40 research outputs found

    Cell composition in BALF of PBS-, LPS-, PLN- and heat inactivated PLN-treated mice.

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    <p>Macrophage and neutrophil counts in BALF from WT mice, 6 hours after inoculation of PBS, LPS (2 pg/mouse), heat inactivated PLN (HIPLN 500 ng/mouse) or PLN (500 ng/mouse). Data are plotted in Box&Whiskers graph (median+interquartile range N = 5 per group). * P<0.05 versus PBS.</p

    PLN activates HEK cells via TLR4.

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    <p>IL-8 production in HEK-293 cells transfected with CD14 and either TLR2 or TLR4 were incubated with medium (control), LPS (100 ng/ml), LTA (5 µg/ml) or PLN (1 µg/ml) for 6 hours. In some experiments polymyxin B (P) was used at 10 µg/ml. Data are mean±SEM (N = 4 per group). * P<0.01 versus control, † P<0.001 versus control, ‡P<0.001 versus LPS.</p

    Inflammatory and cytolytic effects of PLN on mouse alveolar macrophage MH-S cells.

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    <p>MH-S cells were incubated with increasing doses of PLN for 6 hours with/without polymyxin B (10 µg/ml ) and TNF-α, MIP-2 and cell death were determined thereafter. Cell death was measured using MTT assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007993#s2" target="_blank">Methods</a> section. Data are mean±SEM (N = 5 per group). * P<0.05, † P<0.01 versus control.</p

    Roles of TLR2 and TLR4 in lung inflammatory response to high dose PLN <i>in vivo</i>.

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    <p>Macrophage and neutrophil counts, total protein, IL-6, IL-1β, TNF-α and KC concentrations in BALF from WT, TLR2 KO and TLR4 KO mice, 6 hours after inoculation of 500 ng/mouse. Data are plotted in Box&Whiskers graph (median+interquartile range N = 8 per group). * P<0.05, † P<0.01, ‡ P<0.001 versus WT mice. Dotted line indicates mean value of PBS-treated mice.</p

    Leukocyte influx in the kidneys.

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    <p>Quantifications of immunostainings for detection of (<b>A</b>) granulocytes, (<b>B</b>) macrophages, (<b>C</b>) T lymphocytes in paraffin renal sections of WT (white bars) and CD44 KO (black bars) mice. (<b>A</b>) Number of Ly6G-positive cells per HPF (x20). (<b>B</b>,<b>C</b>) Data expressed as percent positive area of the total areal analyzed. Mean <u>+</u> SEM, n=8, *=p<0.05, WT vs CD44 KO; #=p<0.05, vs sham; in (<b>A</b>) all groups # vs sham.</p

    CD44-Deficiency Attenuates the Immunologic Responses to LPS and Delays the Onset of Endotoxic Shock-Induced Renal Inflammation and Dysfunction

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    <div><p>Acute kidney injury (AKI) is a common complication during systemic inflammatory response syndrome (SIRS), a potentially deadly clinical condition characterized by whole-body inflammatory state and organ dysfunction. CD44 is a ubiquitously expressed cell-surface transmembrane receptor with multiple functions in inflammatory processes, including sterile renal inflammation. The present study aimed to assess the role of CD44 in endotoxic shock-induced kidney inflammation and dysfunction by using CD44 KO and WT mice exposed intraperitoneally to LPS for 2, 4, and 24 hours . Upon LPS administration, CD44 expression in WT kidneys was augmented at all time-points. At 2 and 4 hours, CD44 KO animals showed a preserved renal function in comparison to WT mice. In absence of CD44, the pro-inflammatory cytokine levels in plasma and kidneys were lower, while renal expression of the anti-inflammatory cytokine IL-10 was higher. The cytokine levels were associated with decreased leukocyte influx and endothelial activation in CD44 KO kidneys. Furthermore, <i>in vitro</i> assays demonstrated a role of CD44 in enhancing macrophage cytokine responses to LPS and leukocyte migration. In conclusion, our study demonstrates that lack of CD44 impairs the early pro-inflammatory cytokine response to LPS, diminishes leukocyte migration/chemotaxis and endothelial activation, hence, delays endotoxic shock-induced AKI. </p> </div

    Migratory ability of blood leukocytes in absence of CD44.

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    <p>Transwell migration assay of (<b>A</b>) monocytes and (<b>B</b>) granulocytes from WT (white bars) and CD44 KO (black bars) blood. Immunofluorescent staining for flow cytometric analysis of (<b>A</b>) CD11b and (<b>B</b>) Ly6G after 24 hours culture in Transwell plate with or without 200ng/ml MIP-2 and MCP-1 in the lower chamber. Data expressed as percent cells in the lower chamber of the total number of cells. Mean <u>+</u> SEM, n=3, *=p<0.05, WT vs CD44 KO; ###=p<0.001, vs control. </p

    Macrophage response to LPS in presence/absence of CD44.

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    <p><i>In </i><i>vitro</i> LPS (100ng/ml) stimulation of WT (white bars) and CD44 KO (black bars) bone marrow-derived macrophages (BMM) for 4 and 24 hours. Secretion levels (pg/ml) of (<b>A</b>) TNF-α and (<b>B</b>) IL-6 detected by ELISA. (<b>C</b>) Gene expression of IL-1β and (<b>D</b>) IFNβ normalized for TBP transcript levels, assessed by Q-PCR. (<b>E</b>) ELISA measurement of IL-10 supernatant concentration (pg/ml). Mean <u>+</u> SEM, n=10, *=p<0.05, ***=p<0.001. (<b>F</b>) WB assay of cell lysates from WT and CD44 KO BMM for detection of cytoplasmatic phosphorylated (p)-IκBα and nuclear p65. β-actin or LaminA/C used as loading controls. Fold-increase in protein expression in respect to control is indicated. </p

    Cytokines in the kidneys.

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    <p>Expression (pg/mg proteins) of (<b>A</b>) MCP-1, (<b>B</b>) IL-1β, (<b>C</b>) TNF-α, (<b>D</b>) IL-6 and (<b>E</b>) IL-10 in kidneys of WT (white bars) and CD44 KO (black bars) mice measured by ELISA. Data shown as mean <u>+</u> SEM, n=8, *=p<0.05.</p

    Renal CD44 and HA expression.

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    <p>(<b>A</b>) Expression of CD44 in kidneys in saline- and LPS-treated WT mice assessed by Q-PCR. Data normalized for TBP transcript expression. Mean <u>+</u> SEM, n=8, *=p<0.05 versus (vs) sham. (<b>B</b>) Representative micrographs (x10 magnification upper panel, x40 lower panel) of CD44 immunostaining in renal sections of WT sham and 24 hours LPS-treated mice. (<b>C</b>) Quantification of HABP immunostaining in kidneys of WT (white bars) and CD44 KO (black bars) mice. Data expressed as percent positive area of the total area analyzed. Mean <u>+</u> SEM, n=8, *=p<0.05. </p
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