Heterologous expression of <i>T. cruzi</i> Ecto-NTPDase-1 and the effect of ecto-ATPase inhibitors on purified protein.

<p>A) SDS-PAGE stained with coomassie blue. A 15 µL sample of each step of production and purification of Ecto-NTPDase-1 was applied in each lane, showing the purified protein with approx. 66 kDa. MW, molecular weight markers. B) ATPDase activity of purified Ecto-NTPDase-1 in the presence of ecto-ATPase inhibitors. The results represent the percent activity with inhibitor related to the activities in the absence of inhibitors. Data are mean±SE of two independent experiments, each assayed in triplicate.</p

<i>T. cruzi</i> infectivity and ecto-ATPase/ADPase ratio decrease during in vitro cultivation.

<p>A) Ecto-ATPDase (solid bars) and Ecto-ADPDase (open bars) activities from live trypomastigotes from different cellular passages (P1, P3 and P4). P1-1 and P1-2 are the first and second massive exits of parasites from the 1^st passage; in the same way P3-1 and P3-2 are the first and second massive exits of parasites for the 3^rd passage. Data are mean±SE of triplicate assays from one experiment. The inset shows the ATPase/ADPase hydrolytic activities ratio. B) Microphotograph of infected VERO cells culture after 24 h of parasite-cell interaction at the 3^rd to 4^th passage. Spherical bodies are non-internalized amastigote-like parasites. C) Zoom from box section shown in B. Black arrow exemplifies a non-internalized amastigote-like and white arrow a non-internalized trypomastigote parasite.</p

Effect of inhibitors in the ecto-ATPDase activity of Y strain P1 trypomastigotes.

<p>Parasites were pre-incubated for 10 min with different concentrations of Suramin (Panel A), ARL 67156 (Panel B) or Gadolinium (Panel C) and ecto-ATPDase activities were measured. Ecto-ATPase (Δ) and ecto-ADPase (□) activities are expressed as percentage of control activity (without inhibitors). Data are mean±standard error of two independent experiments in triplicate.</p

Effect of apyrase inhibitors on <i>in vitro</i> infectivity of trypomastigotes.

<p>Percent inhibition of infection was calculated for each inhibitor assay relative to control data. Data reflect the mean±SE from three analyzed slides. Asterisks indicate significant differences (p<0.05) between the control without inhibitor and the test with ATPDase inhibitor.</p

Ecto-nucleotidase activities of trypomastigotes from different strains/clone of <i>T. cruzi</i>.

<p>Trypomastigotes were obtained from the first VERO cells passage (P1). The Ecto-ATPDase activities were measured at 37°C during 1 hour. Data are mean±SE of two independent experiments in triplicate.</p

Course of infection of <i>Leishmania (V.) braziliensis</i> isolates in C57BL/6J mice.

<p>C57BL/6J mice were inoculated in the footpad of the left hind leg with 10⁷ promastigotes obtained from mucosal (ML)/mucocutaneous (MCL) lesions (A) or cutaneous lesions (CL) (B). Lesion sizes were measured weekly. The lesion size was defined as the difference between the infected and uninfected contralateral footpad from two independent experiments with four mice per group with the exception of isolates RS, SAP, IMG3 and RPL5 (one experiment each). Each line represents a distinct isolate and was drawn based on the mean lesion size for each time point. Error bars were not included to facilitate visualization. Eleven weeks after infection, animals were sacrificed and the relationship between lesion size and clinical form (C) as well the parasite load by limiting dilution technique was determined (D). Each point represents a different isolate. The line represents the mean of the group. Statistical analysis was performed by Students's <i>t</i>-test.</p

Promastigotes with high ecto-nucleotidase activity inhibit NO production by activated macrophages.

<p>J774-macrophages were infected with promastigotes of <i>L. (V.) braziliensis</i> isolates (5 parasites/cell) for 3 and 72 hr, in presence or not of IFN-γ/LPS. (A) Percentage of infected cells and ATPase activity of promastigotes (nmolPi/10⁸ parasites/hr). (B) Number of parasites per infected macrophage. (C) NO production in 72 hr supernatants. Bars represent the mean+SD of two independent experiments performed in duplicates. (*) indicates statistical difference (p<0.05). Statistical analysis was performed by Students's <i>t</i>-test.</p

<i>L. (V.) braziliensis</i> isolates differ in their ability to hydrolyze adenine nucleotides.

<p>Promastigotes were isolated on the 5th day of culture and incubated with ATP (A), ADP (B) and AMP (C) for 1 hr at 30°C. Enzymatic activity was evaluated by the measurement of inorganic phosphate released. Bars represent the mean+standard deviation (SD) of three or more independent experiments performed in triplicates. Significant differences are shown below each graph. Statistical analysis was performed by one-way ANOVA followed by Bonferroni post-test.</p

Peak of lesion development correlates with source of <i>Leishmania (V.) braziliensis</i> isolates.

<p>C57BL/6J mice were inoculated in the footpad of the left hind leg with 10⁷ promastigotes. Lesion sizes were measured weekly. The lesion size was defined as the difference between the infected and uninfected contralateral footpad from two independent experiments with four mice per group with the exception of isolates RS, SAP, IMG3 and RPL5 (one experiment each). Each point represents the time at which lesion development reached its maximum size for each isolate obtained from mucosal (ML)/mucocutaneous (MCL) lesions or cutaneous lesions (CL). The line represents the mean of the group. Statistical analysis was performed by Students's <i>t</i>-test.</p

Ecto-nucleotidase activity and infectivity in J774-macrophages of amastigotes from <i>L. (V.) braziliensis</i> isolates.

<p>(A–C) Amastigotes were isolated from infected J774 macrophages and incubated with ATP (A), ADP (B) and AMP (C) for 1 hr at 30°C in pH 7.2 or pH 5.5. Enzymatic activity was evaluated by the measurement of inorganic phosphate released. Bars represent the mean+standard deviation (SD) of three or more independent experiments performed in triplicates. (D–F) J774-macrophages were infected with amastigotes of <i>L. (V.) braziliensis</i> isolates (5 parasites/cell) for 3 and 72 hr, in presence or not of IFN-γ/LPS. (D) Percentage of infected cells. (E) Number of parasites per infected macrophage. (F) NO production in 72 hr supernatants. Bars represent the mean+SD of two or more independent experiments performed in duplicates.</p