We have investigated the influences of gamma interferon (IFN-␥) and interleukin-12 (IL-12

Studies in humans and in experimental models suggest the involvement of the immune system for efficacy of drug treatment against protozoan parasites. This study tested this hypothesis by using various cytokine and inducible nitric oxide synthase (iNOS) knockout (KO) mice infected with Trypanosoma cruzi and treated with benznidazole. In contrast with the 100% parasitologic cure rate achieved in wild-type animals, benznidazole failed to cure 100%, 42%, 35%, and 28% of interferon-gamma, interleukin-12 (protein 40), protein 55-tumor necrosis factor receptor, and iNOS KO mice, respectively. These results suggest that activation of the immune system by the parasite and endogenous interferon-gamma play a major role in the efficacy of benznidazole against infection with T. cruzi.

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We investigated the influence of CD4؉ T lymphocytes, CD8 ؉ T lymphocytes, and B lymphocytes on the efficacy of posaconazole (POS) and the reference drug benznidazole (BZ) during treatment of acute Trypanosoma cruzi infection in a murine model. Wild-type mice infected with T. cruzi and treated with POS or BZ presented no parasitemia, 100% survival, and 86 to 89% cure rates, defined as the percentages of animals with negative hemocultures at the end of the observation period. CD4؉ -T-lymphocyte-knockout (KO) mice infected with T. cruzi and treated with BZ or POS controlled parasitemia during treatment, although circulating parasites reappeared after drug pressure cessation, leading to only a 6% survival rate and no cure. CD8؉ -Tlymphocyte-KO mice infected with T. cruzi and treated with POS or BZ had intermediate results, displaying discrete parasitemia after the treatment was ended, 81 and 86% survival, and cure rates of 31 and 66%, respectively. B-lymphocyte-KO mice infected with T. cruzi and treated with BZ relapsed with parasitemia 1 week after the end of treatment and had a 67% survival rate and only a 22% cure rate. In contrast, the activity of POS was much less affected in these animals, with permanent suppression of parasitemia, 100% survival, and a 71% cure rate. Our results demonstrate that abrogation of different lymphocytes' activities has distinct effects on the efficacy of POS and BZ in this experimental model, probably reflecting different parasite stages preferentially targeted by the two drugs and distinct cooperation patterns with the host immune system.

•NO is considered to be a key macrophage-derived cytotoxic effector during Trypanosoma cruzi infection. On the other hand, the microbicidal properties of reactive oxygen species (ROS) are well recognized, but little importance has been attributed to them during in vivo infection with T. cruzi. In order to investigate the role of ROS in T. cruzi infection, mice deficient in NADPH phagocyte oxidase (gp91phox −/− or phox KO) were infected with Y strain of T. cruzi and the course of infection was followed. phox KO mice had similar parasitemia, similar tissue parasitism and similar levels of IFN-γ and TNF in serum and spleen cell culture supernatants, when compared to wild-type controls. However, all phox KO mice succumbed to infection between day 15 and 21 after inoculation with the parasite, while 60% of wild-type mice were alive 50 days after infection. Further investigation demonstrated increased serum levels of nitrite and nitrate (NOx) at day 15 of infection in phox KO animals, associated with a drop in blood pressure. Treatment with a NOS2 inhibitor corrected the blood pressure, implicating NOS2 in this phenomenon. We postulate that superoxide reacts with •NO in vivo, preventing blood pressure drops in wild type mice. Hence, whilst superoxide from phagocytes did not play a critical role in parasite control in the phox KO animals, its production would have an important protective effect against blood pressure decline during infection with T. cruzi.

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The natural lignans veraguensin and grandisin have been reported to be active against Trypanosoma cruzi bloodstream forms. Aiming at the total synthesis of these and related compounds, we prepared three 2-arylfurans and eight 2,5-diarylfurans. They were evaluated for their potential as T. cruzi trypanothione reductase (TR) 60% at 20 µg/ml (59 and 90 µM, respectively). On the other hand, none of the compounds was significantly active against the parasite bloodstream forms even at 250 µg/ml (0.6-1.5 mM).Key words: tropical diseases -Chagas disease -arylfurans -trypanothione reductase Chagas disease, caused by the flagellate protozoan Trypanosoma cruzi, affects 18 million people in Latin America and is responsible for 13,000 deaths every year (WHO 2002). The treatment relies on only two available drugs, nifurtimox and benznidazole, which are relatively efficient in the acute phase of the disease, but almost ineffective in the chronic phase. Nowadays, one of the most important mechanisms of Chagas disease transmission in many countries is by blood transfusion (Schmuñis 1991). In highly endemic areas it is strongly recommended the use of chemoprophylatic measures such as the addition of gentian violet to clear trypomastigotes from blood banked for transfusion (Moraes-Souza et al. 1995). Although effective, this triphenylmethane dye is not well accepted because of undesirable effects such as coloring the skin and possible mutagenicity (Wendel 1993). Thus, new drugs to treat or prevent Chagas disease are still needed.Trypanosoma cruzi enzymes such as the trypanothione reductase (TR) represent a potential drug targets because they play an essential role in the life of this parasite. TR and its substrate trypanothione, the disulfide of a glutathione-spermidine conjugate [N 1 , N 8 -bis(glutathionyl)spermidine, T(SH) 2 ] 1, help to protect the parasite from oxidative stress by maintaining an intracellular reducing environment in a manner analogous to glutathione reductase (GR) and glutathione [L-γ-glutamyl-Lcysteiylglycine, GSH] 2 (Fig. 1a) in mammalian cells (Schmidt & Krauth-Siegel 2002). TR catalyses the NADPHdependent reduction of trypanothione disulfide TS 2 to its dithiol form, T(SH) 2 . Trypanothione may be oxidized back to TS 2 (Fig. 1b) following reaction with potentially damaging radicals and oxidants generated by aerobic metabolism. Another aspect that makes TR an even more attractive target is its structural differences from the human counterpart GR. GR has a narrow positively charged active site, to accommodate the glycine carboxylates of its substrate glutathione, whereas TR has a wider, noncharged, and more hydrophobic active site (Bond et al. 1999). These differences allowed the discovery of several promising selective inhibitors of TR (Schmidt & KrauthSiegel 2002).Lignans is a class of natural products that possess important biological properties (Jensen et al. 1993). Lopes et al. (1998) showed that the tetrahydrofuran lignans veraguensin 3 and grandisin 4 (Fig. 2) were active in vitro at 5 µg/ml aga...