RESUMO
T cell-mediated immune response plays a crucial role in controlling Trypanosoma cruzi infection and parasite burden, but it is also involved in the clinical onset and progression of chronic Chagas' disease. Therefore, the study of T cells is central to the understanding of the immune response against the parasite and its implications for the infected organism. The complexity of the parasite-host interactions hampers the identification and characterization of T cell-activating epitopes. We approached this issue by combining in silico and in vitro methods to interrogate patients' T cells specificity. Fifty T. cruzi peptides predicted to bind a broad range of class I and II HLA molecules were selected for in vitro screening against PBMC samples from a cohort of chronic Chagas' disease patients, using IFN-γ secretion as a readout. Seven of these peptides were shown to activate this type of T cell response, and four out of these contain class I and II epitopes that, to our knowledge, are first described in this study. The remaining three contain sequences that had been previously demonstrated to induce CD8+ T cell response in Chagas' disease patients, or bind HLA-A*02:01, but are, in this study, demonstrated to engage CD4+ T cells. We also assessed the degree of differentiation of activated T cells and looked into the HLA variants that might restrict the recognition of these peptides in the context of human T. cruzi infection.
Assuntos
Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos/imunologia , Cardiomiopatia Chagásica/imunologia , Epitopos de Linfócito T/imunologia , Trypanosoma cruzi/imunologia , Antígenos de Protozoários/metabolismo , Argentina , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/imunologia , Cardiomiopatia Chagásica/sangue , Cardiomiopatia Chagásica/parasitologia , Simulação por Computador , ELISPOT , Epitopos de Linfócito T/metabolismo , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imunidade Celular , Memória Imunológica , Testes de Liberação de Interferon-gama , Ativação Linfocitária , Masculino , Trypanosoma cruzi/metabolismoRESUMO
Several studies have proposed different genetic markers of susceptibility to develop chronic Chagas cardiomyopathy (CCC). Many genes may be involved, each one making a small contribution. For this reason, an appropriate approach for this problematic is to study a large number of single nucleotide polymorphisms (SNPs) in individuals sharing a genetic background. Our aim was to analyze two CCR2 and seven CCR5 SNPs and their association to CCC in Argentina. A case-control study was carried out in 480 T. cruzi seropositive adults from Argentinean Gran Chaco endemic region (Wichi and Creole) and patients from Buenos Aires health centres. They were classified according to the Consensus on Chagas-Mazza Disease as non-demonstrated (non-DC group) or demonstrated (DC group) cardiomyopathy, i.e. asymptomatic or with CCC patients, respectively. Since, after allelic analysis, 2 out of 9 studied SNPs did not fit Hardy-Weinberg equilibrium in the unaffected non-DC group from Wichi patients, we analyzed them as a separate population. Only rs1800024T and rs41469351T in CCR5 gene showed significant differences within non-Wichi population (Creole + patients from Buenos Aires centres), being the former associated to protection, and the latter to risk of CCC. No evidence of association was observed between any of the analyzed CCR2-CCR5 gene polymorphisms and the development of CCC; however, the HHE haplotype was associated with protection in Wichi population. Our findings support the hypothesis that CCR2-CCR5 genes and their haplotypes are associated with CCC; however, depending on the population studied, different associations can be found. Therefore, the evolutionary context, in which the genes or haplotypes are associated with diseases, acquires special relevance.
Assuntos
Cardiomiopatia Chagásica/genética , Predisposição Genética para Doença , Receptores CCR2/genética , Receptores CCR5/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Argentina , Estudos de Casos e Controles , Feminino , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Adulto JovemRESUMO
This work evaluated a serial blood sampling procedure to enhance the sensitivity of duplex real-time quantitative PCR (qPCR) for baseline detection and quantification of parasitic loads and posttreatment identification of failure in the context of clinical trials for treatment of chronic Chagas disease, namely, DNDi-CH-E1224-001 (ClinicalTrials.gov registration no. NCT01489228) and the MSF-DNDi PCR Sampling Optimization Study (NCT01678599). Patients from Cochabamba (n = 294), Tarija (n = 257), and Aiquile (n = 220) were enrolled. Three serial blood samples were collected at each time point, and qPCR triplicates were tested for each sample. The first two samples were collected during the same day and the third one 7 days later. A patient was considered PCR positive if at least one qPCR replicate was detectable. Cumulative results of multiple samples and qPCR replicates enhanced the proportion of pretreatment sample positivity from 54.8% to 76.2%, 59.5% to 77.8%, and 73.5% to 90.2% in Cochabamba, Tarija, and Aiquile cohorts, respectively. This strategy increased the detection of treatment failure from 72.9% to 91.7%, 77.8% to 88.9%, and 42.9% to 69.1% for E1224 low-, short-, and high-dosage regimens, respectively, and from 4.6% to 15.9% and 9.5% to 32.1% for the benznidazole arm in the DNDi-CH-E1224-001 and MSF-DNDi studies, respectively. The addition of the third blood sample and third qPCR replicate in patients with nondetectable PCR results in the first two samples gave a small, non-statistically significant improvement in qPCR positivity. No change in clinical sensitivity was seen with a blood volume increase from 5 to 10 ml. The monitoring of patients treated with placebo in the DNDi-CH-E1224-001 trial revealed fluctuations in parasitic loads and occasionally nondetectable results. In conclusion, a serial sampling strategy enhanced PCR sensitivity to detecting treatment failure during follow-up and has the potential for improving recruitment capacity in Chagas disease trials, which require an initial positive qPCR result for patient admission.
Assuntos
Doença de Chagas/tratamento farmacológico , DNA de Protozoário/sangue , Monitorização Fisiológica/métodos , Carga Parasitária/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adolescente , Adulto , Humanos , Pessoa de Meia-Idade , Nitroimidazóis/uso terapêutico , Placebos/administração & dosagem , Tiazóis/uso terapêutico , Resultado do Tratamento , Triazóis/uso terapêutico , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/efeitos dos fármacos , Adulto JovemRESUMO
BACKGROUND: Chagas disease is caused by Trypanosoma cruzi, a parasite endemic to Latin America. Most infections occur in children by vector or congenital transmission. Trypanosoma cruzi establishes a complexity of specific molecular parasite-host cell interactions to invade the host. However, most studies have been mainly focused on the interaction between the parasite and different cell types, but not on the infection and invasion on a tissue level. During congenital transmission, T. cruzi must cross the placental barrier, composed of epithelial and connective tissues, in order to infect the developing fetus. Here we aimed to study the global changes of transcriptome in the placental tissue after a T. cruzi challenge. RESULTS: Strong changes in gene expression profiling were found in the different experimental conditions, involving the reprogramming of gene expression in genes involved in the innate immune response. CONCLUSIONS: Trypanosoma cruzi induces strong changes in genes involved in a wide range of pathways, especially those involved in immune response against infections.
Assuntos
Doença de Chagas/congênito , Doença de Chagas/parasitologia , Perfilação da Expressão Gênica , Interações Hospedeiro-Parasita/genética , Imunidade Inata/genética , Placenta/parasitologia , Doença de Chagas/imunologia , Doença de Chagas/transmissão , Criança , Regulação para Baixo , Feminino , Expressão Gênica , Interações Hospedeiro-Parasita/imunologia , Humanos , Placenta/citologia , Gravidez , Análise Serial de Tecidos , Trypanosoma cruzi/imunologia , Trypanosoma cruzi/fisiologia , Regulação para CimaRESUMO
Congenital infection of Trypanosoma cruzi allows transmission of this parasite through generations. Despite the problematic that this entails, little is known about the placenta environment genetic response produced against infection. We performed functional genomics by microarray analysis in C57Bl/6J mice comparing placentas from uninfected animals and from animals infected with two different T. cruzi strains: K98, a clone of the non-lethal myotropic CA-I strain (TcI), and VD (TcVI), isolated from a human case of congenital infection. Analysis of networks by GeneMANIA of differentially expressed genes showed that "Secretory Granule" was a pathway down-regulated in both infected groups, whereas "Innate Immune Response" and "Response to Interferon-gamma" were pathways up-regulated in VD infection but not in K98. Applying another approach, the GSEA algorithm that detects small changes in predetermined gene sets, we found that metabolic processes, transcription and macromolecular transport were down-regulated in infected placentas environment and some pathways related to cascade signaling had opposite regulation: over-represented in VD and down-regulated in K98 group. We also have found a stronger tropism to the placental organ by VD strain, by detection of parasite DNA and RNA, suggesting living parasites. Our study is the first one to describe in a murine model the genetic response of placental environment to T. cruzi infection and suggests the development of a strong immune response, parasite genotype-dependent, to the detriment of cellular metabolism, which may contribute to control infection preventing the risk of congenital transmission.
Assuntos
Doença de Chagas/parasitologia , Genótipo , Placenta/patologia , Placenta/parasitologia , Complicações Infecciosas na Gravidez/parasitologia , Trypanosoma cruzi/classificação , Trypanosoma cruzi/genética , Animais , Doença de Chagas/patologia , Doença Crônica , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Camundongos Endogâmicos C57BL , Análise em Microsséries , Gravidez , Complicações Infecciosas na Gravidez/patologiaRESUMO
Trypanosoma cruzi, the causative agent of Chagas disease, is divided into six Discrete Typing Units (DTUs): TcI-TcVI. We aimed to identify T. cruzi DTUs in Latin-American migrants in the Barcelona area (Spain) and to assess different molecular typing approaches for the characterization of T. cruzi genotypes. Seventy-five peripheral blood samples were analyzed by two real-time PCR methods (qPCR) based on satellite DNA (SatDNA) and kinetoplastid DNA (kDNA). The 20 samples testing positive in both methods, all belonging to Bolivian individuals, were submitted to DTU characterization using two PCR-based flowcharts: multiplex qPCR using TaqMan probes (MTq-PCR), and conventional PCR. These samples were also studied by sequencing the SatDNA and classified as type I (TcI/III), type II (TcII/IV) and type I/II hybrid (TcV/VI). Ten out of the 20 samples gave positive results in the flowcharts: TcV (5 samples), TcII/V/VI (3) and mixed infections by TcV plus TcII (1) and TcV plus TcII/VI (1). By SatDNA sequencing, we classified the 20 samples, 19 as type I/II and one as type I. The most frequent DTU identified by both flowcharts, and suggested by SatDNA sequencing in the remaining samples with low parasitic loads, TcV, is common in Bolivia and predominant in peripheral blood. The mixed infection by TcV-TcII was detected for the first time simultaneously in Bolivian migrants. PCR-based flowcharts are very useful to characterize DTUs during acute infection. SatDNA sequence analysis cannot discriminate T. cruzi populations at the level of a single DTU but it enabled us to increase the number of characterized cases in chronically infected patients.
Assuntos
Doença de Chagas/etnologia , Doença de Chagas/parasitologia , DNA de Protozoário/genética , Migrantes , Trypanosoma cruzi/classificação , Trypanosoma cruzi/genética , Adolescente , Adulto , Bolívia/epidemiologia , Doença de Chagas/sangue , Doença de Chagas/epidemiologia , Criança , Coinfecção/epidemiologia , Coinfecção/parasitologia , Feminino , Variação Genética , Genótipo , Humanos , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Tipagem Molecular , Carga Parasitária , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Espanha/epidemiologia , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
This study aimed to evaluate well-documented diagnostic antigens, named B13, 1F8 and JL7 recombinant proteins, as potential markers of seroconversion in treated chagasic patients. Prospective study, involving 203 patients treated with benznidazole, was conducted from endemic areas of northern Argentina. Follow-up was possible in 107 out of them and blood samples were taken for serology and PCR assays before and 2, 3, 6, 12, 24 and 36 months after treatment initiation. Reactivity against Trypanosoma cruzi lysate and recombinant antigens was measured by ELISA. The rate of decrease of antibody titers showed nonlinear kinetics with an abrupt drop within the first three months after initiation of treatment for all studied antigens, followed by a plateau displaying a low decay until the end of follow-up. At this point, anti-B13, anti-1F8 and anti-JL7 titers were relatively close to the cut-off line, while anti-T. cruzi antibodies still remained positive. At baseline, 60.8% (45/74) of analysed patients tested positive for parasite DNA by PCR and during the follow-up period in 34 out of 45 positive samples (75.5%) could not be detected T. cruzi DNA. Our results suggest that these antigens might be useful as early markers for monitoring antiparasitic treatment in chronic Chagas disease.
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Adulto Jovem , Anticorpos Antiprotozoários/sangue , Doença de Chagas/tratamento farmacológico , Nitroimidazóis/uso terapêutico , Tripanossomicidas/uso terapêutico , Antígenos de Protozoários/imunologia , Argentina , Doença de Chagas/sangue , Doença Crônica , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática , Seguimentos , Estudos Prospectivos , Fatores de TempoRESUMO
This study aimed to evaluate well-documented diagnostic antigens, named B13, 1F8 and JL7 recombinant proteins, as potential markers of seroconversion in treated chagasic patients. Prospective study, involving 203 patients treated with benznidazole, was conducted from endemic areas of northern Argentina. Follow-up was possible in 107 out of them and blood samples were taken for serology and PCR assays before and 2, 3, 6, 12, 24 and 36 months after treatment initiation. Reactivity against Trypanosoma cruzi lysate and recombinant antigens was measured by ELISA. The rate of decrease of antibody titers showed nonlinear kinetics with an abrupt drop within the first three months after initiation of treatment for all studied antigens, followed by a plateau displaying a low decay until the end of follow-up. At this point, anti-B13, anti-1F8 and anti-JL7 titers were relatively close to the cut-off line, while anti-T. cruzi antibodies still remained positive. At baseline, 60.8% (45/74) of analysed patients tested positive for parasite DNA by PCR and during the follow-up period in 34 out of 45 positive samples (75.5%) could not be detected T. cruzi DNA. Our results suggest that these antigens might be useful as early markers for monitoring antiparasitic treatment in chronic Chagas disease.
Assuntos
Anticorpos Antiprotozoários/sangue , Doença de Chagas/tratamento farmacológico , Nitroimidazóis/uso terapêutico , Tripanossomicidas/uso terapêutico , Adulto , Antígenos de Protozoários/imunologia , Argentina , Doença de Chagas/sangue , Doença Crônica , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de Tempo , Adulto JovemRESUMO
An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.
Assuntos
Doença de Chagas/sangue , DNA de Protozoário/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Trypanosoma cruzi/genética , Doença de Chagas/diagnóstico , Doença de Chagas/genética , Doença de Chagas/parasitologia , DNA de Protozoário/isolamento & purificação , Humanos , Cooperação Internacional , Ensaio de Proficiência Laboratorial , Tipagem Molecular , Parasitemia/sangue , Parasitemia/diagnóstico , Parasitemia/genética , Sensibilidade e Especificidade , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
The effects of replacing dietary saturated fat by different monounsaturated fatty acid (ω-9MUFA) sources on serum lipids, body fat and bone in growing hypercholesterolemic rats were studied. Rats received one of the six different diets: AIN-93G (control, C); extra virgin olive oil (OO) + C; high-oleic sunflower oil (HOSO) + C or atherogenic diet (AT) for 8 weeks; the remaining two groups received AT for 3 weeks and then, the saturated fat was replaced by an oil mixture of soybean oil added with OO or HOSO for 5 weeks. Rats consuming MUFA-rich diets showed the highest body fat, hepatic index and epididymal, intestinal and perirenal fat, and triglycerides. T-chol and non-HDL-chol were increased in HOSO rats but decreased in OO rats. Bone mineral content and density were higher in both OO and HOSO groups than in AT rats. This study casts caution to the generalization of the benefits of MUFA for the treatment of hypercholesterolemia.