Table 1.
Half-maximal inhibitory concentrations (IC50 values) of TPF against L. infantum and L. major promastigotes.
Fig 1.
Effect of TPF on the viability of L. infantum and L. major promastigotes.
Fig 2.
Growth kinetics of Leishmania spp. promastigotes exposed to TPF.
Fig 3.
Leishmania spp. promastigote proliferation determined by CFSE staining.
L. infantum (A) and L. major (B) early exponential-phase promastigotes were treated with IC50 and 2 x IC50 concentrations of TPF and their proliferation rate was qualitatively monitored at 24 h intervals for 3 consecutive days by CFSE staining and subsequent analysis of fluorescence intensity in FACS. HePC (IC50)-treated and untreated parasites were used as positive and negative control groups, respectively. The results are expressed as mean fluorescence intensity values ± SD of three independent experiments. Symbols of * and ¥ indicate statistically significant differences compared to negative and positive control groups, respectively, while ≠ indicates significant differences between TPF groups.
Fig 4.
Microscopy images of Leishmania spp. promastigotes.
Fig 5.
Analysis of cell cycle arrest in TPF-treated L. infantum and L. major promastigotes.
Fig 6.
PS externalization in TPF-treated L. infantum and L. major promastigotes.
Exponential-phase L. infantum (A, C) and L. major (B, D) promastigotes were either left untreated (negative control) or were treated with IC50 and 2 x IC50 concentrations of TPF and HePC (IC50, positive control) for 24, 48 and 72 h. At the end of the aforementioned time-points, parasites were double stained with annexin V-FITC and PI and were analyzed by FACS. The results are presented as mean values of % annexin V+ parasites ± SD in bar diagrams (A, B) representative of three independent experiments and as flow cytometric dot plots with respective quadrants (C, D), representative of one experiment. Symbols of * and ¥ indicate statistically significant differences compared to negative and positive control groups, respectively while ≠ indicates significant differences between TPF groups.
Fig 7.
Evaluation of mitochondrial membrane potential in Leishmania spp. promastigotes treated with TPF.
Fig 8.
ROS production in TPF-treated Leishmania spp. promastigotes.
Fig 9.
Therapeutic effect of TPF in a murine experimental model of cutaneous leishmaniasis.
(A) Definition of treatment schedule and experimental groups. BALB/c mice were subcutaneously infected into the right hind footpad with 106 L. major promastigotes and one week later, were either treated with TPF (20 mg/kg b.w., ip) or HePC (15 mg/kg b.w., oral gavage) for up to 28 days or left untreated (control group). (B) Time course of L. major infection. The increase in footpad swelling was monitored weekly by measuring the increase of footpad thickness (in mm) as compared with the uninfected contralateral footpad. Results are expressed as mean values ± SD. (C) Parasite load estimation in popliteal lymph nodes of TPF-treated mice. Parasite load was determined at five weeks post-treatment termination by a limiting dilution assay. Values represent the mean value ± SD of five animals per experimental group. (D) The IgG1 and IgG2a reciprocal end-point titers against L. major SLA were determined by ELISA at five weeks post-treatment termination (n = 5 mice per group) and represented as whisker (min to max) plots. * indicates the statistical differences between IgG1 and IgG2a anti-SLA titers among experimental groups. (E) Effect of TPF treatment on the percentage of IFN-γ-producing CD4+ T cells. Spleen cells from each experimental animal were isolated five weeks post-treatment termination and intracellular staining was performed. Flow cytometric analysis was conducted and total splenocytes were first gated on a forward scatter (FSC-H)/side scatter (SSC-H) plot and then subgated on the CD4+ population. Results of cytokine profiles are presented in bar diagram showing the percentage of IFN-γ-producing CD4+ T cells. Symbol of * indicates statistically significant differences among the experimental groups. (F) Quantitative analysis of Tbx21 and GATA-3 mRNA in spleen cells. The expression of Tbx21 and GATA-3 mRNA was evaluated by real-time PCR and the expression level of the GAPDH housekeeping gene from the same samples was used as normalizer. All expression levels were computed via the ΔΔCt method. Symbol of * indicates statistically significant differences among the experimental groups.