Effect of LipESP on human DCs maturation.

<p>Immature DCs were stimulated with LipESP for 48h and analysis of CD40, HLA-DR, CD86 and CD80 expression was performed by cytometry. Non-stimulated or LPS-stimulated DCs were considered as control cultures. Results are expressed as mean ± standard deviation of percentages of positive cells (n = 8). *: The results are statistically significant (p<0.05), when compared to control cells.</p

Effect of LipESP on LPS-, IFN-γ-, or IFN-γ/LPS-induced cytokine production by human DCs.

<p>Production of (a) IL-12p70, (b) IL-10 and (c) TNF-α were measured in DCs supernatant by ELISA after 48 h of stimulation by LipESP in presence or absence of LPS or IFN-γ or IFN-γ/LPS. Results are expressed as mean ± standard deviation (n = 10). * Statistically significant difference between non stimulated and LipESP-, LPS-, IFN-γ and IFN-γ/LPS-stimulated DCs (p<0·05); ** statistically significant difference between LPS-stimulated and LipESP/LPS-co-treated DCs (p<0·05); *** statistically significant difference between IFN-γ stimulated and LipESP/IFN-γ-co-treated DCs (p<0·05).</p

Effect of LipESP on LPS-, IFN-γ-, or IFN-γ/LPS-induced cytokine production by human monocytes.

<p>Production of (a) IL-12p70, (b) IL-10 and (c) TNF-α were measured in monocytes supernatant by ELISA after 24 h of stimulation by LipESP in presence or absence of LPS (for IL-10 and TNF-α analysis) or after 12 h of priming with IFN-γ followed by 24 additional hours of stimulation by LipESP with or without LPS co-treatment (for IL-12 analysis). Results are expressed as mean ± standard deviation (n = 9). * Statistically significant difference between non stimulated and LipESP-, LPS-, and IFN-γ/LPS-stimulated monocytes (p<0·05); ** Statistically significant difference between IFN-γ stimulated and LipESP/IFN-γ-co-treated monocytes (p<0·05) for IL-12p70 and between LPS-stimulated and LipESP-LPS-co-treated monocytes (p<0·05) for IL-10 and TNF-α; *** Statistically significant difference between IFN-γ/LPS stimulated and LipESP/IFN-γ/LPS-co-treated monocytes (p<0·05).</p

Effect of LipESP on human DCs differentiation.

<p>After 6 days of differentiation in presence or absence of LipESP, cells were harvested and labeled with appropriate antibodies. Cells with media alone represent DCs differentiation in the absence of LipESP and are considered as control cultures. Results are expressed as mean ± standard deviation of percentages of positive cells (n = 7). *: The results are statistically significant (p<0.05), when compared to control cells.</p

GrB induction by the selected peptides.

<p>(A) PBMCs from HLA-A*0201⁺ (black mark) and HLA-A*0201^- (white mark) -healed ZCL individuals were stimulated with selected peptides separately. The supernatant was collected after 5 days of incubation and assayed for GrB production. (B) Peptides E2, E6, F6, G2, G3, and G4 induced the highest levels of GrB.</p

Binding affinity of <i>Lm</i>ES peptides to HLA-A*0201 molecules.

<p>The affinities of selected peptides were determined by class I HLA stabilization assay. (A) Results for individual peptides. T2 cells were initially incubated with 100μg (final concentration) of each of the peptides/mL for 16h at 37°C, followed by incubation at 37°C for 2h in presence of Brefeldine A. HLA-A2 expression on these cells was analyzed by flow cytometry using the BB7.2 antibody. MHC stabilization efficiency for each peptide was calculated as the percentage increase of the mean fluorescence above that of the negative controls. Results were expressed as relative fluorescence intensity (RFI). (B) Box plot results. ++: RFI ranges from 200 to 300%, +: RFI ranges from 100 to 200%, and -: RFI ranges from 0 to 100%.</p

Total Soluble <i>Leishmania</i> antigen (TSLA) specific IFN-γ, granzyme B, TNF-α responses.

<p>IFN-γ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092708#pone-0092708-g002" target="_blank">Fig. 2a</a>), granzyme B (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092708#pone-0092708-g002" target="_blank">Fig. 2b</a>), were detected and quantified from culture supernatants of PBMC exposed for 120h to local TSLA (10 µg/ml) and TSLA Ldd8 (10 µg/ml), by Cytokine Beads Array test (CBA) using Flow cytometry. Statistically significant differences between stimulated and non stimulated cultures and between groups (p≤0.03) are showed.</p

<i>La</i>PSA-38S specific IFN-γ, granzyme B, TNF-α and IL-10 responses.

<p>IFN-γ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092708#pone-0092708-g003" target="_blank">Fig. 3a</a>), granzyme B (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092708#pone-0092708-g003" target="_blank">Fig. 3b</a>), TNF-α (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092708#pone-0092708-g003" target="_blank">Fig. 3c</a>) and IL-10 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092708#pone-0092708-g003" target="_blank">Fig. 3d</a>) were detected and quantified from culture supernatants of PBMC exposed for 120 h to SLA (10 µg/ml) or <i>La</i>PSA-38S (10 µg/ml) using Cytokine Beads Array test (CBA). Data were analyzed by Flow cytometry. PHA (10 µg/ml) was used for all cultures as positive control (data not shown). Statistically significant differences between stimulated and non stimulated cultures (p≤0.003) and between groups (p≤0.01) are showed.</p

Total IgG prevalence against <i>La</i>PSA-38S in different endemic areas of leishmaniasis.

<p>The cut-off value of reactivity for <i>La</i>PSA-38S antigen was calculated as the mean plus 3 SD of the OD values observed in naive controls from each country. These cut-off values allowed us to distinguish between positive and negative sera and consequently to estimate performance parameters of each ELISA test.</p

SDS-PAGE and silver staining of purified recombinant <i>La</i>-PSA38S protein expressed in <i>L. tarentolae</i>.

<p>Purified <i>La</i>-PSA38S was separated by electrophoresis in a 12% SDS-PAGE and silver stained under reducing (R) and non reducing (NR) conditions. The arrow indicates the position of <i>La</i>-PSA-38S (45 kDa) and Molecular Weight markers were marked in kDa.</p