14 research outputs found

    Administration of mAb-<i>iFt</i> immune complexes reverses the anti-inflammatory properties of LVS in mouse PECs <i>ex vivo</i>.

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    <p>C57BL/6 mice were immunized i.n. with PBS, i<i>Ft</i> (2x10<sup>7</sup> CFUs), or mAb-<i>iFt</i>, boosted on day 21 and challenged on day 35 with 10,000 CFUs of <i>Ft</i> LVS. After 48 hours post—LVS challenge, the PECs of immunized mice were harvested and cultured in the presence or absence of <i>Ft</i>. LVS at 1:10 and 1:100 MOI for 24 hrs. The cytokine production was measured as previously described. Results are representative of three independent experiments. (*) P-value < 0.1; (**) P-value < 0.05; bars represent the SD.</p

    Anti-inflammatory effect of <i>F</i>. <i>tularensis</i> LPS on mouse PECs is IFN-γ and IL-10 dependent.

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    <p>PECs from naïve and IL-10 genetically deficient C57BL/6 mice were obtained and resuspended in cell culture media. PECs were cultured in a 96-well plate at 2 x 10<sup>5</sup> cells/well with either <i>Ft</i>-LPS or <i>E</i>. <i>coli</i>-LPS at 1 ng/mL in the presence or absence of recombinant IFN-γ at 100 U/ml. Cells cultured with PBS were used as a control. The cytokine production was measured using BD Biosciences Cytometric Bead Array (CBA) following vendor instructions. Results are representative of three independent experiments. (*) P-value < 0.1, (**) P-value < 0.05, bars represent SD.</p

    <i>tularensis</i> LPS contributes to the anti-inflammatory properties of <i>F</i>. <i>tularensis</i> LVS during the early stages of infection.

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    <p><b><i>F</i>.</b> PECs from C57BL/6 mice were obtained and cultured in a 96-well plate at 2 x 10<sup>5</sup> cells/well in the presence or absence of either <i>Ft</i>-LPS or <i>E</i>. <i>coli</i>-LPS at 1 ng/mL, 10 ng/mL and 20 ng/ml. Cells incubated with PBS were used as a control. Levels of IL-12p70, TNF-α and IL-10 were measured using BD Biosciences Cytometric Bead Array (CBA) following vendor instructions. Results are representative of three independent experiments. (*) P-value < 0.1; (**) P-value < 0.05; bars represent SD.</p

    Administration of mAb-i<i>Ft</i> immune complexes reverses the anti-inflammatory properties of LVS in the lungs of immunized mice.

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    <p>C57BL/6 mice were immunized i.n. with PBS, i<i>Ft</i> (2x10<sup>7</sup> CFUs), or mAb-i<i>Ft</i>, boosted on day 21 and challenged on day 35 with 10,000 CFUs of <i>Ft</i> LVS. Lung tissue homogenates were obtained from immunized mice 24, 48 and 96 hours post-infection as indicated above and spun down at 15,000g for 30 minutes at room temperature to remove tissue debris. Cytokine levels were detected by using the IL-6, IL-10, TNF-α and IFN-γ ELISA kits and following vendor instructions (Biolegend). Results are representative of three independent experiments. (*) P-value < 0.1; (**) P-value < 0.05; bars represent the SD.</p

    FcγR targeting drives polarization of mouse macrophages towards the AM1 phenotype at the early stages of LVS infection.

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    <p>Lungs of immunized mice were harvested 24, 48 and 96 hours post-infection. For cell surface marker staining, cells were fluorescently labeled with antibodies against CD11b, F4/80, MHC class II, B7.1, B7.2, CCR7, or their corresponding isotype controls were added. Cells were then analyzed by flow cytometry on an LSRII flow cytometer (BD Biosciences). Results are representative of three independent experiments. (*) P-value < 0.1; (**) P-value < 0.05; bars represent the SD.</p

    Immunization with mAb-i<i>Ft</i> immune complexes increases the activation of PECs following LVS challenge.

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    <p>C57BL/6 mice were immunized i.n. with PBS, i<i>Ft</i> (2x10<sup>7</sup> CFUs), or mAb-i<i>Ft</i>, boosted on day 21 and challenged on day 35 with 10,000 CFUs of <i>Ft</i> LVS. On day 2 post-infection the peritoneal exudate cells of immunized mice were harvested and the expression of F4/80, MHC class II, B7.1 (CD80), and B7.2 (CD86) were analyzed by flow cytometry. Results are representative of three independent experiments. (*) P-value < 0.1; (**) P-value < 0.05; bars represent the SD.</p

    Mice immunized with the <i>emrA1</i> mutant are protected against 1000LD100–10,000LD100 challenge dose of <i>Ft</i> LVS.

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    <p>C57BL/6 mice (n = 5–10 per group) were immunized i.n. with 1×10<sup>6</sup> CFU of the <i>emrA1</i> mutant. <b>(A, B)</b> On day 42 of the primary immunization mice were challenged i.n. with 1×10<sup>7</sup> CFU of wild type <i>Ft</i> LVS. Age matched unvaccinated mice challenged with a similar dose of <i>Ft</i> LVS were kept as controls. (<b>C, D)</b> On day 42 of the primary immunization mice were challenged i.n. with 1×10<sup>8</sup> CFU of wild type <i>Ft</i> LVS. Age matched unvaccinated mice challenged with a similar dose of <i>Ft</i> LVS were kept as controls. (<b>E, F)</b> On day 75 of the primary immunization mice were challenged i.n. with 1×10<sup>7</sup> CFU of wild type <i>Ft</i> LVS. Age matched unvaccinated mice challenged with a similar dose of <i>Ft</i> LVS were kept as controls. The Challenged mice were observed for morbidity and mortality for a period of 21 days post-challenge (<b>A, C, E</b>). The mice were weighed at the indicated times post-challenge to monitor the progression of infection (<b>B, D, F</b>). The survival results are expressed as Kaplan-Meier survival curves and P values were determined by Log-rank test. Body weights of mice are expressed percent body weights.</p

    Immunization with the <i>emrA1</i> mutant results in minimal weight loss, rapid bacterial clearance, and histopathological lesions in lung, liver and spleen.

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    <p>C57BL/6 mice were immunized i.n. with 1×10<sup>6</sup> CFU of the <i>emrA1</i> mutant. Mice infected with equal numbers of wild type <i>Ft</i> LVS were used as controls. <b>(A)</b> The immunized mice were weighed at the indicated times post-immunization to track the progress of infection. <b>(B)</b> On days 1, 5, 7, 14 and 21 post-immunization, mice (n = 4 per group/time point) were euthanized and bacterial burdens were quantified in their lung, liver and spleen. Bacterial counts in organs are expressed as Log<sub>10</sub>CFU/mL. The <i>P</i> values were determined using one way ANOVA. *<i>P<0</i>.<i>05; **P<0</i>.<i>01; ***P<0</i>.<i>001</i>. <b>(C)</b> Excised lungs, livers and spleens were preserved in 10% formalin, paraffin embedded, sliced into 5 μM thin sections and stained with Hematoxylene & Eosin. Stained sections were observed for histopathological lesions under a light microscope (Magnification 100×). # = <i>Ft</i> LVS infected mice succumbed to infection.</p

    The <i>emrA1</i> mutant vaccinated mice induce sustained production of pro-inflammatory cytokines and a potent antibody response following lethal <i>Ft</i> LVS challenge.

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    <p>C57BL/6 mice immunized i.n. with 1×10<sup>6</sup> CFU of the <i>emrA1</i> mutant were challenged i.n. with 1×10<sup>7</sup> CFU of wild type <i>Ft</i> LVS 42 days post-immunization. <b>(A-D)</b> On days 5, 7 and 14 post-challenge, mice (n = 3 per group/time point) were euthanized and their excised lungs were homogenized. Clear lung homogenates were used for quantification of indicated pro-inflammatory cytokines using flow cytometric analysis. The data are represented as Mean ± S.D. <b>(E)</b> On day 21 post-challenge, mice (n = 3 per group) were anesthetized and bled retroorbitally to obtain serum. <i>Ft</i> specific total IgG, IgG2a, IgG2b, IgG1 and IgA levels in serum samples were determined by ELISA. The data are represented as Mean ± S.D. of absorbance values measured at 450 nm. Red arrows indicate antibody titers. ND = Not detected.</p

    Mice immunized with the <i>emrA1</i> mutant induce regulated production of pro-inflammatory cytokines and a potent antibody response.

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    <p>C57BL/6 mice were immunized i.n. with <i>1</i>×10<sup>6</sup> CFU of the <i>emrA1</i> mutant or <i>Ft</i> LVS. On days 1, 5, 7 and 14 post-immunization, mice (n = 4 per group/time point) were euthanized and their excised lungs and spleens were homogenized. Clear lung <b>(A-D)</b> and spleen <b>(E-H)</b> homogenates were used for quantification of indicated pro-inflammatory cytokines using flow cytometric analysis. The data are represented as Mean ± S.D. The <i>P</i> values were determined using one way ANOVA. *<i>P<0</i>.<i>05; **P<0</i>.<i>01</i>. <b>(I)</b> On day 42 post-immunization, mice (n = 3 per group/ time point) were anesthetized and bled retroorbitally to obtain serum. <i>Ft</i> specific total IgG, IgG2a, IgG2b, IgG1 and IgA levels in serum samples were determined by ELISA. The data are represented as Mean ± S.D. of absorbance values measured at 450 nm. Red arrows indicate antibody titers. # = <i>Ft</i> LVS immunized mice succumbed to infection; ND = Not detected.</p
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