Epidemiological characteristics of the subjects enrolled.

<p>BCG, Bacillus Calmette-Guerin. IQR, Interquartile range.</p

Expanded “non-conventional” Treg in HIV-TB and IRIS patients.

<p>Analysis of CD4+FoxP3+CD25+ and CD4+FoxP3+CD25- populations within Treg in <b>A;</b> HIV-TB (n = 11), <b>B;</b> IRIS (n = 5), <b>C;</b> HIV-LTB (n = 4), <b>D;</b> HD (n = 10) and <b>E;</b> HIV (n = 7) individuals. Horizontal lines inside the boxes indicate the means ± SEM of each group. Asterisks denote comparisons between each sub-population. *: p < 0.05; **: p < 0.01; ***: p< 0.001. <b>F.</b> Spearman correlation analysis between plasma DHEA and the percentage of CD4+FoxP3+CD25- lymphocytes from all grouped patients (HIV+, HIV-TB, IRIS, HIV-LTB and HD). Results of statistical analysis are shown in the graphic.</p

Modulation of <i>M. tuberculosis</i>-induced IFN-γ production by adrenal hormones.

<p><b>A.</b> Absolute numbers of IFN-γ producer cells from PBMC of HIV+, HIV-TB, IRIS, HIV-LTB and HD patients, which have been stimulated with <i>M. tuberculosis</i> antigen for 16 hours. Horizontal lines indicate the mean and comparisons between groups and statistically significant differences are shown. SFU, Spots forming units. <b>B.</b> Percentage of IFN-γ producer cells relative to <i>M. tuberculosis</i> in PBMC of HIV-TB patients (n = 12). PBMC (10⁵ cells/well) were stimulated in the presence of <i>M. tuberculosis</i> with or without addition of DHEA and/or cortisol at the indicated concentrations. Each bar illustrates the mean ± SEM of the percentage for IFN-γ producer cells relative to <i>M. tuberculosis</i> for the each group, calculated as follows: % of IFN-γ relative to <i>Mtb</i> = ([(<i>Mtb</i> hormone-Media)-(<i>Mtb</i>-Media)]/(<i>Mtb</i>-Media))×100. Asterisks indicate comparisons between each condition against <i>Mtb</i> specific response. *: <i>p</i> < 0.05; **: <i>p</i> < 0.01.</p

Cortisol, dehidroepindrosterone-sulfate (DHEA-s), DHEA levels and Cortisol/DHEA ratio in HIV, HIV-TB, IRIS, HIV-LTB and HD.

<p><b>A.</b> DHEA, <b>B.</b> DHEA-s, <b>C.</b> Cortisol and <b>D.</b> Cortisol/DHEA (ratio) levels in plasma of HIV+, HIV-TB, IRIS, HIV-LTB, and HD individuals. Bars indicate the mean ± SEM for each group. Horizontal lines indicate comparisons between groups and statistically significant differences. DHEA was measured by radioimmunoassay, DHEA-s by immunochemoluminiscence tests and Cortisol by electrochemiluminescence. HIV+ individuals n = 10, HIV-TB n = 21, IRIS n = 6, HIV-LTB n = 5, and HD n = 16. *: <i>p</i> < 0.05; **: <i>p</i> < 0.01; ***: <i>p</i> < 0.001.</p

DHEA modulates the expression of the FoxP3 transcription factor in coinfected individuals. A and B.

<p>Percentages of Treg cells (defined as CD4+FoxP3+CD25+) in PBMC from <b>A.</b> HIV-TB and <b>B.</b> HD individuals stimulated with <i>M. tuberculosis</i> antigen in the presence or absence of DHEA at the indicated concentrations for 3 days. Bars indicate the mean ± SEM for each experimental condition. <b>C and D.</b> Relative FoxP3 Median florescence intensity (MFI) in Treg lymphocytes from <b>C.</b> HIV-TB patients and <b>D.</b> HD volunteers after culturing PBMC as detailed in A. Bars indicate the mean ± SEM for each treatment. Data are representative of four different experiments. *: <i>p</i> < 0.05; **: <i>p</i> < 0.01. <b>E.</b> Representative flow cytometry graphs depicting the results obtained from culturing PBMC from HIV-TB and HD individuals as indicated above.</p

Sensitivity values of the different serological tests^a.

a<p>The analysis was performed considering as positive reference controls sera obtained from patients with culture-confirmed brucellosis (52 sera) and from culture-negative/serologically-positive patients with clinical diagnosis of brucellosis (86 sera).</p>b<p>%I, percentage of inhibition.</p>c<p>GBA, glycoconjugate-beads assay.</p><p>TP, true positives; FN, false negatives; Se, sensitivity (TP/TP+FN)×100.</p

Glycoconjugate-magnetic beads assay analysis of serum samples obtained from healthy individuals and patients of different clinical groups.

<p>A) Dotplot of the glycoconjugate-magnetic beads assay results. Serum samples of blood culture-positive patients (52 sera), culture-negative/serologically-positive patients with clinical diagnosis of brucellosis (86 sera), blood donors (240 sera), healthy individuals occupational-exposed to the pathogen (30 sera), patients with febrile syndrome (34 sera) and patients with other diseases (46 sera) were tested as indicated in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002048#s2" target="_blank">Materials and Methods</a>. The mean and standard deviation for each group are indicated. B) Analysis of glycoconjugate-magnetic beads assay results classifying the samples into two categories. Non-brucellosis category includes samples obtained from blood donors, healthy individuals occupational-exposed to the pathogen, patients with febrile syndrome and patients with other diseases. Brucellosis category includes samples of blood culture-positive and culture-negative/serologically-positive patients with clinical diagnosis of brucellosis. For each category reactivity values are arrange in increasing order. The horizontal lines indicate the cutoff values calculated by ROC analysis for which maximal sensitivity (Se) or specificity (Sp) is achieved. The inset shows a zoom of the graph part displaying the limit between categories.</p

Receiver-operating characteristic (ROC) analysis of glycoconjugate-magnetic beads assay results.

<p>A) ROC plot. The analysis was carried out considering as positive controls sera of patients with culture-confirmed brucellosis and culture-negative/serologically-positive patients with clinical diagnosis of brucellosis (138 sera), and as negative controls serum samples from blood-donors, healthy individuals occupational-exposed to the pathogen, patients with febrile syndrome and patients with other diseases (350 sera). AUC, area under the ROC curve. Values in parentheses indicate the 95% confidence interval. B) Plot of the diagnostic sensitivity (Se) and specificity (Sp) of the assay as a function of the cutoff value. The dot vertical line indicates the cutoff value that concurrently optimizes Se and Sp (cutoff = 14.18%, Se = 99.28% and Sp = 99.43%). The dash vertical lines indicate the cutoff values for which maximal Se or Sp is achieved (cutoff = 13.20%, Se = 100% and Sp = 98.57%; cutoff = 16.15%, Se = 93.48% and Sp = 100%).</p

OAg-AcrA glycoconjugate as an antigen for the diagnosis of human brucellosis.

<p>A) 10% SDS-PAGE analysis of purified non-glycosylated (NG) and glycosylated AcrA (G) by Coomassie brilliant blue and immunoblot using anti-histidine tag and anti-<i>Brucella O</i>-antigen monoclonal antibodies. B) Glycoconjugate magnetic beads-based immunoassay for detection of antibodies against the <i>O</i>-polysaccharide fraction of lipopolysaccharide (LPS). Magnetic beads coated with OAg-AcrA were incubated with the indicated human serum samples. Bound antibodies were detected using Cy5-conjugated goat anti human IgM/G antibodies.Positive and negative controls; human sera included as positive and negative controls in each assay run. The figures in parenthesis correspond to the identification of the samples according to <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002048#pntd.0002048.s002" target="_blank">Tables S1</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002048#pntd.0002048.s003" target="_blank">S2</a>. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002048#s3" target="_blank">Results</a> are expressed as percentage of reactivity of the positive control serum. The bar graph data represents the means and standard deviation for two separate determinations. C) Western blot analysis of the same human serum samples.</p