Fast high yield of pure Leishmania (Leishmania) infantum axenic amastigotes and their infectivity to mouse macrophages.

Leishmania (L.) infantum (syn. Leishmania chagasi) is a dimorphic protozoan parasite that lives in promastigote and amastigote form in its sandfly vector and mammalian hosts, respectively. Here, we describe an in vitro culture system for the generation of a pure population of L. infantum axenic amastigotes after only 4 days incubation in culture medium supplemented with fetal calf serum, human urine, L: -glutamine, and HEPES at 37 masculineC (pH 5.5). Ultrastrutural analysis and infection assays in two macrophage populations (Kupffer cells (KUP) and peritoneal macrophages (PM)) infected with axenic amastigotes demonstrated that they maintained morphological and biochemical (A2 expression) features and a similar infection pattern to tissue-derived L. infantum amastigotes. The susceptibility of the macrophage lines to axenic or tissue-derived amastigotes and promastigotes was investigated. We found a completely different susceptibility profile for KUP and PM. Liver macrophages, both KUP and immigrant macrophages, are intimately involved in the response to L. infantum infection; this difference in susceptibility is probably related to their capacity to eliminate these parasites. Our in vitro system was thus able to generate axenic amastigotes that resemble tissue-derived amastigotes both in morphology and infectivity pattern; this will help in further investigation of the biological characteristics of the host-parasite relationship as well as the process of pathogenesis.

Trypanosoma cruzi induces changes in cardiac connexin43 expression.

Gap junction proteins (connexins) are required for myocardial function, since they allow intercellular transmission of current carrying ions and signaling molecules. Previous studies demonstrated that rat cardiac myocytes infected with Trypanosoma cruzi lost gap junctional communication and decreased automaticity. We infected mouse cardiac myocytes with trypomastigotes of the Y strain of T. cruzi and observed alterations in connexin43 (Cx43) distribution. One hour post infection Cx43 levels were significantly increased. However, at longer time points post infection there was a significant loss of Cx43 staining in membranes of infected cardiac myocytes. Interestingly, there was also a significant reduction in myocardial Cx43 protein levels during acute infection. These data indicate that T. cruzi infection alters Cx43 expression both in vitro and in vivo. Disruptions in Cx43 may contribute to the pathogenesis of cardiac electrical alterations observed in T. cruzi infection.

Leishmania chagasi: cytotoxic effect of infected macrophages on parenchymal liver cells.

Leishmania (Leishmania) chagasi, the ethiological agent of New World visceral leishmaniasis, causes morphological and functional injury to the liver. To investigate the role of macrophage-released leishmanicidal factors in hepatocyte damage, we used a co-culture model of hepatocytes and L. chagasi promastigote-infected peritoneal macrophages obtained from C57BL/6 or BALB/c mice. C57BL/6 macrophages killed intracellular parasites more efficiently than BALB/c macrophages, leading to higher number of intracellular amastigotes in the BALB/c culture during the entire course of infection. Early TNF-alpha production led to macrophages activation resulting in parasite growth control. Hepatic transaminases and lactate dehydrogenase were present at high levels in the supernatants of both co-cultures; concurrently, parasites were eliminated from infected macrophages. Nitric oxide production was higher in C57BL/6 co-cultures than in BALB/c co-cultures. Inhibitors of the oxidative burst and secreted proteinases protected hepatocytes against toxicity, and treatment with an inducible nitric oxide synthase inhibitor fully impeded the enzyme release. Our data suggest that the intracellular cytotoxic effects elicited by macrophages for parasite destruction are directly associated with hepatocyte damage, and that nitric oxide plays a pivotal role in this phenomenon.