Clinical and immunologic data from patients with diffuse cutaneous leishmaniasis (DCL) and localized cutaneous leishmaniasis (LCL).

<p>LST, Leishmanin Skin Test. –, no nodules; +, few; ++, moderate; +++, intense; ++++, very intense.</p

PS exposure on the <i>L. amazonensis</i> amastigotes surface.

<p>Thioglycollate-induced peritoneal macrophages derived from F1 (BALB/C X C57BL/6) mice were infected with different isolates obtained from patients with LCL (BA69, BA73, BA115, BA 125, and M2269) (□ ) and DCL (BA106, BA276, BA336, BA700, and BA760) (▪ ) at a 3∶1 parasite-to-cell ratio. After 24 h of infection, amastigotes were purified for PS exposure analysis by flow-cytometry, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036595#s2" target="_blank">Methods</a>. One representative experiment of at least five independent repeats is shown. Boxes represent median values and interquartile interval from different isolates mentioned above. Differences were checked using Unpaired t test.</p

<i>Leishmania amazonensis</i> isolates used in this study.

<p>(*) Code recommended for the <i>Leishmania</i> strain nomenclature, which includes the following data: host, country of origin, year when it was isolated, and original code (WHO, 1984).</p

<i>Leishmania</i> isolate infectivity and PS exposure.

<p>Peritoneal macrophages derived from F1 mice were infected with different isolates obtained from patients with LCL (BA69, BA73, BA 125, and M2269) (□) and DCL (BA276, BA336, and BA700) (▪). After 5, 24 and 72 h of infection, cells were fixed and stained. The percentage of infected macrophages (<b>A</b>) and infectivity index (<b>B</b>) were defined under a microscope. Parasite burden was measured by quantitative PCR at 24 and 72 h (<b>C</b>). Boxes represent median values and interquartile interval from different isolates mentioned above. The correlations between PS exposure at 24 h with percentage of infected macrophages and infectivity index in 72 h are showed in (<b>D</b>) and (<b>E</b>), respectively. The four lower points in X axis (PS exposure) represents LCL isolates while the three higher points are from DCL isolates. One representative experiment of at least three independent repeats is shown. Kruskal-Wallis was used with Dunn's Multiple Comparison post-test. Spearman test was used to verify the correlations. The r values are plotted in each graph.</p

PS exposure on <i>L. amazonensis</i> isolates correlates with clinical parameters of the disease.

<p>Correlation between clinical parameters and PS exposure in isolates from DCL (BA276, BA336, BA700 and BA760) and LCL (BA69, BA73, BA 115, and BA125) at 24 h post-infection. The four lower points in X axis (PS exposure) represents LCL isolates while the four higher points are from DCL isolates. Spearman test was used to verify the significance in the correlations between PS exposure in 24 h with lesions number (<b>A</b>) and time of disease (<b>B</b>). The r values are plotted in each correlation graph.</p

Parasitophorous vacuole analysis.

<p>Photomicrographs of macrophages from F₁ mice, infected with DCL (BA276) and LCL (BA125) isolates after 72 h of culture (<b>A</b>). Arrows point to individual PVs. Magnification 400X. The average sizes of the PVs induced by different isolates from patients with LCL (BA69, BA73 and BA 125) (□) and DCL (BA276, BA336, and BA700) (▪) after 72 h of infection were measured using Image-Pro Plus 6.0 (<b>B</b>). Data are shown as the area in μm² of PVs in each tested isolate. Boxes represent median values and interquartile interval of PV sizes pooled from different isolates mentioned above. The correlation between PS exposure at 24 h and PV area showed in (<b>C</b>). The three lower points in X axis (PS exposure) represents LCL isolates while the three higher points are from DCL isolates. Differences were checked using unpaired t test. Spearman test was used to verify the significance in the correlations between PV area and PS exposure. The r value is plotted in the correlation graph. PS exposure on the <i>L. amazonensis</i> amastigotes surface from nude mice (<b>Figures </b><b>D–F</b>). Mean fluorescence intensity of annexin V staining on lesion-derived amastigotes (LV79) purified from BALB/c WT or BALB/c ^nu/nu mice (<b>D</b>). Graph corresponds to one representative experiment out of three. H&E staining of histological slides of infected footpads obtained from BALB/c WT or BALB/c nude mice 5 weeks post infection (<b>E</b>). Peritoneal macrophages derived from BALB/c mice were infected with lesion-derived amastigotes (LV79) purified from BALB/c WT or BALB/c ^nu/nu mice 5 weeks post infection. After 24 h of infection cells were fixed and stained. The infectivity index (<b>F</b>) was defined by microscopic analysis. Representative experiment of two repeats. Differences were checked using unpaired t test.</p

sCD163 is elevated in the serum of lepromatous leprosy patients.

<p>(A) Sera of leprosy patients with various clinical presentations were collected and sCD163 concentrations measured by ELISA. Household contacts without symptoms or signs of leprosy (Contacts) were used as a control group. The mean ± SD sCD163 levels in patients with indeterminate leprosy (IL) (n = 9; 114 ± 49,57 ng/mL), true tuberculoid leprosy (TT) (n = 14; 90,29 ± 44,06 ng/mL), borderline leprosy (BL) (n = 14; 97,71 ± 47,97 ng/mL) and lepromatous leprosy (LL) (n = 10; 177,6 ± 62,18 ng/mL), as well as contacts (n = 23; 90,78 ± 31,55 ng/mL) were compared by Mann-Whitney test. ROC curves comparing sCD163 concentrations from TT versus LL (B) and Contacts versus LL patients (C) were constructed and are shown.</p

CD163 expression is induced by <i>Leishmania</i> infection of neutrophils.

<p>(A) Gating strategy for the analysis of neutrophil phenotypes. Neutrophils were purified from healthy donors and infected with GFP-expressing <i>L</i>. <i>amazonensis</i> (5 parasites: 1 neutrophil) in RPMI 1640 plus 10% FBS. (B) 3h after infection, neutrophils were characterized by flow cytometry and data analyzed by FlowJo software (in duplicate, n = 5 experiments). Green and black bars represent, respectively, the GFP positive and negative cells. The white bars represent non-exposed group (unstimulated). The mean ± SD of parental percentage of GFP+, GFP- and non-exposed cells were compared by Friedman paired test with Dunn’s post test.</p

Variable cytokine production by CD163+ and CD163- monocyte/macrophages.

<p>(A) Gating strategy for analysis of the cytokine profiles of CD163+ and CD163- monocyte/macrophages. PBMC were collected from healthy donors and the adherent cells cultured for 5 days in RPMI 1640 plus 20% FBS. The cells were incubated with <i>L</i>. <i>amazonensis</i> strain (10 parasites: 1 macrophage) and <i>L</i>. <i>infantum-</i>isolate 1 (5:1), and incubated with antibodies specific for intracellular cytokines, prior to analysis by flow cytometry (n = 6 experiments, in duplicate). (B) Frequency of IL-4+ cells, (C) MFI of IL-4-PerCPCy5.5, (D) iMFI of IL-4 analysis, (E) Frequency of IL-10+ cells, (F) MFI of IL-10-APC, (G) iMFI of IL-10 analysis, (H) Frequency of IL-12+ cells, (I) MFI of IL-12-APC, (J) iMFI of IL-12 analysis, (K) Frequency of TNF-α+ cells, (L) MFI of TNF-α-PerCPCy5.5, (M) iMFI of TNF-α analysis.</p

sCD163 levels correlate with severity of VL.

<p>(A) sCD163 levels were measured in sera of VL patients of different clinical status. The mean ± SD sCD163 levels of patients with classical VL at D0 (D0-Classic, n = 33) (152,1 ± 67,86 ng/mL), D30 (n = 19) (98,79 ± 58,58 ng/mL) and of patients of severe VL at D0 (D0-SVL) (n = 13) (241,5 ± 76,88 ng/mL) were compared by Mann-Whitney test. Sera from <i>Leishmania</i>-infected individuals without symptoms or signs of VL (DTH+, n = 11, 72,55 ± 25,68 ng/mL) and healthy individuals from non-endemic regions (HC, n = 8, 49,0 ± 23,71 ng/mL) were included as control groups. Spearman correlation analyses between sCD163 concentrations were performed versus (B) spleen size, (C) liver size and (D) neutrophil count. (E) Paired analysis of sCD163 levels of VL patients before and after treatment (n = 15, p = 0.0455, paired t test). ROC curves of sCD163 concentration comparing HC versus (F) D0-Classic, (G) D0-Classic versus D30 and (H) D0 versus D0-SVL group.</p