Differential apoptosis-like cell death in amastigote and trypomastigote forms from Trypanosoma cruzi-infected heart cells in vitro.

Apoptosis, type-I of programmed cell death (PCD-I), is not restricted to multicellular organisms since many apoptotic features have been described in different trypanosomatids, including Trypanosoma cruzi. Our present aim was to monitor, by different morphological markers, the occurrence of apoptosis-like death in amastigotes and trypomastigotes of T.cruzi (Y strain) during the infection of heart culture cells. We documented the differential occurrence of PCD-I in amastigotes and trypomastigotes, with distinct death rates noticed between these two parasite-distinct forms. Fluorescence microscopy and flow cytometry analysis using different hall markers of apoptosis (phosphatidylserine exposure, collapse of mitochondrial membrane potential and DNA fragmentation) showed that amastigotes present higher levels of apoptosis-like cell death as compared to trypomastigotes. It is possible that the higher levels of PCD-I in these highly multiplicative forms may contribute to the control of the parasite burden within the host cells. On the other hand, the apoptosis-like occurrence in the infective but non-proliferative stage of the parasite (trypomastigotes) may play a role in parasite evasion mechanisms as suggested for other parasites.

Phenyl substitution of furamidine markedly potentiates its anti-parasitic activity against Trypanosoma cruzi and Leishmania amazonensis.

Furamidine (DB75) and related unfused aromatic diamidines have proven useful for the treatment of parasitic infections. These compounds were primarily developed to combat infections by Pneumocystis carinii and African trypanosomes but they are also active against other parasites. Here we have investigated the in vitro effects of DB75 and its phenyl-substituted analog DB569 on two kinetoplastid haemoflagellates Trypanosomatidae: Trypanosoma cruzi and Leishmania (L) amazonensis. The phenyl-amidine compound DB569 has equivalent DNA binding properties compared to DB75 but it was selected on the basis of its distinct tumor cell distribution properties. We found that DB569 is significantly more potent than DB75 at reducing the proliferation of the parasites, using either isolated parasites in cultures or with cardiomyocyte and macrophage host cells. DB569 is effective towards the intracellular forms of T. cruzi (IC(50) in the low-micromolar range) and it exhibits trypanocidal dose-dependent effects against trypomastigote forms of T. cruzi parasites obtained from the Y strain and Dm28c clone, which belong to two different biodemes. Fluorescence microscopy experiments indicated that both diamidines were mostly localized in the nucleus of the mammalian host cells and within the nuclei and kinetoplast of the parasites. Electron microscopy studies showed that the treatment of the parasites with DB75 and DB569 induces important alterations of the parasite nucleus and kinetoplast, at sites where their DNA target is localized. Altogether, the data suggest that the phenyl-substituted furamidine analogue DB569 is a potential new candidate for the treatment of the Chagas' disease and Leishmaniasis.

Host and parasite apoptosis following Trypanosoma cruzi infection in in vitro and in vivo models.

The mechanism of cell death which occurs during Chagas' cardiopathy is disputed. To address this issue we analyzed the molecular pathways implicated in the death of cardiomyocytes during T. cruzi invasion and found that they undergo apoptosis during both in vitro and in vivo infections. However, the death rates and onset were related to the parasite stocks belonging to different biodemes, which can be correlated to the different histological inflammation findings that have already been reported. Our in vitro data provide additional support for this hypothesis since higher levels and earlier apoptosis induction were noted during the interaction with the Dm28c (type I) as compared to the Y and CL stocks (type II). Modifications of the surface carbohydrates of the infected cardiomyocytes were observed and these molecular events may be acting as "eat me" tags for their final engulfment by macrophages and/or other non-professional phagocytes. The analysis of other host cell types showed that the in vitro infection of fibroblasts did not result in host apoptosis even when a highly infective stock was used. Conversely, infected macrophages undergo apoptosis but at a higher degree than cardiomyocytes. Apoptotic intracellular parasites were observed to varied extents depending on the T. cruzi stock, which was related to the parasite invasion and proliferation. In summary, our results show that during T. cruzi infection, the extent of apoptosis varies according to the host cell type and the parasite stocks. The apoptosis of both host and T. cruzi can contribute to the silent spreading and persistence of the parasite without triggering an exacerbated inflammatory response.