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1.
Rev Soc Bras Med Trop ; 52: e20190133, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31508781

RESUMO

INTRODUCTION: Chagas disease (CD) is an important public health problem in Brazil and worldwide. Aging and obesity are important matters in patients with CD, as is hypovitaminosis D3, which can decrease the quality of life of these patients. Immunomodulation mediated by vitamin D3, especially the production of antimicrobial peptides such as cathelicidin LL-37, might be related to the severity and symptoms of CD. This study aimed to determine the serum levels of vitamin D and LL-37 and VDR gene polymorphisms in patients with chronic CD. METHODS: This study included male patients with cardiac and indeterminate clinical forms of CD. Clinical, anthropometric, and blood parameters were obtained. Serum levels of 25(OH)D3 and LL-37 were determined by chemiluminescence and enzyme-linked immunosorbent assay respectively. Fok (rs731236), Bsm (rs1544410), Apa (rs7975232), and Taq (rs731236) polymorphisms of the VDR gene were investigated by PCR-RFLP. RESULTS: Sixty-four patients were included in the study: 18 of the cardiac form and 46 of the indeterminate form. No differences in age, ethnicity, BMI, arterial hypertension, diabetes mellitus, or dyslipidemias were observed between groups. However, the serum levels of 25(OH)D3, but not of LL-37, were lower in the cardiac form group. The association among polymorphisms, vitamin D, and clinical form was not significant. CONCLUSIONS: Decreased levels of vitamin D suggest an association with the cardiac form of CD. Studies investigating the roles of vitamin D and LL-37 in the immune response and their associations with VDR polymorphisms and disease susceptibility are necessary.


Assuntos
Peptídeos Catiônicos Antimicrobianos/sangue , Doença de Chagas/sangue , Doença de Chagas/genética , Colecalciferol/sangue , Polimorfismo Genético/genética , Receptores de Calcitriol/genética , Biomarcadores/sangue , Humanos , Masculino , Pessoa de Meia-Idade
2.
Parasitol Res ; 118(8): 2343-2351, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31236660

RESUMO

Chagas disease, caused by the protozoan Trypanosoma cruzi (T. cruzi), although discovered more than a century ago, is still a not very well-elucidated aspect. Individuals in the chronic phase of the disease may present asymptomatic clinical form or symptomatologies related to the cardiac, digestive systems, or both (mixed clinical form). SNPs (single nucleotide polymorphisms) have been identified as important markers because they constitute about 90% of the variation in the human genome. One of them is localized to the ACAT-1 gene (cholesterol acyltransferase 1) (rs1044925) and has been linked to lipid disorders. Some studies have suggested the interaction between T. cruzi and the lipid metabolism of the host. Therefore, the objective of the present study was to evaluate the association between the ACAT-1 gene rs1044925 SNP in relation to clinical manifestations in patients with chronic Chagas disease. A total of 135 individuals with chronic Chagas disease, 86 (63.7%) asymptomatic individuals and 49 (36.3%) symptomatic patients (22 with cardiac clinical form, 18 with digestive form and 9 with mixed form) participated in the study. To evaluate the polymorphism, the PCR-RFLP technique were used. There was a significant difference and a higher frequency of AA and AC genotypes (p = 0.047 and p = 0.016, respectively) of the ACAT-1 gene in asymptomatic chagasic individuals. The result suggests a protective character of the AA and AC genotypes of the rs1044925 SNP in relation to the presence of symptomatic clinical manifestations of the disease in chronic chagasic individuals.


Assuntos
Doença de Chagas/genética , Polimorfismo de Nucleotídeo Único , Esterol O-Aciltransferase/genética , Idoso , Doenças Assintomáticas , Doença de Chagas/parasitologia , Doença Crônica , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Fragmento de Restrição , Trypanosoma cruzi/fisiologia
3.
Acta Trop ; 197: 105062, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31201776

RESUMO

Genetic approaches have been proposed for improving the understanding of the causes of differential susceptibility to Trypanosoma cruzi infection and Chagas disease outcome. Polymorphisms in genes involved in the immune/inflammatory response are being studied in order to clarify their possible role in the occurrence or severity of the cardiac and/or gastrointestinal complications. However still today, the number of significant associated genes is limited and the pathophysiological mechanisms underlying this condition are unknown. This article review the information currently available from the published scientific literature regarding the genetic variants of molecules of the immune system and other variants that can contribute to the clinical presentation of the disease. Genomic medicine will improve our knowledge about the molecular basis of Chagas disease, will open new avenues for developing biomarkers of disease progression, new therapeutic strategies to suit the requirements of individual patients, and will contribute to the control of one of the infections with the greatest socio-economic impact in the Americas.


Assuntos
Doença de Chagas/genética , Doença de Chagas/tratamento farmacológico , Progressão da Doença , Genômica , Humanos , Polimorfismo Genético
4.
Genome Biol Evol ; 11(7): 1952-1957, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31218350

RESUMO

Chagas disease was described by Carlos Chagas, who first identified the parasite Trypanosoma cruzi from a 2-year-old girl called Berenice. Many T. cruzi sequencing projects based on short reads have demonstrated that genome assembly and downstream comparative analyses are extremely challenging in this species, given that half of its genome is composed of repetitive sequences. Here, we report de novo assemblies, annotation, and comparative analyses of the Berenice strain using a combination of Illumina short reads and MinION long reads. Our work demonstrates that Nanopore sequencing improves T. cruzi assembly contiguity and increases the assembly size in ∼16 Mb. Specifically, we found that assembly improvement also refines the completeness of coding regions for both single-copy genes and repetitive transposable elements. Beyond its historical and epidemiological importance, Berenice constitutes a fundamental resource because it now constitutes a high-quality assembly available for TcII (clade C), a prevalent lineage causing human infections in South America. The availability of Berenice genome expands the known genetic diversity of these parasites and reinforces the idea that T. cruzi is intraspecifically divided in three main clades. Finally, this work represents the introduction of Nanopore technology to resolve complex protozoan genomes, supporting its subsequent application for improving trypanosomatid and other highly repetitive genomes.


Assuntos
Doença de Chagas/parasitologia , Trypanosoma cruzi/genética , Doença de Chagas/genética , Genoma de Protozoário/genética , Sequenciamento de Nucleotídeos em Larga Escala , Nanoporos , Filogenia , Análise de Sequência de DNA
5.
Parasit Vectors ; 12(1): 260, 2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31126327

RESUMO

BACKGROUND: Chagas disease is a protozoan infection caused by Trypanosoma cruzi. The disease has a chronic course in which 20-30% of the patients would develop progressive damage to the cardiovascular system and the gastrointestinal tube. We are still unable to predict who will develop end-organ damage but there are some acquired and genetic risk factors already known. RESULTS: We reviewed data from 833 patients with serologically confirmed Chagas disease in this retrospective study. Patients were classified as siblings or non-siblings (controls) and the results of pre-treatment blood PCR assay, end-organ damage (cardiac and/or gastrointestinal), and the presence of delayed type hypersensitivity (DTH) skin involvement in patients treated with benznidazole were analyzed. Siblings were grouped by family and we randomly generated groups of 2 or 3 persons with the remaining controls. We classified the results of each variable as concordant or discordant and compared the concordance in these results among the sibling groups with that among control groups. We identified 71 groups of siblings and randomly generated 299 groups of non-related patients. Pre-treatment blood PCR concordance was significantly higher (19%) among siblings compared to controls (P = 0.02), probably due to a higher frequency in pre-treatment positive results. No other statistically significant differences were found. CONCLUSIONS: A significant difference was found in the concordance of pre-treatment blood PCR for T. cruzi among siblings compared to non-related controls.


Assuntos
Doença de Chagas/etnologia , Doença de Chagas/genética , Irmãos , Adulto , Bolívia , Cardiomiopatia Chagásica/diagnóstico , Cardiomiopatia Chagásica/etiologia , Doença de Chagas/complicações , Doença Crônica , Feminino , Gastroenteropatias/diagnóstico , Gastroenteropatias/parasitologia , Humanos , Masculino , Estudos Retrospectivos , Fatores de Risco , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi
6.
PLoS Negl Trop Dis ; 13(4): e0007324, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30995222

RESUMO

Chagas Disease (CD) is an anthropozoonosis caused by Trypanosoma cruzi. With complex pathophysiology and variable clinical presentation, CD outcome can be influenced by parasite persistence and the host immune response. Complement activation is one of the primary defense mechanisms against pathogens, which can be initiated via pathogen recognition by pattern recognition molecules (PRMs). Collectin-11 is a multifunctional soluble PRM lectin, widely distributed throughout the body, with important participation in host defense, homeostasis, and embryogenesis. In complex with mannose-binding lectin-associated serine proteases (MASPs), collectin-11 may initiate the activation of complement, playing a role against pathogens, including T. cruzi. In this study, collectin-11 plasma levels and COLEC11 variants in exon 7 were assessed in a Brazilian cohort of 251 patients with chronic CD and 108 healthy controls. Gene-gene interactions between COLEC11 and MASP2 variants were analyzed. Collectin-11 levels were significantly decreased in CD patients compared to controls (p<0.0001). The allele rs7567833G, the genotypes rs7567833AG and rs7567833GG, and the COLEC11*GGC haplotype were related to T. cruzi infection and clinical progression towards symptomatic CD. COLEC11 and MASP2*CD risk genotypes were associated with cardiomyopathy (p = 0.014; OR 9.3, 95% CI 1.2-74) and with the cardiodigestive form of CD (p = 0.005; OR 15.2, 95% CI 1.7-137), suggesting that both loci act synergistically in immune modulation of the disease. The decreased levels of collectin-11 in CD patients may be associated with the disease process. The COLEC11 variant rs7567833G and also the COLEC11 and MASP2*CD risk genotype interaction were associated with the pathophysiology of CD.


Assuntos
Doença de Chagas/genética , Doença de Chagas/fisiopatologia , Colectinas/genética , Epistasia Genética , Serina Proteases Associadas a Proteína de Ligação a Manose/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Brasil , Estudos de Casos e Controles , Colectinas/sangue , Feminino , Predisposição Genética para Doença , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único
7.
Methods Mol Biol ; 1955: 203-214, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30868529

RESUMO

MicroRNAs (miRNAs) are a class of small noncoding RNAs (typically 19-23 nucleotides) which act by annealing to partially complementary binding sites present on the 3' untranslated regions (UTR) of messenger RNAs (mRNAs) leading to inhibition of protein translation or by inducing mRNA decay. Since their discovery, miRNAs have come to be recognized as master regulators of gene expression in plant and mammals, controlling tissue-specific protein expression. Up to one-third of mammalian mRNAs are susceptible to miRNA-mediated regulation. It has been shown that miRNAs are determinants of the physiology and pathophysiology of the cardiovascular system, and altered expression of muscle- and/or cardiac-specific miRNAs in myocardial tissue is involved in heart development and cardiovascular diseases, including myocardial hypertrophy, heart failure, and fibrosis. The analysis of miRNA expression pattern provides important information, as well as is a starting point to understand miRNA function in different tissues, during development, and in disease. Several techniques can be used for miRNA profiling analysis like high-throughput sequencing, microarrays, and real-time PCR using microfluidic low-density arrays. This chapter describes the complete methodology to perform miRNA profiling using the stem-loop reverse-transcription (RT)-based TaqMan® MicroRNA low-density arrays (TLDA) method. This methodology was used to perform miRNA profiling in the heart of T. cruzi acutely infected mice.


Assuntos
Doença de Chagas/genética , Perfilação da Expressão Gênica/métodos , Coração/parasitologia , MicroRNAs/genética , Transcriptoma , Trypanosoma cruzi/fisiologia , Animais , Doença de Chagas/parasitologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Interações Hospedeiro-Parasita , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Trypanosoma cruzi/isolamento & purificação
8.
Free Radic Biol Med ; 130: 408-418, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30445126

RESUMO

Chagas disease is caused by the hemoflagellate protozoa Trypanosoma cruzi and is one of the most important neglected tropical diseases, especially in Latin American countries, where there is an association between low-income populations and mortality. The nitroderivatives used in current chemotherapy are far from ideal and present severe limitations, justifying the continuous search for alternative drugs. Since the1990s, our group has been investigating the trypanocidal activity of natural naphthoquinones and their derivatives, and three naphthoimidazoles (N1, N2 and N3) derived from ß-lapachone were found to be most effective in vitro. Analysis of their mechanism of action via cellular, molecular and proteomic approaches indicates that the parasite mitochondrion contains one of the primary targets of these compounds, trypanothione synthetase (involved in trypanothione production), which is overexpressed after treatment with these compounds. Here, we further evaluated the participation of the mitochondria and reactive oxygen species (ROS) in the anti-T. cruzi action of naphthoimidazoles. Preincubation of epimastigotes and trypomastigotes with antioxidants (α-tocopherol and urate) strongly protected the parasites from the trypanocidal effect of naphthoimidazoles, decreasing the ROS levels produced and reverting the mitochondrial swelling phenotype. The addition of pro-oxidants (menadione and H2O2) before the treatment induced an increase in parasite lysis. Despite the O2 uptake and mitochondrial complex activity being strongly reduced by N1, N2 and N3, urate partially restored the mitochondrial metabolism only in N1-treated parasites. In parallel, MitoTEMPO, a mitochondrial-targeted antioxidant, protected the functionality of the mitochondria in N2- and N3-treated parasites. In addition, the trypanothione reductase activity was remarkably increased after treatment with N1 and N3, and molecular docking demonstrated that these two compounds were positioned in pockets of this enzyme. Based on our findings, the direct impairment of the mitochondrial electron transport chain by N2 and N3 led to an oxidative misbalance, which exacerbated ROS generation and led to parasite death. Although other mechanisms cannot be discounted, mainly in N1-treated parasites, further investigations are required.


Assuntos
Doença de Chagas/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Naftoquinonas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Doença de Chagas/genética , Doença de Chagas/parasitologia , Humanos , Peróxido de Hidrogênio , Imidazóis/química , Imidazóis/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/patologia , Dilatação Mitocondrial/efeitos dos fármacos , Naftoquinonas/química , Compostos Organofosforados/farmacologia , Piperidinas/farmacologia , Proteômica , Espécies Reativas de Oxigênio/metabolismo , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/patogenicidade
9.
Front Immunol ; 9: 2790, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30555475

RESUMO

Arginase-1 (Arg-1) is a marker for alternatively activated macrophages (AAM) and is mainly induced by the type 2 cytokines interleukin (IL)-4 and IL-13 through the common IL-4 receptor-alpha (Rα) subunit. Both, Arg-1 and AAM undermine macrophage effector functions against intracellular parasites and are therefore implicated in the susceptibility to infection with Trypanosoma cruzi, the causative agent of Chagas' disease. However, the involvement of Arg-1 in promoting intracellular replication of T. cruzi in AAM has not been proven so far in vivo. Because Arg-1 is only moderately expressed in T. cruzi-infected wildtype mice, we elucidated the role of Arg-1 and AAM during infection in IL-13-overexpressing (IL-13tg) mice, which are characterized by an inflammation-induced development of AAM and an accompanied elevated expression of Arg-1. In comparison to wildtype littermates, IL-13tg mice were highly susceptible to T. cruzi infection with enhanced parasitemia and impaired survival. Importantly, T. cruzi-infected IL-13tg mice developed an elevated alternative macrophage activation with increased arginase activity. To proof the hypothesis, that Arg-1 accounts for the increased susceptibility of IL-13tg mice, we blocked arginase activity in infected IL-13tg mice. Because this arginase inhibition resulted in a decreased susceptibility to experimental Chagas disease our study supports in summary the conclusion that IL-13/IL-4Rα-driven Arg-1 expression contributes to the permissiveness of the host to T. cruzi infection.


Assuntos
Arginase/imunologia , Doença de Chagas/imunologia , Interleucina-13/imunologia , Trypanosoma cruzi/imunologia , Animais , Arginase/genética , Doença de Chagas/genética , Suscetibilidade a Doenças , Interleucina-13/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Trypanosoma cruzi/genética
10.
PLoS Negl Trop Dis ; 12(12): e0006998, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30517089

RESUMO

BACKGROUND: The severity of cardiac disease in chronic Chagas disease patients is associated with different features of T-cell exhaustion. Here, we assessed whether the ability of T cells to secrete IFN-γ in response to T. cruzi was linked to disruption in immune homeostasis and inflammation in patients with chronic Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: PBMCs from chronic Chagas disease patients and uninfected controls were examined for frequencies of T. cruzi-responsive IFN-γ-producing cells by ELISPOT and cellular expression and function of IL-7R using flow cytometry. Serum levels of IL-7, IL-21, IL-27, soluble IL-7R, and inflammatory cytokines were also evaluated by ELISA or CBA techniques. Patients possessing T. cruzi-specific IFN-γ-producing cells (i.e. IFN-γ producers) had higher levels of memory T cells capable of modulating the alpha chain of IL-7R and an efficient response to IL-7 compared to that in patients lacking (i.e. IFN-γ nonproducers) parasite-specific T-cell responses. IFN-γ producers also showed low levels of soluble IL-7R, high basal expression of Bcl-2 in T cells and low basal frequencies of activated CD25+ T cells. Modulation of IL-7R was inversely associated with serum IL-6 levels and positively associated with serum IL-8 levels. Circulating IL-21 and IL-27 levels were not associated with the frequency of IFN-γ producing cells but were reduced in less severe clinical forms of the disease. In vitro stimulation of PBMCs with IL-7 or IL-27 enhanced IFN-γ production in IFN-γ producers but not in IFN-γ nonproducers. CONCLUSIONS/SIGNIFICANCE: Alterations of the IL-7/IL-7R axis and in the levels of inflammatory cytokines were linked to impaired T. cruzi-specific IFN-γ production. These alterations might be responsible of the process of immune exhaustion observed in chronic Chagas disease.


Assuntos
Doença de Chagas/sangue , Interferon gama/sangue , Interleucina-7/sangue , Receptores de Interleucina-7/metabolismo , Trypanosoma cruzi/fisiologia , Adulto , Idoso , Doença de Chagas/genética , Doença de Chagas/parasitologia , Doença Crônica , ELISPOT , Feminino , Humanos , Interferon gama/genética , Interleucina-7/genética , Interleucinas/sangue , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Receptores de Interleucina-7/genética , Linfócitos T/metabolismo , Trypanosoma cruzi/genética , Adulto Jovem
11.
PLoS One ; 13(9): e0203462, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30183775

RESUMO

Trypanosoma cruzi is a genetically heterogeneous group of organisms that cause Chagas disease. It has been long suspected that the clinical outcome of the disease and response to therapeutic agents are, at least in part, related to the genetic characteristics of the parasite. Herein, we sought to validate the significance of the genotype of T. cruzi isolates recovered from patients with different clinical forms of Chagas disease living in Argentina on their biological behaviour and susceptibility to drugs. Genotype identification of the newly established isolates confirmed the reported predominance of TcV, with a minor frequency of TcI. Epimastigote sensitivity assays demonstrated marked dissimilar responses to benznidazole, nifurtimox, pentamidine and dihydroartemisinin in vitro. Two TcV isolates exhibiting divergent response to benznidazole in epimastigote assays were further tested for the expression of anti-oxidant proteins. Benznidazole-resistant BOL-FC10A epimastigotes had decreased expression of Old Yellow Enzyme and cytosolic superoxide dismutase, and overexpression of mitochondrial superoxide dismutase and tryparedoxin- 1, compared to benznidazole-susceptible AR-SE23C parasites. Drug sensitivity assays on intracellular amastigotes and trypomastigotes reproduced the higher susceptibility of AR-SE23C over BOL-FC10A parasites to benznidazole observed in epimastigotes assays. However, the susceptibility/resistance profile of amastigotes and trypomastigotes to nifurtimox, pentamidine and dihydroartemisinin varied markedly with respect to that of epimastigotes. C3H/He mice infected with AR-SE23C trypomastigotes had higher levels of parasitemia and mortality rate during the acute phase of infection compared to mice infected with BOL-FC10A trypomastigotes. Treatment of infected mice with benznidazole or nifurtimox was efficient to reduce patent parasitemia induced by either isolate. Nevertheless, qPCR performed at 70 dpi revealed parasite DNA in the blood of mice infected with AR-SE23C but not in BOL-FC10A infected mice. These results demonstrate high level of intra-type diversity which may represent an important obstacle for the testing of chemotherapeutic agents.


Assuntos
Doença de Chagas/metabolismo , Resistência a Medicamentos/efeitos dos fármacos , Genótipo , Fenótipo , Tripanossomicidas/farmacologia , Trypanosoma cruzi/metabolismo , Adulto , Animais , Argentina , Doença de Chagas/tratamento farmacológico , Doença de Chagas/genética , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/genética , Células Vero
12.
J Biol Chem ; 293(45): 17402-17417, 2018 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-30232153

RESUMO

In vertebrate cells, mitochondrial Ca2+ uptake by the mitochondrial calcium uniporter (MCU) leads to Ca2+-mediated stimulation of an intramitochondrial pyruvate dehydrogenase phosphatase (PDP). This enzyme dephosphorylates serine residues in the E1α subunit of pyruvate dehydrogenase (PDH), thereby activating PDH and resulting in increased ATP production. Although a phosphorylation/dephosphorylation cycle for the E1α subunit of PDH from nonvertebrate organisms has been described, the Ca2+-mediated PDP activation has not been studied. In this work, we investigated the Ca2+ sensitivity of two recombinant PDPs from the protozoan human parasites Trypanosoma cruzi (TcPDP) and T. brucei (TbPDP) and generated a TcPDP-KO cell line to establish TcPDP's role in cell bioenergetics and survival. Moreover, the mitochondrial localization of the TcPDP was studied by CRISPR/Cas9-mediated endogenous tagging. Our results indicate that TcPDP and TbPDP both are Ca2+-sensitive phosphatases. Of note, TcPDP-KO epimastigotes exhibited increased levels of phosphorylated TcPDH, slower growth and lower oxygen consumption rates than control cells, an increased AMP/ATP ratio and autophagy under starvation conditions, and reduced differentiation into infective metacyclic forms. Furthermore, TcPDP-KO trypomastigotes were impaired in infecting cultured host cells. We conclude that TcPDP is a Ca2+-stimulated mitochondrial phosphatase that dephosphorylates TcPDH and is required for normal growth, differentiation, infectivity, and energy metabolism in T. cruzi Our results support the view that one of the main roles of the MCU is linked to the regulation of intramitochondrial dehydrogenases.


Assuntos
Doença de Chagas/enzimologia , Metabolismo Energético , Cetona Oxirredutases/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/enzimologia , Linhagem Celular , Doença de Chagas/genética , Doença de Chagas/patologia , Técnicas de Silenciamento de Genes , Humanos , Cetona Oxirredutases/genética , Fosforilação/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética
13.
Biosci Rep ; 38(5)2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-30181380

RESUMO

Chagas disease remains a serious health problem for countries where the most common mode of transmission is infection contracted from the feces of a Triatominae insect vector. In México, 32 species of Triatoma have been identified; amongst them, Triatoma (Meccus) pallidipennis is an endemic species reported to have high percentages of infection with T. cruzi Defensins, cysteine-rich cationic peptides, are a family of antimicrobial peptides (AMPs); the synthesis of these molecules is crucial for insect's immune defense. In the present study, the genes encoding defensins in T. pallidipennis were sequenced with the purpose of identifying the variability of these genes in a Mexican vector of T. cruzi We found 12 different genes encoding three mature peptides, all of which had the typical folding of a functional insect defensin. In this work two Defensins type 1 and one type 4 were identified. The pro-peptide domain was highly variable and the mature peptide was not. This is the first report focus on variability of defensins from an epidemiologically important Triatoma in Mexico.


Assuntos
Doença de Chagas/genética , Defensinas/genética , Peptídeos/genética , Triatoma/genética , Animais , Doença de Chagas/epidemiologia , Doença de Chagas/transmissão , Doenças Endêmicas/prevenção & controle , Fezes , Humanos , Insetos Vetores/genética , México/epidemiologia , Peptídeos/química , Dobramento de Proteína , Triatoma/patogenicidade
14.
Free Radic Biol Med ; 129: 227-236, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30248443

RESUMO

Elderly organisms are more susceptible to infectious diseases. However, the impact of aging on antiparasitic mechanisms, especially the nitric oxide pathway, is poorly understood. Using an integrated in vivo and in vitro model, we compared the severity of Trypanosoma cruzi infection in young and elderly (8 or 72 weeks old) mice. Forty C57BL/6 mice were randomized into four groups: Y-inf, young infected; Yn-inf, young uninfected; A-inf, aged infected; An-inf, aged uninfected. Parasitemia was measured daily, and animals were euthanized after 15 days of infection. Trypanosoma cruzi-induced inflammatory processes were analyzed in blood and heart samples, as well as in bone marrow-derived macrophages (BMDMs) co-cultured with splenocytes isolated from young or elderly mice. Our results indicated upregulated IgG2b and IL-17 production in elderly animals, which was not sufficient to reduce parasitemia, parasitic load and myocarditis to levels observed in young animals. The higher susceptibility of elderly mice to T. cruzi infection was accompanied by reduced cardiac inducible nitric oxide synthase (iNOS) gene expression, nitric oxide (NO) and IFN-γ levels, as well as an antagonistic upregulation of arginase-1 expression and arginase activity. The same responses were observed when BMDMs co-cultured with splenocytes from elderly mice were stimulated with T. cruzi antigens. Our findings indicate that elderly mice were more susceptible to T. cruzi infection, which was potentially related to an attenuated response to antigenic stimulation, inhibition of iNOS gene expression and NO production, and antagonistic upregulation of arginase gene expression and activity, which created favorable conditions for heart parasitism and myocarditis development.


Assuntos
Envelhecimento/genética , Arginase/genética , Cardiomiopatia Chagásica/genética , Doença de Chagas/genética , Óxido Nítrico Sintase Tipo II/genética , Parasitemia/genética , Trypanosoma cruzi/patogenicidade , Envelhecimento/imunologia , Animais , Antígenos de Protozoários/farmacologia , Arginase/sangue , Cardiomiopatia Chagásica/imunologia , Cardiomiopatia Chagásica/parasitologia , Doença de Chagas/imunologia , Doença de Chagas/parasitologia , Técnicas de Cocultura , Regulação da Expressão Gênica , Coração/parasitologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/genética , Interferon gama/sangue , Interferon gama/genética , Interleucina-10/sangue , Interleucina-10/genética , Interleucina-17/sangue , Interleucina-17/genética , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/sangue , Parasitemia/imunologia , Índice de Gravidade de Doença , Transdução de Sinais , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/parasitologia , Trypanosoma cruzi/imunologia
15.
Parasitol Res ; 117(9): 3009-3013, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29922960

RESUMO

Chagas disease, caused by the protozoan Trypanosoma cruzi, is considered to be a multifactorial disease associated with host and parasite genetics, which influence clinical aspects of the disease and other host conditions. In order to understand better the evolution of the disease, this study intended to evaluation of parasite and host genetics in two generations of a family with Chagas disease from the Alto Paranaiba region, Minas Gerais, Brazil, comprising a mother and her five daughters. Several features were evaluated, including the characterization of T. cruzi directly from the blood of patients, host polymorphisms of genes related to cardiomyopathy (TNF, WISP1, CCR5, and TGF-ß1) and clinical aspects of the patients. To verify the intraspecific variability of the parasite, the characterization was done directly from human blood using the PCR-LSSP technique and analyzed based on Dice coefficient and unweighted pair group analysis (UPGMA). The host polymorphism was evaluated by PCR-RFLP. The global results showed low variability of the parasites characterized from blood of patients, through Shannon index (0.492) and mean heterozygosity value per locus (0.322). All six patients presented the same genetic polymorphism profile for TNF, WISP1, and TGF-ß1, and only one patient was homozygous to CCR5, which suggests that there is no association between the clinical aspects of the patients and their genetic profiles. In conclusion, the findings confirm that the understanding of the clinical evolution of Chagas disease goes beyond the genetic aspects of the parasite and the host.


Assuntos
Proteínas de Sinalização Intercelular CCN/genética , Doença de Chagas/genética , Doença de Chagas/patologia , Proteínas Proto-Oncogênicas/genética , Receptores CCR6/genética , Fator de Crescimento Transformador beta1/genética , Trypanosoma cruzi/genética , Fator de Necrose Tumoral alfa/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Brasil , Doença de Chagas/parasitologia , DNA de Protozoário/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo Genético/genética , Polimorfismo de Fragmento de Restrição
16.
Front Immunol ; 9: 1173, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29896197

RESUMO

Recombinant filamentous fd bacteriophages (rfd) expressing antigenic peptides were shown to induce cell-mediated immune responses in the absence of added adjuvant, being a promising delivery system for vaccination. Here, we tested the capacity of rfd phages to protect against infection with the human protozoan Trypanosoma cruzi, the etiologic agent of Chagas Disease. For this, C57BL/6 (B6) and Tlr9-/- mice were vaccinated with rfd phages expressing the OVA257-264 peptide or the T. cruzi-immunodominant peptides PA8 and TSKB20 and challenged with either the T. cruzi Y-OVA or Y-strain, respectively. We found that vaccination with rfd phages induces anti-PA8 and anti-TSKB20 IgG production, expansion of Ag-specific IFN-γ, TNF-α, and Granzyme B-producing CD8+ T cells, as well as in vivo Ag-specific cytotoxic responses. Moreover, the fd-TSKB20 vaccine was able to protect against mortality induced by a high-dose inoculum of the parasite. Although vaccination with rfd phages successfully reduced both parasitemia and parasite load in the myocardium of WT B6 mice, Tlr9-/- animals were not protected against infection. Thus, our data extend previous studies, demonstrating that rfd phages induce Ag-specific IgG and CD8+ T cell-mediated responses and confer protection against an important human parasite infection, through a TLR9-dependent mechanism.


Assuntos
Bacteriófago M13 , Doença de Chagas , Regulação da Expressão Gênica , Vacinas Protozoárias , Receptor Toll-Like 9 , Trypanosoma cruzi , Vacinação , Animais , Bacteriófago M13/genética , Bacteriófago M13/imunologia , Doença de Chagas/genética , Doença de Chagas/imunologia , Doença de Chagas/prevenção & controle , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Camundongos , Camundongos Knockout , Vacinas Protozoárias/genética , Vacinas Protozoárias/imunologia , Vacinas Protozoárias/farmacologia , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologia
17.
PLoS Negl Trop Dis ; 12(5): e0006480, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29750791

RESUMO

BACKGROUND: Chagas disease is caused by Trypanosoma cruzi. The persistence of the parasite is associated with the disease chronicity and the impairment of the cellular immune response. It has been reported that the CD4+CD8+ T cell population expands in chronic Chagas disease patients. Few studies have focused on this subset of cells, and very little is known about the impact of antiparasitic treatment on this population. METHODOLOGY: Thirty-eight chronic Chagas disease patients (20 asymptomatic and 18 symptomatic) and twelve healthy controls were enrolled in this study. Peripheral blood mononuclear cells were stimulated with soluble T. cruzi antigens to analyze the production of cytokines and cytotoxic molecules by CD4+CD8+ T cells before and after benznidazole treatment. Additionally, expression and co-expression of five inhibitory receptors in these patients after treatment were studied using a multiparameter flow cytometry technique. PRINCIPAL FINDINGS: The frequency of CD4+CD8+ T cells was higher in chronic Chagas disease patients compared with healthy donors. Furthermore, a higher ratio of CD4+CD8low/CD4+CD8high subpopulations was observed in chronic Chagas disease patients than in healthy donors. Additionally, CD4+CD8+ T cells from these patients expressed and co-expressed higher levels of inhibitory receptors in direct proportion to the severity of the pathology. Benznidazole treatment reduced the frequency of CD4+CD8+ T cells and decreased the ratio of CD4+CD8low/CD4+CD8high subpopulations. The co-expression level of the inhibitory receptor was reduced after treatment simultaneously with the enhancement of the multifunctional capacity of CD4+CD8+ T cells. After treatment, an increase in the frequency of T. cruzi antigen-specific CD4+CD8+ T cells expressing IL-2 and TNF-α was also observed. CONCLUSIONS: CD4+CD8+ T cells could play an important role in the control of T. cruzi infection since they were able to produce effector molecules for parasite control. Benznidazole treatment partially reversed the exhaustion process caused by T. cruzi infection in these cells with an improvement in the functional response of the T. cruzi antigen-specific CD4+CD8+ T cells.


Assuntos
Antiprotozoários/administração & dosagem , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Doença de Chagas/tratamento farmacológico , Nitroimidazóis/administração & dosagem , Trypanosoma cruzi/efeitos dos fármacos , Adulto , Anticorpos Antiprotozoários/imunologia , Doença de Chagas/genética , Doença de Chagas/imunologia , Doença de Chagas/parasitologia , Doença Crônica/terapia , Citocinas/imunologia , Feminino , Humanos , Interleucina-2/genética , Interleucina-2/imunologia , Leucócitos Mononucleares/imunologia , Masculino , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
18.
Dis Markers ; 2018: 4579198, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29670670

RESUMO

Transforming growth factor ß1 (TGF-ß1) is an important mediator in Chagas disease. Furthermore, patients with higher TGF-ß1 serum levels show a worse clinical outcome. Gene polymorphism may account for differences in cytokine production during infectious diseases. We tested whether TGFB1 polymorphisms could be associated with Chagas disease susceptibility and severity in a Brazilian population. We investigated five single-nucleotide polymorphisms (-800 G>A, -509 C>T, +10 T>C, +25 G>C, and +263 C>T). 152 patients with Chagas disease (53 with the indeterminate form and 99 with the cardiac form) and 48 noninfected subjects were included. Genotypes CT and TT at position -509 of the TGFB1 gene were more frequent in Chagas disease patients than in noninfected subjects. Genotypes TC and CC at codon +10 of the TGFB1 gene were also more frequent in Chagas disease patients than in noninfected subjects. We found no significant differences in the distribution of the studied TGFB1 polymorphisms between patients with the indeterminate or cardiac form of Chagas disease. Therefore, -509 C>T and +10 T>C TGFB1 polymorphisms are associated with Chagas disease susceptibility in a Brazilian population.


Assuntos
Doença de Chagas/genética , Polimorfismo de Nucleotídeo Único , Fator de Crescimento Transformador beta/genética , Adulto , Idoso , Brasil , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
19.
Sci Rep ; 8(1): 4140, 2018 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-29515202

RESUMO

Trypanosoma cruzi is the agent of Chagas disease, transmitted by hematophagous triatomine vectors. Establishing transmission cycles is key to understand the epidemiology of the disease, but integrative assessments of ecological interactions shaping parasite transmission are still limited. Current approaches also lack sensitivity to assess the full extent of this ecological diversity. Here we developed a metabarcoding approach based on next-generation sequencing to identify triatomine gut microbiome, vertebrate feeding hosts, and parasite diversity and their potential interactions. We detected a dynamic microbiome in Triatoma dimidiata, including 23 bacterial orders, which differed according to blood sources. Fourteen vertebrate species served as blood sources, corresponding to domestic, synantropic and sylvatic species, although four (human, dog, cow and mice) accounted for over 50% of blood sources. Importantly, bugs fed on multiple hosts, with up to 11 hosts identified per bug, indicating very frequent host-switching. A high clonal diversity of T. cruzi was detected, with up to 20 haplotypes per bug. This analysis provided much greater sensitivity to detect multiple blood meals and multiclonal infections with T. cruzi, which should be taken into account to develop transmission networks, and characterize the risk for human infection, eventually leading to a better control of disease transmission.


Assuntos
Biodiversidade , Doença de Chagas , Código de Barras de DNA Taxonômico , Insetos Vetores , Triatoma , Trypanosoma cruzi/genética , Animais , Bovinos , Doença de Chagas/genética , Doença de Chagas/transmissão , Cães , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Insetos Vetores/genética , Insetos Vetores/parasitologia , Camundongos , Triatoma/genética , Triatoma/parasitologia
20.
Am J Pathol ; 188(6): 1345-1353, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29545200

RESUMO

Trypanosoma cruzi infection in women of reproductive age is associated with congenital transmission and adverse pregnancy outcomes. The placenta is a key barrier to infection. Gene expression profiles of term placental environment from T. cruzi-seropositive (SP) and -seronegative (SN) mothers were characterized by RNA-Seq. Nine pools of placental RNA paired samples were used: three from SN and six from SP tissues. Each pool consisted of female/male newborns and vaginal/cesarean delivery binomials. No newborn was congenitally infected. T. cruzi satellite DNA quantitative PCR in placental tissues and maternal and neonatal blood, and parasite 18S quantitative RT-PCR from placental RNA were negative, except in three SP women's bloodstream. To identify pathways associated with maternal T. cruzi infection, a gene-set association analysis was implemented: SP placental samples showed overexpression of inflammatory response and lymphocytic activation, whereas numerous biosynthetic processes were down-regulated. About 42 genes showed a significant fold-change between SP and SN groups. KISS1 and CGB5 were down-regulated, whereas KIF12, HLA-G, PRG2, TAC3, FN1, and ATXN3L were up-regulated. Several expressed genes in SP placentas encode proteins associated with preeclampsia and miscarriage. This first transcriptomics study in human term placental environment shows a placental response that may affect the fetus while protecting it from parasite infection; this host response could be responsible for the low rate of congenital transmission in chronic Chagas disease.


Assuntos
Doença de Chagas/genética , Feto/metabolismo , Regulação da Expressão Gênica , Placenta/metabolismo , Complicações Infecciosas na Gravidez/genética , Trypanosoma cruzi/genética , Adolescente , Adulto , Doença de Chagas/complicações , Doença de Chagas/parasitologia , Feminino , Feto/parasitologia , Perfilação da Expressão Gênica , Humanos , Recém-Nascido , Masculino , Placenta/parasitologia , Gravidez , Complicações Infecciosas na Gravidez/parasitologia , Adulto Jovem
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