Figure 3. Effect of aspartyl PIs on Leishmania aspartyl peptidase activity. L. amazonensis soluble extract was incubated with 2 mM HIV-1 peptidase substrate for 15 min at 37uC in 100 mM sodium acetate, 1 M sodium chloride, 1 mM EDTA, 1 mM DTT, 10% DMSO, 1 mg/ml BSA, pH 4.7, in the absence (control) or in the presence of different concentrations of the following PIs: nelfinavir, amprenavir, lopinavir and pepstatin A (0.1 to 10 mM). The control (16.664.5 arbitrary fluorescence units) was taken as 100%. The values represent the mean6standard deviation of three independent experiments performed in triplicate. Symbol denotes systems treated with PIs that had a rate of substrate hydrolysis significantly different from the control (P,0.01; Student’s t test). doi:10.1371/journal.pone.0004918.g003 restricted to cutaneous leishmaniasis has often been observed in patients with Leishmania–HIV co-infection [2]. HIV infection also reduces the likelihood of a therapeutic response, and greatly increases the probability of relapse [25–27]. At the same time, Figure 4. Effect of the pre-treatment of promastigotes with HIV PIs on the Leishmania amazonensis–macrophage interaction. The promastigotes of L. amazonensis were treated or not with nelfinavir, amprenavir or lopinavir (as indicated) for 1 hour prior to macrophage– parasite interactions, then the cells were washed with PBS. Parasites maintained their viability under this experimental condition (see Methods section). Macrophages were then infected with promastigote forms for 1 hour at 37uC, and macrophage monolayers were washed with PBS to remove unbound parasites. The association index was determined after 1 hour of infection by light microscopy, counting at least 200 cells in each of duplicated coverslips. Each bar represents the mean6standard error of at least three independent experiments. doi:10.1371/journal.pone.0004918.g004 HIV PIs on L. amazonensis Figure 5. Toxicity of HIV PIs in mouse peritoneal macrophages and susceptibility of intracellular parasites to HIV PIs in macrophages. Initially, the macrophages (16105 cells) were incubated in a 96-well plate for 24 hours in the absence or in the presence of different concentrations (as indicated) of the following PIs: nelfinavir, amprenavir and lopinavir. After this period, the viability of the macrophage cells was determined spectrophotometrically at 490 nm by means of MTT assay. The dotted line separates the graphic in two portions: minor than and equal or major than 95% of macrophage viability. Control represents untreated macrophages (A). After that, we studied the susceptibility of intracellular parasites to HIV PIs in macrophages. Macrophages were infected with promastigotes of L. amazonensis for 1 hour at 37uC, followed by exhaustive washing in PBS and then the interaction was incubated for additional 24 hours in the absence (control), or in the presence of nelfinavir, amprenavir or lopinavir as indicated. The concentrations employed correspond to those in which more than 95% of the macrophage cells were viable as demonstrated in (A). Finally, the association index was determined by light microscopy, counting at least 200 cells in each of duplicated coverslips. Control system is shown as 100%. Each bar represents the mean6standard error of at least three independent experiments (B). doi:10.1371/journal.pone.0004918.g005 visceral leishmaniasis promotes the clinical progression of HIV disease and the development of AIDS-defining conditions. Both diseases exert a synergistic detrimental effect on the cellular immune response because they target similar immune cells [2]. Patients with HIV infection are surviving longer and with an improved quality of life as a result of the institution of HAART, regimens that usually consist of a PI combined with at least two other antiretroviral drugs [28,29]. PIs bind competitively to the cleavage site within the HIV-1 aspartyl peptidase enzyme. They render the enzyme non-functional and lead to the release of PLoS ONE | www.plosone.org 7 March 2009 | Volume 4 | Issue 3 | e4918