BHT pathways and inhibitors.

<p><b>A,</b> Morphological signaling pathways in <i>C. albicans</i>. For simplicity, only the Efg1, Cph1, Cph2/Tec1, and Rim101 pathways are shown. <b>B,</b> BHT inhibitors used in this study.</p

Chemical epistasis with <i>efg1</i>-T206E mutant and <i>ADH1pr-CPH1</i> overexpression.

<p><b>A,</b> Wild-type strain SC5314 (white bars) and <i>efg1</i>-T206E mutant strain AV55 (black bars) were grown in Spider media with the indicated BHT inhibitors (100 µM final concentration) for 6 h at 37°C prior to quantification of morphological phenotype as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025395#pone-0025395-g002" target="_blank">Figure 2</a>. Asterisks indicates a <i>P</i> value of <0.05 compared to the corresponding wild-type SC5314 plus BHT inhibitor control. <b>B, </b><i>ADH1pr-CPH1</i> strain CDH72-1 was assayed in the presence of the indicated BHT inhibitors as in A. Asterisks indicates a <i>P</i> value of <0.05 compared to the wild-type SC5314 plus BHT inhibitor control.</p

Chemical epistasis with cAMP and <i>ras1</i>-G13V mutant.

<p><b>A,</b> Wild-type strain SC5314 was incubated in the absence (white bars) and presence (black bars) of 10 mM <i>N</i>⁶,2′-<i>O</i>-dibutyryl-cAMP (db-cAMP) for 6 h at 37°C. Quantification of morphological phenotypes was as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025395#pone-0025395-g002" target="_blank">Figure 2</a>. Asterisks indicates a <i>P</i> value of <0.05 compared to the corresponding –db-cAMP control. <b>B,</b> The <i>ras1</i>-G13V constitutively active mutant strain DH409 was incubated in Spider media with the indicated BHT inhibitors for 6 h at 37°C as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025395#pone-0025395-g003" target="_blank">Figure 3</a>. Asterisks indicates a <i>P</i> value of <0.05 compared to the corresponding wild-type SC5314 plus BHT inhibitor control.</p

Primary Vaccination with Low Dose Live Dengue 1 Virus Generates a Proinflammatory, Multifunctional T Cell Response in Humans

<div><p>The four dengue virus serotypes (DENV-1–DENV-4) have a large impact on global health, causing 50–100 million cases of dengue fever annually. Herein, we describe the first kinetic T cell response to a low-dose DENV-1 vaccination study (10 PFU) in humans. Using flow cytometry, we found that proinflammatory cytokines, IFNγ, TNFα, and IL-2, were generated by DENV-1-specific CD4⁺ cells 21 days post-DENV-1 exposure, and their production continued through the latest time-point, day 42 (<em>p</em><0.0001 for all cytokines). No statistically significant changes were observed at any time-points for IL-10 (<em>p</em> = 0.19), a regulatory cytokine, indicating that the response to DENV-1 was primarily proinflammatory in nature. We also observed little T cell cross-reactivity to the other 3 DENV serotypes. The percentage of multifunctional T cells (T cells making ≥2 cytokines simultaneously) increased with time post-DENV-1 exposure (<em>p</em><0.0001). The presence of multifunctional T cells together with neutralizing antibody data suggest that the immune response generated to the vaccine may be protective. This work provides an initial framework for defining primary T cell responses to each DENV serotype and will enhance the evaluation of a tetravalent DENV vaccine.</p> </div

Multifunctional T cells are produced following exposure to 10 PFU of a DENV-1 monovalent vaccine.

<p><b>A.</b> Distribution of cytokines produced by multifunctional or monofunctional T cells. The percent of multifunctional CD4⁺ T cells producing IFNγ (black line), TNFα (black dashes), and/or IL-2 (black dots) over time relative to the number of CD4⁺ T cells making a single cytokine: IFNγ (gray line), TNFα (gray dashes) or IL-2 (gray dots). Error bars represent standard error, n = 9–11 depending on time-point. <b>B.</b> IFNγ, IL2, and TNFα production in CD4⁺ T cells as a function of time and co-production. Each day is represented with a single color. Black dots correspond to the response from a single subject. Shaded bars represent the interquartile range. Line separates multifunctional T cells (left) from monofunctional T cells (right). A “+” or “−” indicates whether a particular cytokine (TNFα, IFNγ and/or IL-2) is present. <b>C.</b> Proportions of total CD4⁺ T cells producing IFNγ (blue), IL2 (green), TNFα (red), or different combinations of the three (colors indicated by boxes in <b>B</b>) are shown for 5 post-vaccination time-points (days 8, 14, 21, 28, and 42) relative to pre-vaccination (day 0) for 11 vaccinees. Overlaps in red, green, and/or blue arcs indicate T cells producing the correlated cytokines simultaneously. A “ #” denotes statistically a significant difference between a response on a post-vaccination day and day 0 (<i>p</i><0.05 by Wilcoxon's rank sum test). There are statistically higher percentages of total multifunctional T cells on days 21, 28, and 42 (<i>p</i><0.0001).</p

Proinflammatory cytokines are produced at specific times post-vaccination with live DENV-1.

<p>Cytokine profiles for 12 subjects vaccinated with 10 PFU of live monovalent DENV-1 and 2 placebo recipients. CD4⁺-dependent production of 4 cytokines are shown for pre-vaccination (day 0) and 5 post-vaccination time-points following <i>ex vivo</i> stimulation with homologous, wild type DENV-1 antigen: <b>A.</b> IFNγ, <b>B.</b> TNFα, <b>C.</b> IL-2 and <b>D.</b> IL-10. Responses shown are the percentage of cytokine positive T cells from DENV-1-stimulated PBMCs with the background percentage of cytokine-positive T cells in the negative control (Vero) subtracted. Clinical information for each subject is shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001742#pntd-0001742-t001" target="_blank"><b>Table 1</b></a>. Significantly higher levels of IFNγ and TNFα were observed on days 21, 28 and 42 (<i>p</i><0.05 for IFNγ and <i>p</i><0.01 for TNFα), and on days 21 and 28 for IL-2 (<i>p</i><0.01). For <b>A–C</b>, <i>p</i><0.0001 for group by day differences demonstrating increased cytokines with time compared to day 0.</p

Non-viremic vaccinees may produce more multifunctional T cells than viremic volunteers.

<p><b>A.</b> Multifunctional CD4⁺ T cells produced by non-viremic group. <b>B.</b> Multifunctional T cell profile for viremic group. # denotes statistically higher % CD4⁺ cells producing the cytokine profiles indicated compared to Day 0 (<i>p</i><0.05 by Wilcoxon's rank sum test). Each day is represented by a separate color. A “+” or “−” indicates whether a particular cytokine (TNFα, IFNγ and/or IL-2) is present. Shaded bars denote interquartile ranges.</p