Effect of Gal–1 on phosphatidylserine exposure in <i>T</i>. <i>cruzi</i> infected HL–1 cells.

<p>Cells were incubated with rGal–1 (10 and 50 μg/ml) for 18 h and, then infected with <i>T</i>. <i>cruzi</i>, Tulahuén (A) or Brazil (B) strains. Annexin V assay was performed at 3 dpi. Results expressed as mean ± SEM are representative of two independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey test. *<i>p</i><0.05; **<i>p</i><0.01. Only comparisons between infected groups were shown.</p

Expression and release of Gal–1 in cultures of HL–1 cells infected with <i>T</i>. <i>cruzi</i>.

<p>Cells were infected with trypomastigotes of Tulahuén or Brazil strains, in a parasite:cell ratio of 5:1, and incubated for additional 2 or 5 days. A) Immunoblot analysis of Gal–1 expression in lysates from non-infected (a) and infected (b) HL–1 cells. Immunoreactive protein bands were semiquantified by densitometry. Results are expressed as Arbitrary Units (AU) relative to β-actin. B) RT-qPCR analysis of Gal–1 mRNA expression of non-infected and infected HL–1 cells. Results are expressed as relative to GAPDH mRNA. C) Detection of Gal–1 in the supernatant of non-infected and infected HL–1 using trypomastigotes of the Tulahuén and Brazil strains, as measured by ELISA. D) Detection of LDH activity in the supernatants of non-infected and infected HL–1 cells by using the LDH-UP kit (Weiner Lab, Argentina), following the manufacturer’s instructions. Results are expressed as Units/ml (U/ml). Data represent the mean ± SEM of three (A and B) and two (C and D) independent experiments. Statistical analysis was performed using Student’s <i>t</i> test for data shown in A (a <i>vs</i> b) and using one-way ANOVA followed by Tukey test in the remaining experiments. *<i>p</i><0.05; ***<i>p</i><0.001.</p

Effect of exogenous rGal–1 in <i>T</i>. <i>cruzi</i> infection.

<p>HL–1 cells were incubated with rGal–1 (10 and 50 μg/ml) for 24 h and then infected with trypomastigotes of both strains. After 4 dpi with <i>T</i>. <i>cruzi</i> Tulahuén (A) or Brazil strain (D), cells were fixed and stained with an anti-<i>T</i>. <i>cruzi</i> mouse serum. Representative images are shown in (B) and (E). Similar experiments were performed after 2 dpi with <i>T</i>. <i>cruzi</i> of the Tulahuén (C) or Brazil strains (F). In this case, some wells were treated with 100 mM lactose, added simultaneously with rGal–1. G) HL–1 cells transfected with pcDNA3-Gal–1 vector or empty vector (mock) were infected with trypomastigotes of both strains, in the presence or absence of 100 mM lactose. Cells were fixed and stained after 2 dpi, with an anti-<i>T</i>. <i>cruzi</i> mouse serum. In all cases, the percentage of infected cells was determined by counting an average of 3,500 cells in each slide on 3–5 distinct coverslips in randomly selected fields. Results are expressed as mean ± SEM of triplicates determinations from three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey test. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001.</p

Histopathological findings in <i>Lgals1</i>^<i>-/-</i> and WT mice at 19 dpi with <i>T</i>. <i>cruzi</i> Tulahuén strain.

<p>A) Microphotographs representative of heart and skeletal muscle histopathological abnormalities (H&E). Parasite density (B) and Inflammation Index (C) were calculated as indicated in the Methods section. Bars represent mean ± SEM of 5–7 mice per group. Statistical analysis was performed using Mann-Whitney U test. *<i>p</i><0.05. F: Female mice; M: Male mice.</p

Binding of rGal–1 to <i>T</i>. <i>cruzi</i> trypomastigotes.

<p>A) Fluorescence assay of trypomastigotes incubated with rGal–1 (25 μg/ml) for 1 h, followed by incubation with a mouse anti-Gal–1 Ab labeled with Alexa Fluor 488. Staining with a rabbit polyclonal serum anti-Tc13, a surface protein presented in trypomastigotes, was used as positive control. B) Representative histograms of trypomastigotes of the Tulahuén or Brazil strain incubated with Gal-1-FITC (25 μg/ml). Red lines correspond to parasites treated with Gal-1-FITC, black lines to parasites incubated with streptavidin-FITC used as negative control.</p

Parasitemia levels (A) and survival rate (B) of WT and <i>Lgals1</i>^<i>-/-</i> mice acutely infected with <i>T</i>. <i>cruzi</i> Tulahuén strain, via the intraperitoneal route.

<p>For parasitemia levels, each point represents the mean ± SEM of 5–15 animals per group, and statistical analysis was performed using Mann-Whitney U test. *<i>p</i><0.05, **<i>p</i><0.01 <i>vs</i>. WT mice; ^$<i>p</i><0.05, ^$$<i>p</i><0.01 <i>vs</i>. male mice. For survival rate, statistical analysis was achieved with Log-rank test.</p

Characteristics of patients with chronic Chagas' disease Cardiomyopathy.

a<p>F: female, M: male.</p>b<p>Patient's heart failure was classified according to New York Heart Association (NYHA) Functional Classification. Class I: Patients with cardiac disease with slight functional alterations but resulting in no limitation of ordinary physical activity; however, elevated activity causes symptoms, such as fatigue, palpitation, or dyspnea (shortness of breath); Class II: Patients with cardiac disease resulting in slight limitation of physical activity; comfortable at rest, but ordinary physical activity results in symptoms; Class III: Patients with cardiac disease resulting in marked limitation of physical activity; comfortable at rest, but less than ordinary activity causes symptoms; Class IV: Patients with cardiac disease resulting in marked limitation of physical activity, unable to carry out any physical activity without discomfort; symptoms of cardiac insufficiency at rest.</p><p>Patients with cardiac disease without any functional alteration were classified as Class 0.</p>c<p>The level of antibodies directed to <i>T. cruzi</i> lysate was determined by in-house ELISA as described upon <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002906#s2" target="_blank">Methods</a>.</p>d<p><i>T cruzi</i> lineage was analyzed by TSSA recognition as described upon <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002906#s2" target="_blank">Methods</a>. Results are shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002906#pntd.0002906.s001" target="_blank">Figure S1</a>. ND: not detected.</p

Parasite lysate but not ribosomal proteins trigger PBMC proliferative responses.

<p>PBMC isolated from chronic Chagas' disease Cardiomyopathy patients (CCC; n = 27) and non-infected individuals (NI; n = 20) were seeded at 2.5×10⁵ cells/well and stimulated with <i>T. cruzi</i> lysate, P2β or CP0 proteins (10 µg/ml) or medium alone for 6 days. Cell proliferation was determined by ³H-thymidine incorporation. Results are expressed as Stimulation index, calculated as: (mean cpm of stimulated cultures/mean cpm of non-stimulated cultures (medium only)). Each symbol represents data from a single subject. Statistical analysis was performed using the Mann-Whitney U Test, ***<i>P<</i>0.001.</p

Humoral response against ribosomal P proteins and their C-terminal peptides.

<p>The presence of antibodies directed against P2β and CP0 proteins as well as peptides R13, P015 and H13 in the sera of 27 patients with chronic Chagas' disease Cardiomyopathy patients (CCC) and 20 non-infected individuals (NI) was determined by ELISA as described under <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002906#s2" target="_blank">Methods</a>. Results are expressed as Reactivity index, calculated as: (Optical Density mean value obtained of each serum sample/baseline value). Each symbol represents data from a single subject. Statistical analysis was performed using the Mann-Whitney U Test, <i>***P<</i>0.001, *<i>P<</i>0.05. The line for each of the scatters represents the median.</p

Activation markers on CD4+ and CD8+ T cell subsets upon <i>T. cruzi</i> and ribosomal protein activation.

<p>PBMC isolated from patients with chronic Chagas' disease Cardiomyopathy (CCC; n = 27) and non-infected individuals (NI; n = 20) were seeded at 2.5×10⁶ cells/well and stimulated with <i>T. cruzi</i> lysate, P2β or CP0 proteins (10 µg/ml) or medium alone for 6 days. PBMC were stained with CD3-APC, CD4-PE-Cy5 or CD8-PE-Cy5 and activation marker-specific labeled antibodies (CD25-FITC and HLA-DR-PE) prior to flow cytometry analysis. 10,000–15,000 events in the lymphocyte gate (R1 gate) were acquired using a FACSAria flow cytometer (Becton Dickinson); dead cells were excluded by forward <i>vs</i> side-scatter (FSC/SSC) gating. A) Gate-pathway used to determine the activation expression in the populations graphed in B. B) Results were expressed as the percentage of CD25+ or HLA-DR+ cells in CD3+CD4+ (R2 gate) or CD3+CD8+ (R3 gate) lymphocytes. Horizontal lines represent the median and percentiles 25–75th, vertical lines represent percentiles 5–95th. Statistical analysis was performed using the Mann-Whitney U Test, ***<i>P<</i>0.001, **<i>P<</i>0.01, *<i>P<</i>0.05.</p