HIV PIs on L. amazonensis Figure 1. Effect of HIV PIs on the growth rate of Leishmania amazonensis. The growth pattern of L. amazonensis was followed for parasites cultivated at 26uC in the absence (control) or presence of lopinavir, nelfinavir, amprenavir, saquinavir and indinavir at 50 mM (HIV PIs). Subsequently, lopinavir and nelfinavir were assayed in concentrations ranging from 15 to 50 mM. Amprenavir, saquinavir and indinavir were tested in concentrations ranging from 50 to 500 mM. The PIs were added to the cultures at 0 hour and viable cells were counted daily by trypan blue exclusion and motility. In all experiments, the data from DMSO represents the concentration present in the highest dose of each compound. Data shown are the mean6standard error (S.E.) of three independent experiments performed in triplicate. doi:10.1371/journal.pone.0004918.g001 production by L. amazonensis. Cells were incubated for 4 hours in the presence of nelfinavir, lopinavir and amprenavir at 100 mM and assayed by zymography. At this experimental condition, the parasites maintained their viability, as judged by their morphol- ogy, motility and propidium iodine staining, in which more than 95% of the promastigotes were viable (data not shown). The proteolytic profile of L. amazonensis in gelatin-SDS-PAGE at pH 5.5 supplemented with DTT has been previously described and is composed mainly of metallo- and cysteine peptidases [16,17,22]. The comparison of the peptidase expression by gelatin- SDS-PAGE in parasites incubated or not for 4 hours with the PIs revealed that both the 63-kDa metallopeptidase and the low- molecular mass cysteine peptidases had their activity enhanced when parasites were subjected to PIs (Fig. 6). In order to further determine the influence of the PIs on classical peptidases from L. amazonensis, we have performed western blotting analysis of these molecules employing anti-gp63 or anti-cpb antibodies. In agreement with the gelatin-SDS-PAGE analysis, the gp63 molecule (a 63 kDa polypeptide) and the cpb (17 and 31 kDa) were enhanced when the parasites were incubated with nelfinavir, amprenavir and lopinavir. In all the methods employed, lopinavir promoted the most pronounced effect. For sample loading control, we have revealed the western blotting with anti-a-tubulin, which showed no difference between the experimental systems (Fig. 6). Discussion Leishmaniasis is one of the most neglected tropical diseases [1], and the HIV/AIDS pandemic has changed the natural history of leishmaniasis [23]. The number of co-infections is increasing PLoS ONE | www.plosone.org 5 March 2009 | Volume 4 | Issue 3 | e4918