The murine infection is one of the best characterized experimental models for studying Leishmania-mammalian host cells interplays [48]. Data from infection of peritoneal mouse macrophages by L. amazonensis revealed that the parasites pretreated with PIs had association indexes considerably lower in comparison to control system. Taken together, these results suggest interference in early steps of macrophage infection either through modulation of yet unidentified virulence factors, blockade of ligand/receptor binding, suppression of ligand/receptor expression or due to direct non-specific toxic effects of the inhibitor. It should be pointed out that these interaction inhibition profiles were not caused by a decreased viability of L. amazonensis cells, as assessed by propidium iodide staining. The identification of the molecular mechanism by which the HIV PIs interfere with promastigote interaction with mammalian cells remains an open question. It has been previously demonstrated that inhibitor compounds effectiveness may depend on the developmental stage of the parasite. For instance, L. mexicana amastigotes developing within macrophages during human infection are more sensitive to lactacystin than promastigotes developing in culture medium [49], which may be explained by a distinct action of the drugs on promastigotes alone and on amastigotes in an intracellular environment. In order to assess if the same applies to HIV PIs, we evaluated the effect of nelfinavir, amprenavir and lopinavir on the Leishmania development inside target cells. The drug concen- trations employed in this assay showed no direct toxicity on macrophages, while they drastically reduced the association index at considerably lower doses than those required to interfere with promastigotes. Whether the inhibitors are acting directly in the amastigotes or indirectly, modulating the killing capability of the macrophages, remains an open question. Alternatively, macro- phages could concentrate higher levels of PIs. Accordingly, it was shown in L. chagasi that higher drug concentrations are necessary to interfere with axenic amastigotes when compared to intracel- lular amastigotes [36]. Finally, even the lower doses required to inhibit amastigote development are still higher than those needed for the inhibition of HIV progression in humans. However, we have to consider that HAART consists of a combination of antiretroviral drugs and the pharmacodynamics of an in vitro model is very different from those in humans. Moreover, the HIV PIs were designed to fit viral peptidases and may thus have a lower affinity for aspartyl peptidases of L. amazonensis. Additional in vivo studies will help to elucidate this issue, and also further efforts References 1. World Health Organization (2007) Report of the Fifth Consultative Meeting on Leishmania/HIV Co-infection. WHO, p. 1–38. Addis Ababa, Ethiopia: World Health Organization. 2. Alvar J, Aparicio P, Aseffa A, Den Boer M, Canavate C, et al. (2008) The relationship between leishmaniasis and AIDS: the second 10 years. Clin Microbiol Rev 21: 334–359. 3. Amato VS, Tuon FF, Bacha HA, Neto VA, Nicodemo AC (2008) Mucosal leishmaniasis. Current scenario and prospects for treatment. Acta Trop 105: 1–9. 4. Powderly WG, Landay A, Lederman MM (1998) Recovery of the immune system with antiretroviral therapy. The end of opportunism? JAMA 280: 72–77. 5. Mastroianni CM, Lichtner M, Mengoni F, D’Agostino C, Forcina G, et al. (1999) Improvement in neutrophil and monocyte function during highly active antiretroviral treatment of HIV-1 infected patients. AIDS 13: 883–890. 6. Mezzaroma I, Carlesimo M, Pinter E, Alario C, Sacco G, et al. (1999) Long- term evaluation of T-cell subsets and Tcell function after HAART in advanced stage HIV-1 disease. AIDS 13: 1187–1193. 7. Malhotra U, Berrei MM, Huang Y, Maree J, Brown DJ, et al. (2000) Effect of combination antiretroviral therapy on T-cell immunity in acute human immunodeficiency virus type 1 infection. J Infect Dis 181: 121–131. 8. Desjeux P, Alvar J (2003) Leishmania/HIV co-infections: epidemiology in Europe. Ann Trop Med Parasitol 97: 3–15. HIV PIs on L. amazonensis should be directed to find new PIs that are more specific for these parasite enzymes. In Brazil, the clinical forms found among coinfected cases are 43% mucocutaneous, 37% visceral, and 20% cutaneous leish- maniasis [50], a clinical pattern that differs from that found in southern Europe, where typical visceral leishmaniasis represents 88% of coinfected cases; 8% of cases are atypical because of intestinal, lung, or other parasite colonizations, about 5% are cutaneous, and 0.3% are combined cases of visceral and cutaneous leishmaniasis [2,51]. In the face of leishmaniasis distribution in Brazil, its spread to important cities and the overlap with HIV- infected individuals, it is critical to further comprehend the sophisticated interplays among Leishmania, HIV and macrophages. In addition, there are many unresolved questions related to the management of Leishmania-HIV-coinfected patients. For instance, the efficacy of therapy aimed at controlling each pathogen in coinfected individuals remains largely undefined. The results presented herein add new in vitro insight into the wide spectrum efficacy of HIV PIs and suggest more detailed studies about the synergistic effects of classical antileishmanial compounds and HIV PIs in macrophages coinfected with Leishmania and HIV-1, especially in the light that sodium stibogluconate can stimulate HIV-1 replication in vitro [52], and that Leishmania infection enhances HIV-1 replication [53,54]. Macrophages infected with HIV-1 allow the intracellular growth of an otherwise non- pathogenic protozoan, Blastocrithidia culicis, which illustrates the susceptibility of these cells to parasite infections [55]. Finally, it was recently reported that L. infantum promastigotes enhance HIV-1 replication in monocyte-derived macrophages at late time points in the virus growth curve but also, surprisingly, a reduction in HIV-1 production is seen during the initial days after infection. This early effect is caused by a Leishmania-mediated inhibition of virus entry into monocyte-derived macrophages through the action of lipophosphoglycan (LPG), the major promastigote surface glycolipid. Altogether, these data suggest the establishment of complex interactions in Leishmania-HIV common natural host cells [56]. Author Contributions Conceived and designed the experiments: MCMM MHB AS CMdL. Performed the experiments: LdOS FdAM EFA BdSV MCMM. Analyzed the data: LdOS CRA MCMM MHB AS CMdL. Contributed reagents/ materials/analysis tools: CRA CB MCMM MHB AS CMdL. Wrote the paper: LdOS CRA MCMM MHB AS CMdL. 9. Pozio E, Morales MA (2005) The impact of HIV-protease inhibitors on opportunistic parasites. Trends Parasitol 21: 58–63. 10. Blasi E, Colombari B, Orsi CF, Pinti M, Troiano L, et al. (2004) The human immunodeficiency virus (HIV) protease inhibitor indinavir directly affects the opportunistic fungal pathogen Cryptococcus neoformans. FEMS Immunol Med Microbiol 42: 187–195. 11. Bektic J, Lell CP, Fuchs A, Stoiber H, Speth C, et al. (2001) HIV protease inhibitors attenuate adherence of Candida albicans to epithelial cells in vitro. FEMS Immunol Med Microbiol 31: 65–71. 12. Palmeira VF, Kneipp LF, Rozental S, Alviano CS, Santos AL (2008) Beneficial effects of HIV aspartyl peptidase inhibitors on the human pathogen Fonsecaea pedrosoi: promising compounds to arrest key fungal biological processes and virulence. PLoS ONE 3: e3382. 13. Parikh S, Gut J, Istvan E, Goldberg DE, Havlir DV, et al. (2005) Antimalarial activity of human immunodeficiency virus type 1 protease inhibitors. Antimicrob Agents Chemother 49: 2983–2985. 14. Savoia D, Allice T, Tovo PA (2005) Antileishmanial activity of HIV protease inhibitors. Int J Antimicrob Agents 26: 92–94. 15. Alexander J, Satoskar AR, Russel DG (1999) Leishmania species: Models of intracellular parasitism. J Cell Sci 112: 2993–3002. 16. Mottram JC, Coombs GH, Alexander J (2004) Cysteine peptidases as virulence factors of Leishmania. Curr Opin Microbiol 7: 375–381. PLoS ONE | www.plosone.org 10 March 2009 | Volume 4 | Issue 3 | e4918