Piplartine-Inspired 3,4,5-Trimethoxycinnamates: Trypanocidal, Mechanism of Action, and In Silico Evaluation.

Chagas disease (CD) is one of the main neglected tropical diseases that promote relevant socioeconomic impacts in several countries. The therapeutic options for the treatment of CD are limited, and parasite resistance has been reported. Piplartine is a phenylpropanoid imide that has diverse biological activities, including trypanocidal action. Thus, the objective of the present work was to prepare a collection of thirteen esters analogous to piplartine (1-13) and evaluate their trypanocidal activity against Trypanosoma cruzi. Of the tested analogues, compound 11 ((E)-furan-2-ylmethyl 3-(3,4,5-trimethoxyphenyl)acrylate) showed good activity with IC50 values = 28.21 ± 5.34 µM and 47.02 ± 8.70 µM, against the epimastigote and trypomastigote forms, respectively. In addition, it showed a high rate of selectivity to the parasite. The trypanocidal mechanism of action occurs through the induction of oxidative stress and mitochondrial damage. In addition, scanning electron microscopy showed the formation of pores and leakage of cytoplasmic content. Molecular docking indicated that 11 probably produces a trypanocidal effect through a multi-target mechanism, including affinity with proteins CRK1, MPK13, GSK3B, AKR, UCE-1, and UCE-2, which are important for the survival of the parasite. Therefore, the results suggest chemical characteristics that can serve for the development of new trypanocidal prototypes for researching drugs against Chagas disease.

Nuclear export of replication protein A in the nonreplicative infective forms of Trypanosoma cruzi.

Replication protein A (RPA), a heterotrimeric complex, is the major single-stranded DNA binding protein in eukaryotes. Recently, we characterized RPA from Trypanosoma cruzi, showing that it is involved in DNA replication and DNA damage response in this organism. Better efficiency in differentiation from epimastigote to metacyclic trypomastigote forms was observed in TcRPA-2 subunit heterozygous knockout cells, suggesting that RPA is involved in this process. Here, we show that RPA cellular localization changes during the T. cruzi life cycle, with RPA being detected only in the cytoplasm of the metacyclic and bloodstream trypomastigotes. We also identify a nuclear export signal (NES) in the trypanosomatid RPA-2 subunit. Mutations in the negatively charged residues of RPA-2 NES impair the differentiation process, suggesting that RPA exportation affects parasite differentiation into infective forms.

Revisión de más de 40 años de estudios en pacientes seropositivos para la enfermedad de Chagas en la provincia de Entre Ríos, Argentina / Review of more than 40 years of studies in seropositive patients for Chagas disease in the province of Entre Ríos, Argentina

El presente trabajo es una revisión de más de 40 años de investigación sobre los aspectos epidemiológicos, socioeconómicos y clínicos de la Enfermedad de Chagas en el Departamento Uruguay (Provincia de Entre Ríos). Se resumen las investigaciones sobre la presencia de vectores triatominos en viviendas urbanas y rurales donde se procedió a su identificación y búsqueda de Trypanosoma cruzi. Se describió la seroprevalencia de la enfermedad y su evolución a lo largo del tiempo. Se agregaron también aspectos cardiológicos y gastroenterológicos de pacientes en el período crónico de la enfermedad. La presencia de triatominos derivó en campañas de fumigación y de educación sanitaria, que provocaron un marcado descenso en el número de vectores capturados en los años siguientes. También se destacó el descenso de la seroprevalencia de la enfermedad de Chagas a través del tiempo, debido a las campañas de fumigación, educación sanitaria, controles en banco de sangre y embarazadas, mejora de las técnicas utilizadas y la aparición de tratamientos efectivos contra el parásito. Los estudios cardiológicos y gastroenterológicos de los pacientes crónicos mostraron alteraciones dentro de lo esperado para este estadio de la enfermedad. También se describió la detección de personas infectadas con acceso al tratamiento y los estudios cardiológicos y gastroenterológicos realizados en pacientes en estadio crónico. Por último, se considera que, globalmente, los estudios realizados en la zona han colaborado en lograr que la Provincia de Entre Ríos fuera declarada libre de transmisión vectorial en 2012.

This is a review of more than 40 years of research on the epidemiological, socioeconomic and clinical aspects of Chagas disease in the Department of Uruguay (Entre Ríos province). Research on the presence of triatomine vectors in urban and rural housing is summarized here.These vectors were identified and Trypanosoma cruzi was searched for. . The seroprevalence of the disease and its evolution over time were described. Cardiological and gastroenterological aspects of patients in the chronic period of the disease were also added. The presence of triatomines resulted in fumigation and health education campaigns, which caused a marked decrease in the number of vectors captured in the following years. The decrease in the seroprevalence of Chagas disease over time was also highlighted, due to fumigation campaigns, health education, blood bank and pregnant women controls, improvement of the techniques used and the development of effective treatments against the parasite. Cardiological and gastroenterological studies of chronic patients showed the abnormalities expected for this stage of the disease. The detection of infected persons with access to treatment and cardiological and gastroenterological studies performed in patients with chronic stage were also described. Finally, it is considered that, globally, the studies carried out in the area have helped to ensure that the Province of Entre Ríos be declared free of the vector transmission in 2012.

Este trabalho é uma revisão de mais de 40 anos de pesquisa sobre os aspectos epidemiológicos, socioeconômicos e clínicos da doença de Chagas no Departamento Uruguai (província de Entre Ríos). São resumidas as pesquisas sobre a presença de vetores de triatomíneos em moradias urbanas e rurais, onde foram identificados e pesquisados por Trypanosoma cruzi nelas. A soroprevalência da doença e sua evolução ao longo do tempo foram descritas. Também foram adicionados aspectos cardiológicos e gastroenterológicos dos pacientes no período crônico da doença. A presença de triatomíneos resultou em campanhas de fumigação e educação sanitária, o que causou uma redução acentuada no número de vetores capturados nos anos seguintes. Também foi destacada a diminuição da soroprevalência da doença de Chagas através do tempo, devido às campanhas de fumigação, educação sanitária, controles em bancos de sangue e gestantes, melhora das técnicas utilizadas e surgimento de tratamentos eficazes contra o parasita. Estudos cardiológicos e gastroenterológicos de pacientes crônicos mostraram alterações dentro do esperado para esse estágio da doença. Também foram descritas a detecção de pessoas infectadas com acesso ao tratamento e estudos cardiológicos e gastroenterológicos realizados em pacientes em estágio crônico. Por fim, consideramos que, globalmente, os estudos realizados na área ajudaram a garantir que a Província de Entre Ríos fosse declarada livre de transmissão vetorial em 2012.

Corrigendum: Activation of Human CD11b+ B1 B-Cells by Trypanosoma cruzi-Derived Proteins Is Associated With Protective Immune Response in Human Chagas Disease.

[This corrects the article DOI: 10.3389/fimmu.2018.03015.].

Activation of Human CD11b+ B1 B-Cells by Trypanosoma cruzi-Derived Proteins Is Associated With Protective Immune Response in Human Chagas Disease.

B-cells mediate humoral adaptive immune response via the production of antibodies and cytokines, and by inducing T-cell activation. These functions can be attributed to distinct B-cell subpopulations. Infection with Trypanosoma cruzi, the causative agent of Chagas disease, induces a polyclonal B-cell activation and lytic antibody production, critical for controlling parasitemia. Individuals within the chronic phase of Chagas disease may remain in an asymptomatic form (indeterminate), or develop severe cardiomyopathy (cardiac form) that can lead to death. Currently, there is no effective vaccine to prevent Chagas disease, and no treatment to halt the development of the cardiomyopathy once it is installed. The pathology associated with cardiac Chagas disease is a result of an inflammatory reaction. Thus, discovering characteristics of the host's immune response that favor the maintenance of favorable heart function may unveil important immunotherapeutic targets. Given the importance of B cells in antibody production and parasite control, we investigated T. cruzi-derived antigenic fractions responsible for B-cell activation and whether frequencies and functional characteristics of B-cell subpopulations are associated with different clinical outcomes of human Chagas disease. We stimulated cells from indeterminate (I) and cardiac (C) Chagas patients, as well as non-infected individuals (NI), with T. cruzi-derived protein- (PRO), glycolipid- (GCL) and lipid (LIP)-enriched fractions and determined functional characteristics of B-cell subpopulations. Our results showed that the frequency of B-cells was similar amongst groups. PRO, but not GCL nor LIP, led to an increased frequency of B1 B-cells in I, but not C nor NI. Although stimulation with PRO induced higher TNF expression by B1 B-cells from C and I, as compared to NI, it induced expression of IL-10 in cells from I, but not C. Stimulation with PRO induced an increased frequency of the CD11b+ B1 B-cell subpopulation, which was associated with better cardiac function. Chagas patients displayed increased IgM production, and activation of gamma-delta T-cells, which have been associated with B1 B-cell function. Our data showed that PRO activates CD11b+ B1 B-cells, and that this activation is associated with a beneficial clinical status. These findings may have implications in designing new strategies focusing on B-cell activation to prevent Chagas disease cardiomyopathy.

Caracterización del perfil antigénico de extractos proteicos solubles de cepas de Trypanosoma cruzi / Antigenic profile characterization of soluble protein extracts from two Trypanosoma cruzi strains

El parásito Trypanosoma cruzi es el agente causal de la enfermedad de Chagas. El mismo presenta una amplia diversidad biológica y genética, por lo cual se lo agrupa en 6 unidades taxonómicas discretas. Las cepas Y, CL Brener y una aislada en Paraguay aún no caracterizada en su totalidad fueron empleadas en este estudio con el fin de determinar sus perfiles proteico y antigénico para aportar en la investigación sobre esta enfermedad en nuestro país, aplicando técnicas como SDS-PAGE, ELISA y Western blot. Se observaron semejanzas en los perfiles proteicos de los extractos solubles de Y Lote 73 e Y Lote 74, siendo ambos obtenidos a partir de la misma cepa, así como perfil diferente de éstos con los de CL Brener y Py. Se detectaron bandas de proteínas de pesos moleculares comunes entre extractos, principalmente las consideradas de bajo peso molecular. Se constató la capacidad antigénica al ensayar los 4 extractos frente a sueros chagásicos, no chagásicos, controles positivos y negativos, se evidenció la importancia de confirmar el diagnóstico con pruebas de principios diferentes. Además, se detectaron proteínas antigénicas comunes a los 3 extractos empleados en el Western blot, las que podrían ser estudiadas con mayor profundidad debido a su potencial antigénico, característica de interés para fines diagnósticos.

The parasite Trypanosoma cruzi is the causal agent of Chagas disease.It presents a wide biological and genetic diversity, therefore it is grouped into 6 taxonomic units..The strains Y, CL Brener and one strain isolated in Paraguaywere used in this study in order to determine their protein and antigenic profiles, applying techniques such as SDS-PAGE, ELISA and Western blot. Similarities were observed in the protein prifles of the soluble extracts of Lot 73 Y and Lot 74 Y both obtained from the same strain, and the different profile of these with those of CL Brener and Py strains. Protein bands of common molecular weights, manly those consideredlow, were detected between extracts. By testing the four extracts against chagasic and non-chagasic sera, positive and negative controls, the antigenic capacity was verified and the importance of utilizing different test to confirm diagnosis was shown. In addition antigenic proteins common to the three extracts used in the Western blot were detected and can be further investigated due to their antigenic potential, an interesting characteristic for diagnostic purpose.

Buthionine sulfoximine is a multitarget inhibitor of trypanothione synthesis in Trypanosoma cruzi.

Buthionine sulfoximine (BSO) induces decreased glutathione (GSH) and trypanothione [T(SH)2 ] pools in trypanosomatids, presumably because only gamma-glutamylcysteine synthetase (γECS) is blocked. However, some BSO effects cannot be explained by exclusive γECS inhibition; therefore, its effect on the T(SH)2 metabolism pathway in Trypanosoma cruzi was re-examined. Parasites exposed to BSO did not synthesize T(SH)2 even when supplemented with cysteine or GSH, suggesting trypanothione synthetase (TryS) inhibition by BSO. Indeed, recombinant γECS and TryS, but not GSH synthetase, were inhibited by BSO and kinetics and docking analyses on a TcTryS 3D model suggested BSO binding at the GSH site. Furthermore, parasites overexpressing γECS and TryS showed ~ 50% decreased activities after BSO treatment. These results indicated that BSO is also an inhibitor of TryS.

Estudo do potencial anti-leishmania e anti-trypanosoma cruzi do ergosterol isolado do basidiomiceto pleurotus salmoneostramineus

Considerando a necessidade de novos tratamentos para doenças negligenciadas como a leishmaniose visceral e a doença de Chagas, o presente trabalho realizou o fracionamento do basidiomiceto comestível Pleurotus salmoneos tramineus na busca por substâncias potencialmente antiparasitárias. Dentre as frações ativas, foi isolado um composto denoninado ergosterol, o qual apresentou atividade anti-Leishmania (L.) infantum e anti-Trypanosoma cruzi. O ergosterol foi ativo contra amastigotas intracelulares de Leishmania (L.) infantum, com valor de Concentração Efetiva 50% (CE50) de 125 µM e de 129 µM contra formas tripomastigotas de Trypanosoma cruzi. O estudo da citotoxicidade em células de mamífero resultou em um valor de CE50 de 619 µM. Seu mecanismo de ação em tripomastigotas resultou uma rápida permeabilização da membrana plasmática, com a despolarização do potencial de membrana mitocondrial,levando o parasito à morte. Apesar disso, não se verificou aumento de espécies reativas de oxigênio no parasito, demonstrando que seu mecanismo de ação não envolve a indução de estresse oxidativo. A seleçãode metabólitos secundários antiparasitários presentes na natureza podefornecer futuros protótipos para o desenho de novos fármacos para doenças negligenciadas.

Considering the need for new treatments for neglected diseases as visceralleishmaniasis and Chagas disease, in this work we fractionated the ediblemushroom Pleurotus salmoneostramineus in the search for potentialantiparasitic compounds. Among the active fractions, it was isolated theergosterol, which showed anti-Leishmania (L.) infantum e anti-Trypanosomacruzi activities. The ergosterol was active against intracellular amastigotes ofLeishmania (L.) infantum and trypomastigotes of Trypanosoma cruzi, with50% Inhibitory Concentration (IC50) values of 125 µM and 129 µM,respectively. The cytotoxicity in mammalian cells resulted in an IC50 value of619 µM. Its mechanism of action in Trypanosoma cruzi trypomastigotesresulted in permeabilization of the plasma membrane, as well asdepolarization of mitochondrial membrane potential, leading to parasitedeath. Nevertheless, there was no increase in reactive oxygen species,demonstrating that its mechanism of action does not involve the induction ofoxidative stress in the parasite. The selection of antiparasitic secondarymetabolites present in nature can provide future prototypes for the design ofnew drugs for neglected diseases.

Actividad antiparasitaria de extractos de plantas colombianas de la familia Euphorbiaceae

Introducción: La familia Euphorbiaceae es un grupo heterogéneo de plantas distribuidas en el territorio colombiano utilizadas algunas de ellas, como plantas medicinales. Objetivo: Determinar la actividad tóxica de aceites esenciales (AE) y extractos de plantas obtenidos de la familia Euphorbiaceae contra tripanosomátidos. Materiales y métodos: Los AE de Croton pedicellatus Kunth (AE1) y C.leptostachyus Kunth (AE2) y el extracto de Phyllanthus acuminatus Vahl fueron obtenidos por hidrodestilación asistida por la radiación de microondas y maceración con metanol; se caracterizaron por cromatografía de gases acoplada a espectrometría de masas. Fueron evaluados contra las formas extracelulares e intracelulares de Trypanosoma cruzi, Leishmania (Viannia) panamensis, L. (V.) braziliensis y células Vero y THP-1. La actividad antiparasitaria fue determinada por recuento microscópico y el efecto tóxico en células por la prueba colorimétrica de MTT. Los resultados fueron expresados como la concentración que inhibe (CI50) o destruye (CC50) el 50% de parásitos o células. Resultados: Los componentes mayoritarios de los AE fueron borneol, γ-terpineno, germacreno D y trans-ß-cariofileno. Los AE1 y AE2 inhibieron el crecimiento de epimastigotes de T.cruzi y de promastigotesde L. (V.) panamensis y L. (V.) braziliensis con CI50 entre 7,14-8,78μg/mL y fueron activos contra amastigotes intracelulares de L. (V.) braziliensis (AE1:CI50 36,74 y AE2:19,77μg/mL). El extracto 1 mostró baja actividad contra los parásitos. Los AE y extractosmostraron toxicidad en células THP-1(CC50 9,29-64,12μg/mL) y células Vero (CC50 24,86-3,52μg/mL). Conclusión: Los AE obtenidos de plantas de la familia de Euphorbiaceae mostraron actividad antiparasitaria con toxicidad moderada en células de mamífero.

Introduction: The Euphorbiaceae family is a heterogeneous group of plants distributed in the Colombian territory used for medicinal proposes. Objective: To determine the toxic activity of Euphorbiaceae family essential oils and plant extract against tripanosomatides. Materials and methods: Essential oil from Croton pedicellatus Kunth (EO1) and C.leptostachyus Kunth(EO 2) and plant extract from Phyllanthus acuminatus Vahl (Ext1) were obtained by microwave-assisted hidrodistillation and characterized by gas chromatography coupled with mass spectrometry and methanol maceration. They were assessed against extracellular and intracellular forms of Trypanosoma cruzi, Leishmania(Viannia) panamensis, L.(Viannia)braziliensis and Vero and THP-1 cells. The parasite activity was determined by microscopic counting and cell toxicity by MTT colorimetric test. The results were expressed as the inhibitory (ICz50) or citotoxic (CC50) concentration for 50% of parasites or cells. Results: The EO-major components were borneol, γ-terpinene and trans-ß-caryophyllene. The EO1 and EO2 inhibited both T.cruzi-epimastigotes and L(V.) panamensis and L.(V.) brazilienses promastigotes growth with IC50 between 7.14 to 8.78 μg/mL They were active against intracellular amastigotes of L. (V.) braziliensis (EO1:IC50:36.74 and EO2:19.77μg/mL). The Ext 1 showed low activity against both parasites. The EOs and extracts were partially toxic to THP-1 (CC50 9.29 to 64.12μg/mL) and Vero cells (CC50 24.86 to 63.52μg/mL). Conclusions: The EO obtained from plants of the Euphorbiaceae family showed antiparasitic activity with some toxicity against mammalian cells.

Associação do polimorfismo de ifng +874T/A com a cardiopatia chagásica crônica / Association of ifng +874 T/A polymorphism with chronic chagas' disease

A doença de Chagas é uma zoonose causada pelo parasita hemoflagelado Trypanosoma cruzi. Atualmente afeta cerca de 10 milhões de pessoas nas Américas, e aproximadamente 90 milhões de pessoas se encontram em áreas de risco. A cardiopatia chagásica crônica (CCC) se desenvolve em um terço dos indivíduos infectados e corresponde a uma miocardite crônica fibrosante intensa e os possíveis fatores que podem contribuir para o desenvolvimento desta podem estar relacionados com a carga parasitária, a cepa do parasita, a autoimunidade e fatores genéticos do parasita e do hospedeiro. Citocinas e quimiocinas têm um importante papel no desenvolvimento de uma resposta imune protetora contra o parasita, porém esta pode estar envolvida com o aumento da inflamação encontrada no miocárdio de pacientes com CCC. O principal objetivo deste estudo foi identificar a freqüência dos genótipos e alelos para o SNP no IFNG na posição +874T/A através da técnica de ARMS - PCR, em 102 pacientes soropositivos para o T. cruzi apresentando a CCC, 86 pacientes soropositivos sem cardiopatia aparente e 179 controles soronegativos para T. cruzi. Não há diferença estatística quando pacientes com CCC foram comparados com indivíduos saudáveis, indicando que o polimorfismo para IFNG na posição +874T/A não parece ter influencia na infecção. Contudo, observamos que o genótipo AA foi mais freqüentemente encontrado em pacientes portadores da CCC do que naqueles portadores da forma indeterminada, sugerindo que indivíduos com este genótipo estão mais susceptíveis ao aparecimento de lesões cardíacas e ao adoecimento (p=0,024)...

Chagas disease is a zoonosis caused by the parasite hemoflagellate Trypanosoma cruzi. Currently affects approximately 10 millions people in the Americas, where approximately 90 millions people are at risk areas. The Chronic chagasic cardiomyopathy (CCC) develops in one third of infected individuals and represents a severe chronic myocarditi. Possible factors that may contribute to the development of several cariopathy can be related to parasite load and difference on strains, autoimmunity and the genetics of the parasite and host. Cytokines and chemokinesplay an important role in the development of a protective immune response against the parasite, but it may be involved with increased inflammation found in the myocardium of CCC. The main objective of this study was to identify the frequency of genotypes and alleles for the SNP in the IFNG position +874 T/A by ARMS-PCR technique in 102 patients seropositive for T. cruzi presenting the CCC, 86seropositive patients without apparent heart disease and 179 negative controls for T. cruzi. There was no statistical difference when CCC patients were compared withhealthy subjects, indicating that the polymorphism at the position +874 T/A does not seem to influence the infection. However, we observed that the AA genotype was more frequently found among patients with CCC than in patients without apparent heart disease, suggesting that individuals with this genotype are more susceptible to develop cardiac illness (p=0,024)...