Splenectomy increases host's susceptibility to <i>T. cruzi</i> infection.

<p>BALB/c mice were submitted to surgery to remove the spleen (SX). Sham-operated mice were used as controls. Ten days after surgery, mice were infected intraperitoneally with 2×10⁵ metacyclic trypomastigote forms of Dm28c clone of <i>T. cruzi</i>. Parasitemia was followed during acute phase. In the splenectomized animals, parasitemia was significantly higher, as compared to the sham-operated infected counterparts. Kinetic points with significant differences between SX (<i>n</i> = 07, filled line) and sham-operated (<i>n</i> = 05, dashed line) groups are indicated. Data were compared by Student's <i>t</i> test for independent samples using a Sigma Plot for Windows (version 4.01) package. For parasitemia, data were transformed to parasites/ml for statistical analysis. Data were considered significant if <i>p</i> values were <0.05 (*). Data represent mean±standard error. All experiments and animal handling were conducted according to protocols approved by the Oswaldo Cruz Foundation Committee on the Use of Animals.</p

Differential fluctuations in the cellularity of the thymus, spleen, MLN, and SCLN in the course of acute <i>T. cruzi</i> infection.

<p>Note a lymphocyte expansion in the spleen and SCLN, in parallel to a lymphocyte decrease in the thymus and MLN. BALB/c mice were infected intraperitoneally with 10² blood trypomastigotes of the <i>Tulahuén</i> strain, killed at different days of infection, and cell numbers evaluated by trypan blue exclusion. Erythrocytes were previously depleted in the spleen cell suspensions by treatment with Tris-buffered ammonium chloride. Values represent the mean±standard error; <i>n</i> = 3–5 mice/group in each point. Data recorded on thymus, spleen, MLN, and SCLN from <i>T. cruzi</i>-infected mice (closed squares) were compared to non-infected age-matched controls (open squares) with ANOVA statistical test, using the program SigmaStat (Statistical Software) for Windows. Data were considered significant if <i>p</i> values were <0.05. Data represent mean±standard error. All experiments and animal handling were conducted according to protocols approved by the Oswaldo Cruz Foundation Committee on the Use of Animals.</p

Differential cytokine and apoptosis/proliferation profiles in lymphoid organs of mice undergoing <i>T. cruzi</i> infection.

<p>Differences between normal versus infected mice are indicated with the arrows.</p

The thymus is a target organ in <i>T. cruzi</i> infection.

<p>Panel A shows the number of thymocytes in thymus of <i>T. cruzi</i>-infected mice (C3H/HeJ) during acute (one month) and chronic phases (after three to five months) of infection. Adapted from <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000417#pntd.0000417-LeitedeMoraes3" target="_blank">[32]</a>, *<i>p</i><0.01. Panel B reveals the presence of the amastigote forms of the Colombian strain of <i>T. cruzi</i> within cultured thymic epithelial cells, ascertained by DAPI staining. Note that one cell (arrow) is deeply loaded with trypomastigote forms of the parasite in the cytoplasm. The mouse TEC line (IT-76M1) was cultured with 60 <i>T. cruzi</i> trypomastigotes/TEC for six hours; washed to remove free parasites, and cultured for a further 48 hours. Panel C shows a progressive thymocyte depletion in mice acutely infected with <i>T. cruzi</i>. BALB/c mice were infected intraperitoneally with 10² blood-derived trypomastigotes of the <i>Tulahuén</i> strain, and killed at 14 and 19 days post-infection (d.p.i.). Percentage values of the CD4⁺CD8⁺ thymocytes and respective days post-infection are shown. In normal animals, the percentage of CD4⁺CD8⁺ thymocytes remained the same along with the experimental period.</p

Regional dynamics of immune responses in lymphoid organs following acute experimental <i>T. cruzi</i> infection.

<p>Regional dynamics of immune responses in lymphoid organs following acute experimental <i>T. cruzi</i> infection.</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

Anti-TNF therapy improves the survival of orally infected mice.

<p>Male BALB/c mice were infected with 5x10⁴ tissue culture-derived trypomastigotes forms of <i>T</i>. <i>cruzi</i> (Tulahuén strain) within oral cavity (OI). Anti-TNF treatment with etanercept began after 14 dpi and was performed weekly. A) Parasitemia (mean and SEM of ln parasite/mL) and B) mortality were followed during the acute phase and subjected to statistical analysis. Parasites were counted by light microscopy, and parasitemia calculated by Brener method. Statistical analysis was performed using GraphPad Prism 5. For parasitemia comparisons on days 8, 15, 19 and 22 dpi, one-tailed Mann-Whitney test was used. n: OI+enbrel = 14–23, OI+H₂O = 3–23. Survival was analyzed by Log-rank (Mantel-Cox) (***) and Gehan-Breslow-Wilcoxon (##) tests. n = 20 mice (equivalent to 100%). * p = 0.05; ** p = 0.01; *** p = 0.001.</p

Liver histology during acute <i>Trypanosoma cruzi</i> infection after GI and OI inoculation.

<p>Male BALB/c mice were infected with 5x10⁴ tissue culture-derived trypomastigotes forms of <i>T</i>. <i>cruzi</i> (Tulahuén strain) through gavage (GI) or within oral cavity (OI). A) Hematoxylin and Eosin stained sections demonstrating liver histological lesions in terms of inflammatory foci. For the quantification of inflammatory infiltrate, the relative area of infiltration from 25 fields (200X) was analyzed. Pictures represents cells-rich infiltrated areas. n: GI, 9 dpi = 4, 15–17 dpi = 4, 25 dpi = 5; OI, 9 dpi = 4, 15–17 dpi = 9. B) Immunofluorescence analyses demonstrating the percentage of each subset present within the tissue after 16/17 dpi, CD4⁺, CD8⁺, F4/80⁺ and Ly6G⁺ cells. Numbers represent mean± SEM. n = 3 mice/group (two different section from each mouse). C) ALT and AST activity (17 dpi) in sera. All statistical analyses were performed using one-tailed Mann-Whitney test, GraphPad Prism 5. Comparison between GI and OI groups, and each one of them with uninfected mice. *, p = 0.05; **, p = 0.01; ***, p = 0.001. Bars represent 20 μm. Arrows show inflammatory infiltrates.</p

Cytokine production in GI and OI infected mice.

<p>Male BALB/c mice were infected with 5x10⁴ tissue culture-derived trypomastigotes forms of <i>T</i>. <i>cruzi</i> (Tulahuén strain) through gavage (GI) or within oral cavity (OI). A) In the course of acute infection, serum was isolated and levels of cytokines (IFN-γ, TNF, IL-17, IL-10 and TGF-β) were quantified in uninfected control and infected mice by a multiplex analysis. The results are expressed as the mean values (±SEM) for each group/day post-infection. n: IFN-γ, uninfected (0) = 12; 3 dpi GI = 11, OI = 5; 9 dpi GI = 8, OI = 5; 12 dpi GI = 9, OI = 4; 17 dpi GI = 4, OI = 6. TNF, uninfected (0) = 11; 3 dpi GI = 10, OI = 10; 9, 12 dpi, GI = 3, OI = 3; 17 dpi, GI = 6, OI = 11. IL-17, uninfected (0) = 12; 3 dpi, GI = 10, OI = 10; 9 dpi, GI = 3, OI = 3; 12 dpi, GI = 5, OI = 5; 17 dpi, GI = 6, OI = 14. TGF-β, uninfected (0) = 6; 3 dpi, GI = 4, OI = 4; 9 dpi, GI = 5, OI = 5; 12 dpi, GI = 5, OI = 4; 17 dpi, GI = 2, OI = 5. IL-10 and IL-4, uninfected (0) = 6; 3, 9, 12 dpi, GI = 6, OI = 6; 17 dpi, GI = 3, OI = 8. B) Cytokine gene expression levels were performed by RT-qPCR. Total RNA was isolated from the heart at different days post-infection, and the reaction was performed using SYBR Green Master Mix. HPRT and β-actin were used as housekeeping genes. RT-qPCR data were normalized to the housekeeping genes, and fold changes were determined compared with uninfected controls, using the Expression Suite software. Lymphocytes from subcutaneous lymph nodes of infected mice were stimulated with anti-CD3 and used as positive control of cytokines production (C+ Lymph). Statistical analysis was performed using ΔCt values. n: uninfected (0) = 5–9; 9 dpi GI = 3, OI = 2–3; 17 dpi GI = 2–3, OI = 2–3. Both sets of data were analyzed using one-tailed Mann-Whitney test, GraphPad Prism 5. *, p = 0.05; **, p = 0.01; ***, p = 0.001. *GI versus OI. Kruskal-Wallis (Dunn’s post-test) for group kinetics: A) IFN-γ, TNF and IL-10 increased in both groups compared with uninfected. IL-17 presented a non-significant increase followed by decrease. TGF-β was only elevated in GI group at 12 dpi in sera. B) IFN-γ increased in both groups compared with uninfected. TNF and IL-10 increased in OI whereas TGF-β decreased in GI.</p

F4/80⁺ cells are the major TNF-producing cells.

<p>Male BALB/c mice were infected with 5x10⁴ tissue culture-derived trypomastigotes forms of <i>T</i>. <i>cruzi</i> (Tulahuén strain) through gavage (GI) or within oral cavity (OI). Immunofluorescence analyses demonstrated the percentage of TNF-producing cells among each subset present within the tissue after 16/17 dpi, CD4⁺, CD8⁺, F4/80⁺ and Ly6G⁺ cells. A) heart and B) liver. Numbers represent mean±SEM. n = 3 mice/group (two different sections from each mouse).</p