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Introdução: A esquistossomose é uma doença parasitária negligenciada, crônica e gradualmente debilitante, que mata 200 mil pessoas anualmente, segundo a Organização Mundial de Saúde. As estratégias atuais de controle da esquistossomose são ineficazes e há, portanto, necessidade de desenvolvimento de uma vacina contra a doença. Objetivo: Apresentar as principais pesquisas sobre o desenvolvimento de vacinas contra a esquistossomose. Metodologia: Revisão narrativa de literatura realizada em outubro de 2020, com a seleção de artigos a partir de 2012 nas bases de dados Pubmed e Scielo, utilizando os descritores “schistosomiasis” e “vaccine”. Foi consultada, também, a plataforma Clinical Trials. Foram incluídos artigos sobre vacinas contra Schistosoma mansoni e excluídos artigos referentes exclusivamente a outras espécies e outras formas de tratamento ou prevenção. Resultados: Foram selecionados 26 artigos, os quais apontam que há apenas duas vacinas em testes clínicos, a Sm14 e a Sm-TSP2. A Sm14 (Brasil), com fase II concluída, apresentou 50-68% de proteção contra a esquistossomose. A Sm-TSP2 (EUA), ainda em fase I, apresentou proteção de 65-69%. Além destas, alguns candidatos vacinais ainda estão na fase pré-clínica, como a Sm-p80 (EUA), a SmCB (Canadá), a SmCyp (Brasil) e vacinas quiméricas (Irã) que combinam antígenos através da imunoinformática. Há também propostas de fabricação de vacinas que usam nanotecnologia e nanobastões de ouro na produção de adjuvantes. Discussão: Ainda há muitos desafios para o desenvolvimento dessas vacinas, como a dificuldade de isolar os genes do parasita, a diferença de resposta imune entre os testes em animais e humanos, o movimento antivacina e o desinteresse de grandes indústrias farmacêuticas. Conclusão: Há duas pesquisas em fase de ensaios clínicos atualmente, a Sm14 e a Sm-TSP2, além de várias outras em fase pré-clínica. Apesar de mais estudos serem necessários, essa estratégia de imunização poderia ser responsável pelo controle da doença no futuro, problemática evidenciada nesse trabalho.

Background The palmitate analogue 2-bromopalmitate (2-BP) is a non-selective membrane tethered cysteine alkylator of many membrane-associated enzymes that in the last years emerged as a general inhibitor of protein S-palmitoylation. Palmitoylation is a post-translational protein modification that adds palmitic acid to a cysteine residue through a thioester linkage, promoting membrane localization, protein stability, regulation of enzymatic activity, and the epigenetic regulation of gene expression. Little is known on such important process in the pathogenic protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. Results The effect of 2-BP was analyzed on different developmental forms of Trypanosoma cruzi. The IC50/48 h value for culture epimastigotes was estimated as 130 μM. The IC50/24 h value for metacyclic trypomastigotes was 216 nM, while for intracellular amastigotes it was 242 μM and for cell derived trypomasigotes was 262 μM (IC50/24 h). Our data showed that 2-BP altered T. cruzi: 1) morphology, as assessed by bright field, scanning and transmission electron microscopy; 2) mitochondrial membrane potential, as shown by flow cytometry after incubation with rhodamine-123; 3) endocytosis, as seen after incubation with transferrin or albumin and analysis by flow cytometry/fluorescence microscopy; 4) in vitro metacyclogenesis; and 5) infectivity, as shown by host cell infection assays. On the other hand, lipid stress by incubation with palmitate did not alter epimastigote growth, metacyclic trypomastigotes viability or trypomastigote infectivity. Conclusion Our results indicate that 2-BP inhibits key cellular processes of T. cruzi that may be regulated by palmitoylation of vital proteins and suggest a metacyclic trypomastigote unique target dependency during the parasite development. Electronic supplementary material The online version of this article (10.1186/s12860-018-0170-3) contains supplementary material, which is available to authorized users.

We report the synthesis of a series of diaminated terpenoids containing, as side-chain of the diamine core, the "head-to-tail" prenyl derivatives, with amino amino spacers of variable length. In vitro biological activity of these compounds was evaluated against Mycobacterium tuberculosis and Leishmania amazonensis, and the structure-activity relationships are discussed. Different biological results were observed depending on the terpenic side-chain length. The best results were obtained for trans,trans-farnesol derivatives. Moreover, these results demonstrated that the stereochemistry of the double bond could play an important role in determining antitubercular and antileishmanial activities of these compounds.

Epimastigote forms of Trypanosoma cruzi (the etiologic agent of Chagas disease) internalize and store extracellular macromolecules in lysosome-related organelles (LROs) called reservosomes, which are positive for the cysteine protease cruzipain. Despite the importance of endocytosis for cell proliferation, macromolecule internalization remains poorly understood in the most clinically relevant proliferative form, the intracellular amastigotes found in mammalian hosts. The main obstacle was the lack of a simple method to isolate viable intracellular amastigotes from host cells. In this work we describe the fast and efficient isolation of viable intracellular amastigotes by nitrogen decompression (cavitation), which allowed the analysis of amastigote endocytosis, with direct visualization of internalized cargo inside the cells. The method routinely yielded 5x10(7) amastigotes--with typical shape and positive for the amastigote marker Ssp4--from 5x10(6) infected Vero cells (48 h post-infection). We could visualize the endocytosis of fluorescently-labeled transferrin and albumin by isolated intracellular amastigotes using immunofluorescence microscopy; however, only transferrin endocytosis was detected by flow cytometry (and was also analyzed by western blotting), suggesting that amastigotes internalized relatively low levels of albumin. Transferrin binding to the surface of amastigotes (at 4°C) and its uptake (at 37°C) were confirmed by binding dissociation assays using acetic acid. Importantly, both transferrin and albumin co-localized with cruzipain in amastigote LROs. Our data show that isolated T. cruzi intracellular amastigotes actively ingest macromolecules from the environment and store them in cruzipain-positive LROs functionally related to epimastigote reservosomes.

Background Clathrin-mediated vesicular trafficking, the mechanism by which proteins and lipids are transported between membrane-bound organelles, accounts for a large proportion of import from the plasma membrane (endocytosis) and transport from the trans-Golgi network towards the endosomal system. Clathrin-mediated events are still poorly understood in the protozoan Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. In this study, clathrin heavy (TcCHC) and light (TcCLC) chain gene expression and protein localization were investigated in different developmental forms of T. cruzi (epimastigotes, trypomastigotes and amastigotes), using both polyclonal and monoclonal antibodies raised against T. cruzi recombinant proteins. Results Analysis by confocal microscopy revealed an accumulation of TcCHC and TcCLC at the cell anterior, where the flagellar pocket and Golgi complex are located. TcCLC partially colocalized with the Golgi marker TcRAB7-GFP and with ingested albumin, but did not colocalize with transferrin, a protein mostly ingested via uncoated vesicles at the cytostome/cytopharynx complex. Conclusion Clathrin heavy and light chains are expressed in T. cruzi. Both proteins typically localize anterior to the kinetoplast, at the flagellar pocket and Golgi complex region. Our data also indicate that in T. cruzi epimastigotes clathrin-mediated endocytosis of albumin occurs at the flagellar pocket, while clathrin-independent endocytosis of transferrin occurs at the cytostome/cytopharynx complex.

Reservosomes are large round vesicles located at the posterior end of epimastigote forms of the protozoanTrypanosoma cruzi, the etiological agent of Chagas disease. They are the specific end organelles of the endocytosis pathway ofT. cruzi, and they play key roles in nutrient uptake and cell differentiation. These lysosome-like organelles accumulate ingested macromolecules and contain large amounts of a major cysteine proteinase (cruzipain or GP57/51 protein). Aim of this study was to produce a monoclonal antibody (mAb) against a recombinantT. cruzicruzipain (TcCruzipain) that specifically labels the reservosomes. BALB/c mice were immunized with purified recombinant TcCruzipain to obtain the mAb. After fusion of isolated splenocytes with myeloma cells and screening, a mAb was obtained by limiting dilution and characterized by capture ELISA. We report here the production of a kappa-positive monoclonal IgG antibody (mAb CZP-315.D9) that recognizes recombinant TcCruzipain. This mAb binds preferentially to a protein with a molecular weight of about 50 kDa on western blots and specifically labels reservosomes by immunofluorescence and transmission electron microscopy. The monoclonal CZP-315.D9 constitutes a potentially powerful marker for use in studies on the function of reservosomes ofT. cruzi.

Background Although known to be highly endemic in the Amazon regions of Brazil, the presence of cutaneous leishmaniasis (CL) in the subtropical southern part of the country has largely been ignored. This study was conducted to demonstrate CL is emerging in the Brazilian state of Santa Catarina, as well as to characterize the epidemiological profile and Leishmania species involved. Methodology/Principal Findings For this cross-sectional study, data from all CL cases from Santa Catarina, Brazil, reported to the Brazilian National Notifiable Diseases Information System from 2001 to 2009 were investigated. Amplification of the kDNA minicircle conserved region followed by restriction fragment length polymorphism (PCR-RFLP) was conducted to screen for Leishmania species present in patient biopsy. Overall, 542 CL cases were reported, with majority resulting from autochthonous transmission (n = 401, 73.99%) and occurring in urban zones (n = 422, 77.86%). Age, gender, zone of residence, origin of case, clinical form and case outcome were found to differ significantly by region. Imported cases were over seven times more likely to relapse (95% CI 2.56–21.09). Mapping of cases revealed new endemic areas in northeastern Santa Catarina with two species present. With the exception of three L. (Leishmania) amazonensis cases (1.20%), majority of PCR positive samples were found to be L. (Viannia) braziliensis (n = 248, 98.80%). Conclusions/Significance CL is now endemic in the state of Santa Catarina, Brazil, with case profiles varying significantly by region. L. (V.) braziliensis has been identified as the predominant species in the region.

Background. Chagas disease is caused by the protozoan parasite Trypanosoma cruzi, which exhibits a high genetic variability. TcI, TcII, or mixed TcI/TcII strains may be found during acute human infection while mainly TcII parasites are present at the chronic stage of disease. In a previously studied Chagas disease outbreak, we identified mixed TcI/TcII strains in the vector Triatoma tibiamaculata and only TcII strains in infected humans, indicating that T. cruzi populations may be selected within the human host. Methods. Utilizing molecular typing and cell biology techniques, we investigated the interaction of TcI, TcII, and mixed TcI/TcII strains with macrophages, an important cell population implicated in controlling protozoan infection. Results. TcII but not TcI strains were selected by both human and murine macrophages in vitro and by peritoneal cavity cells in vivo. Biological analysis revealed that, compared with TcI, TcII strains display higher infective and multiplicative ability as well as lower doubling time inside macrophages. However, TcI and TcII strains present similar susceptibility to interferon-c‐activated macrophages in vitro. Conclusions. Taken together, our results reveal the existence of an intracellular selection process in macrophages that favors TcII, but not TcI, when infection occurs with vector-derived mixed TcI/TcII strains.

Bioactivity of fractions and compounds obtained from Polygala sabulosa against Trypanosoma cruzi epimastigote, blood trypomastigote and amastigote forms were evaluated in vitro. Dichloromethane and ethyl acetate fractions showed a strong trypanocidal activity on epimastigotes (IC50 < 10.4 µg/mL). Chromatographic analysis by TLC of these fractions confirmed the presence of previously described compounds (dihydrostyryl-2-pyrones, styryl-2-pyrones and 6-methoxy-7-prenyloxycoumarin). The dichloromethane fraction was fractioned by silica gel column chromatography to afford the compound α-spinasterol and the ethyl acetate fraction yielded apigenin, quercetin and a quercetin-3-O-glucoside, being the first description for the Polygala genus. 4-Methoxy-6-(11,12-methylenedioxy-14-methoxydihydrostyryl)-2-pyrone, 4-methoxy-6-(11,12-dimethoxystyryl)-2-pyrone, 6-methoxy-7-prenyloxycoumarin and quercetin-3-O-glucoside showed a weak activity against blood trypomastigotes (IC50 < 1008.6 µg/mL). The prenylated coumarin was the most active compound against both epimastigote and trypomastigote forms, IC50 10.5 and 88.2 µg/mL, respectively. The hemolytic activity and cell toxicity of each active compound was also assessed. Furthermore, 4-methoxy-6-(11,12-methylenedioxy-14-methoxydihydrostyryl)-2-pyrone and 6-methoxy-7-prenyloxycoumarin reduced 4 times the T. cruzi infection rate for Vero cells at 100 and 50 µg/mL, respectively. These results show for the first time active compounds against T. cruzi in P. sabulosa. A bioatividade das frações e compostos obtidos de Polygala sabulosa contra as formas epimastigota, tripomastigota sanguínea e amastigota de Trypanosoma cruzi foram avaliadas in vitro. Frações diclorometano e acetato de etila mostraram potente atividade tripanocida sob as formas epimastigotas (IC50 < 10,4 µg/mL). Análises por cromatografia em camada delgada destas frações confirmaram a presença de compostos previamente descritos (dihidroestiril-2-pironas, estiril-2-pironas e 6-metoxi-7-preniloxicumarina). Após o fracionamento da fração diclorometano por cromatografia em coluna, obteve-se o composto α-espinasterol e da fração acetato de etila obtiveram-se os compostos apigenina, quercetina e uma quercetina-3- O-glicosídeo, todos descritos pela primeira vez para o gênero Polygala. 4-metoxi-6-(11,12-metilenodioxi-14-metoxidihidroestiril)-2-pirona, 4-metoxi-6-(11,12-dimetoxi-14-metoxiestiril)-2-pirona, 6-metoxi-7-preniloxicumarina e quercetina-3- O-glicosídeo mostraram fraca atividade contra a forma tripomastigota sanguínea (IC50 < 1008,6 µg/mL). A cumarina prenilada foi o composto mais ativo contra ambas as formas epimastigota e tripomastigota, com IC50 10,5 e 88,2 µg/mL, respectivamente. A atividade hemolítica e a toxicidade celular de cada composto foram também avaliadas. Além disso, 4-metoxi-6-(11,12-metilenodioxi-14-metoxidihidroestiril)-2-pirona e 6-metoxi-7-preniloxicumarina reduziram em quatro vezes a infecção em ratos por Vero Cells nas concentrações de 100 e 50 µg/mL respectivamente. Esses resultados mostram, pela primeira vez, a atividade de compostos de P. sabulosa contra T. cruzi.

Os Remédios Florais de Bach (RFB), constituem um método alternativo de tratamento usado largamente na terapêutica de várias patologias em muitos países do mundo. Os RFB são reconhecidos como tratamento natural pela OMS desde 1956. Embora o mecanismo de ação dos RFB ainda não tenha sido elucidado, eles vêm sendo indicados para o tratamento de várias doenças neuropsiquiátricas. O objetivo do presente trabalho foi detectar possíveis efeitos centrais dos RFB em modelos farmacológicos utilizados na pesquisa de substâncias com efeitos ansiolíticos, hipnóticos, antidepressivos e neurolépticos. Para tanto, camundongos receberam um tratamento agudo via oral (0,45 mL) 1 hora antes dos testes. Os resultados mostraram que os florais Gorse e, em conjunto, White chestnut, Agrymony e Vervain exibiram perfis antidepressivo e hipnótico, respectivamente. No modelo de ansiedade foi detectado efeito ansiolítico do floral Agrymony. Entretanto, não foram observados efeitos neurolépticos do floral Clematis. Os resultados nos levam a sugerir que os efeitos centrais dos florais avaliados podem ser parcialmente detectados através de modelos farmacológicos utilizados na pesquisa de agentes psicotrópicos.

Universidade Federal de Santa Catarina. Centro de Ci?ncias Biol?gicas. Departamento de Microbiologia e Parasitologia. Florian?polis, SC, Brazil / UCLA School of Medicine. Department of Microbiology and Immunology. Los Angeles, CA, USA. Universidade Federal de Santa Catarina. Centro de Ci?ncias Biol?gicas. Departamento de Microbiologia e Parasitologia. Florian?polis, SC, Brazil. Universidade de S?o Paulo. Instituto de Ci?ncias Biol?gicas. Departamento de Parasitologia. S?o Paulo, SP, Brazil. Minist?rio da Sa?de. Funda??o Nacional de Sa?de. Instituto Evandro Chagas. Bel?m, PA, Brasil. Universidade Federal de Santa Catarina. Centro de Ci?ncias Biol?gicas. Departamento de Microbiologia e Parasitologia. Florian?polis, SC, Brazil. Universidade Federal de Santa Catarina. Centro de Ci?ncias Biol?gicas. Departamento de Microbiologia e Parasitologia. Florian?polis, SC, Brazil. Universidade Federal do Rio de Janeiro. Centro de Ci?ncias da Sa?de. Departamento de An?lises Cl??nicas e Toxicol?gicas. Rio de Janeiro, RJ, Brazil. Universidade Federal de Santa Catarina. Centro de Ci?ncias Biol?gicas. Departamento de Microbiologia e Parasitologia. Florian?polis, SC, Brazil. Funda??o Oswaldo Cruz. Centro de Pesquisas Ren? Rachou. Laborat?rio de Parasitologia Celular e Molecular. Belo Horizonte, MG, Brazil. UCLA School of Medicine. Department of Microbiology and Immunology. Los Angeles, CA, USA. Four Leishmania sp. samples were isolated from autochthonous human cases of American cutaneous leishmaniasis (ACL) in Santa Catarina State, southern Brazil. These strains were characterized using indirect immunofluorescence with a panel of Leishmania-specific monoclonal antibodies (MAbs), and by PCR amplification and hybridization assay of the mini-exon gene with group specific probes. The results obtained with the MAbs were in agreement with the genetic marker. Two isolates (MHOM/BR/89/JSC89-H1 and MHOM/BR/89/JSC89-H2) were identified as L. (Leishmania) amazonensis and two (MHOM/BR/96/LSC96-H3 and MHOM/BR/97/LSC97-H4) as L. (Viannia) braziliensis. The southernmost autochthonous cases of ACL in Brazil are due to two different Leishmania sp. species, confirming the spreading of ACL on the American continent.

Trypanosoma rangeli is a hemoflagelate parasite that infects domestic and sylvatic animals, as well as man, in Central and South America. T. rangeli has an overlapping distribution with T. cruzi, the etiological agent of Chagas disease, sharing several animal reservoirs and triatomine vectors. We have isolated T. rangeli strains in the State of Santa Catarina, in southern Brazil, which dramatically increased the distribution area of this parasite. This brief review summarizes several studies comparing T. rangeli strains isolated in Santa Catarina with others isolated in Colombia, Honduras and Venezuela. The different methods used include indirect immunofluorescence and western blot assays, lectin agglutination, isoenzyme electrophoresis and random amplified polymorphic DNA analysis, triatomine susceptibility, in vitro cell infection assays, and mini-exon gene analysis.

Background American Trypanosomiasis or Chagas disease is caused by Trypanosoma cruzi which currently infects approximately 16 million people in the Americas causing high morbidity and mortality. Diagnosis of American trypanosomiasis relies on serology, primarily using indirect immunofluorescence assay (IFA) with T. cruzi epimastigote forms. The closely related but nonpathogenic Trypanosoma rangeli has a sympatric distribution with T. cruzi and is carried by the same vectors. As a result false positives are frequently generated. This confounding factor leads to increased diagnostic test costs and where false positives are not caught, endangers human health due to the toxicity of the drugs used to treat Chagas disease. Results In the present study, serologic cross-reactivity between the two species was compared for the currently used epimastigote form and the more pathologically relevant trypomastigote form, using IFA and immunoblotting (IB) assays. Our results reveal an important decrease in cross reactivity when T. rangeli culture-derived trypomastigotes are used in IFA based diagnosis of Chagas disease. Western blot results using sera from both acute and chronic chagasic patients presenting with cardiac, indeterminate or digestive disease revealed similar, but not identical, antigenic profiles. Conclusion This is the first study addressing the serological cross-reactivity between distinct forms and strains of T. rangeli and T. cruzi using sera from distinct phases of the Chagasic infection. Several T. rangeli-specific proteins were detected, which may have potential as diagnostic tools.

Ten chalcones were synthesized and tested as potential leishmanicidal and trypanocidal agents. All tested compounds caused concentration-dependent inhibition of the in vitro growth ofLeishmania braziliensisandTrypanosoma cruziwith no significant toxic effect towards host macrophages. Our results show that the positions of the substituents seem to be critical for their antiprotozoal activities.