Vitamin D treatment does not induce cell death/apoptosis of chicken splenocytes.

<p>Chicken splenocytes were treated with Vitamin D (100nM) or vehicle (DMSO) for 4 hrs. The cells were stained with 7AAD (dead cell marker) and Annexin V (early apoptotic marker) and the data were analysed using flow cytometry. (<b>A</b>) Shows dot plots of the stained cells and the numbers in each quadrant represents percentage of cells. (<b>B</b>) The percentages (as mean ± SD) of live cells (Annexin V-7AAD- cells), apoptotic cells (Annexin V+7AAD- cells) and dead cells (Annexin V+7AAD+ cells) are shown from four independent experiments with three biological replicates in each experiment (12 biological replicates in total). (<b>C</b>) The data represent the percentage of live, apoptotic and dead cells 24hrs post Vitamin D (100 nM) or vehicle (DMSO) treatment. <b>(D)</b> The data represents the percentage of live, apoptotic, dead cells 3 days after Con A (10 μg/ml) stimulation.</p

Degranulation of chicken T cells is not influenced by Vitamin D.

<p>(<b>A</b>) Representative flow cytometry dot plots of spleen mononuclear cells that were cultured in medium containing Vitamin D (100nM or 10 nM) or vehicle only (DMSO) for 4 hours and were stimulated with a T-cell stimulation cocktail of PMA and Ion for an additional 4 hrs. The expression of CD107a in CD3+ and CD3- cells was analysed using flow cytometry. Upper right quadrant and lower right quadrant shows the percentages of CD107a+ cells within CD3+ and CD3- T cells, respectively. The bar graphs represents the percentages of CD3-CD107a+. (<b>B</b>) and CD3+CD107a+ cells (<b>C</b>) in the treated cells. The results are shown as mean ± SD of the study population of CD107a expression (<i>P</i> = NS indicates no statistical significance). † (symbol) represents the cells treated with the Vehicle only (DMSO). Similar data were obtained in four independent experiments.</p

Vitamin D limits ERK1/2 phosphorylation in the resting chicken CD3+ T cells.

<p>Following incubation of splenocytes with Vitamin D or vehicle only for 4hrs, phosphorylation of ERK1/2 (T202/Y204) was evaluated in CD3+ T cells in the resting cells (unstimulated) or cells stimulated with PMA for 5 minutes using flow cytometry. The data represents flow cytometry analysis of ERK 1/2 phosphorylation in cells treated with vehicle (thick line), Vitamin D (thin line) or Isotype control (grey area) in the resting cells and PMA-stimulated T cells.</p

Vitamin D inhibits chicken T-cell proliferation and IFN-γ production <i>ex vivo</i>.

<p>Splenocytes were pre-treated for 4hrs with Vitamin D (100 and 10 nM) or vehicle only (DMSO). (<b>A</b>) BrdU incorporation measured by ELISA assay to quantify T cell proliferation after stimulation with different concentrations of Concanavalin A. The results are presented as absorbance at OD₄₅₀ from three independent experiments with 3–4 biological replicates in each experiment. (B, C) The combined results from proliferation from 6 independent experiments for absorbance at OD₄₅₀ (B) and the percentages of inhibition by Vitamin D are shown. (<b>D</b>) The frequency of IFN-γ producing mononuclear cells stimulated with a T-cell stimulation cocktail of PMA and Ion was detected using a chicken IFN-γ ELISPOT assay. The results are presented as spots forming unit (SFU) per 1.0 x 10⁶ cells from five independent experiments with 3–5 biological replicates in each experiment (19 replicates in total). (<b>E</b>) Represents the percentage of inhibition for IFN-γ production by mononuclear cells after Vitamin D₃ pre-treatment from five independent experiments. Each dot represent a biological replicate. Non-parametric Wilcoxon tests (Mann-Whitney) was used to assess normal distribution and test significance. The results are shown as mean ± SD. † (symbol) represents cells treated with Vehicle only (DMSO). * indicates a statistically significant difference (<i>P</i> < 0.05).</p

Cytokine mRNA expression in cecal tonsil of chickens infected with FAdV-4.

<p>The groups were: uninfected (negative control), and FAdV-4 infected chickens using im inoculation. Target and reference gene expression was quantified by real-time RT-PCR using SYBR Green. Target gene expression is presented relative to β-actin expression and normalized to a calibrator. The results are diagrammed as whisker boxes with medians. Boxes represent interquartile ranges and whiskers indicate extreme values. There was no statistically significant differences detected (P>0.05) by Mann-Whitney test. ● symbols represent values which were identified as outliers. </p

Cytokine mRNA expression in spleen of chickens infected with FAdV-4.

<p>The groups were: Uninfected (negative control), and FAdV-4 infected chickens using im inoculation. Target and reference gene expression was quantified by real-time RT-PCR using SYBR Green. Target gene expression is presented relative to β-actin expression and normalized to a calibrator. The results are diagrammed as whisker boxes with medians. Boxes represent interquartile ranges and whiskers indicate extreme values. The difference in cytokine expression between groups was assessed by Mann-Whitney test and comparisons were considered significant at P≤0.05 (*).● symbols represent values which were identified as outliers. </p

Antibody response to viral proteins in chickens inoculated with FAdV-4 by the oral and intramuscular routes and mock infected as measured by S/P ratios.

<p>Serum samples were collected at 7, 14, 21 and 28 days post-infection. Gray and black lines represent im and orally inoculated chickens, respectively. Dashed gray line and black dotted line represent mock-infected chickens. Statisticaly significant differences among mock infected and infected birds have been detected at 14, 21, and 28 d.p.i.</p

Cytokine mRNA expression in liver of chickens infected with FAdV-4.

<p>The groups were: uninfected (negative control), and FAdV-4 infected chickens using im inoculation. Target and reference gene expression was quantified by real-time RT-PCR using SYBR Green. Target gene expression is presented relative to β-actin expression and normalized to a calibrator. The results are diagrammed as whisker boxes with medians. Boxes represent interquartile ranges and whiskers indicate extreme values. The difference in cytokine expression between groups was assessed by Mann-Whitney test and comparisons were considered significant at P≤0.05 (*).● symbols represent values which were identified as outliers. </p

Learning pipeline: Labeling tweets to misinformation or fact.

Learning pipeline: Labeling tweets to misinformation or fact.</p

Vaccine-related tweets per country (for countries with less than 24,000 tweets).

Vaccine-related tweets per country (for countries with less than 24,000 tweets).</p