The repositioned drugs disulfiram/diethyldithiocarbamate combined to benznidazole: Searching for Chagas disease selective therapy, preventing toxicity and drug resistance.

Chagas disease (CD) affects at least 6 million people in 21 South American countries besides several thousand in other nations all over the world. It is estimated that at least 14,000 people die every year of CD. Since vaccines are not available, chemotherapy remains of pivotal relevance. About 30% of the treated patients cannot complete the therapy because of severe adverse reactions. Thus, the search for novel drugs is required. Here we tested the benznidazole (BZ) combination with the repositioned drug disulfiram (DSF) and its derivative diethyldithiocarbamate (DETC) upon Trypanosoma cruzi in vitro and in vivo. DETC-BZ combination was synergistic diminishing epimastigote proliferation and enhancing selective indexes up to over 10-fold. DETC was effective upon amastigotes of the BZ- partially resistant Y and the BZ-resistant Colombiana strains. The combination reduced proliferation even using low concentrations (e.g., 2.5 µM). Scanning electron microscopy revealed membrane discontinuities and cell body volume reduction. Transmission electron microscopy revealed remarkable enlargement of endoplasmic reticulum cisternae besides, dilated mitochondria with decreased electron density and disorganized kinetoplast DNA. At advanced stages, the cytoplasm vacuolation apparently impaired compartmentation. The fluorescent probe H2-DCFDA indicates the increased production of reactive oxygen species associated with enhanced lipid peroxidation in parasites incubated with DETC. The biochemical measurement indicates the downmodulation of thiol expression. DETC inhibited superoxide dismutase activity on parasites was more pronounced than in infected mice. In order to approach the DETC effects on intracellular infection, peritoneal macrophages were infected with Colombiana trypomastigotes. DETC addition diminished parasite numbers and the DETC-BZ combination was effective, despite the low concentrations used. In the murine infection, the combination significantly enhanced animal survival, decreasing parasitemia over BZ. Histopathology revealed that low doses of BZ-treated animals presented myocardial amastigote, not observed in combination-treated animals. The picrosirius collagen staining showed reduced myocardial fibrosis. Aminotransferase de aspartate, Aminotransferase de alanine, Creatine kinase, and urea plasma levels demonstrated that the combination was non-toxic. As DSF and DETC can reduce the toxicity of other drugs and resistance phenotypes, such a combination may be safe and effective.

Histone deacetylases inhibitors as new potential drugs against Leishmania braziliensis, the main causative agent of new world tegumentary leishmaniasis.

The protozoan parasite Leishmania braziliensis is a major causative agent of the neglected tropical diseases Cutaneous and Mucocutaneous Leishmaniases in the New World. There are no vaccines to prevent the infection and the treatment relies on few drugs that often display high toxicity and costs. Thus, chemotherapeutic alternatives are required. Histone Deacetylases (HDACs) are epigenetic enzymes involved in the control of chromatin structure. In this work, we tested an in-house library of 78 hydroxamic acid derivatives as putative inhibitors of L. braziliensis HDACs (HDACi). The compounds were evaluated in relation to the toxicity to the host cell macrophage and to the leishmanicidal effect against L. braziliensis during in vitro infection. Eight HDACi showed significant leishmanicidal effects and the top 5 compounds showed effective concentrations (EC50) in the range of 4.38 to 10.21 µM and selectivity indexes (SI) from of 6 to 21.7. Analyses by Transmission Electron Microscopy (TEM) indicated induction of apoptotic cell death of L. braziliensis amastigotes with a necrotic phenotype. An altered chromatin condensation pattern and cellular disorganization of intracellular amastigotes was also observed. A tight connection between the mitochondrion and nuclear protrusions, presumably of endoplasmic reticulum origin, was found in parasites but not in the host cell. In flow cytometry (FC) analyses, HDACi promoted parasite cell cycle arrest in the G2-M phase and no changes were found in macrophages. In addition, the direct effect of HDACi against the promastigotes showed apoptosis as the main mechanism of cell death. The FC results corroborate the TEM analyses indicating that the HDACi lead to changes in the cell cycle and induction of apoptosis of L. braziliensis. The production of nitric oxide by the infected macrophages was not altered after treatment with the top 5 compounds. Taken together, our results evidenced new HDACi as promising agents for the development of new treatments for American Tegumentary Leishmaniasis caused by L. braziliensis.

Synthesis of cinnamic acid derivatives and leishmanicidal activity against Leishmania braziliensis.

Leishmania braziliensis is one of the pathogenic agents of cutaneous and mucocutanoeous leishmaniasis. There are no validated vaccines to prevent the infection and the treatment relies on drugs that often present severe side effects, which justify the efforts to find new potential antileishmanial drugs. An alternative to promote the discovery of new drugs would be the association of different chemical groups of bioactive compounds. Here we describe the synthesis and bioactivity evaluation against L. braziliensis of cinnamic acid derivatives possessing isobenzofuranone and 1,2,3-triazole functionalities. We tested 25 compounds at 10 µM concentration against extracellular promastigotes and intracellular amastigotes during macrophage infection. Most compounds were more active against amastigotes than to promastigotes. The derivatives (E)-3-oxo-1,3-dihydroisobenzofuran-5-yl-(3,4,5-trimethoxy) cinnamate (5c), (1-(3,4-difluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl cinnamate (9g), and (1-(2-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl cinnamate (9l) were the most effective presenting over 80% toxicity on L. braziliensis amastigotes. While compound 5c is a cinnamate with an isobenzofuranone portion, 9g and 9l are triazolic cinnamic acid derivatives. The action of these compounds was comparable to amphotericin B used as positive control. Ultrastructural analysis revealed that 5c-treated parasites showed impaired cytokinesis and apoptosis triggering. Taken together, these results highlight the potential of cinnamic acid derivatives in development of novel anti-leishmanial drugs.

Health Promotion through Scientific Literacy: the Experience of the "Science on the Road" Program / Promoção da Saúde pelo Letramento Científico: a Experiência do Programa "Ciência na Estrada" / Promoción de la Salud por el la Alfabetización Cientifica: la Experiencia del Programa "Ciencia en la Carretera"

The itinerant program "Science on the Road: Education and Citizenship" aims at promoting health by popularizing science, focusing primarily on infectious and parasitic diseases. Initiated in the mobile Laboratory of Parasite Biology, Oswaldo Cruz Foundation, Bahia, the proposal was transferred to the Oswaldo Cruz Institute, Fiocruz, and began to work mainly in Rio de Janeiro. We use a micro-bus, equipped with mobile laboratory, where pathogens are focused on microscopes, video systems, resin-made, models, educational games etc. We use a cinema projector in low-income communities with educational and public health problems, including rural settlements, Maroons/Kilombos etc. After the exhibitions, we debate with the population. We have developed an educational instrument called "giant cell" consisting of an inflatable igloo, where cellular organelles/structures are represented as well as projected images of functioning cells, including 3D features. We developed didactic (non-formal) materials, resin or rubber models of pathogens and vectors, as well as vectors embedded in transparent resin to approach endemic diseases of great medical importance in Brazil. We elaborated Makets of houses and terrains representing mosquito breeding grounds (with live larvae), among other insects, venomous animals, rats etc. The use of these materials is always accompanied by discussions about prophylaxis with community engagement. (AU)

O programa itinerante "Ciência na Estrada: educação e cidadania" visa a promoção à saúde pela popularização de ciências enfocando, principalmente, as doenças infecto-parasitárias. Iniciada no Laboratório de Biologia Parasitária, Fundação Oswaldo Cruz, Bahia, a proposta foi transferida para o Instituto Oswaldo Cruz, Fiocruz, passando a atuar, principalmente, no Rio de Janeiro. Utilizamos um micro-ônibus, equipado com laboratório-volante, onde são enfocados patógenos em microscópios, modelos em resina, sistemas de vídeo, jogos educativos etc. Utilizamos um projetor de cinema em comunidades de baixa renda com problemas educacionais e de saúde pública, incluindo assentamentos rurais, quilombos etc. Após as exibições são realizados debates com a população. Desenvolvemos um instrumento educativo denominado "célula gigante" constituído por um iglu inflável, onde são representadas organelas/estruturas celulares bem como projetadas imagens de células em funcionamento, inclusive com recursos 3D. Elaboramos materiais didáticos (não-formais), desenvolvemos modelos em resina ou borracha de patógenos e vetores, bem como de vetores incrustados em resina transparente para abordar doenças endêmicas de grande importância médica no Brasil. Elaboramos maquetes de casas e terrenos representando os criadouros de mosquitos (eventualmente com larvas vivas), entre outros insetos, animais peçonhentos, ratos etc. A utilização destes materiais é sempre acompanhada de discussões sobre profilaxia com engajamento comunitário. (AU)

El programa itinerante "Ciencia en la Carretera: educación y ciudadanía" busca promoción a la salud por la popularización de ciencias enfocando, principalmente, las enfermedades infecto-parasitarias. Criada en el laboratorio de Biología Parasitaria, Fundación Oswaldo Cruz, Bahia, la propuesta fue transferida al Instituto Oswaldo Cruz, Fiocruz, pasando a actuar principalmente en Río de Janeiro. Utilizamos un microbús, equipado con laboratorio, donde se enfocan patógenos en microscopios, sistemas de video, juegos educativos etc. Utilizamos un proyector de cine en comunidades de bajos ingresos con problemas educativos y de salud pública, incluyendo asentamientos rurales, quilombos etc. Después de las exhibiciones se realizan debates con la población. Desarrollamos un instrumento educativo denominado "célula gigante" constituido por un iglu inflable, donde se representan organelas/estructuras celulares así como proyectadas imágenes de células en funcionamiento, incluso con recursos 3D. Elaboramos materiales didácticos (no formales), desarrollamos modelos en resina o caucho de patógenos y vectores, y de vectores incrustados en resina transparente para abordar endémicas enfermedades de gran importancia médica en Brasil. Elaboramos maquetas de casas y terrenos representados los criaderos de mosquitos (con larvas vivas), entre otros insectos, animales venenosos, ratas, etc. La utilización de estos materiales siempre va acompañada de discusiones sobre profilaxis con compromiso comunitario. (AU)

Enteroparasitosis in public schools in Bahia: parasitology learning

Parasitic diseases hamper progress in underdeveloped nations, compromising child physical and cognitive development. In order to control intestinal parasites, the popularization of science may enable prophylactic measures. Questionnaires were used to assess the students' knowledge, attitudes and practices. Health fairs were held as an educational tool and subsequently questionnaires on parasitic diseases and prevention were administered. Stool examinations were performed and infected students were treated. Prevalence of Entamoeba histolytica/ dispar, Ascaris lumbricoides, Trichuris trichiura was 7.1%, 5% and 3.5% respectively in the Anísio Teixeira Institute students and 7.2%, 8%, 3.6% in the Raymundo Matta School students in a suburb. The students at both schools displayed limited knowledge on parasitic disease prevention. The school participation in the prophylaxis of parasitic diseases, was not acknowledged by the students. Reviewing curricula is required, addressing themes related to health, possibly establishing partnerships with health services, universities and/or research centers with the effective involvement of the community performing articulated actions in different health districts, so that education will lead to community empowerment in regard to quotidian issues and improving public health conditions.

Effects of a novel ß-lapachone derivative on Trypanosoma cruzi: Parasite death involving apoptosis, autophagy and necrosis.

Natural products comprise valuable sources for new antiparasitic drugs. Here we tested the effects of a novel ß-lapachone derivative on Trypanosoma cruzi parasite survival and proliferation and used microscopy and cytometry techniques to approach the mechanism(s) underlying parasite death. The selectivity index determination indicate that the compound trypanocidal activity was over ten-fold more cytotoxic to epimastigotes than to macrophages or splenocytes. Scanning electron microscopy analysis revealed that the R72 ß-lapachone derivative affected the T. cruzi morphology and surface topography. General plasma membrane waving and blebbing particularly on the cytostome region were observed in the R72-treated parasites. Transmission electron microscopy observations confirmed the surface damage at the cytostome opening vicinity. We also observed ultrastructural evidence of the autophagic mechanism termed macroautophagy. Some of the autophagosomes involved large portions of the parasite cytoplasm and their fusion/confluence may lead to necrotic parasite death. The remarkably enhanced frequency of autophagy triggering was confirmed by quantitating monodansylcadaverine labeling. Some cells displayed evidence of chromatin pycnosis and nuclear fragmentation were detected. This latter phenomenon was also indicated by DAPI staining of R72-treated cells. The apoptotis induction was suggested to take place in circa one-third of the parasites assessed by annexin V labeling measured by flow cytometry. TUNEL staining corroborated the apoptosis induction. Propidium iodide labeling indicate that at least 10% of the R72-treated parasites suffered necrosis within 24 h. The present data indicate that the ß-lapachone derivative R72 selectively triggers T. cruzi cell death, involving both apoptosis and autophagy-induced necrosis.