Protection in a chronic infection model in vaccinated mice.

<p>Animals were immunized as indicated and challenged 15 days later with <i>T</i>. <i>cruzi</i> K98 strain. <b>a)</b> Parasitemia curve (I) and its AUC (II) during the acute phase of infection. <b>b)</b> Serum level of tissue damage-associated enzymes at 100 dpi: creatine kinase, CK (I) and its cardiac isoform, CK-MB(II); glutamate oxaloacetate transaminase, GOT (III); and lactate dehydrogenase, LDH (IV). <b>c)</b> Electrocardiogram parameters at 100 dpi: Corrected QT interval(cQTi) (I) and PR interval(PRi) (II). <b>d)</b> Parasite load by qPCR in target tissues at 100 dpi. <b>e)</b> Histopathological analysis of skeletal and heart muscle at 100 dpi. Representative muscle sections stained with hematoxilin-eosin at 100x magnification (I) and semi-quantitative analysis of inflammatory infiltrate (II). Results are expressed as mean ± SEM (n = 4–5 per group) and are representative of two independent experiments. Survival statistical analysis was performed with log-rank test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. One-way ANOVA plus Tukey’s post-test. (n.s.: non-significant differences).</p

Overall cell-mediated immune response and CD4⁺ cells functionality in immunized mice.

<p><b>a)</b> DTH reaction. Fifteen days after the last immunization, mice were subcutaneously injected in the hind footpad with rTc80. Footpad thickness was measured before and 48 h after rTc80 administration. The result was expressed as the difference between the two measurements. <b>b)</b> Antigen-specific proliferation. Splenocytes from immunized mice were stimulated with rTc80 and proliferation was assessed by 3H-thymidine uptake. Results were expressed as proliferation index: [cpm (counts per minute) in the presence of the antigen/cpm in the absence of the antigen]. <b>c)</b> Cytokines secretion by splenocytes from immunized mice upon antigen recall (I) IL-2 and (II) IFN-γ, determined by capture ELISA. <b>d)</b> Representative dot plots of intracellular cytokine production by CD4⁺ T cells. (I); Percentage of IFN-γ (II) or TNF-α (III) producing CD4⁺ cells. <b>e)</b> Analysis of CD4⁺ T cells polyfunctionality. I) Percentage of CD4⁺ T cells producing simultaneously IFN-γ and TNF-α. II) Proportion of CD4⁺ T cells with different degrees of functionality. <b>f)</b> Cytokine producing ability of mono and polyfunctional CD4⁺ cells for IFN-γ (I) and TNF-α (II), expressed as mean fluorescence intensity (MFI). Results are expressed as mean ± SEM (n = 5–6 per group) and are representative of at least three independent experiments. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. One-way ANOVA plus Tukey’s post-test (a-e) and Student’s t-test (f).</p

<i>In silico</i> toxicologic profile and <i>in vivo</i> trypanocidal activity of estafietin, a sesquiterpene lactone isolated from Stevia alpina Griseb.

Chagas disease is an infection caused by the protozoan Trypanosoma cruzi, affecting 6-8 million people worldwide. Only two drugs are available for its treatment, having a limited efficacy and adverse side-effects. Estafietin is a sesquiterpene lactone isolated from Stevia alpina with in vitro activity against T. cruzi and low cytotoxicity against mammalian cells. The aim of this work was to predict the toxicologic profile of estafietin by in silico methods and assess its in vivo activity on a murine model of Chagas disease. Estafietin showed low toxicity according to pkCSM web tool and passed the PAINS filter from PAINS-remover web server. The treatment of infected mice with 1 mg/Kg/day of estafietin for five consecutive days administrated by intraperitoneal route significatively decreased parasitemia levels and reduced inflammatory infiltrates and myocyte damage on muscle tissue. These results suggest that estafietin had effect both on acute and chronic stages of the infection.</p

Protection against a lethal <i>T</i>. <i>cruzi</i> challenge.

<p><b>a)</b> Parasitemia curve at different days post-infection (dpi) and b) area under the curves (AUC) of parasitemia. <b>c)</b> Parasitemia at early (9 dpi) and late (23 dpi) stages of acute infection. <b>d)</b> Survival rate curve. Results are expressed as mean ± SEM (n = 4–5 per group) and are representative of at least three independent experiments. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. One-way ANOVA plus Dunnett’s post-test(a, b) or Tukey’s post-test (c) and log-rank test for survival curves (d). Color code from panel ‘a’ legend is also used in the other panels (b, c, d).</p

Tc80-specific CTL activity in immunized mice.

<p><b>a)</b><i>Ex vivo</i> CTL activity. Splenocytes from immunized mice were incubated with cells loaded with the SEAELRKKI peptide (target cells, CFSE⁺). Later on, cell death was assessed with propidium iodide by flow cytometry and lysis percentage was calculated according to the following formula: {[(ÏSE⁺PI⁺/CFSE⁺)_loaded/(ÏSE⁺PI⁺/CFSE⁺)_non-loaded]-1}*100. <b>b)</b> <i>In vivo</i> CTL activity. Nonapeptide-pulsed and CFSE_high-labelled target cells were transferred to immunized mice and 16 h later, target cell lysis was evaluated in splenocytes by flow cytometry. Mice were also administered with non-loaded and CFSE_low-labelled cells for non-specific cell lysis control. (I) Representative histograms gated on CFSE positive cells showing CFSE_low and CFSE_high populations and (II) bar chart expressing the percentage of lysis as {1-[(ÏSE_high/ÏSE_low)_immunized/(ÏSE_high/ÏSE_low)_SaroA]}*100. Results are expressed as mean ± SEM (n = 4–5 per group) and are representative of three independent experiments. *p<0.05. One-way ANOVA plus Dunnett’s post-test.</p

Recombinant Tc80 expression, purification and characterization.

<p><b>a)</b> SDS-PAGE analysis of rTc80 expression and purification. Lane 1: MWM, lane 2: post-induction <i>E</i>. <i>coli</i> lysate, lane 3: Ni⁺⁺-NTA column flow through, lane 4: column wash, lanes 5–7: column rTc80 elution steps. <b>b)</b> Prolyl oligopeptidase activity against the substrate Z-Gly-Pro-AMC measured as relative fluorescent units (RFU) in the presence (red circles) or absence (green squares) of rTc80. A control without substrate was included (blue triangles). <b>c)</b> Immunoblot detection of rTc80 (lane 2) and native Tc80 in an epimastigote <i>T</i>. <i>cruzi</i> lysate (lane 3). Protein were transferred to a PVDF membrane and subsequently incubated with mouse anti-rTc80 antibodies and a peroxidase-conjugated anti-mouse IgG antibody. Lane 1: pre-stained MWM, lane 2: Purified rTc80 revealed with 4-Cl naphthol and lane 3: Native Tc80 revealed with an ECL substrate. <b>d)</b> Indirect immunofluorescence on BHK-21 cells transfected with Tc80-pcDNA3.1⁺ construction (I) or empty pcDNA3.1⁺ (II). Cells were permeabilized and incubated with mouse anti-rTc80 antibodies and subsequently with FITC-conjugated anti mouse IgG antibody.</p

Quantification of deoxymikanolide and mikanolide in <i>M</i>. <i>micrantha</i> and <i>M</i>. <i>variifolia</i> extracts.

<p>Quantification of deoxymikanolide and mikanolide in <i>M</i>. <i>micrantha</i> and <i>M</i>. <i>variifolia</i> extracts.</p

Leishmanicidal activity on <i>L</i>. <i>braziliensis</i> promastigotes of mikanolide, dihidromikanolide, deoxymikanolide and scandenolide.

<p>Parasites were cultured for 72 h in the presence of the compounds (0–50 μg/mL). Growth inhibition of parasites was evaluated by a MTT assay. The percent of inhibition was calculated as {100 − [(DO_595nm of treated parasites/ DO_595nm of untreated parasites) × 100]}. Values represent mean ± SEM from three independent experiments carried out in triplicate.</p

Chemical structures of mikanolide, dihydromikanolide, deoxymikanolide and scandenolide.

<p>Chemical structures of mikanolide, dihydromikanolide, deoxymikanolide and scandenolide.</p

Trypanocidal activities against <i>T</i>. <i>cruzi</i> trypomastigotes of mikanolide, dihydromikanolide, deoxymikanolide and scandenolide.

<p>Bloodstream trypomastigotes were cultured in duplicate in the presence of 0 to 50 μg/mL of mikanolide, dihydromikanolide, deoxymikanolide and scandenolide. Cultures were done in 96-well plates with 3 x 10⁵ parasites/mL during 24 h, and the remaining live parasites were counted in a Neubauer chamber. Symbols represent mean ± SEM.</p