Mechanistic details of the modulation by cAMP of Trypanosoma cruzi host cell invasion remain ill-defined. Here we report that activation of host's Epac1 stimulated invasion, whereas specific pharmacological inhibition or maneuvers that alter Epac1 subcellular localization significantly reduced invasion. Furthermore, while specific activation of host cell PKA showed no effect, its inhibition resulted in an increased invasion, revealing a crosstalk between the PKA and Epac signaling pathways during the process of invasion. Therefore, our data suggests that subcellular localization of Epac might be playing an important role during invasion and that specific activation of the host cell cAMP/Epac1 pathway is required for cAMP-mediated invasion.

The trans-sialidases (TSs) from Trypanosoma cruzi, the agent of Chagas disease, are virulence factors shed to the bloodstream that induce strong alterations in the immune system. Here, we report that both enzymatically active TS (aTS) and its lectinlike isoform (iTS) disturb CD4 T cell physiology, inducing downregulation of Th1 cell functionality and in vivo cell expansion. By using ovalbumin-specific DO11.10 cells as tracers of clones developing the Th1 phenotype, we found that the infection induced significant amounts of gamma interferon (IFN-␥) but low levels of interleukin 2 (IL-2) and increased IL-4 production in vivo, in agreement with a mixed T helper response. The production of cytokines associated with the

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Maternal diabetes increases the risk of embryo malformations. Folic acid and safflower oil supplementations have been shown to reduce embryo malformations in experimental models of diabetes. In this study we here tested whether folic acid and safflower oil supplementations interact to prevent embryo malformations in diabetic rats, and analyzed whether they act through the regulation of matrix metalloproteinases (MMPs), their endogenous inhibitors (TIMPs), and nitric oxide (NO) and reactive oxygen species production. Diabetes was induced by streptozotocin administration prior to mating. From Day 0.5 of pregnancy, rats did or did not receive folic acid (15 mg/kg) and/or a 6% safflower oil-supplemented diet. Embryos and decidua were explanted on Day 10.5 of gestation for further analysis of embryo resorptions and malformations, MMP-2 and MMP-9 activities, TIMP-1 and TIMP-2 levels, NO production and lipid peroxidation. Maternal diabetes induced resorptions and malformations that were prevented by folic acid and safflower oil supplementation. MMP-2 and MMP-9 activities were increased in embryos and decidua from diabetic rats and decreased with safflower oil and folic acid supplementations. In diabetic animals, the embryonic and decidual TIMPs were increased mainly with safflower oil supplementation in decidua and with folic acid in embryos. NO overproduction was decreased in decidua from diabetic rats treated with folic acid alone and in combination with safflower oil. These treatments also prevented increases in embryonic and decidual lipid peroxidation. In conclusion, folic acid and safflower oil supplementations interact and protect the embryos from diabetes-induced damage through several pathways related to a decrease in pro-inflammatory mediators.

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In our search for new safe antiparasitic agents, an enzymatic pathway was applied to synthesize a series of N-pyridinylmethyl amides derived from structurally different carboxylic acids. Thirty derivatives, including 11 new compounds, were prepared through lipase-catalyzed acylation in excellent yields. In order to optimize the synthetic methodology, the impact of different reaction parameters was analyzed. Some compounds were evaluated as antiproliferative agents against Trypanosoma cruzi, the parasite responsible for American trypanosomiasis (Chagas’ disease). Some of them showed significant activity as parasite proliferation inhibitors. Amides derived from 2-aminopicoline and stearic and elaidic acids were as potent as nifurtimox against the amastigote form of T. cruzi, the clinically relevant form of the parasite. Even more, a powerful synergism between nifurtimox and N-(pyridin-2-ylmethyl)stereamide was observed, almost completely inhibiting the proliferation of the parasite. Besides, the obtained compounds showed no toxicity in Vero cells, making them excellent potential candidates as lead drugs.

Trypanosoma cruzi cAMP-mediated invasion has long been described, however, the detailed mechanism of action of the pathway activated by this cyclic nucleotide still remains unknown. We have recently demonstrated a crucial role for Epac in the cAMP-mediated invasion of the host cell. In this work, we gathered evidence indicating that the cAMP/Epac pathway is activated in different cells lines. In accordance, data collected from pull-down experiments designed to identify only the active form of Rap1b (Rap1b-GTP), and infection assays using cells transfected with a constitutively active mutant of Rap1b (Rap1b-G12V), strongly suggest the participation of Rap1b as mediator of the pathway. In addition to the activation of this small GTPase, fluorescence microscopy allowed us to demonstrate the relocalization of Rap1b to the entry site of the parasite. Moreover, phospho-mimetic and non-phosphorylable mutants of Rap1b were used to demonstrate a PKA-dependent antagonistic effect on the pathway, by phosphorylation of Rap1b, and potentially of Epac. Finally, Western Blot analysis was used to determine the involvement of the MEK/ERK signalling downstream of cAMP/Epac/Rap1b-mediated invasion.

The current chemotherapy of Chagas disease needs to be urgently improved. With this aim, a series of 16 hybrids of Cinchona alkaloids and bile acids were prepared by functionalization at position C-2 of the quinoline nucleus by a radical attack of a norcholane substituent via a Barton–Zard decarboxylation reaction. The antitrypanosomal activity of the hybrids was tested on different stages and strains of T. cruzi. In particular, eight out of 16 hybrids presented an IC50 ≤1 μg/mL against trypomastigotes of the CL Brener strain and/or a selectivity index higher than 10. These promising hybrids yielded similar results when tested on trypomastigotes from the RA strain of T. cruzi (discrete typing unit—DTU—VI). Surprisingly, trypomastigotes of the Y strain (DTU II) were more resistant to benznidazole and to most of the hybrids than those of the CL Brener and RA strains. However, the peracetylated and non-acetylated forms of the cinchonine/chenodeoxycholic bile acid conjugate 4f and 5f were the most trypanocidal hybrids against Y strain trypomastigotes, with IC50 values of 0.5 and 0.65 μg/mL, respectively. More importantly, promising results were observed in invasion assays using the Y strain, where hybrids 5f and 4f induced a significant reduction in intracellular amastigotes and on the release of trypomastigotes from infected cells.