HIV PIs on L. amazonensis Figure 6. Effect of HIV PIs on peptidase expression by L. amazonensis. Differential peptidase expression observed in L. amazonensis promastigote forms grown in the absence (control) or in the presence of nelfinavir, amprenavir and lopianvir at 100 mM. Gelatin-SDS-PAGE: The gel strips, containing the equivalent to 56106 cells of parasite extract obtained after 4 hours of incubation with each inhibitor, were incubated at 37uC for 48 hours in 50 mM sodium phosphate buffer (pH 5.5) supplemented with 2 mM DTT. Numbers on the left indicate relative molecular mass of the peptidases, expressed in kilodaltons (kDa). Immunoblotting: anti-gp63 or anti-cpb antibodies were employed to detect gp63- and cpb-like molecules, respectively, in the whole cellular extract from L. amazonensis grown in the absence (control) or in the presence of nelfinavir, lopianvir and amprenavir for 4 hours at 26uC; Anti-tubulin monoclonal antibody was used as a control for sample loading in the gels. The graphics represent the densitometric measurements of the proteolytic halos observed in the gelatin-SDS-PAGE. The values represent mean6standard deviation of three independent measurements. Similar densitometric analyses were performed using the reactive polypeptides detected in the immunobloting assays (data not shown). Symbol denotes systems treated with PIs that had densitometric units significantly different from the control (P,0.05; Student’s t test). doi:10.1371/journal.pone.0004918.g006 immature, non-infectious viral particles [30]. Moreover, it has been demonstrated that PIs have advantageous effects on some opportunistic infections caused by fungal pathogens, and also some protozoa, both known to be the major causes of morbidity and mortality in AIDS subjects, due to restoration of the host immune system and a direct action on the pathogens. Although there are several studies concerning Leishmania/HIV co-infection, some particularities of its epidemiology, pathogenesis, prophylaxis and especially of its treatment remain unclear and undefined [1,31]. Based on these preceding evidences, we studied the implication of distinct HIV PIs on the human protozoan parasite L. amazonensis, focusing on the effects in the peptidase activity, parasite proliferation, ultrastructure and interaction with animal cells. Promastigote forms of L. amazonensis were cultured with pepstatin A, nelfinavir, indinavir, saquinavir, lopinavir and amprenavir up to 96 hours, and the parasite survival was analyzed. Pepstatin A promoted a powerful reduction on the growth of L. amazonensis. Pepstatin-like drugs, however, are not used clinically because of their metabolism in the liver and rapid clearance from blood [32]. Nelfinavir and lopinavir robustly inhibited the L. amazonensis growth at 25 mM, in contrast to the other PIs tested. It should be pointed out that the inhibitory effect PLoS ONE | www.plosone.org 8 March 2009 | Volume 4 | Issue 3 | e4918