27 research outputs found

    Identification of multifunctional IFN-γ, IL-2 and TNF-α CD4<sup>+</sup> cells in natural <i>M. bovis</i> infection.

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    <p>PBMC from naturally <i>M. bovis</i> infected cattle were cultured in the presence of either PPD-B or medium and the co-expression of IFN-γ, IL-2 and TNF-α determined by ICS flow cytometry. Histograms were gated on singlet, live lymphocytes and then all CD4<sup>+</sup> cells analysed for all combinations of cytokine productivity. The upper histograms show the total proportion of CD4<sup>+</sup> cells staining for expression of IFN-γ, IL-2 or TNF-α following stimulation with PPD-B (left panel) or medium control (right panel). Subgating of the IFN-γ<sup>+</sup> and IFN-γ<sup>−</sup> CD4<sup>+</sup> cells provides histograms that represent all possible functionalities for the expressing of the 3 cytokines, as shown in the lower histograms. The numbers indicate percentage of CD4<sup>+</sup> cells and data are representative of 1 of 10 naturally infected cattle.</p

    Characterisation of memory markers reveals a characteristic T<sub>EM</sub> phenotype for multifunctional CD4<sup>+</sup> cells in naturally infected cattle.

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    <p>PBMC from naturally <i>M. bovis</i> infected cattle were stimulated with PPD-B and stained for production of cytokine by ICS flow cytometry. Histograms were first gated on singlet, live lymphocytes and gating strategies applied to allow phenotyping of cells for expression of CD44, CD45RO and CD62L. The quadrant gate defining expression of CD44 and CD62L (top panel), CD45RO and CD62L (middle panel) or CD44 and CD45RO (bottom panel) was determined using the total live-lymphocyte population, as shown for each surface marker combination in the histograms on the far left of the panel. This quadrant gating strategy was used to determine the surface phenotype of total CD4<sup>+</sup>IFN-γ<sup>+</sup>, CD4<sup>+</sup>IL-2<sup>+</sup>, CD4<sup>+</sup>TNF-α<sup>+</sup> and triple functional CD4<sup>+</sup> cells, as shown in the other histograms. The percentage of cytokine producing CD44<sup>hi</sup>CD62L<sup>lo</sup>, CD45RO<sup>+</sup>CD62L<sup>lo</sup> and CD44<sup>hi</sup>CD45RO<sup>+</sup> is shown for each of these CD4<sup>+</sup> populations. The data is from one representative animal of 5 analysed.</p

    Identification of recombinant antibodies with specificity for bIL-2.

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    <p>PBMC from a cow naturally infected with <i>M. bovis</i> were cultured in the presence of PPD-B to allow screening of candidate bIL-2 by ICS flow cytometry. Panel A shows the histogram gating strategy used to interrogate responses in singlet, live CD4<sup>+</sup> lymphocytes. Panel B shows the measurement of detectable IL-2 and/or IFN-γ within the CD4<sup>+</sup> population for each of 6 candidate IL-2 antibody clones. The clone number is shown in the top left corner of each histogram and the percentage of CD4<sup>+</sup> cell in which co-expression of IFN-γ and IL-2 could be detected is shown in the top right of each histogram. Data are representative of 1 of 2 independent experiments.</p

    Functional networks most significanty modulated in PPD-B stimulated PBMC from vaccinated/protected calves.

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    <p>Visualisation of the trend and significance of each network: Red bars = up-regulation; blue bars = down-regulation of network. Horizontal bar: p = 0.05.</p

    Results of RNA-Seq analysis.

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    <p>A. Signficantly modulated genes in the three treatment groups. B. Venn diagrams of genes significantly up-regulated and (C) down-regulated genes after vaccination but prior to <i>M. bovis</i> challenge. A. Fold change compared to unstimulated PBMC (medium controls) of PPD-B stimulated PBMC compared to medium controls from unvaccinated, naïve calves (group 1), vaccinated/non-protected (group 2), and vaccinated/protected calves (group 3).</p

    BCG-vaccinated and control cattle samples mapped to <i>ifn-γ</i> and <i>il-22</i> genes.

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    <p>Visualization by IGB of RNA sequencing reads of representative PPD-B stimulated PBMC from vaccinated-protected, vaccinate-un-protected and non-vaccinated control cattle. Y-axis shows the number of reads covering each base along the transcript in RPKM expression values for each sample. Black track: unvaccinated control cattle; red track: vaccinated/un-protected cattle and blue track: vaccinated/protected cattle. The schematic representation of transcript for (A) <i>ifn-γ</i> and (B) <i>il-22</i> is show in green at the bottom of the each figure; the boxes show the exons of the gene.</p

    Gene expression in PPDB-stimulated PBMC from BCG vaccinated and control cattle.

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    <p>A. Protective efficacy after <i>M. bovis</i> challenge determined by pathology scoring. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003077#s2" target="_blank">Results</a> are expressed as total pathology scores <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003077#ppat.1003077-Vordermeier1" target="_blank">[5]</a>. Filled circles, unvaccinated control calves; open triangles, BCG vaccinated calves. (B, C). Transcription of the genes expressing IFN-γ (B) and IL-22 (C) following <i>in vitro</i> stimulation. PBMC were collected from BCG vaccinated (filled symbols) and controls (open symbols) before challenge (week -1) and after challenge with <i>M. bovis</i> at weeks 2, 4 and 8, and stimulated with PPD-B for 24 hours. cDNA was prepared and gene expression determined by RT-qPCR. Data are expressed as log10 relative expression levels compared to non-stimulated cells. Statistical analysis: 2-way ANOVA with Bonferroni post test, * P<0.05.</p

    Protection and <i>in vitro</i> IFN-γ responses prior to challenge.

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    <p>A. Individual pathology scores are shown for the animals used in this study. Naïve animals = no vaccination, Vacc/NP = vaccinated calves that were not protected; Vacc/P = vaccinated calves that were protected. B. Correlation of IFN-γ protein production in culture supernatants measured by Bovigam ELISA (y-axis) and <i>ifn-γ</i> gene expression as determined by deep sequencing (x-axis). Data are shown from PPD-B stimulated PBMC from individual animals. Supernatants and RNA were prepared after 24 h culture.</p

    Schematic representation of the genes involved in the cytokine-cytokine receptor interaction.

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    <p>Colour codes indicate genes that were significantly modulated in vaccinated/protected calves prior to <i>M. bovis</i> challenge. Red colour: up-regulated genes; blue colour: down-regulated genes. Darkness of colour indicates level of gene modification.</p

    Expression of the most up-regulated genes found in the murine model in cattle using PBMC from uninfected (bTb-free, n = 9) and naturally with <i>M. bovis</i> infected cows (bTb, n = 11).

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    <p>The data are represented as mean (± SEM) fold changes in expression after stimulation of PBMC with bovine PPD-B. Significance level for comparison of results in bTb-free and infected animals: p -value≤0.0033 (*).</p>a<p>Used as positive control.</p><p>NA, not applicable.</p
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