Acute CD increases the cerebral leukocyte-endothelium interaction, the percentage of microvessels presenting platelet-leukocyte aggregates and oxidative stress.

<p>An increased number of rolling cells was found in the cerebral venular segment of infected mice (A). The NI group presented only 3±0.5 cells/min. At 8 dpi, the animals presented 6.3±0.8 cells/min, and at 15 dpi, the number of rolling leukocytes increased to 16±0.6 cells/min. The leukocyte adhesion analysis (B) showed that 1±0.2 cells/min/100 µm adhered to the venular segment in the NI group. This number was 0.4±0.2 cells/min/100 µm at 8 dpi and 8.6±1.7 cells/min/100 µm at 15 dpi. At 15 dpi, a high percentage of venules showed microvascular platelet-leukocyte aggregates (PLAs) (C). The malondialdehyde (MDA) levels in the brain of the non-infected and <i>T. cruzi</i>-infected mice showed an increase in oxidative stress only at 8 dpi (D). A–C and H–K, bar = 100 µm. One-way ANOVA test; <i>p</i><0.05^*, <i>p</i><0.01^**and <i>p</i><0.001^***, comparing the infected animals at 15 dpi with the NI group; <i>p</i><0.001^### and <i>p</i><0.01^##, comparing the infected animals at 15 and 8 dpi. Values are the means ± SEM (n = 4–8 animals/group); dpi: days post-infection; NI: non-infected.</p

Acute CD causes cerebral functional capillary rarefaction.

<p>Perfused cerebral arterioles and capillaries (arrows) can be observed by the fluorescence of FITC-dextran in the non-infected (A) and <i>T. cruzi</i>-infected animals at 8 (B) and 15 (C) dpi. A collapse in the microcirculation can be observed at 15 dpi (C). In (D), the graph shows a significant reduction in the number of perfused blood vessels (capillary density) in the infected animals at 15 dpi (405±31.4 capillaries/mm²) compared with the non-infected controls (514±1 capillaries/mm²) and with the <i>T. cruzi</i>-infected mice at 8 dpi (535±31.2 capillaries/mm²). Quantitative data are expressed as means ± SEM (n = 5–8/group). One-way ANOVA test; bar = 100 µm; dpi: days post-infection; <i>p</i><0.05: * comparing the infected animals at 15 dpi with NI group; # comparing 15 to 8 dpi. dpi: days post infection. NI: non-infected.</p

Rhodamine-labeled leukocytes in cerebral venules in acute CD.

<p>The images show venules of the non-infected (A) and <i>T. cruzi</i>-infected (B) mice at 8 and 15 dpi (C and D). The leukocyte-endothelium interaction (arrows) in the venules of the infected animals can be observed. Note the microvascular platelet-leukocyte aggregates (arrowhead) at 15 dpi in the infected animals (C and D). dpi: days post-infection; NI: non-infected.</p

Swiss Webster mice infected with the Y strain of <i>T. cruzi</i>-developed acute CD.

<p>Mice were infected with 10⁴ blood trypomastigote forms, and the following parameters were evaluated in a kinetic study: (A) parasitemia, (B) weight and (C) survival rate. The parasitemia peak occurred at 8 dpi (A). <i>T. cruzi</i> infection induced a significant body weight decrease in a time-dependent manner, starting at 12 dpi. At 22 dpi, the average weight of the NI mice was 31.4±1.9 g, while that of the <i>T. cruzi</i>-infected group was 23.8±2.5 g (B). At 22 dpi, only 20% of the infected animals survived (C). Quantitative data are expressed as the means ± SEM (n = 20). One-way ANOVA test, <i>p</i><0.05^* and <i>p</i><0.001^***, comparing the infected group at 15 dpi with the NI group; dpi: days post-infection; NI: non-infected.</p

Effect of Z-Phe-Ala-FMK, a cysteine protease inhibitor, on TGF-β activation.

<p>Active TGF-β was measured by ELISA after incubation of (A) parasite lysate equivalent to 2.5 × 10⁶ epimastigotes or (B) 100 μg ml^-1 of purified cruzipain for 1 h at 28°C with or without the cysteine peptidase inhibitor, Z-Phe-Ala-FMK, at different concentrations (0.1 to 10 μM). Results are expressed as fold induction, recombinant latent TGF-β (LTGFβ) incubated alone for 1 h at 28°C has been taken as 1 (white bars). Data are the mean ± SD. (*<i>P</i> < 0.05, ** <i>P</i> < 0.01). n = 3.</p

Inhibition of cysteine proteases and TGF-β activities impairs <i>T</i>. <i>cruzi</i> infection and replication.

<p>(A) Different concentrations of Z-Phe-Ala-FMK (1 to 100 μM) or (C) neutralizing anti-TGF-β antibody (1 to 10 μg ml^-1) were added to Vero cell cultures and their effects over the processes of host cell invasion by trypomastigotes of <i>T</i>. <i>cruzi</i> (Y strain) and (B and D) intracellular parasite growth were evaluated. Results are expressed as the percentage of infected cells or as the ratio of parasite number per infected cell, both determined by counting 400 cells per slide in two distinct slides from three independent experiments. Data are the mean ± SD. (*<i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001). n = 3.</p

Transfected parasites overexpressing chagasin prevent TGF-β activation.

<p>(A) Live transfected Dm28c epimastigotes overexpressing chagasin (pCHAG), empty vector (pTEX) or wild type Dm28c (WT) were incubated with latent TGF-β (100 ng ml^-1) (LTGF-β) for 1 h and the activated TGF-β was measured by ELISA. Insert: Dm28c epimastigotes that were not incubated with LTGF-β do not present significant levels of active TGF-β (pg/ml) as compared to the levels of activated LTGF-β incubated for 1h at 37°C—represented by a white bar in Fig 3A). (B) Expression pattern of cruzipain in lysates from <i>T</i>. <i>cruzi</i> Dm28c epimastigotes wild type (WT), transfected with empty vector (pTEX) or overexpressing chagasin (pCHAG) by Western blot. PageRuler Plus Prestained Protein Ladder (Thermo Scientific) was used as the molecular mass marker (MW). Results are expressed as fold induction, recombinant latent TGF-β incubated alone for 1 h at 28°C has been taken as 1. Data are the mean ± SD (*<i>P</i> < 0.05, ** <i>P</i> < 0.01 when compared with WT and ^#<i>P</i> < 0.05, ^##<i>P</i> < 0.01 when compared with pTEX). n = 3.</p

Cruzipain increases <i>T</i>. <i>cruzi</i> invasion through TGF-β activation.

<p>(A) Vero cells (1 × 10⁵ cells per well) were infected with trypomastigote forms of <i>T</i>. <i>cruzi</i> (Y strain) that were added to cultures in a mixture containing purified cruzipain (CZP, 10 μg ml^-1) or anti-TGF-βantibodies (10 μg ml^-1) or FMK (50 μM) or CZP (10 μg ml^-1) in a combination with anti-TGF-β antibody (10 μg ml^-1) or FMK (50 μM). (B) Parasites were pre-treated with different combinations of compounds before addition to Vero cell culture: trypomastigotes were treated or not with Z-Phe-Ala-FMK (FMK, 50 μM) for 30 min at 37°C and then incubated or not with recombinant latent TGF-β (LTGFβ 100 ng ml^-1) or with LTGFβ (100 ng ml^-1) plus CZP (10 μg ml^-1) for 1 h at 37°C. Vero cells were then infected with pre-treated parasites. Cells were fixed 48 h post infection and stained with Giemsa. Representative images are shown (C to H). Arrows indicate infected cells. The percentage of infected cells was determined by counting 400 cells per slide in two distinct slides. Data are the mean ± SD (*<i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001 when compared with controls and ^#<i>P</i> < 0.05, ^##<i>P</i> < 0.01, ^###<i>P</i> < 0.001 when compared with CZP (A) and LTGFβ (B). n = 3.</p

Unraveling Chagas disease transmission through the oral route: Gateways to <i>Trypanosoma cruzi</i> infection and target tissues

<div><p>Oral transmission of <i>Trypanosoma cruzi</i>, the causative agent of Chagas disease, is the most important route of infection in Brazilian Amazon and Venezuela. Other South American countries have also reported outbreaks associated with food consumption. A recent study showed the importance of parasite contact with oral cavity to induce a highly severe acute disease in mice. However, it remains uncertain the primary site of parasite entry and multiplication due to an oral infection. Here, we evaluated the presence of <i>T</i>. <i>cruzi</i> Dm28c luciferase (Dm28c-luc) parasites in orally infected mice, by bioluminescence and quantitative real-time PCR. <i>In vivo</i> bioluminescent images indicated the nasomaxillary region as the site of parasite invasion in the host, becoming consistently infected throughout the acute phase. At later moments, 7 and 21 days post-infection (dpi), luminescent signal is denser in the thorax, abdomen and genital region, because of parasite dissemination in different tissues. <i>Ex vivo</i> analysis demonstrated that the nasomaxillary region, heart, mandibular lymph nodes, liver, spleen, brain, epididymal fat associated to male sex organs, salivary glands, cheek muscle, mesenteric fat and lymph nodes, stomach, esophagus, small and large intestine are target tissues at latter moments of infection. In the same line, amastigote nests of Dm28c GFP <i>T</i>. <i>cruzi</i> were detected in the nasal cavity of 6 dpi mice. Parasite quantification by real-time qPCR at 7 and 21 dpi showed predominant <i>T</i>. <i>cruzi</i> detection and expansion in mouse nasal cavity. Moreover, <i>T</i>. <i>cruzi</i> DNA was also observed in the mandibular lymph nodes, pituitary gland, heart, liver, small intestine and spleen at 7 dpi, and further, disseminated to other tissues, such as the brain, stomach, esophagus and large intestine at 21 dpi. Our results clearly demonstrated that oral cavity and adjacent compartments is the main target region in oral <i>T</i>. <i>cruzi</i> infection leading to parasite multiplication at the nasal cavity.</p></div

Amastigote nests detection by fluorescence microscopy of nasal cavity from mice infected with Dm28c-GFP.

<p>Male BALB/c mice were infected with 1x10⁶ trypomastigotes forms of <i>T</i>. <i>cruzi</i> expressing GFP reporter gene (Dm28c-GFP). At 6 dpi, the nasal cavity tissues were removed, frozen and sections were prepared for fluorescence microscopy analysis. (A) Representative fluorescence image of the nasal cavity from uninfected mice. (B) Representative fluorescence image of the nasal cavity from infected mice. Several amastigote nests (green) can be observed in the nasal cavity. Data represent analysis from an experiment with n = 2.</p