Table 1.
Leishmania amazonensis isolates used in this study.
Table 2.
Clinical and immunologic data from patients with diffuse cutaneous leishmaniasis (DCL) and localized cutaneous leishmaniasis (LCL).
Figure 1.
PS exposure on the L. amazonensis amastigotes surface.
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 Methods. 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.
Figure 2.
Leishmania isolate infectivity and PS exposure.
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 (A) and infectivity index (B) were defined under a microscope. Parasite burden was measured by quantitative PCR at 24 and 72 h (C). 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 (D) and (E), 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.
Figure 3.
Cytokine production by infected macrophages with different Leishmania amazonensis isolates.
Peritoneal macrophages of F1 mice were infected with isolates obtained from LCL (BA69, BA73, BA 125, and M2269) (□) and DCL (BA276, BA336, and BA700) patients in a serum-free medium containing 100 ng/ml LPS. Negative control (NC) represents uninfected macrophages. Supernatants were collected after 24 and 72 h and active TGF-β1 (A) production was assayed by ELISA. IL- 10 (B) and TNF-α (C) production were assessed by Cytometric Bead Array (CBA). The TGF-β/TNF-α (D) and IL-10/TNF-α (E) ratios. Boxes represent median values and interquartile interval of the ratios from different isolates mentioned above. The correlations between PS exposure at 24 h with TGF-β/TNF-α and IL-10/TNF-α at 24 h are showed in (F) and (G), respectively. The four lower points in X axis (PS exposure) represents LCL isolates while the three higher points are from DCL isolates. Differences were checked using Kruskal-Wallis with Dunn's multiple comparison post test. Spearman test was used to verify the significance in the correlations between cytokine ratio and PS exposure. The r values are plotted in each correlation graph.
Figure 4.
Parasitophorous vacuole analysis.
Photomicrographs of macrophages from F1 mice, infected with DCL (BA276) and LCL (BA125) isolates after 72 h of culture (A). 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). 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 (C). 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 L. amazonensis amastigotes surface from nude mice (Figures D–F). Mean fluorescence intensity of annexin V staining on lesion-derived amastigotes (LV79) purified from BALB/c WT or BALB/c nu/nu mice (D). 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 (E). 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 (F) was defined by microscopic analysis. Representative experiment of two repeats. Differences were checked using unpaired t test.
Figure 5.
PS exposure on L. amazonensis isolates correlates with clinical parameters of the disease.
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 (A) and time of disease (B). The r values are plotted in each correlation graph.