Host cell cAMP-Epac-Rap1b pathway inhibition by hawthorn extract as a potential target against Trypanosoma cruzi infection.

Although the two drugs currently available for the treatment of Chagas disease, Benznidazole and Nifurtimox, have proven to be effective in the acute phase of the disease, the 60-90-day treatment leads to high toxicity and unwanted side effects, presenting, in addition, a low efficacy in the chronic phase of the disease. For this reason, new therapies that are more effective are needed. In this regard, we have recently shown that the inhibition of the Epac-Rap1b pathway suppressed the cAMP-mediated host cell invasion by Trypanosoma cruzi. Interestingly, it has been described that vitexin, a natural flavone that protects against ischemia-reperfusion damage, acts by inhibiting the expression of Epac and Rap1 proteins. Vitexin can be found in plants of the genus Crataegus spp., traditionally known as hawthorn, which are of great interest considering their highly documented use as cardio-protectors. Pre-treating cells with an extract of Crataegus oxyacantha produced levels of T. cruzi invasion comparable to the ones observed for the commercially available Epac1-specific inhibitor, ESI-09. In addition, extract-treated cells exhibited a decrease in the activation of Rap1b, suggesting that the effects of the extract would be mediated by the inhibition of the cAMP-Epac-Rap1 signaling pathway. Using HPLC-HRMS2, we could confirm the presence of vitexin, and other flavones that could act as inhibitors of Epac/Rap1b, in the extracts of C. oxyacantha. Most significantly, when cells were treated with the extract of C. oxyacantha in conjunction with Nifurtimox, an increased modulation of invasion was observed.

Host Cell Rap1b mediates cAMP-dependent invasion by Trypanosoma cruzi.

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.

Lipase-Catalyzed Synthesis and Biological Evaluation of N-Picolineamides as Trypanosoma cruzi Antiproliferative Agents.

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.

Carbohydrate-Derived Polytriazole Nanoparticles Enhance the Anti-Inflammatory Activity of Cilostazol.

Poly(amide-triazole) and poly(ester-triazole) synthesized from d-galactose as a renewable resource were applied for the synthesis of nanoparticles (NPs) by the emulsification/solvent evaporation method. The NPs were characterized as stable, spherical particles, and none of their components, including the stabilizer poly(vinyl alcohol), were cytotoxic for normal rat kidney cells. These NPs proved to be useful for the efficient encapsulation of cilostazol (CLZ), an antiplatelet and vasodilator drug currently used for the treatment of intermittent claudication, which is associated with undesired side-effects. In this context, the nanoencapsulation of CLZ was expected to improve its therapeutic administration. The carbohydrate-derived polymeric NPs were designed taking into account that the triazole rings of the polymer backbone could have attractive interactions with the tetrazole ring of CLZ. The activity of the nanoencapsulated CLZ was measured using a matrix metalloproteinase model in a lipopolysaccharide-induced inflammation system. Interestingly, the encapsulated drug exhibited enhanced anti-inflammatory activity in comparison with the free drug. The results are very promising since the stable, noncytotoxic NP systems efficiently reduced the inflammation response at low CLZ doses. In summary, the NPs were obtained through an innovative methodology that combines a carbohydrate-derived synthetic polymer, designed to interact with the drug, ease of preparation, adequate biological performance, and environmentally friendly production.

Sialic acid removal by trans-sialidase modulates MMP-2 activity during Trypanosoma cruzi infection.

Matrix metalloproteinases (MMPs) not only play a relevant role in homeostatic processes but are also involved in several pathological mechanisms associated with infectious diseases. As their clinical relevance in Chagas disease has recently been highlighted, we studied the modulation of circulating MMPs by Trypanosoma cruzi infection. We found that virulent parasites from Discrete Typing Units (DTU) VI induced higher proMMP-2 and MMP-2 activity in blood, whereas both low (DTU I) and high virulence parasites induced a significant decrease in proMMP-9 plasma activity. Moreover, trans-sialidase, a relevant T. cruzi virulence factor, is involved in MMP-2 activity modulation both in vivo and in vitro. It removes α2,3-linked sialyl residues from cell surface glycoconjugates, which then triggers the PKC/MEK/ERK signaling pathway. Additionally, bacterial sialidases specific for this sialyl residue linkage displayed similar MMP modulation profiles and triggered the same signaling pathways. This novel pathogenic mechanism, dependent on sialic acid removal by the neuraminidase activity of trans-sialidase, can be exploited by different pathogens expressing sialidases with similar specificity. Thus, here we present a new pathogen strategy through the regulation of the MMP network.

Hybrids of Cinchona Alkaloids and Bile Acids as Antiparasitic Agents Against Trypanosoma cruzi.

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.

Altered FOXO1 activation in the programming of cardiovascular alterations by maternal diabetes.

Maternal diabetes programs cardiovascular alterations in the adult offspring but the mechanisms involved remain unclarified. Here, we addresed whether maternal diabetes programs cardiac alterations related to extracellular matrix remodeling in the adult offspring, as well as the role of forkhead box transcription factor 1 (FOXO1) in the induction of these alterations. The heart from adult offspring from control and streptozotocin-induced diabetic rats was evaluated. Increased glycemia, triglyceridemia and insulinemia and markers of cardiomyopathy were found in the offspring from diabetic rats. In the heart, an increase in active FOXO1 and mRNA levels of its target genes, Mmp-2 and Ctgf, genes related to an altered extracellular matrix remodeling, together with an increase in collagen deposition and a decrease in the connexin43 levels, were found in the offspring from diabetic rats. Altogether, these results suggest an important role of FOXO1 activation in the cardiac alterations induced by intrauterine programming in maternal diabetes.

Host Epac1 is required for cAMP-mediated invasion by Trypanosoma cruzi.

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.

Trypanosoma cruzi trans-sialidase prevents elicitation of Th1 cell response via interleukin 10 and downregulates Th1 effector cells.

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-γ) 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 Th2 phenotype was prevented by passive transfer of anti-TS neutralizing antibodies. TSs also reduced the T cell receptor signaling as assayed by Zap-70 phosphorylation. TSs also reduced IL-2 and IFN-γ secretion, with a concomitant increase in IL-4 production and then an unbalancing of the CD4 T cell response toward the Th2 phenotype. This effect was prevented by using anti-IL-10 neutralizing antibodies or IL-10(-/-) antigen-presenting cells, supporting the subversion of this regulatory pathway. In support, TSs stimulated IL-10 secretion by antigen-presenting cells during their interaction with CD4 T cells. When polarized cells were stimulated in the presence of TSs, the secretion of IL-2 and IFN-γ was strongly downregulated in Th1 cells, while IL-2 production was upregulated in Th2 cells. Although the Th1 response is associated with host survival, it may simultaneously induce extensive damage to infected tissues. Thus, by delaying the elicitation of the Th1 response and limiting its effector properties, TSs restrain the cell response, supporting T. cruzi colonization and persistence while favoring host survival.

Effects of maternal dietary olive oil on pathways involved in diabetic embryopathy.

Maternal diabetes induces a pro-oxidant/pro-inflammatory intrauterine environment related to the induction of congenital anomalies. Peroxisome proliferator activated receptors (PPARs) are transcription factors that regulate antioxidant and anti-inflammatory pathways. We investigated whether maternal diets supplemented with olive oil, enriched in oleic acid, a PPAR agonist, can regulate the expression of PPAR system genes, levels of lipoperoxidation and activity of matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) in embryos and decidua from diabetic rats. The embryos and decidua from diabetic rats showed reduced expression of PPARs and increased concentration of lipoperoxidation, MMPs and TIMPs, whereas the maternal treatments enriched in olive oil increased PPARδ in embryos and PPARγ and PPARγ-coactivator-1α expression in decidua, and increased TIMPs concentrations and decreased lipoperoxidation and MMPs activity in both tissues. Thus, maternal diets enriched in olive oil can regulate embryonic and decidual PPAR system genes expression and reduce the pro-oxidant/pro-inflammatory environment during rat early organogenesis.