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

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

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

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

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

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

Evaluation of clinical specimens using LAMP assay.

<p>A. Visualization by Naked Eye: 1. positive control; 2. CCD6 Chronic Chagas Disease (case 6); 3. NI8: non-infected patient, (case 8); 4. AI-TxRID 2: acute infection of transplanted recipient from infected donor (case 2); 5. RCD 1: reactivated Chagas disease (case 1); 6. CCD1: chronic Chagas disease 1 (case 1); 7. CI 4: congenital Chagas disease (case 4); 8. negative control. B. Detection of LAMP reaction using Genie III Fluorimeter. 1: positive control; 2 to 7: clinical specimens indicated in A; 7: Negative control. The Y Axis denotes Fluorescence and X axis denotes Tt (time when fluorescence passes the threshold).</p

Inclusivity of LAMP assay tested in purified DNA samples from <i>T</i>. <i>cruzi</i> strains representative of the different discrete type units.

<p>Left panels: Visualization by the naked eye of <i>T</i>. <i>cruzi</i> DNA stocks representative of different DTUs. Tc I (A: 1.0 x10^-2 fg/test; B: 1.0 x10^-3 fg/test); Tc II (C: 2.5 fg/test; D: 2.5x10^-1 fg/test); Tc III (E: 7.5 x 10⁻² fg/test; F: 7.5 x 10⁻³ fg/test); Tc IV (G: 5.0 x 10⁻¹ fg/test, H: 5.0 x 10⁻² fg/test); Tc V (I: 1.5 x 10⁻¹ fg/test; J: 1.5 x 10⁻² fg/test); Tc VI (K: 1.0 x 10⁻¹ fg/test; L. 1.0 x 10⁻² fg/test) Right panels: Amplification plots obtained in the LAMP reaction after analyzing the samples in a Rotor Gene 3000 thermocycler. Y axis represents fluorescence and x axis represents Cts (Threshold cycles). Only the highest dilution giving amplification and the next dilution giving non detectable results are shown.</p

Analytical sensitivity of LAMP assay.

<p>Top panel: Fluorescence observed with the naked eye from serial dilutions obtained from 3 different aliquots of DNA from <i>CL</i> Brener stock (DTU VI). Bottom panel: Fluorescence observed with the naked eye from serial dilutions obtained from 3 different aliquots of DNA from Sylvio X10 stock.The aliquots were expressed in fg/μL. A: 0; B: 1.0 x 10⁻³; C: 1.0 x 10⁻²; D: 1.0 x 10⁻¹, E: 1. NC: Non template control.</p

INCLUSIVITY in <i>Trypanosoma cruzi</i> strains representative of different DTUs (fg/test).

<p>INCLUSIVITY in <i>Trypanosoma cruzi</i> strains representative of different DTUs (fg/test).</p