3+CD4+ T cells, (B) CD69 on CD4+ T cells, (C) CD3+CD8+ T cells, (D) CD69 on CD8+ T cells, (E) Intracellular IFN-γ expression in CD8+ T cells or (F) RSV M2 MHCI pentamer expression on CD8+ T cells. <i>* P<0.05 compared to non-infected control mice, # P<0.05 compared to RSV infected mice, n = 4–6 mice per group.</i></p

    Selective expansion of cDC and pDC affects RSV-induced airway hyperresponsiveness and immunological responses in the lung.

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    <p>(A) Eight days after RSV infection, airway responses were measured in control (cont), RSV-infected (RSV), Flt3L treated and RSV-infected (Flt3L+RSV) and Flt3L treated, pDC depleted, RSV infected (Flt3L+120G8+RSV) mice after one dose of methacholine <i>(125 mg/kg)</i> and compared to basal measurements. Data are represented as mean airway resistance in cm H<sub>2</sub>O/ml/sec±SEM. (B) Lung lymph node T cell cytokine production in response to anti-CD3 stimulation. Eight days after infection, lung lymph nodes were isolated from control (Cont), RSV-infected (RSV), Flt3L treated and RSV-infected (Flt3L+RSV) and Flt3L treated, pDC depleted, RSV infected (Flt3L+120G8+RSV) mice and stimulated with anti-CD3 at a concentration of 5×10<sup>6</sup> cells/ml. Data are represented as mean ng/ml±SEM. (C) Cytokine messenger RNA levels in the lungs of mice. Six days after infection, mRNA was isolated from lungs of control (Cont), RSV-infected (RSV), Flt3L treated and RSV-infected (Flt3L+RSV) and Flt3L treated, pDC depleted, RSV infected (Flt3L+120G8+RSV) mice and analyzed using quantitative real-time PCR by Taqman. Each sample was normalized using a GAPDH control and the data show average fold increase to control±SEM. * <i>P</i><0.01 compared to non-infected control mice, # <i>P</i><0.05 compared to RSV infected mice, n = 5 mice per group. This experiment was repeated twice with similar numbers of mice and displayed comparable results.</p

    Differential activation profiles of pDC and cDC by RSV.

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    <p>Bone marrow from BALB/c mice was cultured with Flt3L or GM-CSF for preferential expansion of pDC and cDC, respectively, as described. Isolated cell populations (1.10<sup>6</sup>/ml) were subjected to RSV infection (MOI-2.0) for 24 hrs and the supernatants and cells were collected to assess cytokine levels by luminex (A) and surface activation markers by flow cytometry (B). The mean fluorescent intensity (MFI) was assessed using the entire cell population in control vs. RSV-infected cells. The data represent mean±SEM of 4 repeat experiments.</p

    CSE increases the production of CCL3 and CXCL2 by TLR4 and MyD88 dependent manner.

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    <p>cDC were prepared by culturing BM cells from Balb/c mice preincubated with anti-TLR4 antibody (20 µg/ml) or isotype control (20 µg/ml) for 1 h and then stimulated with CSE for 16 h and amount of CCL3 (A) and CXCL2 ( B) were determined by ELISA. cDCs were prepared by culturing BM cells from Balb/c mice and age- and sex-matched MyD88-deficient mice. CSE or LPS were incubated for 16 h and supernatant were harvested for determination of CCL3 (C) and CXCL2 (D) by ELISA. White bars represent cDCs treated with medium, dotted bars are cDCs treated with LPS and black bars represent cDCs treated with CSE. Data are representative of three independent experiments, showing the means±SEM from triplicate cultures. * represent significant differences compared with medium-treated cells (***p<0.001).</p

    CSE increases cDC-induced CD8<sup>+</sup>T cell but inhibits CD4<sup>+</sup>T cell proliferation.

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    <p>cDCs from Balb/c mice were incubated with medium (white bars) or CSE ( black bars) and coincubated with allogenic T cells from C57BL/6 mice [CD8<sup>+</sup> (A) and CD4<sup>+</sup>T cells (B)] in a MLR. Presented are pooled data from eight individual experiments using cDCs generated from eight isolations. Values are represented as mean±SEM. A statistically significant modulation of proliferation of T cells with CSE-primed cDCs occurred (<sup>*</sup><i>p</i><0.05 and ** p<0.01 when compared to medium-treated cDCs). The supernatants of MLR were collected for the measurement of IL-2 by ELISA (inserted graphs in A & B). Presented are pooled data from eight individual experiments using cDCs generated from eight isolations. Values are represented as mean±SEM. * Indicates significant differences between medium-treated cells (* p<0.05).</p

    CSE induces the expression of mRNA and the production of chemokines in cDCs.

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    <p>The supernatants of CSE-exposed cDCs were tested for the production and release of CCL3 (A) and CXCL2 (B) ELISA (upper panels) and cell pellets were tested for CCL3 and CXCL2 mRNA levels by real time PCR (upper panels). White bars represent cDCs treated with medium, black bars represent cDCs treated with CSE and gray bars cDCs treated with NAC and CSE. Data are representative of three independent experiments, showing the means±SEM from triplicate cultures. * represents significant differences compared with medium-treated cells (*p<0.05; ***p<0.001). <sup>#</sup> indicates significant differences between cells treated with CSE in combination with NAC and cells treated with CSE.</p

    Alveolar macrophages in <i>ex vivo</i> lung cultures.

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    <p>Double staining for influenza A virus nucleoprotein (brown nuclear staining) and a macrophage marker (red/pink, cytoplasmic staining) 24 hours after infection with seasonal H3N2 virus (a), HPAIV H5N1 virus (b) or pandemic H1N1 virus (c). The arrowhead indicates influenza virus staining in alveolar epithelial cells and the arrow indicates an alveolar macrophage (AM). Seasonal H3N2 virus infection results in infection of alveolar epithelial cells (arrowhead) but not of AM (arrow). HPAIV H5N1 infection results in the infection of alveolar epithelial cells (arrowhead) and infection of AM (arrow). Pandemic H1N1 virus infection results in infection of alveolar epithelial cells (arrowhead) but not of AM (arrow). Percentages of AM infected after 24 hpi with seasonal H3N2 virus, HPAIV H5N1 or pH1N1 virus infection (d). Mean values are represented by horizontal lines. * indicates a statistical (p<0.05) difference.</p

    CSE increases the production of intracellular ROS in cDC.

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    <p>cDCs were incubated with CSE , with or without NAC or PMA (as a control) and ROS generation was assayed by FACS analysis. The mean fluorescent intensity (MFI) of the following groups are indicated in the figure: control: unlabelled CSE-treated cells (green line), unstimulated: control labeled cells (red line), CSE: CSE-stimulated labeled cells (blue line), CSE+NAC: CSE-stimulated labeled cells treated with NAC (black line), PMA: PMA-stimulated labeled cells (light blue line).</p

    Virus production in alveolar macrophages and monocyte-derived macrophages after H3N2, H5N1 or H1N1 virus infection.

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    <p>Virus production of alveolar macrophages (AM) (a), macrophages cultured in the presence of human serum (MM) (b), and macrophages cultured in the presence of GM-CSF (GM-MM) (c), after infection with seasonal H3N2 virus, HPAIV H5N1 or pandemic H1N1 virus. Geometric mean titers were calculated from independent experiments; error bars indicate standard deviation. * indicates a statistical (p<0.05) difference. ** indicates a statistical (p<0.01) difference.</p
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