were analyzed statistically using Student’s t test using EPI-INFO 6.04 (Database and Statistics Program for Public Health) computer software. P values of 0.05 or less were considered statistically significant. Results Effect of HIV PIs on the growth In order to establish whether the aspartyl HIV PIs might have any effect on L. amazonensis multiplication, we performed experiments in which each PI was added to replicating promastigote forms (56107 cells) at different concentrations and cell growth was monitored for 4 days in vitro. Only lopinavir and nelfinavir significantly reduced leishmanial development at 50 mM (P,0.05) (Fig. 1, HIV PIs). After this preliminary screen, each inhibitor was tested in the appropriate concentration range. All the PIs inhibited the parasite growth in a dose-dependent manner (Fig. 1). Nelfinavir and lopinavir induced a powerful reduction in the cellular growth rate from 20 and 25 mM concentration on, respectively. The IC50 after 48 hours was 15.1261.1 mM and 16.4760.8 mM, respectively. Amprenavir significantly impaired parasite multiplication only after 48 hours of growth at 100 mM (P,0.05). This drug at 250 mM virtually blocked parasite proliferation, while at 500 mM, parasite death was detected (Fig. 1, Amprenavir). The IC50 after 48 hours was 62.062.1 mM. Indinavir only at the highest concentration and after 72 hours of cultivation significantly altered parasite growth (Fig. 1, Indinavir). Conversely, saquinavir at the highest concen- tration used only marginally diminished the L. amazonensis proliferation (Fig. 1, Saquinavir). Pepstatin A was assayed in parallel for comparative purposes. This inhibitor powerfully diminished parasite growth at 50 mM (data not shown). DMSO did not significantly affect parasite growth behavior. Effect of HIV PIs on the ultrastructure Based on the efficacy of the HIV aspartyl PIs in diminishing the growth of L. amazonensis, particularly nelfinavir and lopinavir, we next investigated the effect of these inhibitors in the Leishmania ultrastructure, by transmission electron microscopy. For this purpose, the morphology of non-treated cells (Fig. 2A) was compared with the ultrastructure of PIs-treated protozoa (Fig. 2B– J). Results showed blebs detaching from the entire cell surface (Fig. 2B–C, arrowheads), including the flagellar membrane (Fig. 2D, arrowheads), after 4 hours of treatment using the IC50 concentration (15.12 mM to nelfinavir and 16.47 mM to lopinavir). Interestingly cells treated with nelfinavir, but not with lopinavir, presented cytoplasm shrinking (Fig. 2B,E, ), a higher number of vesicles, which according to their electron-density, probably corresponds to lipid-containing compartments (Fig. 2E,F, v) or acidocalciosomes (Fig. 2G, w). Cells treated with both drugs presented the nucleus surrounded by endoplasmic reticulum (Fig. 2G–H, black arrows), mitochondrial swelling (Fig. 2F, white arrowhead) and myelin-like structures (Fig. 2H, larger arrow). After 24 hours of treatment with lopinavir, extensive blocks of condensed chromatin were observed close to the nuclear envelope (Fig. 2I–J, white arrows), which is suggestive of apoptosis, as well as enlarged vesicles (Fig. 2J, ). These effects were commonly visualized in the drug-treated population. Effect of HIV PIs on Leishmania aspartyl peptidase The effect of distinct HIV aspartyl PIs on the hydrolytic activity of L. amazonensis extract over HIV-1 peptidase substrate was determined. To this end, nelfinavir, amprenavir and lopinavir, as well as pepstatin A were tested in concentrations ranging from 0.1 HIV PIs on L. amazonensis to 10 mM. The inhibitory capability of these compounds was very divergent (Fig. 3). Lopinavir was by far the most effective inhibitor, reducing the proteolytic hydrolysis of the substrate by approxi- mately 90% at 1 mM, and virtually abolishing the proteolytic activity at 10 mM, while at 0.1 mM, the inhibitor exerted no significant effect in relation to control. Nelfinavir exhibited an inhibition of approximately 98% at 10 mM, however, at 1 mM, the inhibitor was ineffective, while amprenavir even at 10 mM did not significantly inhibited the hydrolysis of the substrate by L. amazonensis extract. The inhibition of hydrolysis by lopinavir was comparable to that exerted by pepstatin A, which is a prototypal inhibitor of aspartyl-type peptidases (Fig. 3). Effect of HIV PIs on the interaction of Leishmania- macrophage cells The effects of PIs on the L. amazonensis-macrophage interaction are shown in Fig. 4 and 5B. The promastigote forms of the parasite were treated for 1 hour before interaction with the PIs that showed more effectiveness in suppressing parasite proliferation in vitro, i.e., nelfinavir, lopinavir and amprenavir. At the drug concentra- tions tested (50 and 100 mM for nelfinavir and lopinavir; and 100 and 200 mM for amprenavir), the parasites maintained their viability after the treatment for 1 hour with each PI, as judged by their morphology, motility and propidium iodine staining, in which more than 95% of the promastigotes were viable (data not shown). Then, the drugs were washed away and the parasites were allowed to interact with macrophages for 1 hour, free parasites were removed and the cells incubated for an additional hour, then the association indexes were determined. We showed that nelfinavir was the most potent inhibitor of the interaction process between L. amazonensis and macrophage cells, showing a clear dose-dependent inhibition profile, where the inhibition increased from 86 to 93% (in relation to control) as nelfinavir concentration rose from 50 to 100 mM. When parasites were pretreated with 100 or 200 mM of amprenavir, the association indexes were, respectively, 75 and 81% lower as compared to the control system. Lopinavir was also capable of interfering in the interaction process; however, a dose-dependent inhibition profile was not prominent, being around 80% either at 50 or 100 mM (Fig. 4). Given that PIs can interfere in the early steps of parasite infection, since the inhibitors were added exclusively to Leishmania promastigotes and that the interaction process was stopped with only 1 hour, we resolved to investigate the association index of L. amazonensis with macrophage cells during the in vitro treatment for 24 hours with aspartyl PIs used in the chemotherapy of HIV. First of all, we tested the effect of these three PIs alone on the mouse peritoneal macrophages viability by using the MTT assay. A significant deleterious effect was only observed with lopinavir at 25 mM, while amprenavir also at this concentration was in the boundary between non-toxic and toxic effects (Fig. 5A). Therefore, in the next section of these experiments, we used the PIs at the maximal concentrations in which more than 95% of macrophages were viable after 24 hours of treatment. Finally, the macrophages were preinfected with L. amazonensis for 1 hour, unbound parasites were washed away, and then treated with nelfinavir, lopinavir or amprenavir for 24 hours. Our results evidenced that all the tested aspartyl PIs notably reduced intracellular number of Leishmania in macrophages in a dose dependent manner. Effect of HIV PIs on the expression of classical leishmanial peptidases In this set of experiments we aimed to assess if the stress induced by the PIs would led to any change in the pattern of peptidase PLoS ONE | www.plosone.org 4 March 2009 | Volume 4 | Issue 3 | e4918