Crystal structure of dihydroorotate dehydrogenase from Leishmania major

Dihydroorotate dehydrogenase (DHODH) is the fourth enzyme in the de novo pyrimidine biosynthetic pathway and has been exploited as the target for therapy against proliferative and parasitic diseases. In this study, we report the crystal structures of DHODH from Leishmania major, the species of Leishmania associated with zoonotic cutaneous leishmaniasis, in its apo form and in complex with orotate and fumarate molecules. Both orotate and fumarate were found to bind to the same active site and exploit similar interactions, consistent with a ping-pong mechanism described for class 1A DHODHs. Analysis of LmDHODH structures reveals that rearrangements in the conformation of the catalytic loop have direct influence on the dimeric interface. This is the first structural evidence of a relationship between the dimeric form and the catalytic mechanism. According to our analysis, the high sequence and structural similarity observed among trypanosomatid DHODH suggest that a single strategy of structure-based inhibitor design can be used to validate DHODH as a druggable target against multiple neglected tropical diseases such as Leishmaniasis, Sleeping sickness and Chagas' diseases. (C) 2012 Elsevier Masson SAS. All rights reserved.

Deficient Regulatory T Cell Activity and Low Frequency of IL-17-Producing T Cells Correlate with the Extent of Cardiomyopathy in Human Chagas' Disease

Background: Myocardium damage during Chagas' disease results from the immunological imbalance between pro-and production of anti-inflammatory cytokines and has been explained based on the Th1-Th2 dichotomy and regulatory T cell activity. Recently, we demonstrated that IL-17 produced during experimental T. cruzi infection regulates Th1 cells differentiation and parasite induced myocarditis. Here, we investigated the role of IL-17 and regulatory T cell during human Chagas' disease. Methodology/Principal Findings: First, we observed CD4(+)IL-17(+) T cells in culture of peripheral blood mononuclear cells (PBMC) from Chagas' disease patients and we evaluated Th1, Th2, Th17 cytokine profile production in the PBMC cells from Chagas' disease patients (cardiomyopathy-free, and with mild, moderate or severe cardiomyopathy) cultured with T. cruzi antigen. Cultures of PBMC from patients with moderate and severe cardiomyopathy produced high levels of TNF-alpha, IFN-gamma and low levels of IL-10, when compared to mild cardiomyopathy or cardiomyopathy-free patients. Flow cytometry analysis showed higher CD4(+)IL-17(+) cells in PBMC cultured from patients without or with mild cardiomyopathy, in comparison to patients with moderate or severe cardiomyopathy. We then analyzed the presence and function of regulatory T cells in all patients. All groups of Chagas' disease patients presented the same frequency of CD4(+)CD25(+) regulatory T cells. However, CD4(+)CD25(+) T cells from patients with mild cardiomyopathy or cardiomyopathy-free showed higher suppressive activity than those with moderate and severe cardiomyopathy. IFN-gamma levels during chronic Chagas' disease are inversely correlated to the LVEF (P = 0.007, r = -0.614), while regulatory T cell activity is directly correlated with LVEF (P = 0.022, r = 0.500). Conclusion/Significance: These results indicate that reduced production of the cytokines IL-10 and IL-17 in association with high levels of IFN-gamma and TNF-alpha is correlated with the severity of the Chagas' disease cardiomyopathy, and the immunological imbalance observed may be causally related with deficient suppressor activity of regulatory T cells that controls myocardial inflammation.

Aortic distensibility measured by pulse-wave velocity is not modified in patients with Chagas' disease

Abstract Background Experimental studies demonstrate that infection with trypanosoma cruzi causes vasculitis. The inflammatory lesion process could hypothetically lead to decreased distensibility of large and small arteries in advanced Chagas' disease. We tested this hypothesis. Methods and results We evaluated carotid-femoral pulse-wave velocity (PWV) in 53 Chagas' disease patients compared with 31 healthy volunteers (control group). The 53 patients were classified into 3 groups: 1) 16 with indeterminate form of Chagas' disease; 2) 18 with Chagas' disease, electrocardiographic abnormalities, and normal systolic function; 3) 19 with Chagas' disease, systolic dysfunction, and mild-to-moderate congestive heart failure. No difference was noted between the 4 groups regarding carotid-femoral PWV (8.4 ± 1.1 vs 8.2 ± 1.5 vs 8.2 ± 1.4 vs 8.7 ± 1.6 m/s, P = 0.6) or pulse pressure (39.5 ± 7.6 vs 39.3 ± 8.1 vs 39.5 ± 7.4 vs 39.7 ± 6.9 mm Hg, P = 0.9). A positive, significant, similar correlation occurred between PWV and age in patients with Chagas' disease (r = 0.42, P = 0.002), in controls (r = 0.48, P = 0.006), and also between PWV and systolic blood pressure in both groups (patients with Chagas' disease, r = 0.38, P = 0.005; healthy subjects, r = 0.36, P = 0.043). Conclusion Carotid femoral pulse-wave velocity is not modified in patients with Chagas' disease, suggesting that elastic properties of large arteries are not affected in this disorder.

In vitro trypanocidal evaluation of pinane derivatives from essential oils of ripe fruits from Schinus terebinthifolius Raddi (Anacardiaceae)

Essential oils of ripe fruits from Schinus terebinthifolius (Anacardiaceae), obtained using a pilot extractor and a Clevenger apparatus were chemically characterized. Due the high amount of (-)- α-pinene in both oils, this monoterpene was tested against the protozoan parasite Trypanosoma cruzi, showing a moderate potential (IC50 63.56 µg/mL) when compared to benznidazole (IC50 43.14 µg/mL). Otherwise, (-)- α-pinene oxide did not showed anti-trypanosomal activity (IC50 > 400 µg/mL) while (-)-pinane showed an IC50 of 56.50 µg/mL. The obtained results indicated that the epoxydation of α-pinene results to the loss of the anti-parasitic activity while its hydrogenation product, contributed slightly to the increased activity.

The effect of beta-blockade on myocardial remodelling in Chagas' cardiomyopathy

OBJECTIVE: Chagas' disease has spread throughout Latin America because of the high rate of migration among these countries. Approximately 30% of Chagas' patients will develop cardiomyopathy, and 10% of these will develop severe cardiac damage leading to heart failure. Beta-blockade improves symptoms and survival in heart failure patients; however, its efficacy has not been well established in Chagas' disease. We evaluated the role of carvedilol in cardiac remodeling and mortality in a Chagas' cardiomyopathy animal model. METHODS: We studied Trypanosoma cruzi infection in 55 Syrian hamsters that were divided into three groups: control (15), infected (20), and infected + carvedilol (20). Animals underwent echocardiography, electrocardiography, and morphometry for collagen evaluation in ventricles stained with picrosirius red. RESULTS: The left ventricular diastolic diameter did not change between groups, although it was slightly larger in infected groups, as was left ventricular systolic diameter. Fractional shortening also did not change between groups, although it was slightly lower in infected groups. Collagen accumulation in the interstitial myocardial space was significantly higher in infected groups and was not attenuated by carvedilol. The same response was observed in the perivascular space. The survival curve showed significantly better survival in the control group compared with the infected groups; but no benefit of carvedilol was observed during the study. However, in the acute phase (up to 100 days of infection), carvedilol did reduce mortality. CONCLUSION: Carvedilol did not attenuate cardiac remodeling or mortality in this model of Chagas' cardiomyopathy. The treatment did improve survival in the acute phase of the disease.

Base racional e plano de estudo prospectivo para avaliar o efeito de terapêutica antiplaquetária e vasodilatadora microcirculatória em pacientes com cardiopatia chagásica crônica e distúrbios microvasculares coronários

INTRODUÇÃO: Há evidência, embasada por estudos em modelos experimentais de infecção pelo Trypanosoma cruzi, e também por investigações histopatológicas em humanos com a doença de Chagas, de que distúrbios de natureza isquêmica participem da patogênese de lesões miocárdicas na fase crônica da moléstia. Esses distúrbios isquêmicos derivam de desregulação microcirculatória. Dor precordial atípica é sintoma comum em pacientes na fase crônica da doença de Chagas. Em substancial proporção desses pacientes, apesar da inexistência de obstruções coronárias angiograficamente detectáveis, documenta-se com cintilografia miocárdica a ocorrência de distúrbios perfusionais durante o estresse, que são reversíveis após repouso. MÉTODOS: Estudo unicêntrico, prospectivo, de coorte única, com intervenção terapêutica seguida de reavaliação quantitativa, após 90 dias, da área ventricular apresentando alterações perfusionais isquêmicas inicialmente detectadas em pacientes cardiopatas chagásicos com coronárias angiograficamente normais. A cintilografia miocárdica de perfusão será executada com o método SPECT, antes e após 90 dias da intervenção terapêutica, tendo o sestamibi-Tc99m como radiotraçador e o esforço físico ou o estímulo vasodilatador com dipiridamol como estressores. A intervenção terapêutica consistirá de ácido acetilsalicílico (dose de 100 mg diária) associado a verapamil (dose diária de 160 mg, em duas tomadas de 80 mg). O desfecho primário do estudo será redução > 50% da área ventricular de isquemia miocárdica reversível calculada pelo mapa polar da cintilografia miocárdica de perfusão. CONCLUSÕES: Este é o primeiro estudo de intervenção terapêutica para atenuar ou reverter alterações miocárdicas isquêmicas de origem microvascular em pacientes com cardiopatia chagásica crônica.

Atypical human infections by animal Trypanosomes

The two classical forms of human trypanosomoses are sleeping sickness due to Trypanosoma brucei gambiense or T. brucei rhodesiense, and Chagas disease due to T. cruzi. However, a number of atypical human infections caused by other T. species (or sub-species) have been reported, namely due to T. brucei brucei, T. vivax, T. congolense, T. evansi, T. lewisi, and T. lewisi-like. These cases are reviewed here. Some infections were transient in nature, while others required treatments that were successful in most cases, although two cases were fatal. A recent case of infection due to T. evansi was related to a lack of apolipoprotein L-I, but T. lewisi infections were not related to immunosuppression or specific human genetic profiles. Out of 19 patients, eight were confirmed between 1974 and 2010, thanks to improved molecular techniques. However, the number of cases of atypical human trypanosomoses might be underestimated. Thus, improvement, evaluation of new diagnostic tests, and field investigations are required for detection and confirmation of these atypical cases.

Genetic characterization, population history and evolutionary medicine perspective in two native south american populations: Yanesha and Wichi

Two Amerindian populations from the Peruvian Amazon (Yanesha) and from rural lowlands of the Argentinean Gran Chaco (Wichi) were analyzed. They represent two case study of the South American genetic variability. The Yanesha represent a model of population isolated for long-time in the Amazon rainforest, characterized by environmental and altitudinal stratifications. The Wichi represent a model of population living in an area recently colonized by European populations (the Criollos are the population of the admixed descendents), whose aim is to depict the native ancestral gene pool and the degree of admixture, in relation to the very high prevalence of Chagas disease. The methods used for the genotyping are common, concerning the Y chromosome markers (male lineage) and the mitochondrial markers (maternal lineage). The determination of the phylogeographic diagnostic polymorphisms was carried out by the classical techniques of PCR, restriction enzymes, sequencing and specific mini-sequencing. New method for the detection of the protozoa Trypanosoma cruzi was developed by means of the nested PCR. The main results show patterns of genetic stratification in Yanesha forest communities, referable to different migrations at different times, estimated by Bayesian analyses. In particular Yanesha were considered as a population of transition between the Amazon basin and the Andean Cordillera, evaluating the potential migration routes and the separation of clusters of community in relation to different genetic bio-ancestry. As the Wichi, the gene pool analyzed appears clearly differentiated by the admixed sympatric Criollos, due to strict social practices (deeply analyzed with the support of cultural anthropological tools) that have preserved the native identity at a diachronic level. A pattern of distribution of the seropositivity in relation to the different phylogenetic lineages (the adaptation in evolutionary terms) does not appear, neither Amerindian nor European, but in relation to environmental and living conditions of the two distinct subpopulations.

Procerenone: a Fatty Acid Triterpenoid from the Fruit Pericarp of Omphalocarpum procerum (Sapotaceae)

Phytochemical investigation of a dichloromethane-methanol (1:1) extract of the fruit pericarp of Omphalocarpum procerum which exhibited antiplasmodial activity during preliminary screening led to the isolation of the new fatty ester triterpenoid 3β-hexadecanoyloxy-28-hydroxyolean-12-en-11-one (1), together with five known compounds 2-6. The structure of the new compound as well as those of the known compounds was established by means of spectroscopic methods and by comparison with previously reported data. Compounds 1- 4 were evaluated in-vitro for their cytotoxicity against L6 cell lines and antiprotozoal activities against Plasmodium falciparum, Leishmania donovani, Trypanosoma brucei rhodesiense and Trypanosoma cruzi (species responsible for human malaria, visceral leishmaniasis, African trypanosomiasis and Chagas disease, respectively). The tested compounds showed weak to moderate antiprotozoal activity and, no significant effect was detected regarding their cytotoxic potency.

Genome profiling of sterol synthesis shows convergent evolution in parasites and guides chemotherapeutic attack

Sterols are an essential class of lipids in eukaryotes, where they serve as structural components of membranes and play important roles as signaling molecules. Sterols are also of high pharmacological significance: cholesterol-lowering drugs are blockbusters in human health, and inhibitors of ergosterol biosynthesis are widely used as antifungals. Inhibitors of ergosterol synthesis are also being developed for Chagas's disease, caused by Trypanosoma cruzi. Here we develop an in silico pipeline to globally evaluate sterol metabolism and perform comparative genomics. We generate a library of hidden Markov model-based profiles for 42 sterol biosynthetic enzymes, which allows expressing the genomic makeup of a given species as a numerical vector. Hierarchical clustering of these vectors functionally groups eukaryote proteomes and reveals convergent evolution, in particular metabolic reduction in obligate endoparasites. We experimentally explore sterol metabolism by testing a set of sterol biosynthesis inhibitors against trypanosomatids, Plasmodium falciparum, Giardia, and mammalian cells, and by quantifying the expression levels of sterol biosynthetic genes during the different life stages of T. cruzi and Trypanosoma brucei. The phenotypic data correlate with genomic makeup for simvastatin, which showed activity against trypanosomatids. Other findings, such as the activity of terbinafine against Giardia, are not in agreement with the genotypic profile.

Inhibition of apoptosis by intracellular protozoan parasites

Protozoan parasites which reside inside a host cell avoid direct destruction by the immune system of the host. The infected cell, however, still has the capacity to counteract the invasive pathogen by initiating its own death, a process which is called programmed cell death or apoptosis. Apoptotic cells are recognised and phagocytosed by macrophages and the parasite is potentially eliminated together with the infected cell. This potent defence mechanism of the host cell puts strong selective pressure on the parasites which have, in turn, evolved strategies to modulate the apoptotic program of the host cell to their favour. Within the last decade, the existence of cellular signalling pathways which inhibit the apoptotic machinery has been demonstrated. It is not surprising that intracellular pathogens subvert these pathways to ensure their own survival in the infected cell. Molecular mechanisms which interfere with apoptotic pathways have been studied extensively for viruses and parasitic bacteria, but protozoan parasites have come into focus only recently. Intracellular protozoan parasites which have been reported to inhibit the apoptotic program of the host cell, are Toxoplasma gondii, Trypanosoma cruzi, Leishmania sp., Theileria sp., Cryptosporidium parvum, and the microsporidian Nosema algerae. Although these parasites differ in their mechanism of host cell entry and in their final intracellular localisation, they might activate similar pathways in their host cells to inhibit apoptosis. In this respect, two families of molecules, which are known for their capacity to interrupt the apoptotic program, are currently discussed in the literature. First, the expression of heat shock proteins is often induced upon parasite infection and can directly interfere with molecules of the cellular death machinery. Secondly, a more indirect effect is attributed to the parasite-dependent activation of NF-kappaB, a transcription factor that regulates the transcription of anti-apoptotic molecules.

Seroprevalence of Chagas' disease in Texas among shelter dogs in Houston and the Rio Grande Valley region and its implication for human infection

Chagas’ disease, also called American Trypanosomiasis, is a vector-borne disease caused by the protozoan parasite Trypanosoma cruzi. T. cruzi is spread by triatomine insects, commonly referred to as ‘kissing bugs.’ After the insect takes a blood meal from its animal or human host, it usually defecates near the bite wound. The parasite is present in the feces, and when rubbed into the bite wound or mucous membranes by the host, infection ensues. Chagas’ disease is highly endemic in Central and South America where it originated. Many people in these endemic areas live in poor conditions surrounded by animals, mainly dogs, that can serve as a possible link to human infection. In Chagas’ endemic countries, dogs can be used as a sentinel to infer risk for human infection. In Texas, the prevalence of Chagas’ and risk for human infection is largely unknown. This study aimed to determine the prevalence of Chagas’ disease in shelter dogs in Houston, Texas and the Rio Grande Valley region by using an immunochromatographic assay (Chagas’ Stat-Pak) to test for the presence of T. cruzi antibodies. Of the 822 samples tested, 26 were found to be positive (3.2%). In both locations, Chagas’ prevalence increased over time. This study found that dogs, especially strays, can serve as sentinels for disease activity. Public health authorities can implement this strategy to understand the level of Chagas’ activity in a defined geographic area and prevent human infection.^

Chagas, la enfermedad silenciosa

Este video es parte de una serie de producciones realizadas por alumnos de la Carrera de Comunicación Social de la UNCuyo, como trabajo final de la Cátedra de Televisión y Medios Audiovisuales en el año 2006. Los mismos participaron de una muestra audiovisual en el Cine de la Universidad en agosto de 2007. Son trabajos periodísticos y documentales de alumnos avanzados de la carrera, en formato profesional, abordando temáticas sociales, políticas, culturales y de arte, haciendo visible realidades y personajes de la provincia. Según alumnos y docentes, estos videos “van al encuentro del público mendocino para que nos conozca, critique, reflexione, sonría y se emocione…" CHAGAS, LA ENFERMEDAD SILENCIOSA, documental sobre los distintos aspectos de esta enfermedad: el contagio, sus etapas, la marginación y los estados psicológicos del enfermo.

Evidence for clonal propagation in natural isolates of Plasmodium falciparum from Venezuela

We have analyzed 75 isolates of Plasmodium falciparum, collected in Venezuela during both the dry (November) and rainy (May–July) seasons, with a range of genetic markers including antigen genes and 14 random amplified polymorphic DNA (RAPD) primers. Thirteen P. falciparum stocks from Kenya and four other Plasmodium species are included in the analysis for comparison. Cross-hybridization shows that the 14 RAPD primers reveal 14 separate regions of the parasite's genome. The P. falciparum isolates are a monophyletic clade, significantly different from the other Plasmodium species. We identify three RAPD characters that could be useful as “tags” for rapid species identification. The Venezuelan genotypes fall into two discrete genetic subdivisions associated with either the dry or the rainy season; the isolates collected in the rainy season exhibit greater genetic diversity. There is significant linkage disequilibrium in each seasonal subsample and in the full sample. In contrast, no linkage disequilibrium is detected in the African sample. These results support the hypothesis that the population structure of P. falciparum in Venezuela, but not in Africa, is predominantly clonal. However, the impact of genetic recombination on Venezuelan P. falciparum seems higher than in parasitic species with long-term clonal evolution like Trypanosoma cruzi, the agent of Chagas' disease. The genetic structure of the Venezuelan samples is similar to that of Escherichia coli, a bacterium that propagates clonally, with occasional genetic recombination.

Molecular and cytotoxic effects of camptothecin, a topoisomerase I inhibitor, on trypanosomes and Leishmania.

Parasites pose a threat to the health and lives of many millions of human beings. Among the pathogenic protozoa, Trypanosoma brucei, Trypanosoma cruzi, and Leishmania donovani are hemoflagellates that cause particularly serious diseases (sleeping sickness, Chagas disease, and leishmaniasis, respectively). The drugs currently available to treat these infections are limited by marginal efficacy, severe toxicity, and spreading drug resistance. Camptothecin is an established antitumor drug and a well-characterized inhibitor of eukaryotic DNA topoisomerase I. When trypanosomes or leishmania are treated with camptothecin and then lysed with SDS, both nuclear and mitochondrial DNA are cleaved and covalently linked to protein. This is consistent with the existence of drug-sensitive topoisomerase I activity in both compartments. Camptothecin also inhibits the incorporation of [3H]thymidine in these parasites. These molecular effects are cytotoxic to cells in vitro, with EC50 values for T. brucei, T. cruzi, and L. donovani, of 1.5, 1.6, and 3.2 microM, respectively. For these parasites, camptothecin is an important lead for much-needed new chemotherapy, as well as a valuable tool for studying topoisomerase I activity.