Pentoxifylline reduces inflammation and prevents myocardial perfusion derangements in experimental chronic Chagas' cardiomyopathy.

BACKGROUND: Myocardial perfusion defect (MPD) is common in chronic Chagas cardiomyopathy (CCC) and is associated with inflammation and development of left ventricular systolic dysfunction. We tested the hypothesis that pentoxifylline (PTX) could reduce inflammation and prevent the development of MPD in a model of CCC in hamsters. METHODS AND RESULTS: We investigated with echocardiogram and rest myocardial perfusion scintigraphy at baseline (6-months after T. cruzi infection/saline) and post-treatment (after additional 2-months of PTX/saline administration), female Syrian hamsters assigned to 3 groups: T. cruzi-infected animals treated with PTX (CH + PTX) or saline (CH + SLN); and uninfected control animals (CO). At the baseline, all groups showed similar left ventricular ejection fraction (LVEF) and MPD areas. At post-treatment evaluation, there was a significant increase of MPD in CH + SLN group (0.8 ± 1.6 to 9.4 ± 9.7%), but not in CH + PTX (1.9 ± 3.0% to 2.7 ± 2.7%) that exhibited MPD area similar to CO (0.0 ± 0.0% to 0.0 ± 0.0%). The LVEF decreased in both infected groups. Histological analysis showed a reduced inflammatory infiltrate in CH + PTX group (395.7 ± 88.3 cell/mm2), as compared to CH + SLN (515.1 ± 133.0 cell/mm2), but larger than CO (193.0 ± 25.7 cell/mm2). The fibrosis and TNF-α expression was higher in both infected groups. CONCLUSIONS: The prolonged use of PTX is associated with positive effects, including prevention of MPD development and reduction of inflammation in the chronic hamster model of CCC.

Prolonged dipyridamole administration reduces myocardial perfusion defects in experimental chronic Chagas cardiomyopathy.

BACKGROUND: Myocardial perfusion defects (MPD) due to coronary microvascular dysfunction is frequent in chronic Chagas cardiomyopathy (CCC) and may be involved with development of myocardial damage. We investigated whether MPD precedes left ventricular systolic dysfunction and tested the hypothesis that prolonged use of dipyridamole (DIPY) could reduce MPD in an experimental model of CCC in hamsters. METHODS AND RESULTS: We investigated female hamsters 6-months after T. cruzi infection (baseline condition) and control animals, divided into T. cruzi-infected animals treated with DIPY (CH + DIPY) or placebo (CH + PLB); and uninfected animals treated with DIPY (CO + DIPY) or placebo (CO + PLB). The animals were submitted to echocardiogram and rest SPECT-Sestamibi-Tc99m myocardial perfusion scintigraphy. Next, the animals were treated with DIPY (4 mg/kg bid, intraperitoneal) or saline for 30 days, and reevaluated with the same imaging methods. At baseline, the CH + PLB and CH + DIPY groups showed larger areas of perfusion defect (13.2 ± 13.2% and 17.3 ± 13.2%, respectively) compared with CO + PLB and CO + DIPY (3.8 ± 2.2% e 3.5 ± 2.7%, respectively), P < .05. After treatment, we observed: reduction of perfusion defects only in the CH + DIPY group (17.3 ± 13.2% to 6.8 ± 7.6%, P = .001) and reduction of LVEF in CH + DIPY and CH + PLB groups (from 65.3 ± 9.0% to 53.6 ± 6.9% and from 69.3 ± 5.0% to 54.4 ± 8.6%, respectively, P < .001). Quantitative histology revealed greater extents of inflammation and interstitial fibrosis in both Chagas groups, compared with control group (P < .001), but no difference between Chagas groups (P > .05). CONCLUSIONS: The prolonged use of DIPY in this experimental model of CCC has reduced the rest myocardial perfusion defects, supporting the notion that those areas correspond to viable hypoperfused myocardium.

Structural analysis and shape-based identification of novel inhibitors targeting the Trypanosoma cruzi proteasome.

There is an urgent need to develop new, safer, and more effective drugs against Chagas disease (CD) as well as related kinetoplastid diseases. Targeting and inhibiting the Trypanosoma cruzi proteasome has emerged as a promising therapeutic approach in this context. To expand the chemical space for this class of inhibitors, we performed virtual screening campaigns with emphasis on shape-based similarity and ADMET prioritization. We describe the ideation and application of robustly validated shape queries for these campaigns, which furnished 44 compounds for biological evaluation. Five hit compounds demonstrated in vitro antitrypanosomal activity by potential inhibition of T. cruzi proteasome and notable chemical diversities, particularly, LCQFTC11. Structural insights were achieved by homology modeling, sequence/structure alignment, proteasome-species comparison, docking, molecular dynamics, and MMGBSA binding affinity estimations. These methods confirmed key interactions as well as the stability of LCQFTC11 at the ß4/ß5 subunits' binding site of the T. cruzi proteasome, consistent with known inhibitors. Our results warrant future assay confirmation of our hit as a T. cruzi proteasome inhibitor. Importantly, we also shed light into dynamic details for a proteasome inhibition mechanism that shall be further investigated. We expect to contribute to the development of viable CD drug candidates through such a relevant approach.

Ligand-based virtual screening, molecular dynamics, and biological evaluation of repurposed drugs as inhibitors of Trypanosoma cruzi proteasome.

Chagas disease is a well-known Neglected Tropical Disease, mostly endemic in continental Latin America, but that has spread to North America and Europe. Unfortunately, current treatments against such disease are ineffective and produce known and undesirable side effects. To find novel effective drug candidates to treat Chagas disease, we uniquely explore the Trypanosoma cruzi proteasome as a recent biological target and, also, apply drug repurposing through different computational methodologies. For this, we initially applied protein homology modeling to build a robust model of proteasome ß4/ß5 subunits, since there is no crystallographic structure of this target. Then, we used it on a drug repurposing via a virtual screening campaign starting with more than 8,000 drugs and including the methodologies: ligand-based similarity, toxicity predictions, and molecular docking. Three drugs were selected concerning their favorable interactions at the protein binding site and subsequently submitted to molecular dynamics simulations, which allowed us to elucidate their behavior and compare such theoretical results with experimental ones, obtained in biological assays also described in this paper.Communicated by Ramaswamy H. Sarma.

Trypanosoma cruzi Sirtuin 2 as a Relevant Druggable Target: New Inhibitors Developed by Computer-Aided Drug Design.

Trypanosoma cruzi, the etiological agent of Chagas disease, relies on finely coordinated epigenetic regulation during the transition between hosts. Herein we targeted the silent information regulator 2 (Sir2) enzyme, a NAD+-dependent class III histone deacetylase, to interfere with the parasites' cell cycle. A combination of molecular modelling with on-target experimental validation was used to discover new inhibitors from commercially available compound libraries. We selected six inhibitors from the virtual screening, which were validated on the recombinant Sir2 enzyme. The most potent inhibitor (CDMS-01, IC50 = 40 µM) was chosen as a potential lead compound.

Organometallic Gold(III) Complex [Au(Hdamp)(L14)]+ (L1 = SNS-Donating Thiosemicarbazone) as a Candidate to New Formulations against Chagas Disease.

Chagas disease remains a serious public health concern with unsatisfactory treatment outcomes due to strain-specific drug resistance and various side effects. To identify new therapeutic drugs against Trypanosoma cruzi, we evaluated both the in vitro and in vivo activity of the organometallic gold(III) complex [Au(III)(Hdamp)(L14)]Cl (L1 = SNS-donating thiosemicarbazone), henceforth denoted 4-Cl. Our results demonstrated that 4-Cl was more effective than benznidazole (Bz) in eliminating both the extracellular trypomastigote and intracellular amastigote forms of the parasite without cytotoxic effects on mammalian cells. In in vivo assays, 4-Cl in PBS solution loses the protonation and becomes the 4-neutral. 4-Neutral reduced parasitaemia and tissue parasitism in addition to protecting the liver and heart from tissue damage at 2.8 mg/kg/day. All these changes resulted in the survival of 100% of the mice treated with the gold complex during the acute phase. Analyzing the surviving animals of the acute infection, the parasite load after 150 days of infection was equivalent to those treated with the standard dose of Bz without demonstrating the hepatotoxicity of the latter. In addition, we identified a modulation of interferon gamma (IFN-γ) levels that may be targeting the disease's positive outcome. To the best of our knowledge, this is the first gold organometallic study that shows promise in an in vivo experimental model against Chagas disease.

Synthesis and 2D-QSAR studies of neolignan-based diaryl-tetrahydrofuran and -furan analogues with remarkable activity against Trypanosoma cruzi and assessment of the trypanothione reductase activity.

Two series of diaryl-tetrahydrofuran and -furan were synthesised and screened for anti-trypanosomal activity against trypomastigote and amastigote forms of Trypanosoma cruzi, the causative agent of Chagas disease. Based on evidence that modification of a natural product may result in a more effective drug than the natural product itself, and using known neolignan inhibitors veraguensin 1 and grandisin 2 as templates to synthesise simpler analogues, remarkable anti-trypanosomal activity and selectivity were found for 3,5-dimethoxylated diaryl-furan 5c and 2,4-dimethoxylated diaryl-tetrahydrofuran 4e analogues with EC50 0.01 µM and EC50 0.75 µM, respectively, the former being 260-fold more potent than veraguensin 1 and 150-fold better than benznidazole, the current available drugs for Chagas disease treatment. The ability of the most potent anti-trypanosomal compounds to penetrate LLC-MK2 cells infected with T. cruzi amastigotes parasite was tested, which revealed 4e and 5e analogues as the most effective, causing no damage to mammalian cells. In particular, the majority of the derivatives were non-toxic against mice spleen cells. 2D-QSAR studies show the rigid central core and the position of dimethoxy-aryl substituents dramatically affect the anti-trypanosomal activity. The mode of action of the most active anti-trypanosomal derivatives was investigated by exploring the anti-oxidant functions of Trypanothione reductase (TR). As a result, diarylfuran series displayed the strongest inhibition, highlighting compounds 5d-e (IC50 19.2 and 17.7 µM) and 5f-g (IC50 8.9 and 7.4 µM), respectively, with similar or 2-fold higher than the reference inhibitor clomipramine (IC50 15.2 µM).

Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice.

Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important public health problem and veterinary concern. Although there is no vaccine for human toxoplasmosis, many attempts have been made to develop one. Promising vaccine candidates utilize proteins, or their genes, from microneme organelle of T. gondii that are involved in the initial stages of host cell invasion by the parasite. In the present study, we used different recombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6) or combinations of these proteins (TgMIC1-4 and TgMIC1-4-6) to evaluate the immune response and protection against experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinant TgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated by reduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4 or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% of mice, respectively, survived to the acute phase of infection. In addition, these vaccinated mice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and 68%, respectively. The protective effect was related to the cellular and humoral immune responses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serum antibodies. Our results demonstrate that microneme proteins are potential vaccines against T. gondii, since their inoculation prevents or decreases the deleterious effects of the infection.

Seroprevalence and risk factors for Toxoplasma gondii infection in pigs in southern Piauí.

This study is aimed to assess the prevalence and risk factors associated with T. gondii infection in pigs. We evaluated 143 pigs, in 10 randomly-chosen farms located in Southern Piauí. The pig's blood serum was analyzed through ELISA in detection of anti-T. gondii antibodies. A seroprevalence of 25.5% was observed in the pigs that reacted against T. gondii antigens. The data from the records demonstrated an association with some factors such as: age, diet, type of management, breed and presence of cats in the farms with a prevalence of T. gondii. With the exception of sex, all others features represent risk factors for T. gondii infection. Furthermore, our data contributed to the understanding of the T. gondii seroprevalence in pig farms located in Southern Piauí.

The involvement of heparin in retinal infection by Toxoplasma gondii in a chick model revealed an ontogenetic-dependent pattern.

This work aimed to test the influence of heparin on the susceptibility of retinal cells to Toxoplasma gondii infection. Primary cultures of retinas from chick embryos of 8 (E8) or 11 (E11) days and fibroblasts (control) were used. To determine the influence of heparin in T. gondii infection, tachyzoites of the RH strain were treated with heparin before addition in the culture. A monoclonal anti-heparin antibody was used to analyze the heparin distribution on fibroblast and retinal cell surfaces. Our results showed that retinal cells (E8 and E11) had a higher infection rate than fibroblasts (91% and 24% versus 13%, respectively). Pre-treatment of T. gondii with heparin decreased infection of E8 retinal cells when compared with non-treated parasites (45% versus 91%, respectively), but not of E11 cells (35% versus 48%). In accordance, retinal cells presented an intense heparin staining by immunofluorescence assay. In conclusion, retinal cells from chick embryos were more susceptible to infection by T. gondii compared to fibroblasts and, pre-treatment of tachyzoites with heparin decreased the number of infected cells and parasite burden particularly for E8 retinal cells.

Lysophosphatidylcholine triggers TLR2- and TLR4-mediated signaling pathways but counteracts LPS-induced NO synthesis in peritoneal macrophages by inhibiting NF-κB translocation and MAPK/ERK phosphorylation.

BACKGROUND: Lysophosphatidylcholine (LPC) is the main phospholipid component of oxidized low-density lipoprotein (oxLDL) and is usually noted as a marker of several human diseases, such as atherosclerosis, cancer and diabetes. Some studies suggest that oxLDL modulates Toll-like receptor (TLR) signaling. However, effector molecules that are present in oxLDL particles and can trigger TLR signaling are not yet clear. LPC was previously described as an attenuator of sepsis and as an immune suppressor. In the present study, we have evaluated the role of LPC as a dual modulator of the TLR-mediated signaling pathway. METHODOLOGY/PRINCIPAL FINDINGS: HEK 293A cells were transfected with TLR expression constructs and stimulated with LPC molecules with different fatty acid chain lengths and saturation levels. All LPC molecules activated both TLR4 and TLR2-1 signaling, as evaluated by NF-қB activation and IL-8 production. These data were confirmed by Western blot analysis of NF-қB translocation in isolated nuclei of peritoneal murine macrophages. However, LPC counteracted the TLR4 signaling induced by LPS. In this case, NF-қB translocation, nitric oxide (NO) synthesis and the expression of inducible nitric oxide synthase (iNOS) were blocked. Moreover, LPC activated the MAP Kinases p38 and JNK, but not ERK, in murine macrophages. Interestingly, LPC blocked LPS-induced ERK activation in peritoneal macrophages but not in TLR-transfected cells. CONCLUSIONS/SIGNIFICANCE: The above results indicate that LPC is a dual-activity ligand molecule. It is able to trigger a classical proinflammatory phenotype by activating TLR4- and TLR2-1-mediated signaling. However, in the presence of classical TLR ligands, LPC counteracts some of the TLR-mediated intracellular responses, ultimately inducing an anti-inflammatory phenotype; LPC may thus play a role in the regulation of cell immune responses and disease progression.

Seroprevalence and risk factors for Toxoplasma gondii infection in pigs in southern Piauí / Soroprevalência e fatores de risco para infecção por Toxoplasma gondii em suínos no Sul do Piauí

This study is aimed to assess the prevalence and risk factors associated with T. gondii infection in pigs. We evaluated 143 pigs, in 10 randomly-chosen farms located in Southern Piauí. The pig's blood serum was analyzed through ELISA in detection of anti-T. gondii antibodies. A seroprevalence of 25.5% was observed in the pigs that reacted against T. gondii antigens. The data from the records demonstrated an association with some factors such as: age, diet, type of management, breed and presence of cats in the farms with a prevalence of T. gondii. With the exception of sex, all others features represent risk factors for T. gondii infection. Furthermore, our data contributed to the understanding of the T. gondii seroprevalence in pig farms located in Southern Piauí.

Este estudo teve como objetivo avaliar a soroprevalência e os fatores de risco associados a infecção por T. gondii em suínos. Foram avaliados 143 suínos em 10 propriedades localizadas no Sul do Estado do Piauí. Os soros dos suínos foram analisados para a detecção de anticorpos anti-T. gondii pela técnica de ELISA. Encontrou-se uma soroprevalência de 25,5% em suínos reativos para antígenos de T. gondii. Por meio de fichas, alguns fatores como idade, dieta, raça, sistema de criação e presença de gatos foram associados à presença de T. gondii. Com exceção do sexo, todas as outras características representaram fatores de risco para a infecção com o parasita. Os dados contribuem para a compreensão da soroprevalência de T. gondii de suínos criados no Sul do Estado do Piauí.