Comparison of serum cytokine concentrations in mild, nonfatal severe, and fatal leptospirosis.

<p>Box plots of serum cytokine concentrations among studied patients with mild, nonfatal severe, and fatal leptospirosis (gray, yellow, and red boxes, respectively). The bottom, median, and top lines of the box mark the 25th, 50th, and 75th percentiles, respectively. The vertical line with whiskers shows the range of values. Dots show individual data points. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i>≤0.001.</p

Characteristics of included patients.

<p>N, number; IQR, inter-quartile range.</p>a<p><i>P</i><0.05 comparing patients with mild disease against all hospitalized patients.</p>b<p><i>P</i><0.05 comparing hospitalized patients with severe nonfatal disease against hospitalized patients with fatal disease.</p>c<p>Any bleeding, including pulmonary or gastrointestinal hemorrhage, mild hemoptysis, epistaxis, or gingival bleeding.</p

Immunologic predictors of death among hospitalized patients.

<p>–, Not selected for entry into multivariable model. Bold font signifies significant association with death. Odds ratio is expressed per log increment of cytokine concentration.</p

High frequencies of DN αβ T-cells are associated with TB severity.

<p>Representative contour plots showing the gate strategy used for the analysis of CD4 (middle left), CD8 (middle center), DN (middle right) αβ-T cells and the expression of CD69 (upper panels) and HLA-DR (lower panels) on DN αβ-T cells (A). Percentages of CD4⁺ (left panels), CD8⁺ (middle panels) and DN (right panels) αβ T-cells in healthy donors (HD, open symbols), TB (total TB, black symbols), nsTB (non-severe TB, light gray symbols) and sTB patients (severe TB, dark gray) were measured before treatment (B). The percentage of CD69 (C) and HLA-DR (D) expression within CD4⁺ (left panels), CD8⁺ (middle panels) and DN (right panels) αβ T-cells in HD, TB, nsTB and sTB patients were analyzed ex vivo. The boxes represent the means.</p

Higher frequencies of IFN-γ producing DN αβ T-cells are found in nsTB patients.

<p>Representative contour plots showing the proportions of IFN-γ producing CD4 (left panel), CD8 (middle panel) and DN (right panel) αβ-T cells (A). The percentages of IFN-γ (B), TNF-α (C) and IL-10 (D) expression within CD4⁺ (left panels), CD8⁺ (middle panels) and DN (right panels) αβ T-cells in healthy donors (HD, open symbols), TB (total TB, black symbols), nsTB (non-severe TB, light gray symbols) and sTB patients (severe TB, dark gray) were measured before treatment. PBMCs were stimulated with (MTB-Ag) for 48 hours. The boxes represent the means.</p

Advanced TB patients display decreased proportions of DN γδ T-cells.

<p>Representative contour plots showing the gate strategy used for the analysis of CD4 (middle left), CD8 (middle center), DN (middle right) γδ-T cells and the expression of CD69 (upper panels) and HLA-DR (lower panels) on DN γδ -T cells (A). Percentages of CD4⁺ (left panels), CD8⁺ (middle panels) and DN (right panels) γδ T-cells in healthy donors (HD, open symbols), TB (total TB, black symbols), nsTB (non-severe TB, light gray symbols) and sTB patients (severe TB, dark gray) were measured before treatment (B). The percentage of CD69 (C) and HLA-DR (D) expression within CD4⁺ (left panels), CD8⁺ (middle panels) and DN (right panels) γδ T-cells in HD, TB, nsTB and sTB patients were analyzed ex vivo. The boxes represent the means.</p

Characterization of monocyte subsets in malaria patients.

<p>(A) Representative dot plots showing the gate strategy for the identification of CD14⁺CD16⁻ (green gate), CD14⁺CD16⁺ (red gate), CD14^loCD16⁺ (blue gate) monocyte subsets. CD14⁺CD16⁻, CD14⁺CD16⁺ and CD14^loCD16⁺ monocytes are represented by green, red and blue symbols. (B) Frequencies of CD14⁺CD16⁻, CD14⁺CD16⁺ and CD14^lo monocytes within PBMC from <i>P. vivax</i>-infected patients before (BT, filled symbols) and 30–45 days after treatment (AT, open symbols) (n = 28). (C) Mean fluorescence intensity (MFI) of CCR2 (BT, n = 28 and AT, n = 18), CX3CR1 (BT, n = 26 and AT, n = 19), CCR7 (BT, n = 28 and AT, n = 19) and LFA-1 (BT, n = 15 and AT, n = 11) (from the top to the bottom) was evaluated on monocyte subsets (CD14⁺CD16⁻ (left panel), CD14⁺CD16⁺ (middle panel), CD14^loCD16⁺ (right panel)) from <i>P. vivax</i>-infected subjects, before and 30–45 days after treatment. Dotted lines represent medians of a given measurements from healthy donors. (D) Scattered dot plots (left panels) and representative histograms (right panels) showing MFI of the molecules described above on CD14⁺CD16⁻, CD14⁺CD16⁺ and CD14^loCD16⁺ monocytes from <i>P. vivax</i>-infected patients before treatment (open histograms). Levels of the molecules above were measured by flow cytometry. Circles indicate individual patients and lines represent median values and interquartile ranges. (E) Levels of molecules expressed by the monocyte subsets analyzed according to D. * <i>p</i><0.05, **0.05><i>p</i>>0.01, ***<i>p</i><0.01.</p

Cytokine profile of spleen T-lymphocytes from mice before (0) and at 7, 14, 28 and 42 days after infection with metacyclic (MT) or blood trypomastigotes (BT) of <i>Trypanosoma cruzi</i>.

<p>“Gray scale” diagrams were used to represent the cytokine pattern and the cytokine balance within T-cell subsets besides the overall cytokine balance with T-cells, highlighting the predominance of “low” cytokine-producers (white square), “high” TNF-α or IFN-γ producers (black square), “high” IL-10⁺ producers (light gray square) or “high” mixed cytokine-producers (dark gray square). Pie charts represent the percentage of animals displaying a given T-cells overall cytokine balance selectively amongst the “high” cytokine-producers. ND = Not detected.</p

Circulating monocyte subpopulations display distinct morphology, mitochondrial and NADPH subunit content.

<p>FACS-sorted CD14⁺CD16⁻, CD14⁺CD16⁺ and CD14^loCD16⁺ monocytes from healthy donors and <i>P. vivax</i>-infected patients were fixed and prepared for electron microscopy. Monocyte subsets from a single healthy donor (A, upper panel) and a single patient (A, lower panel) shown are representative of the analysis of at least six cells of each monocyte subpopulation per patient and of the analysis of 5 controls and 6 patients. Mitochondria (white arrows) and vesicles (black arrows). Scale bar, 2 µm. (B) Mitochondria area from CD14⁺CD16⁻ (green circles), CD14⁺CD16⁺ (red circles) and CD14^loCD16⁺ (blue circles) monocytes was assessed using ImageJ. Circles indicate individual mitochondria. (C) Mitochondria content in CD14⁺CD16⁻ (green bars), CD14⁺CD16⁺ (red bars) and CD14^loCD16⁺ (blue bars) monocytes from <i>P. vivax</i>-infected patients was measured based on Mitotracker reactivity (n = 11). (D) Mean fluorescence intensity (MFI) of p47phox and p67phox within CD14⁺CD16⁻, CD14⁺CD16⁺, CD14^loCD16⁺ monocytes from <i>P. vivax</i>-infected patients was measured by flow cytometry 3 hours after culture in medium alone or <i>P. vivax</i>-infected reticulocytes (n = 4). Results are representative of 2 independent experiments. Symbols represent individual subject. *<i>p</i><0.05, ***<i>p</i><0.01.</p

Morphometric analysis and photomicrographs of the area of <i>T. cruzi</i> immunoreactions in the heart at 28 days after mice infection with metacyclic (MT; light gray bar) or blood trypomastigotes (BT; black bar) of <i>Trypanosoma cruzi</i>.

<p>Analysis of the area of <i>T. cruzi</i> amastigotes were identified by immunohistochemistry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032912#s4" target="_blank">Material and Methods</a>. The results are expressed as parasited area ± standard error. Photomicrographs of the heart parasitism demonstrating isolated amastigotes in MT and typical amastigote nests in BT.</p