S1P modulates F-actin polymerization, AKT, ERK and Rac1 activity.

<p><b>(A)</b> Modulation of the actin cytoskeleton after stimulation by different S1P concentrations in CEM cells pre-treated or not with W146 (100 μM). Results are represented as [(MFI after addition of the ligand) / (MFI before addition of the ligand)] x 100. The MFI values obtained before the addition of S1P were arbitrarily set as 100% and correspond to the time zero. White circles correspond to cells previously treated with W146 (100 μM) and the black circles correspond to cells treated with RPMI-BSA 0.1%. Results are expressed by mean ± SEM and were analyzed by unpaired Student’s <i>t</i> test (n = 3). Differences were considered statistically significant when * p˂0.05, ** p ˂0.01 or *** p ˂0.001 <b>(B)</b> AKT and <b>(C)</b> ERK1/2 activation after stimulation of CEM cells, with different S1P concentrations, being pre-treated or not with W146 (100 μM). Protein extracts were analyzed by Western-blot with AKT, phosphorylated-AKT, ERK1/2 and phosphorylated-ERK-1/2 specific antibodies. Representative western-blots images are shown (n = 3). <b>(D)</b> Rac1 activity after stimulation of CEM cells, pre-treated or not with W146 (100 μM), with different S1P concentrations was accessed by G-LISA. Optical density (OD) was detected in 490 nm. Results are represented as [(OD after addition of the ligand) / (OD before addition of the ligand)] x 100. OD values obtained before the addition of S1P were arbitrarily set as 100% and correspond to the time zero. Black circles correspond pre-treatment with RPMI-BSA 0.1% and white circles correspond to pre-treatment with W146 (100 μM). Results and are expressed as mean ± SEM. Differences between distinct time points and control (time zero) were analyzed by One-way ANOVA, followed by Dunnett post-test and were considered statistically significant when # p˂0.05, ## p ˂0.01 or ### p ˂0.001. Differences between pre-treatment with RPMI-BSA 0.1% and pre-treatment with W146 (100 μM) were analyzed by unpaired Student T test (n = 1, with 2 biological replicates in duplicate). Differences were considered statistically significant when * p˂0.05, ** p ˂0.01 or *** p ˂0.001.</p

High S1P concentrations induce S1P1-dependent fugetaxis of CEM cells.

<p><b>(A)</b> CEM cells were serum-starved for 2 h, applied to Transwell™ chambers containing different S1P concentrations and incubated for 4 hours. S1P was added to the upper chamber to evaluate fugetaxis; only RPMI-BSA 0.1% was added to the bottom chambers. Results were analyzed by One-way ANOVA, followed by Tukey post-test (n = 3). <b>(B)</b> CEM cells were serum-starved for 2 h and pre-treated or not with W146 (100 μM). Cells were then applied to Transwell™ chambers containing different S1P concentrations and incubated for 4 hours. S1P was added to the upper chamber to evaluate repulsive responses. Black bars correspond to T-ALL blasts pre-treated with RPMI-BSA 0.1% alone and white bars correspond to T-ALL blasts pre-treated with W146. Results were analyzed by unpaired Student’s <i>t</i> test. Results are expressed as mean ± SEM and differences were considered statistically significant when * p˂0.05, ** p ˂0.01 or *** p ˂0.001 (n = 3).</p

S1P1 is involved in S1P-driven chemotactic responses of T-ALL blasts.

<p>T-ALL blasts were serum-starved for 2 h and pre-treated or not with W146 (100 μM). <b>(A)</b> Cells were applied to Transwell™ chambers with S1P 1, 10, 100, 500 or 1000 nM and incubated for 4 hours (n = 3). <b>(B)</b> Migratory response of CEM cells toward S1P 100, 2500, 5000 and 10000 nM (n = 3). Values correspond to a specific migration after subtracting the numbers of migrating cells obtained in wells with culture medium only. Black bars correspond to T-ALL blasts pre-treated with RPMI-BSA 0.1% and white bars correspond to T-ALL blasts pre-treated with W146. Results are expressed as mean ± SEM and were analyzed by unpaired Student’s <i>t</i> test. Differences were considered statistically significant when * p˂0.05, ** p ˂0.01 or *** p ˂0.001.</p

S1P3 is not involved in S1P-driven chemotactic responses of CEM cells.

<p><b>(A)</b> CEM cells were serum-starved for 2 h and pre-treated or not with W146 (100 μM) and/or BM-241 (100 μM). Cells were applied in Transwell™ chambers containing different S1P concentrations and incubated for 4 hours. Black bars correspond to pre-treatment with RPMI-BSA 0.1%; white bars correspond to pre-treatment with W146; grid bars correspond to pre-treatment with BML-241; and chess bars correspond to pre-treatment with W146 plus BML-241. Results were analyzed by Two-way ANOVA, followed by Bonferroni post-test (n = 3). <b>(B)</b> S1P receptors mRNA expression were analyzed by real time quantitative PCR, compared with the control Abelson (Abl) gene (2^-ΔCt) in SU-DHL-1 cells (n = 4). <b>(C)</b> S1P1 and S1P3 mRNA expression was analyzed by real time quantitative PCR, compared with the control Abelson (Abl). Fold change analysis were done using HPB-ALL as calibrator to normalize the expression of S1P1 and S1P3 on SU-DHL-1 cells. Statistical analysis was made with ΔCt values and significant differences are related to HPB-ALL cells. Results were analyzed by Student’s <i>t</i> test (n = 4). <b>(D)</b> SU-DHL-1 cells were serum-starved for 2 h, applied to Transwell™ chambers containing different S1P concentrations and incubated for 4 hours. Results were analyzed by One-way ANOVA, followed by Tukey post-test (n = 3). <b>(E)</b> SU-DHL-1 cells were serum-starved for 2 h and treated or not with W146 (100 μM). Cells were applied to Transwell™ chambers containing different S1P concentrations and incubated for 4 hours. Values correspond to specific migration after subtracting the numbers of migrating cells in culture medium only. Black bars correspond to pre-treatment with RPMI-BSA 0.1% alone and white bars correspond to pre-treatment with W146. Results were analyzed by unpaired Student’s <i>t</i> test (n = 3). Results are expressed as mean ± SEM and differences were considered statistically significant when * p˂0.05, ** p ˂0.01 or *** p ˂0.001.</p

S1P-induced migratory responses in checkerboard assays.

<p>CEM cells were serum-starved for 2 h, applied to Transwell™ chambers containing different S1P concentrations in a checkerboard format and incubated for 4 hours. <b>(A)</b> S1P 100 nM or <b>(B)</b> S1P 5000 nM were added to upper and/or lower chamber as RPMI-BSA 0,1%. Results were analyzed by One-way ANOVA, followed by Tukey post-test and are expressed as mean ± SEM. Differences between wells with medium alone in upper and lower chambers and wells with S1P in upper and/or lower chamber were considered statistically significant when * p˂0.05, ** p ˂0.01 or *** p ˂0.001. Differences between wells with S1P in lower (100 nM) or upper (5000 nM) chambers and wells with S1P in both upper and lower chambers were considered statistically significant when # p˂0.05, ## p ˂0.01 or ### p ˂0.001 (n = 3).</p

Different S1P concentrations induce chemotactic responses of T-ALL blasts.

<p>T-ALL blasts were serum-starved for 2 h and inserted into Transwell™ chambers with different S1P concentrations for 4 hours. Results are expressed as mean ± SEM and were analyzed by One-way ANOVA, followed by Tukey post-test. Differences were considered statistically significant when * p˂0.05, ** p ˂0.01 or *** p ˂0.001 (n = 3).</p

S1P receptors mRNA expression in human T-ALL blasts.

<p><b>(A)</b> Bars show S1P receptors mRNA expression analyzed by real time quantitative PCR, compared with the control Abelson (Abl) gene (2^-ΔCt) in T-ALL (n = 1–2, with 3–6 biological replicates). <b>(B)</b> Bars shows S1P1 or (C) S1P3 mRNA expression analyzed by real time quantitative PCR, compared with the control Abelson (Abl). Fold change analysis were done using HPB-ALL as calibrator to normalize the expression of the receptors on the other T-ALL blasts. Statistical analysis was made with ΔCt values and significant differences are related to HPB-ALL cells. Results are expressed as mean ± SEM and were analyzed by Student’s <i>t</i> test and differences were considered statistically significant when p<0.05 (*), p< 0.01 (**) or p< 0.001(***) (n = 1–2, with 3–6 biological replicates).</p

Serum cytokine profile of NGP5B and NGP5B+CpG immunizations.

<p>(A-D) Th1 cytokines IL-12p40, IL-2, IFN-γ, and TNF-α. (E-G) Th2 cytokines IL-4, IL-10, and IL-5. (H) IFN-γ/IL4 ratio. (I) IFN-γ/IL10 ratio. Two-tailed unpaired Student’s <i>t</i>-test (compared with Naïve group): (*), <i>P</i><0.05; (***), <i>P</i><0.0001. (A-I) Error bars indicate S.E.M. of triplicate determinations.</p

Anti-NGP5B antibody response is specific against terminal α-Gal residues.

<p>(A-B) Chemiluminescent ELISA reactivity of mouse serum pools obtained at Boost 3 (n = 6) and endpoint (n = 3) from α1,3GalT-KO mice vaccinated with NGP5B, NGP5B+CpG, CpG, or PBS. Immunized groups are indicated in the legend and antigens on the microplate are shown in the Y-axis. (C) Chemiluminescent ELISA reactivity of mouse serum obtained at Boost 2. NGP5B (125 ng/well) was treated or not with green-coffee bean α-galactosidase. One-way ANOVA (compared with untreated sample): (**), <i>P<</i>0.01; (***), <i>P</i><0.001; (****), <i>P</i><0.0001. (A-C) Error bars indicate S.E.M. of triplicate determinations.</p

α-Gal-NGPs as biomarkers of active CL in patients with <i>L</i>. <i>major</i> infection.

<p>Assessment by chemiluminescent ELISA of α-Gal-containing NGPs and controls (Cysteine-BSA and Galβ-BSA) were immobilized on a microplate and reacted with pools of sera (at 1:100 dilution) from patients with active or cured CL, or heterologous skin (non-CL) infections (n = 5 per group, randomly selected) from an endemic region (Saudi Arabia), as described [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006039#pntd.0006039.ref026" target="_blank">26</a>]. RLU, Relative luminescence units. Error bars indicate S.E.M. of triplicate determinations. The fold difference in reactivity between active CL vs. cured CL, and active CL vs. heterologous infection are indicated. Two-way ANOVA with Tukey’s multiple comparisons: ns, non-significant; (*), <i>P</i><0.05; (**), <i>P</i><0.01; (***), <i>P</i><0.001; (****), <i>P</i><0.0001.</p