C57BL/6 mice chronically infected with the <i>LiΔHSP70-II</i> showed a Th1 like response against <i>Leishmania</i>.

<p>C57BL/6 mice (n = 8) were infected with 1 × 10⁷ <i>LiΔHSP70-II</i> promastigotes in the right footpad (s.c.). <i>LiΔHSP70-II</i> parasite burdens were analyzed at week 4 or at week 12 after vaccination by limiting dilution in the spleen (Sp, parasites per total organ), liver (Liv; parasites per g), bone marrow (BM; parasites per 1 × 10⁷ cells), in the right popliteal lymph node (RP; parasites per total lymph node) or right footpad (RFP; parasites per total footpad) (A). Scatter plots are shown including the mean ± standard deviation (SD). No statistical differences were found in the parasite burdens in the RP from week 4 to week 12 whereas a significant decrease was shown in the RFP (unpaired Student t-test). At week 4 or at week 12 after vaccination, animals were euthanized and spleen cell cultures and sera were prepared. The IgG1 and IgG2c reciprocal end-point titer against <i>L</i>. <i>infantum</i> SLA was determined by ELISA and represented as whisker (min to max) plots (B). * (P < 0.05) indicates the statistical differences between IgG1 and IgG2c anti-SLA titers within each group (Mann-Whitney test). For cytokine determinations spleen cells (C) or RP cells (D) were cultured in the absence (Med) or in the presence <i>L</i>. <i>infantum</i> SLA. Levels of IFN-γ, IL-10 and IL-4 were assessed by ELISA in culture supernatants. Mean ± standard deviations (SD) are shown in C and mean ± standard error of mean (SEM) are shown in D. * (P < 0.01) shows the statistical differences between SLA-stimulated and non-stimulated cells (unpaired Student t-test). No parasite loads or SLA-dependent cytokines were detected in saline immunized mice. Results are representative of two independent experiments.</p

External aspect of mycetomas showing the characteristic painless tumor with sinuses.

A) Different appearance of mycetoma lesions located on the feet B) Mycetoma lesions in other body locations.</p

Evolution of parasite burdens and immune response induced after <i>LiΔHSP70-II</i> infection in BALB/c mice.

<p>Mice (n = 8 per group) were inoculated with PBS (Saline) or with 1 × 10⁷ <i>LiΔHSP70-II</i> promastigotes in the vein tail (i.v.) (A, B) or in the right footpad (s.c.) (C, D, E). In A and C, scatter plots of <i>LiΔHSP70-II</i> parasite burdens are shown including the mean ± standard deviation (SD). Parasite loads were analyzed by limiting dilution at week 12 (12 wk) for i.v. inoculated animals (A) and at week 4 (4 wk) and 12 wk for s.c. inoculated ones (C) in the spleen (parasites per total organ), liver (parasites per g) or bone marrow (parasite per 1 × 10⁷ cells) (A). In (C) parasite burdens in the right popliteal lymph node (RP, parasites per total lymph node) and in the right footpad (RFP, parasites per total footpad) are shown. * (<i>P</i> < 0.05) indicates the statistical differences in 4 wk and 12 wk parasite burdens determined by a Mann-Whitney test. Spleen cell cultures for i.v. (B) or s.c. (D), or right popliteal lymph node cell cultures for s.c. (E) vaccinated animals were established at the indicated times after the inoculation of the <i>LiΔHSP70-II</i> line. The presence of cytokines in supernatants was measured after growing cells in the absence (Med) or in the presence of <i>L</i>. <i>infantum</i> SLA. In B and D, data show the mean ± SD of at least 8 mice per group. In E, data show the mean ± standard error of mean (SEM) of two independent assays performed with pooled cells from 8 mice. * <i>P</i> < 0 .05 shows statistical differences between SLA-stimulated and non-stimulated cells (unpaired Student t-test). The IgG1 and IgG2a reciprocal end-point titers against <i>L</i>. <i>infantum</i> SLA were analyzed by ELISA at the indicated times for i.v and s.c. inoculated mice, and represented as whisker (min to max) plots (F). * (<i>P</i> < 0.05) indicates the statistical differences between IgG1 and IgG2a anti-SLA titers (Kruskal-Wallis test and Dunn's Multiple Comparison post-test). No parasite loads or SLA-specific antibodies or cytokines were detected in mice receiving saline. Results are representative of at least two independent experiments.</p

Evolution of <i>L</i>. <i>major</i> infection in C57BL/6 vaccinated mice.

<p>Animals (n = 8 per group) inoculated with PBS (Saline) or with 1 × 10⁷ <i>LiΔHSP70-II</i> promastigotes in the right footpad (s.c.) were challenged with 1 × 10³ metacyclic <i>L</i>. <i>major</i> promastigotes in the dermis of both ears at week 4 or at week 12 after vaccination. (A) Ear lesion diameter was monitored weekly (16 ears from week 1 to 5 and 8 ears from week 6 to 13). Mean ± standard deviation (SD) is shown. <i>L</i>. <i>major</i> parasite burdens were determined at week 5 after <i>L</i>. <i>major</i> challenge by limiting dilution in the ears (n = 8 per group) (B) or in the retromandibular draining lymph node (n = 8 per group) (C). Scatter plots with the individual number of parasite per total organ are shown including the mean ± standard deviation (SD), * (P < 0.05) shows the statistical differences determined by the one-way ANOVA test followed by the Tukey post-test. For cytokine determinations the DLNs (D) or the spleen (E) from mice sacrificed at week 5 after <i>L</i>. <i>major</i> challenge (n = 4 mice) cells were cultured in the absence (Med) or in the presence <i>L</i>. <i>major</i> SLA. Levels of IFN-γ and IL-10 were assessed by ELISA in culture supernatants. The means ± SD are shown. * (P < 0.01) shows the statistical differences among saline and short-term or long-term vaccinated mice groups (unpaired Student t-test). The IgG1 and IgG2c reciprocal end-point titer against <i>L</i>. <i>major</i> SLA was determined by ELISA at week 5 after <i>L</i>. <i>major</i> challenge (n = 8 mice per group) and represented as whisker (min to max) plots (F). * (P < 0.05) indicates the statistical differences between IgG1 and IgG2c anti-SLA titers within each group (Mann-Whitney test). Results are representative of at least two independent experiments.</p

Analysis of splenic T cell populations in vaccinated mice.

<p>Mice (n = 4 per group) were inoculated with PBS (Saline; s.c.) or with 1 × 10⁷ <i>LiΔHSP70-II</i> promastigotes in the vein tail (i.v.) or in the right footpad (s.c.). After 4 weeks (vaccinated) and 12 weeks (saline and vaccinated), T cells from the spleen were studied in pool by flow cytometry. Animals were age matched at the moment of the analysis. In (A) analyzed CD4⁺ T cells were: antigen-experienced cells CD44^high, central memory T cells (Tcm) CD44^highCD62L^high and effector T cells or effector memory T cells (Teff/Tem) CD44^highCD62L^low. Spleen cells (B) or right popliteal lymph node cells (C) of the indicated groups were ex vivo stimulated by anti-CD3/anti-CD28 and treated with brefeldin A to block cytokine secretion. Cells were characterized by using anti-CD4 and anti-CD8 antibodies as well as by intracellular staining for IFN-γ. The ratio between the percentages of CD4⁺ or CD8⁺ producing IFN-γ cells in vaccinated animals versus saline ones, derived from two independent experiments is shown (mean ± standard error of mean).</p

Humoral and cellular parasite induced responses after <i>L</i>. <i>major</i> challenge.

<p>BALB/c mice (n = 8 per group) inoculated with PBS (Saline) or with 1 × 10⁷ <i>LiΔHSP70-II</i> promastigotes in the vein tail (i.v.) (A, C, D, E) or in the right footpad (s.c.) (B, F, G, H) were infected with 5 × 10⁴ stationary-phase <i>L</i>. <i>major</i> promastigotes in the left footpad at week 4 or at week 12 after vaccination. Animals were euthanized 8 weeks after <i>L</i>. <i>major</i> challenge. IgG1 and IgG2a reciprocal end-point titers against <i>L</i>. <i>major</i> SLA were determined by ELISA (A, B). Results are shown as whisker (min to max) plots of at least 8 mice per group. * (P < 0.05) indicates the statistical differences between IgG1 and IgG2a anti-SLA titers within each group or differences in IgG2a between short- and long-term protected mice (Mann-Whitney test). For cytokine determinations spleen cells were cultured in the absence (Med) or in the presence of <i>L</i>. <i>major</i> SLA. Levels of IFN-γ (C, F), IL-10 (D, G) and IL-4 (E, H) were assessed by ELISA in culture supernatants and shown as whisker (min to max) plots of at least 8 mice per group. * (P < 0.01) shows the statistical differences of saline and short-term or long-term vaccinated mice groups, or the statistical differences between short-term and long-term vaccinated mice groups (Mann-Whitney test). Results are representative of two independent experiments.</p

Mice vaccinated with the attenuated <i>LiΔHSP70-II</i> line showed protection against an infective challenge with <i>L</i>. <i>major</i>.

<p>BALB/c mice (n = 8 per group) inoculated with PBS (Saline) or with 1 × 10⁷ <i>LiΔHSP70-II</i> promastigotes in the vein tail (i.v.) (A, B) or in the right footpad (s.c.) (C, D) were challenged with 5 × 10⁴ stationary-phase <i>L</i>. <i>major</i> promastigotes in the left footpad at week 4 (s.c.) or at week 12 after vaccination (i.v. and s.c.). Footpad swelling was monitored weekly. Mean ± standard deviation (SD) is shown (A, C). <i>L</i>. <i>major</i> parasite burdens were determined by limiting dilution in the spleen, liver and in the draining lymph node (left popliteal). Scatter plots with the individual number of parasite per total organ (spleen or lymph nodes) or per g of liver are shown including the mean ± SD (B, D). * (P < 0 .05) shows the statistical differences determined by the one-way ANOVA test followed by the Tukey post-test. Results are representative of two independent experiments.</p

S1 Fig -

Size of lesions in relation to: A) type of mycetoma, B) color of grains and C) time of evolution. (PPTX)</p

Large mycetoma affecting the right foot of a 15-year-old patient.

A) Aspect of the foot at direct examination. B) Radiology of the same area showing the involvement of bones and deep tissues. The patient needed amputation to avoid the spreading of mycetoma in the lower limb.</p

Protective effect of the blue-blocking filter on retinal response to damaging light.

<p>Representative ERG traces from unprotected (A) and protected (B) groups of mice, obtained before (grey traces) and after (black traces) exposure to white light (5,000 lux) for seven days. Arrows indicate the amplitudes measured for each different ERG wave. In the unprotected group (A), amplitudes of scotopic (rod-driven, mixed rod-cone and Oscillatory Potentials) and photopic (cone-driven) parameters are strongly reduced upon light-induced damage, while in the protected group (B), this reduction is attenuated.</p