Sesquiterpene lactones affect the redox system of trypanosoma cruzi

Chagas disease is caused by Trypanosoma cruzi (T. cruzi) and affects millions of people worldwide, mostly in Latin America. Despite its sanitary importance, there are currently only two drugs available for its treatment: benznidazole and nifurtimox, both exhibiting serious adverse effects on patients. In order to complete its life cycle, T. cruzi faces extreme environmental conditions ?i.e. oxidative stress- as it propagates from an insect vector to a mammalian host, driving the transition from non-infective epimastigote to the infective form trypomastigote. It is known that the antioxidant defense system in the trypanosomatids is different from that in mammalian cells since the parasites have exclusive molecules and reducing enzymes. Because of this, the parasite redox machinery is an attractive target for antiparasitic therapies. The sesquiterpene lactone dehydroleucodine (DhL), is a trypanocidal molecule containing an alpha-methylene group that could react with sulfhydryl groups of key redox enzymes. This study was focused on elucidating the DhL mechanism of action and extended to ten DhL derivatives (DC-X1 to DC-X10) obtained by chemical substitutions on the methylene group. We firstly confirmed an antiproliferative effect of DhL and its chemical derivatives, being DC- X6 one of the most active. The effect of DhL and DC-X6 was blocked by reduced glutathione, suggesting that compounds are reactive to sulfhydryl groups of certain molecules. Moreover, parasites overexpressing reducing enzymes, such as Tc-CPX, showed a protective effect against these STLs. Consistent with these results, both STLs increased ROS concentration in the wild type parasites. These results indicate that STLs induce oxidative stress on the parasites, possibly by affecting some crucial enzymes of the redox system.Fil: Gomez, Jessica Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Histología y Embriología D/mend Dr.m.burgos; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales. Departamento de Biología; ArgentinaFil: Guarise, C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Histología y Embriología D/mend Dr.m.burgos; ArgentinaFil: Tello Faral, P.. Instituto Pasteur de Montevideo; UruguayFil: Robello, Carlos. Instituto Pasteur de Montevideo; UruguayFil: Caballero, P.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Histología y Embriología D/mend Dr.m.burgos; ArgentinaFil: Cifuente, Diego Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Investigaciones en Tecnología Química. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Investigaciones en Tecnología Química; ArgentinaFil: Sosa, M. A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Histología y Embriología D/mend Dr.m.burgos; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Barrera, Patricia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Histología y Embriología D/mend Dr.m.burgos; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaXXXVII Reunión Científica Anual de la Sociedad de Biología de CuyoSan LuisArgentinaSociedad de Biología de Cuy

Trypanothione synthetase confers growth, survival advantage and resistance to anti-protozoal drugs in Trypanosoma cruzi

Background: Chagas cardiomyopathy, caused by Trypanosoma cruzi infection, continues to be a neglected illness, and has a major impact on global health. The parasite undergoes several stages of morphological and biochemical changes during its life cycle, and utilizes an elaborated antioxidant network to overcome the oxidants barrier and establish infection in vector and mammalian hosts. Trypanothione synthetase (TryS) catalyzes the biosynthesis of glutathione-spermidine adduct trypanothione (T(SH)2) that is the principal intracellular thiol-redox metabolite in trypanosomatids. Methods and Results: We utilized genetic overexpression (TryShi) and pharmacological inhibition approaches to examine the role of TryS in T. cruzi proliferation, tolerance to oxidative stress and resistance to anti-protozoal drugs. Our data showed the expression and activity of TryS was increased in all morphological stages of TryShi (vs. control) parasites. In comparison to controls, the TryShi epimastigotes (insect stage) recorded shorter doubling time, and both epimastigotes and infective trypomastigotes of TryShi exhibited 36-71% higher resistance to H2O2 (50-1000 M) and heavy metal (1-500 M) toxicity. Treatment with TryS inhibitors (5-30 M) abolished the proliferation and survival advantages against H2O2 pressure in a dose-dependent manner in both TryShi and control parasites. Further, epimastigote and trypomastigote forms of TryShi (vs. control) T. cruzi tolerated higher doses of benznidazole and nifurtimox, the drugs currently administered for acute Chagas disease treatment. Conclusions: TryS is essential for proliferation and survival of T. cruzi under normal and oxidant stress conditions, and provides an advantage to the parasite to develop resistance against currently used antitrypanosomal drugs. TryS indispensability has been chemically validated with inhibitors that may be useful for drug combination therapy against Chagas disease.Fil: Mesias, Andrea Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Sasoni, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Pérez Brandan, Cecilia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Orban, Oliver C. F.. Technische Universitat Carolo Wilhelmina Zu Braunschweig.; AlemaniaFil: Kunick, Conrad. Technische Universitat Carolo Wilhelmina Zu Braunschweig.; AlemaniaFil: Robello, Carlos. Instituto Pasteur de Montevideo; UruguayFil: Comini, Marcelo. Instituto Pasteur de Montevideo; UruguayFil: Garg, Nisha J.. University of Texas Medical Branch; Estados UnidosFil: Zago, María Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentin

Longitudinal Association between Late-Life Depression (LLD) and Frailty:Findings from a Prospective Cohort Study (MiMiCS-FRAIL)

OBJECTIVES: The aim of the present study was to investigate whether late-life depression (LLD) is associated with incident frailty over time. DESIGN: Prospective cohort study, one-year follow-up. SETTING: Geriatric outpatient clinic, Southwestern of Brazil. PARTICIPANTS: 181 follow-up participants aged 60 years or over. MEASUREMENTS: Depressive disorders were classified as Major Depressive disorder (MDD) or Subthreshold Depression (STD) according to DSM-5 criteria. Depressive symptoms were assessed with validated versions of 15-item Geriatric Depression Scale (GDS-15) and 9-item Patient Health Questionnaire (PHQ-9). We performed binary logistic regressions to estimate the odds ratio (OR) for frailty in LLD adjusting for multiple confounders. Participants who were frail at baseline were excluded from the analyses according to measures of frailty (FRAIL questionnaire and 36-item Frailty Index, FI-36). We also estimated the risk ratio or relative risk (RR) and the risk difference (RD) for incident frailty. RESULTS: We observed a 2 to 4-fold increased risk for incident frailty among participants with LLD. The presence of a depressive disorder was significantly associated with the onset of frailty (adjusted OR for FRAIL and FI-36: 3.07 [95% CI = 1.03 - 9.17] and 3.76 [95% CI = 1.09 - 12.97], respectively. Notably, the risk for frailty due to LLD was significantly higher with the FI-36 compared to the FRAIL (RR: 3.03 versus 2.23). RD was of 17.3% and 12.7% with the FRAIL and the FI-36, respectively. CONCLUSION: Our data support the association between LLD and incident frailty over one year among geriatric outpatients, reinforcing longitudinal evidence from population-based studies

The association between cardiovascular risk factors and major cardiovascular diseases decreases with increasing frailty levels in geriatric outpatients

BACKGROUND: Frailty marks a process of increasing dysregulation of physiological systems which increases the risk of adverse health outcomes. This study examines the hypothesis that the association between multiple cardiovascular risk factors (CVRF) and cardiovascular diseases (CVD) becomes stronger with increasing frailty severity. METHODS: Cross-sectional analysis of 339 older adults (55.2% women; aged 75.2 ± 9.1 years) from an outpatient geriatric clinic from a middle-income country. The frailty index (FI) was calculated as the proportion of 30 possible health deficits. We assessed hypertension, diabetes, obesity, dyslipidemia, sedentarism and smoking as CVRF (determinants) and myocardial infarction, stroke, heart failure as CVD. Poisson regression models adjusted for age, sex, and education was applied to estimate the association between frailty as well as CVRF (independent variables) with CVD (dependent variable). RESULTS: Of the 339 patients, 18,3% were frail (FI ≥ 0.25) and 32.7% had at least one CVD. Both frailty and CVRF were significantly associated with CVD (PR = 1.03, 95% CI 1.01 to 1.05; p = 0.001, and PR = 1.46, 95% 1.24 to 1.71; p < 0.001, respectively) adjusted for covariates. The strength of the association between CVRF and CVD decreased with increasing frailty levels, as indicated by a significant interaction term of frailty and CVRF (p < 0.001). CONCLUSION: Frailty and CVRF are both associated with CVD, but the impact of CVRF decreases in the presence of frailty. When confirmed in longitudinal studies, randomized controlled trials or causal inference methods like Mendelian randomization should be applied to assess whether a shift from traditional CVRF to frailty would improve cardiovascular outcome in the oldest old

Preclinical Studies in Anti-Trypanosomatidae Drug Development

The trypanosomatid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania are the causative agents of human African trypanosomiasis, Chagas Disease and Leishmaniasis, respectively. These infections primarily affect poor, rural communities in the developing world, and are responsible for trapping sufferers and their families in a disease/poverty cycle. The development of new chemotherapies is a priority given that existing drug treatments are problematic. In our search for novel anti-trypanosomatid agents, we assess the growth-inhibitory properties of >450 compounds from in-house and/or “Pathogen Box” (PBox) libraries against L. infantum, L. amazonensis, L.braziliensis, T. cruzi and T. brucei and evaluate the toxicities of the most promising agents towards murine macrophages. Screens using the in-house series identified 17 structures with activity against and selective toward Leishmania: Compounds displayed 50% inhibitory concentrations between 0.09 and 25 μM and had selectivity index values >10. For the PBox library, ~20% of chemicals exhibited anti-parasitic properties including five structures whose activity against L. infantum had not been reported before. These five compounds displayed no toxicity towards murine macrophages over the range tested with three being active in an in vivo murine model of the cutaneous disease, with 100% survival of infected animals. Additionally, the oral combination of three of them in the in vivo Chagas disease murine model demonstrated full control of the parasitemia. Interestingly, phenotyping revealed that the reference strain responds differently to the five PBox-derived chemicals relative to parasites isolated from a dog. Together, our data identified one drug candidate that displays activity against Leishmania and other Trypanosomatidae in vitro and in vivo, while exhibiting low toxicity to cultured mammalian cells and low in vivo acute toxicity

Genome-wide diversity and differentiation in New World populations of the human malaria parasite Plasmodium vivax.

BACKGROUND: The Americas were the last continent colonized by humans carrying malaria parasites. Plasmodium falciparum from the New World shows very little genetic diversity and greater linkage disequilibrium, compared with its African counterparts, and is clearly subdivided into local, highly divergent populations. However, limited available data have revealed extensive genetic diversity in American populations of another major human malaria parasite, P. vivax. METHODS: We used an improved sample preparation strategy and next-generation sequencing to characterize 9 high-quality P. vivax genome sequences from northwestern Brazil. These new data were compared with publicly available sequences from recently sampled clinical P. vivax isolates from Brazil (BRA, total n = 11 sequences), Peru (PER, n = 23), Colombia (COL, n = 31), and Mexico (MEX, n = 19). PRINCIPAL FINDINGS/CONCLUSIONS: We found that New World populations of P. vivax are as diverse (nucleotide diversity π between 5.2 × 10-4 and 6.2 × 10-4) as P. vivax populations from Southeast Asia, where malaria transmission is substantially more intense. They display several non-synonymous nucleotide substitutions (some of them previously undescribed) in genes known or suspected to be involved in antimalarial drug resistance, such as dhfr, dhps, mdr1, mrp1, and mrp-2, but not in the chloroquine resistance transporter ortholog (crt-o) gene. Moreover, P. vivax in the Americas is much less geographically substructured than local P. falciparum populations, with relatively little between-population genome-wide differentiation (pairwise FST values ranging between 0.025 and 0.092). Finally, P. vivax populations show a rapid decline in linkage disequilibrium with increasing distance between pairs of polymorphic sites, consistent with very frequent outcrossing. We hypothesize that the high diversity of present-day P. vivax lineages in the Americas originated from successive migratory waves and subsequent admixture between parasite lineages from geographically diverse sites. Further genome-wide analyses are required to test the demographic scenario suggested by our data

Deltamethrin Resistance Mechanisms in Aedes aegypti Populations from Three French Overseas Territories Worldwide

BACKGROUND:Aedes aegypti is a cosmopolite mosquito, vector of arboviruses. The worldwide studies of its insecticide resistance have demonstrated a strong loss of susceptibility to pyrethroids, the major class of insecticide used for vector control. French overseas territories such as French Guiana (South America), Guadeloupe islands (Lesser Antilles) as well as New Caledonia (Pacific Ocean), have encountered such resistance. METHODOLOGY/PRINCIPAL FINDINGS:We initiated a research program on the pyrethroid resistance in French Guiana, Guadeloupe and New Caledonia. Aedes aegypti populations were tested for their deltamethrin resistance level then screened by an improved microarray developed to specifically study metabolic resistance mechanisms. Cytochrome P450 genes were implicated in conferring resistance. CYP6BB2, CYP6M11, CYP6N12, CYP9J9, CYP9J10 and CCE3 genes were upregulated in the resistant populations and were common to other populations at a regional scale. The implication of these genes in resistance phenomenon is therefore strongly suggested. Other genes from detoxification pathways were also differentially regulated. Screening for target site mutations on the voltage-gated sodium channel gene demonstrated the presence of I1016 and C1534. CONCLUSION /SIGNIFICANCE:This study highlighted the presence of a common set of differentially up-regulated detoxifying genes, mainly cytochrome P450 genes in all three populations. GUA and GUY populations shared a higher number of those genes compared to CAL. Two kdr mutations well known to be associated to pyrethroid resistance were also detected in those two populations but not in CAL. Different selective pressures and genetic backgrounds can explain such differences. These results are also compared with those obtained from other parts of the world and are discussed in the context of integrative research on vector competence

Research Priorities for Neglected Infectious Diseases in Latin America and the Caribbean Region

Dujardin, J. C. et al. 5 p.-1 tab.Global priorities for research in neglected infectious diseases (NIDs) can be assessed in different ways, but it is important to realize that regional priorities may significantly differ one from another. The region of Latin America and the Caribbean (LAC) is—along with Africa and Asia—more affected by NIDs than other regions of the world. Some of the Latin American NIDs are common to other continents, while others are very specific or disproportionately affect the Latin American region [1– 3] (Table 1). Because of its huge ecological diversity, ongoing environmental changes, and massive migrations, LAC is also a catalyst for the (re-)emergence and spreading of NIDs, both inside and outside the subcontinent. Following a colloquium on NIDs in LAC held in Lima, Peru, between 12 and 14 November 2009, a thematic workshop was organized with the support of the European Commission (EC). It involved 29 scientists (16 from the Americas, two from the Democratic Republic of Congo and India, respectively, and nine from Europe) working on different NIDs and representing several research areas from basic to applied. This report summarizes the consensus comments of the expert group after oral and written consultation. It is envisaged that this document should stimulate a debate within the scientific community and serve as a recommendation for future actions by international or regional funding agencies in the area of NIDs in LACThis work was supported by the Directorate-General for Development Cooperation of the Belgian Government (framework agreement 03, project 95502) and the European CommissionPeer reviewe

Analyses of 32 Loci Clarify Phylogenetic Relationships among Trypanosoma cruzi Lineages and Support a Single Hybridization prior to Human Contact

Trypanosoma cruzi is the protozoan parasite that causes Chagas disease, a major health problem in Latin America. The genetic diversity of this parasite has been traditionally divided in two major groups: T. cruzi I and II, which can be further divided in six major genetic subdivisions (subgroups TcI-TcVI). T. cruzi I and II seem to differ in important biological characteristics, and are thought to represent a natural division relevant for epidemiological studies and development of prophylaxis. Having a correct reconstruction of the evolutionary history of T. cruzi is essential for understanding the potential connection between the genetic and phenotypic variability of T. cruzi with the different manifestations of Chagas disease. Here we present results from a comprehensive phylogenetic analysis of T. cruzi using more than 26 Kb of aligned sequence data. We show strong evidence that T. cruzi II (TcII-VI) is not a natural evolutionary group but a paraphyletic lineage and that all major lineages of T. cruzi evolved recently (<3 million years ago [mya]). Furthermore, the sequence data is consistent with one major hybridization event having occurred in this species recently (< 1 mya) but well before T. cruzi entered in contact with humans in South America