Expression of inhibitory receptors and polyfunctional responses of T cells are linked to the risk of congenital transmission of T. cruzi.

Congenital T. cruzi infections involve multiple factors in which complex interactions between the parasite and the immune system of pregnant women play important roles. In this study, we used an experimental murine model of chronic infection with T. cruzi to evaluate the changes in the expression of inhibitory receptors and the polyfunctionality of T cells during gestation and their association with congenital transmission rate of T. cruzi infection. The results showed that pregnant naïve mice had a higher percentage of CD4+ and CD8+ T cells that expressed inhibitory receptors than cells from non-pregnant naïve mice. However, in mice chronically infected with T. cruzi, gestation induced a significant decrease in the frequency of T cells that expressed or co-expressed inhibitory receptors, as well as an increase in the frequency of polyfunctional CD4+ and CD8+ T cells. This different behavior may be due to the breakdown in the infected mice of the gestation-induced immune homeostasis, probably to control the parasite load. Remarkably, it was observed that the mothers that transmitted the parasite had a higher frequency of T cells that expressed and co-expressed inhibitory receptors as well as a lower frequency of polyfunctional parasite-specific T cells than those that did not transmit it, even though the parasitemia load was similar in both groups. All together these data suggest that the maternal immune profile of the CD4+ and CD8+ T cells could be a determining factor in the congenital transmission of T. cruzi.

Effect of secondary anchor amino acid substitutions on the immunogenic properties of an HLA-A*0201-restricted T cell epitope derived from the Trypanosoma cruzi KMP-11 protein.

The TcTLE peptide (TLEEFSAKL) is a CD8(+) T cell HLA-A*0201-restricted epitope derived from the Trypanosoma cruzi KMP-11 protein that is efficiently processed, presented and recognized by CD8(+) T cells from chagasic patients. Since the immunogenic properties of wild-type epitopes may be enhanced by suitable substitutions in secondary anchor residues, we have studied the effect of introducing specific mutations at position 3, 6 and 7 of the TcTLE peptide. Mutations (E3L, S6V and A7F) were chosen on the basis of in silico predictions and in vitro assays were performed to determine the TcTLE-modified peptide binding capacity to the HLA-A*0201 molecule. In addition, the functional activity of peptide-specific CD8(+) T cells in HLA-A2(+) chagasic patients was also interrogated. In contrast to bioinformatics predictions, the TcTLE-modified peptide was found to have lower binding affinity and stability than the original peptide. Nevertheless, CD8(+) T cells from chronic chagasic patients recognized the TcTLE-modified peptide producing TNF-α and INF-γ and expressing CD107a/b, though in less extension than the response triggered by the original peptide. Overall, although the amino acids at positions 3, 6 and 7 of TcTLE are critical for the peptide affinity, they have a limited effect on the immunogenic properties of the TcTLE epitope.

Mammalian cellular culture models of Trypanosoma cruzi infection: a review of the published literature.

Cellular culture infection with Trypanosoma cruzi is a tool used to dissect the biological mechanisms behind Chagas disease as well as to screen potential trypanocidal compounds. Data on these models are highly heterogeneous, which represents a challenge when attempting to compare different studies. The purpose of this review is to provide an overview of the cell culture infectivity assays performed to date. Scientific journal databases were searched for articles in which cultured cells were infected with any Trypanosoma cruzi strain or isolate regardless of the study's goal. From these articles the cell type, parasite genotype, culture conditions and infectivity results were extracted. This review represents an initial step toward the unification of infectivity model data. Important differences were detected when comparing the pathophysiology of Chagas disease with the experimental conditions used in the analyzed studies. While Trypanosoma cruzi preferentially infects stromal cells in vivo, most of the assays employ epithelial cell lines. Furthermore, the most commonly used parasite strain (Tulahuen-TcVI) is associated with chagasic cardiomyopathy only in the Southern Cone of South America. Suggestions to overcome these discrepancies include the use of stromal cell lines and parasite genotypes associated with the known characteristics of the natural history of Chagas disease.

Diseño de un panel multicolor para evaluar moléculas intracelulares y de superficie mediante citometría de flujo / Design of a multicolor panel to assess intracellular and surface molecules by flow cytometry

Introducción. La citometría de flujo permite detectar la presencia de moléculas intracelulares y de superficie, de forma simultánea sobre cada célula. Objetivo. Describir un método para la construcción armónica de un panel multicolor con 11 parámetros para el análisis fenotípico y funcional de linfocitos T (LT) CD8 + por citometría de flujo. Materiales y métodos. Para la construcción del panel multicolor, se seleccionaron las moléculas y se titularon los conjugados con fluorocromos para la determinación de CD3, CD8, CCR7, CD28, CD27, CD45RA, CD95 y CD127, en células mononucleares de sangre periférica. Para la evaluación del panel, se hizo la construcción progresiva adicionando uno a uno los conjugados y la fluorescencia menos uno (FMO). Este método fue aplicado para células ex vivo y para evaluar la producción de IFN ? , IL-2 y TNFa frente al estímulo con la enterotoxina B de Staphylococcus aureus (SEB) y al antígeno crudo de Trypanosoma cruzi . Finalmente, se procedió al análisis de las subpoblaciones de LT CD8 + ex vivo en individuos sanos. Resultados. La evaluación de las moléculas con los conjugados no mostró interferencia en las señales de fluorescencia. Las frecuencias de las subpoblaciones de LT CD8 + evaluadas fueron cercanas a los valores reportados en otros estudios. Además, se observó que la frecuencia de LT CD8 + productores de IFN ? , IL-2 y TNFa fue mayor a las seis horas de cultivo con SEB y con el antígeno crudo de T. cruzi . Conclusiones. El método aplicado para la construcción del panel multicolor permite obtener frecuencias de las subpoblaciones de LT CD8 + que corresponden a lo reportado en la literatura científica.

Introduction: Flow cytometry allows simultaneous detection of surface and intracellular molecules on each cell. Objective: To describe a method for building up a harmonic multicolor panel with 11 flow cytometry parameters for phenotypic and functional analysis on CD8 + T lymphocytes. Materials and methods: For the multicolor panel construction, we selected the molecules and titred conjugated antibodies with fluorochromes for CD3, CD8, CCR7, CD28, CD27, CD45RA, CD95 and CD127 determination in peripheral blood mononuclear cells (PBMC). To evaluate the panel, the conjugated antibodies were gradually added one by one and fluorescence minus one (FMO) test was performed. This method was applied to assess ex vivo subpopulations of T cells and the production of intracellular IFN ? , IL-2 and TNF a using polyclonal stimulation with enterotoxin B from Staphylococcus aureus (SEB) and antigen-specific cells with crude Trypanosoma cruzi antigen. Finally, the ex vivo CD8 + T lymphocyte subpopulations frequencies were analyzed in healthy individuals. Results: The evaluation of the selected molecules and conjugates did not show interference in the fluorescence signals and detection. The frequencies of CD8 + T cells evaluated were similar to the values reported in other studies. Additionally, we observed that the frequency of CD8 + T lymphocytes producing IFN ? , IL-2 and TNF a was higher 6 hours after culture with SEB and crude T. cruzi lysate. Conclusions: The method used for the construction of a multicolor panel allows obtaining frequencies of CD8 + T lymphocyte subpopulations corresponding to those reported in the literature.

A human astrocytoma cell line is highly susceptible to infection with Trypanosoma cruzi.

Astrocytes play a vital role in neuronal protection, homeostasis, vascular interchange and the local immune response. Some viruses and parasites can cross the blood-brain barrier and infect glia. Trypanosoma cruzi, the aetiological agent of Chagas disease, can seriously compromise the central nervous system, mainly in immune-suppressed individuals, but also during the acute phase of the infection. In this report, the infective capacity of T. cruzi in a human astrocyte tumour-derived cell line was studied. Astrocytes exposed to trypomastigotes (1:10 ratio) produced intracellular amastigotes and new trypomastigotes emerged by day 4 post-infection (p.i.). At day 6 p.i., 93% of the cells were infected. Using flow cytometry, changes were observed in both the expression of major histocompatibility complex class I and II molecules and the chemokine secretion pattern of astrocytes exposed to the parasite. Blocking the low-density lipoprotein receptor on astrocytes did not reduce parasite intracellular infection. Thus, T. cruzi can infect astrocytes and modulate the immune response during central nervous system infection.

A human astrocytoma cell line is highly susceptible to infection with Trypanosoma cruzi

Astrocytes play a vital role in neuronal protection, homeostasis, vascular interchange and the local immune response. Some viruses and parasites can cross the blood-brain barrier and infect glia. Trypanosoma cruzi, the aetiological agent of Chagas disease, can seriously compromise the central nervous system, mainly in immune-suppressed individuals, but also during the acute phase of the infection. In this report, the infective capacity of T. cruzi in a human astrocyte tumour-derived cell line was studied. Astrocytes exposed to trypomastigotes (1:10 ratio) produced intracellular amastigotes and new trypomastigotes emerged by day 4 post-infection (p.i.). At day 6 p.i., 93% of the cells were infected. Using flow cytometry, changes were observed in both the expression of major histocompatibility complex class I and II molecules and the chemokine secretion pattern of astrocytes exposed to the parasite. Blocking the low-density lipoprotein receptor on astrocytes did not reduce parasite intracellular infection. Thus, T. cruzi can infect astrocytes and modulate the immune response during central nervous system infection.

[Design of a multicolor panel to assess intracellular and surface molecules by flow cytometry]. / Diseño de un panel multicolor para evaluar moléculas intracelulares y de superficie mediante citometría de flujo.

INTRODUCTION: Flow cytometry allows simultaneous detection of surface and intracellular molecules on each cell. OBJECTIVE: To describe a method for building up a harmonic multicolor panel with 11 flow cytometry parameters for phenotypic and functional analysis on CD8 + T lymphocytes. MATERIALS AND METHODS: For the multicolor panel construction, we selected the molecules and titred conjugated antibodies with fluorochromes for CD3, CD8, CCR7, CD28, CD27, CD45RA, CD95 and CD127 determination in peripheral blood mononuclear cells (PBMC). To evaluate the panel, the conjugated antibodies were gradually added one by one and fluorescence minus one (FMO) test was performed. This method was applied to assess ex vivo subpopulations of T cells and the production of intracellular IFN ? , IL-2 and TNF a using polyclonal stimulation with enterotoxin B from Staphylococcus aureus (SEB) and antigen-specific cells with crude Trypanosoma cruzi antigen. Finally, the ex vivo CD8 + T lymphocyte subpopulations frequencies were analyzed in healthy individuals. RESULTS: The evaluation of the selected molecules and conjugates did not show interference in the fluorescence signals and detection. The frequencies of CD8 + T cells evaluated were similar to the values reported in other studies. Additionally, we observed that the frequency of CD8 + T lymphocytes producing IFN ? , IL-2 and TNF a was higher 6 hours after culture with SEB and crude T. cruzi lysate. CONCLUSIONS: The method used for the construction of a multicolor panel allows obtaining frequencies of CD8 + T lymphocyte subpopulations corresponding to those reported in the literature.

Chagasic patients are able to respond against a viral antigen from influenza virus.

BACKGROUND: Trypanosoma cruzi, the etiological agent of Chagas' disease, is an obligate intracellular parasite which induces a CD8+ T cell immune response with secretion of cytokines and release of cytotoxic granules. Although an immune-suppressive effect of T. cruzi on the acute phase of the disease has been described, little is known about the capacity of CD8+ T cell from chronic chagasic patients to respond to a non-T. cruzi microbial antigen. METHODS: In the present paper, the frequency, phenotype and the functional activity of the CD8+ T cells specific from Flu-MP*, an influenza virus epitope, were determined in 13 chagasic patients and 5 healthy donors. RESULTS: The results show that Flu-MP* peptide specific CD8+ T cells were found with similar frequencies in both groups. In addition, Flu-MP* specific CD8+ T cells were distributed in the early or intermediate/late differentiation stages without showing enrichment of a specific sub-population. The mentioned Flu-MP* specific CD8+ T cells from chagasic patients were predominately TEM (CCR7- CD62L-), producing IL-2, IFNγ, CD107a/b and perforin, and did not present significant differences when compared with those from healthy donors. CONCLUSIONS: Our results support the hypothesis that there is no CD8+ T cell nonspecific immune-suppression during chronic Chagas disease infection. Nonetheless, other viral antigens must be studied in order to confirm our findings.

Rabbit serum against K1 peptide, an immunogenic epitope of the Trypanosoma cruzi KMP-11, decreases parasite invasion to cells.

KMP-11 is a highly conserved protein of Trypanosoma cruzi implicated in parasite's motility. Here we show that K1, a peptide derived from KMP-11, induced polyclonal antibodies capable of decreasing T. cruzi infection in vitro. Rabbit sera rose against K1 peptide showed recognition of the recombinant protein by ELISA and Western blot and also of the native protein in both epimastigotes and trypomastigotes as evaluated by immunofluorescence test and flow cytometry. Invasion assays showed a significant reduction of trypomastigotes infection of eukaryotic cells when parasites were pre-incubated with anti-K1 rabbit serum. Computational modeling predicted that the K1 sequence conserved its α-helical configuration into the protein, and some of the amino acid residues appear accessible for recognition by antibodies in vivo. Taken together, these results support the idea that the K1 peptide induces antibodies than can have a potential role in protective immunity in Chagas disease.

Expression of inhibitory receptors on CD4+ and CD8+ T cells from healthy Colombian donors / Expresión de receptores Inhibidores en linfocitos T CD4+ y CD8+ de donantes sanos Colombianos / Expressão de receptores inibitórios sobre linfócitos T CD4+ e CD8+ de doadores saudáveis Colombianos

T cell activation involves positive cellular signals that promote effector functions and negative signals that contribute to the regulation of these responses. These regulatory signals are generated upon activation of receptors on T cells that include CD160, 2B4, Programmed Death-1 and CTLA-4. Objective. To evaluate the expression of inhibitory receptors like CD160, 2B4, Programmed Death-1 and CTLA-4 on CD4+ and CD8+ T cells from healthy Colombian donors. Materials and methods. Peripheral blood mononuclear cells from 30 healthy donors from Bogotá (Colombia) were obtained via Ficoll-Hypaque density gradient and cells were stained with specific conjugated antibodies previously titrated. Results. The CD160, 2B4, and Programmed Death-1 inhibitory markers were detected on CD4+ T cells with expression levels of 0.35%, 1.04%, and 1.35%, respectively. On CD8+ T cells, these markers were expressed at higher levels: 16%, 8.97%, and 4.3%, respectively. In contrast to the other receptors, CTLA-4 frequency of expression showed no significant difference between CD4+ (1.56%) and CD8+ (1.53%) T cells. Frequency of CD160/2B4 and CTLA-4/ Programmed Death-1 coexpression was 0.18% and 0.09% on CD4+ cells, and 4.02% and 0.2% on CD8+ T cells. Conclusions. This is the first report showing the frequency of inhibitory receptors such as CD160, 2B4, Programmed Death-1, and CTLA-4 on CD4+ and CD8+ T cells from healthy Colombian donors. Our findings serve as a baseline for the analysis and comparison of these receptors in Colombian populations with different disease conditions.

La activación de células T involucra señales positivas que promueven funciones efectoras y señales negativas que contribuyen a la regulación de la respuesta inmune. Estas actividades regulatorias son generadas por la activación de receptores de las células T e incluyen moléculas como CD160, 2B4, PD-1 y CTLA-4. Objetivo. Evaluar la expresión de los receptores inhibitorios CD160, 2B4, PD-1 y CTLA-4 en células T CD4+ y CD8+ de donantes sanos colombianos. Materiales y métodos. Se obtuvieron células mononucleares de sangre periférica de 30 donantes sanos provenientes de Bogotá (Colombia) mediante gradiente de densidad por Ficoll-Hypaque, las células fueron marcadas con anticuerpos conjugados a fluorocromos previamente titulados. Resultados. CD160, 2B4, y PD-1 presentaron porcentajes de expresión en células T CD4+ de 0.35%, 1.04% y 1.35% respectivamente. En células T CD8+ estos receptores mostraron niveles de expresión más altos con porcentajes de 16.3%, 8.97% y 4.3% respectivamente. A diferencia de los otros receptores, CTLA-4 no mostró diferencias entre células T CD4+ (1.56%) y CD8+ (1.53%). Los porcentajes de co-expresión de CD160/2B4 y CTLA-4/PD-1 en células T CD4+ fueron de 0.18% y 0.09%, en células T CD8+ se observaron porcentajes de expresión de 4.02% y 0.2%. Conclusión. Este es el primer reporte que muestra la frecuencia de expresión de receptores inhibitorios tales como CD160, 2B4, PD-1 y CTLA-4 en células T CD4+ y CD8+ de donantes sanos colombianos. Estos hallazgos representan una línea de base para el análisis y la comparación de estos receptores en la población colombiana con diferentes enfermedades.

A ativação de células T envolve sinais positivos que promovem funções efetoras e sinais negativos que contribuem para a regulação da resposta imune. Estas atividades reguladoras são geradas através da ativação de receptores de células T e incluem moléculas tais como CD160, 2B4, PD-1 e CTLA-4. Objetivo. Avaliar a expressão de receptores inibitórios CD160 e 2B4, PD-1 e CTLA-4 em células T CD4+ e CD8+ de doadores saudáveis colombianos. Materiais e métodos. Foram obtidas células mononucleares do sangue periférico de 30 doadores saudáveis provenientes de Bogotá (Colômbia) a partir de gradiente de densidade por Ficoll-Hypaque, as células foram marcadas com anticorpos conjugados com fluorocromos previamente titulados. Resultados. CD160, 2B4, e PD-1 mostraram percentagens de expressão em células T CD4+ de 0,35%, 1,04% e 1,35%, respectivamente. Em células T CD8+ estes receptores mostraram níveis de expressão mais elevados com percentagens de 16,3%, 8,97% e 4,3% respectivamente. Ao contrário de outros receptores, CTLA-4 não apresentou diferenças entre células T CD4+ (1,56%) e CD8+ (1,53%). As percentagens de co-expressão de CD160/2B4 e CTLA-4/PD-1 em células T CD4+ foram de 0,18% e 0,09%; em células T CD8+ foram observadas percentagens de expressão de 4,02% e 0,2%. Conclusão. Este é o primeiro relatório que apresenta a frequência de expressão de receptores inibitórios tais como CD160, 2B4, PD-1 e CTLA-4 em células T CD4+ e CD8+ de doadores saudáveis colombianos. Estes resultados representam uma linha de base para análise e comparação desses receptores na população colombiana com diferentes doenças.

[Using S35-S36 and TcH2AF-R primer-based PCR tests to follow-up a Chagas´ disease patient who had undergone a heart transplant]. / Seguimiento de paciente con enfermedad de Chagas y trasplante de corazón mediante las PCR S35-S36 y TcH2AF-R.

INTRODUCTION: Cardiomyopathy is the most common clinical form of Chagas' disease in Colombia, and one treatment option is a heart transplant. Tracking the behavior of the Chagas' parasite, Trypanosoma cruzi, is a priority due to the risk of post-transplant reactivation of the infection. OBJECTIVE: A case is presented of a patient who had suffered from dilated chagasic cardiopathy and cardiac failure, and had subsequently undergone heart transplant. The case was monitored by PCR, histopathological and echocardiographic examinations. MATERIALS AND METHODS: Blood samples were drawn before and after the transplant, and post-transplant endomyocardial biopsies were taken. The extracted DNA was amplified with the TcH2AF-R and S35-S36 primers. Parasitemia was examined by the microhematocrit test. In addition, histopathological studies determined the parasite presence and transplant rejection status. Echocardiograms were administered to evaluate cardiac function. RESULTS: Of the blood samples taken 83 and 48 days pre-transplant, the latter was positive by the S35-S36 PCR test. PCR tests in blood with both primers were negative up to the second month post-transplant. However, both PCR tests were positive by the third month post-transplant. Thereupon, the patient was treated with nifurtimox. Both tests presented negative results in blood 35 days after treatment was started and remained negative thereafter at 0, 3, 10 and 12 months post-treatment. The pathology, microhematocrit, and PCR test results from biopsies were negative on all the specified dates. CONCLUSIONS: PCR tests were used as indicators of a reactivation of trypanosomid infection in the patient. After treatment administration, PCR tests became negative. The patient's clinical evolution was favorable.

Seguimiento de pacientes con enfermedad de chagas y trasplante de corazón mediante las PCR S35-S36 y TcH2AF-R / Using S35-S36 and TcH2AF-R primer-based PCR tests to follow-up a Chagas’ disease patient who had undergone a heart transplant

Introducción. La cardiomiopatía es la forma clínica más común de la enfermedad de Chagas en Colombia, siendo el trasplante una opción para su tratamiento. Debido al riesgo de reactivación de la infección posterior al trasplante, es prioritario vigilar el comportamiento del parásito. Objetivo. Presentar el caso de un paciente con cardiopatía chagásica dilatada y falla cardiaca, a quien se le practicó trasplante de corazón y se le hizo seguimiento mediante PCR, análisis histopatológicos y ecocardiográficos. Materiales y métodos. Se tomaron muestras de sangre antes de la intervención y después de ella y de biopsias endomiocárdicas posteriores al trasplante. El ADN extraído fue amplificado con los iniciadores TcH2AF-R y S35-S36. La parasitemia se examinó mediante la técnica de microhematocrito. Se practicaron estudios histopatológicos para determinar la presencia del parásito o el rechazo del trasplante y, ecocardiográficos, para evaluar la función cardiaca. Resultados. De las muestras de sangre tomadas a los 83 y 48 días previos al trasplante, la última fue positiva por la PCR S35-S36. Hasta el segundo mes después del trasplante, ambas PCR fueron negativas. Al tercer mes después del trasplante, ambas PCR fueron positivas, por lo cual se inició tratamiento con nifurtimox. Tras 35 días de haberse iniciado el tratamiento, ambas pruebas presentaron resultados negativos, al igual que las tomadas a los 0, 3, 10 y 12 meses posteriores. Los resultados de la histopatología, del microhematocrito y de las PCR de las biopsias, fueron negativos en todas las fechas. Conclusiones. Las PCR permitieron sospechar la reactivación de la infección en el paciente, se le administró el tratamiento y posterioremente las pruebas se tornaron negativas. La evolución clínica del paciente ha sido favorable.

Introduction. Cardiomyopathy is the most common clinical form of Chagas’ disease in Colombia, and one treatment option is a heart transplant. Tracking the behavior of the Chagas’ parasite, Trypanosoma cruzi, is a priority due to the risk of post-transplant reactivation of the infection. Objective. A case is presented of a patient who had suffered from dilated chagasic cardiopathy and cardiac failure, and had subsequently undergone heart transplant. The case was monitored by PCR, histopathological and echocardiographic examinations. Materials and methods. Blood samples were drawn before and after the transplant, and post-transplant endomyocardial biopsies were taken. The extracted DNA was amplified with the TcH2AF-R and S35-S36 primers. Parasitemia was examined by the microhematocrit test. In addition, histopathological studies determined the parasite presence and transplant rejection status. Echocardiograms were administered to evaluate cardiac function. Results. Of the blood samples taken 83 and 48 days pre-transplant, the latter was positive by the S35-S36 PCR test. PCR tests in blood with both primers were negative up to the second month post-transplant. However, both PCR tests were positive by the third month post-transplant. Thereupon, the patient was treated with nifurtimox. Both tests presented negative results in blood 35 days after treatment was started and remained negative thereafter at 0, 3, 10 and 12 months post-treatment. The pathology, microhematocrit, and PCR test results from biopsies were negative on all the specified dates. Conclusions. PCR tests were used as indicators of a reactivation of trypanosomid infection in the patient. After treatment administration, PCR tests became negative. The patient’s clinical evolution was favorable.

Secuencia parcial del genoma del maxicírculo de Leishmania braziliensis, comparación con otros tripanosomátidos / Maxicircle genome partial sequence of Leishmania braziliensis: assembling and comparison with other trypanosomatids / Seqüência do genoma parcial do maxicirculo de Leishmania braziliensis: montagem e comparação com outros tripanossomatídeos

Objetivo. Con el fin de aportar nueva información relevante para estudios de genotipificación y filogenética del género Leishmania, en este estudio se determinó y comparó la secuencia del maxicírculo de Leishmania braziliensis, cepa MHOM-BR-75-M2904, con las secuencias del maxicírculo reportadas para otras especies de tripanosomátidos. Materiales y métodos. La búsqueda de las secuencias del maxicírculo se realizó en las bases de datos de secuencias no ensambladas del GeneDB versión 2.1, así como en el GenBank, utilizando los genes ND8 y RPS12 de L. braziliensis como sonda inicial. Estas secuencias se ensamblaron y se compararon con sus homólogas en otros tripanosomátidos mediante el uso de herramientas bioinformáticas como LALIGN y ClustalW2. El tamaño total del maxicírculo se determinó mediante ensayos de Southern blot. Resultados. Se ensamblaron dos fragmentos del maxicírculo de L. braziliensis de 6535 y 4257 nucleótidos, cuyos genes presentaron elevada sintenia y similitud en sus secuencias con los previamente reportados en otras especies de Leishmania. Similitud que se extiende incluso a los patrones de edición de estas moléculas. Conclusiones. A pesar de ser L. braziliensis la especie más divergente del género Leishmania en cuanto a su genoma nuclear, el marxicírculo presenta una elevada conservación. Resultado que sugiere que el patrón de edición presente en las diferentes especies de Leishmania hasta ahora estudiadas se conserva también en el subgénero Viannia, lo que indica una elevada conservación en la edición de los transcritos mitocondriales a nivel de género.

Objective. With the aim to provide new insights for genotyping and phylogenetic studies of the Leishmania genus, in this study the sequence of the maxicircle in Leishmania braziliensis, strain MHOM-BR-75-M2904, was determined and compared with those reported in other trypanosomatids species. Materials and methods. Searches for maxicircle sequences were performed in the unassembled sequences of GeneDB database version 2.1, as well as in the GenBank, using the ND8 and RPS12 genes of L. braziliensis as the initial probes. These sequences were assembled and compared with the homologous sequences of trypanosomatids using the bioinformatics tools LALIGN and ClustalW2. The size of maxicircle was determined by Southern blot assays. Results. Two maxicircle fragments of 6535 and 4257 nucleotides were assembled. The sequences of these genes showed high synteny and similarity with the sequences in other Leishmania species. This similarity even was extended to the editing patterns of these molecules. Conclusions. Although L. braziliensis is the most divergent species of the Leishmania genus in their nuclear genome, the maxicicircle has a high conservation. This result suggests that the pattern of editing present in the different Leishmania species studied has been conserved also in the subgenus Viannia. These results indicate a high conservation in the editing of mitochondrial transcripts at the genus level.

Objetivo. Com o fim de contribuir nova informação relevante para estudos de genotipagem e filogenética do género Leishmania, neste estudo determinou-se a sequência do maxicirculo de Leishmania braziliensis, cepa MHOM-BR-75-M2904, comparandosecom as seqüências do maxicirculo reportadas para outras espécies de tripanossomatídeos. Materiais e Métodos. A busca das seqüências do maxicirculo foi realizada nas bases de dados para seqüências não alinhadas no GeneDB versão 2.1, assim como no GeneBank, utilizando o genes ND8 e RPS12 de L. braziliensis como sonda inicial. Essas seqüências foram alinhadas e comparadas com as suas homologas em outros tripanossomatídeos, mediante o uso de ferramentas bioinformáticas como L-ALIGN e ClustalW2. O tamanho total do maxicirculo foi determinado mediante ensaios de Southern blot. Resultados. Foram alinhados dois fragmentos do maxicirculo de L. braziliensis de 6535 e 4257 nucleotídeos, cujos genes apresentaram elevada sintenia e similaridade nas suas seqüências com os genes previamente reportados nas outras espécies de Leishmania. A similaridade vista estende-se, inclusive, aos padrões de edição para estas moléculas. Conclusões. Apesar de L. braziliensis ser a espécie mais divergente do gênero Leishmania, no que se refere ao seu genoma nuclear, o maxicirculo apresenta uma alta conservação. Esse resultado sugere que o padrão de edição apresentado nas espécies de Leishmania até agora estudadas, é conservado também no subgênero Viannia, o que indica uma alta conservação na edição dos transcritos mitocôndriais ao nível de gênero.

Characterising the KMP-11 and HSP-70 recombinant antigens' humoral immune response profile in chagasic patients.

BACKGROUND: Antigen specificity and IgG subclass could be significant in the natural history of Chagas' disease. The relationship between the different stages of human Chagas' disease and the profiles of total IgG and its subclasses were thus analysed here; they were directed against a crude T. cruzi extract and three recombinant antigens: the T. cruzi kinetoplastid membrane protein-11 (rKMP-11), an internal fragment of the T. cruzi HSP-70 protein 192-433, and the entire Trypanosoma rangeli HSP-70 protein. METHODS: Seventeen Brazilian acute chagasic patients, 50 Colombian chronic chagasic patients (21 indeterminate and 29 cardiopathic patients) and 30 healthy individuals were included. Total IgG and its subtypes directed against the above-mentioned recombinant antigens were determined by ELISA tests. RESULTS: The T. cruzi KMP-11 and T. rangeli HSP-70 recombinant proteins were able to distinguish both acute from chronic chagasic patients and infected people from healthy individuals. Specific antibodies to T. cruzi crude antigen in acute patients came from IgG3 and IgG4 subclasses whereas IgG1 and IgG3 were the prevalent isotypes in indeterminate and chronic chagasic patients. By contrast, the specific prominent antibodies in all disease stages against T. cruzi KMP-11 and T. rangeli HSP-70 recombinant antigens were the IgG1 subclass. CONCLUSION: T. cruzi KMP-11 and the T. rangeli HSP-70 recombinant proteins may be explored together in the immunodiagnosis of Chagas' disease. Polarising the IgG1 subclass of the IgG response to T. cruzi KMP-11 and T. rangeli HSP-70 recombinant proteins could have important biological effects, taking into account that this is a complement fixing antibody.

Evaluación de las pruebas de PCR TcH2AF-R y S35-S36 para la detección de Trypanosoma cruzi en tejido cardiaco de ratón / Evaluation of TcH2AF-R and S35-S36 primers in PCR tests for the detection of Trypanosoma cruzi in mouse cardiac tissue

Introducción. El trasplante es una opción terapéutica en la cardiomiopatía chagásica. Para la detección temprana de una posible reactivación de la infección, se propone el uso de pruebas de reacción en cadena de la polimerasa (PCR) a partir de biopsias endomiocárdicas; el modelo de ratón es una aproximación preliminar para evaluar la aplicación de éstas. Objetivo. Evaluar la aplicación de las pruebas de PCR basadas en los iniciadores TcH2AF-R y S35-S36 para la detección de T. cruzi en tejido cardiaco de ratones infectados con el parásito. Materiales y métodos. Se infectaron por vía intraperitoneal dos grupos de ratones ICR de 15 y 10 individuos con 0,3 ml de PBS que contenían 1x106 tripomastigotes de la cepa MHOM/CO/2001/D.A. (T. cruzi I) o 1x104 tripomastigotes de la cepa MHOM/BR/00/Y (T. cruzi II), respectivamente. El seguimiento de la parasitemia se realizó mediante microhematocrito y presencia de parásitos en el corazón a los 30, 60 (modelo agudo), 100 y 150 (modelo crónico) días por medio de histopatología y de las PCR TcH2AF-R y S35-S36. Resultados. La histopatología mostró alteraciones en el miocardio y presencia de amastigotes en los modelos agudo y crónico. En contraste al microhematocrito y al análisis histopatológico, la PCR S35-S36 permitió la detección de ambas cepas del parásito. La PCR TcH2AF-R detectó la cepa T. cruzi I con un desempeño superior al microhematocrito y al análisis histopatológico. Conclusiones. El uso de ambas pruebas de PCR puede ser útil en la confirmación de la reactivación de la infección postrasplante.

[Evaluation of TcH2AF-R and S35-S36 primers in PCR tests for the detection of Trypanosoma cruzi in mouse cardiac tissue]. / Evaluación de las pruebas de PCR TcH2AF-R y S35-S36 para la detección de Trypanosoma cruzi en tejido cardiaco de ratón.

INTRODUCTION: Heart transplant is a therapeutic option in the treatment of chagasic cardiomyopathy. For early detection of Chagas reactivation cases, the use of PCR tests using endomyocardial biopsies has been proposed. Development of an animal model will be the first step in evaluating the applicability of this approach. OBJECTIVE: PCR tests based on the TcH2AF-R and S35-S36 primers were evaluated for the detection of T. cruzi in heart tissue of mice experimentally infected with the parasite. MATERIALS AND METHODS: Two groups of ICR mice of 15 and 10 individuals were infected by intraperitoneal injection with 0.3 ml of PBS containing 1 x 10(6) trypomastigotes of the MHOM/CO/2001/D.A. (T. cruzi I) strain or 1 x 10(4) trypomastigotes of MHOM/BR/00/Y (T. cruzi II) strain. Parasitemia and cardiac parasitic infection were determined at 30, 60 (acute model), 100 and 150 (chronic model) days by means of histopathological examination and by PCR, using the TcH2AF-R and S35-S36 primers. RESULTS: The histopathological findings revealed alterations in the heart and the presence of intracellular amastigotes in acute and chronic models. In contrast to parasitemia levels and histopathological analyses, S35-S36 PCR detected infections in mice that were infected with either parasite strain. TcH2AF-R PCR detected T. cruzi I-infected mice earlier and more frequently than inspection for parasitemia or histopathological examination. CONCLUSIONS: Applying PCR tests with both primers proved superior for Chagas disease confirmation over currently standard detection methods.

[Acute Chagas disease in Colombia: a rarely suspected disease. Report of 10 cases presented during the 2002-2005 period]. / Enfermedad de Chagas aguda en Colombia, una entidad poco sospechada. Informe de 10 casos presentados en el periodo 2002 a 2005.

INTRODUCTION: In Colombia, reported cases of acute Chagas disease are sporadic. OBJECTIVE: Ten cases were described that had been reported to the Parasitology Laboratory of the Colombian National Health Institute between December 2002 and November 2005. MATERIALS AND METHODS: Information from clinical records, epidemiological report forms, laboratory and blood tests was collated. In addition the following data were compiled: demographic variables, clinical findings, results of laboratory tests and other exams (such as peripheral blood smears), IFAT for IgG antibodies, isolation in culture medium, inoculation in mice, polymerase chain reaction tests and isoenzyme eletrophoresis. RESULTS: All the cases presented in known endemic areas for Chagas disease transmission in Colombia. Three cases were from Putumayo Province, two each from the provinces of Arauca, Casanare, Norte de Santander and one from Santander Province. The probable mode of transmission was vector-borne. Seven cases presented in adults aged 18 to 50, three in children aged 6 months to 2 years. Seven were male and three were female. The most frequent symptom was fever in nine cases. Signs of portal of entry were rare; only one patient presented a possible Romahia's sign. Three patients presented myocarditis, two acute cardiac failure and one cardiac tamponade. Parasitemia was evident in nine cases; five had positive IgG serological tests; five cases were confirmed through parasite isolation; isoenzyme electrophoresis showed Trypanosoma cruzi group I. CONCLUSIONS: Clinical variability prevailed. In none of the cases was a clinical diagnosis suspected. The diagnosis was made and confirmed through laboratory tests alone. The results highlight the importance of including this disease in the differential diagnosis of febrile syndrome in endemic regions due to its good response to etiological treatment and thereby preventing its progression to the chronic phase.

La región intergénica del gen H2A apoya las subpoblaciones KP1(-) y KP1(+) de Trypanosoma rangeli / The intergenic region of the histone h2a gene supports two major lineages of Trypanosoma rangeli

Introducción. Con base en la amplificación del ADN de los minicírculos del cinetoplasto y de los genes miniexón, Trypanosoma rangeli ha sido clasificado en las subpoblaciones KP1(-) y KP1(+). Objetivo. Comparar la región intergénica de los genes H2A entre cepas KP1(+) y KP1(-) de T. rangeli, con el fin de aportar evidencias a dicha división. Materiales y métodos. Se amplificó, clonó y determinó la secuencia de la región intergénica de los genes h2a de las cepas KP1(-) Tre y 5048 y de la cepa Choachí [KP1(+)]. Dichas secuencias, junto con las reportadas para las cepas C23 [KP1(-)] y H14 [KP1(+)], fueron utilizadas para la reconstrucción de árboles filogenéticos basados en los métodos de neighbor-joining, máxima parsimonia y máxima verosimilitud, utilizando la cepa Y de Trypanosoma cruzi como grupo raíz externo. Resultados. Se evidenció heterogeneidad intraespecífica en el tamaño de la región estudiada, soportados por valores bootstrap de 85 por ciento (neighbor-joining), 66 por ciento (máxima parsimonia) y 57 por ciento (máxima verosimilitud), los resultados indican que las cepas KP1(-) se agrupan aparte, claramente diferenciadas de las cepas KP1(+), las cuales presentan una mayor heterogeneidad intraespecífica en tamaño y secuencia. Además, se encontró mayor proximidad filogenética entre T. rangeli y T. cruzi que entre T. rangeli y Trypanosoma brucei. Conclusiones. Los análisis filogenéticos basados en la región intergénica de los genes h2a de las cepas estudiadas apoyan la división de T. rangeli en las subpoblaciones KP1(-) y KP1(+). Sin embargo, se requiere estudiar un mayor número de cepas para confirmar estos hallazgos.

Introduction. Trypanosoma rangeli has been classified in the KP1(+) and KP1(-) subpopulations, based on the mini-exon gene and kinetoplast DNA minicircle amplification profiles. Objective. The intergenic region of the histone h2a gene was compared between KP1(+) and KP1(-) strains of T. rangeli to substantiate this classification. Materials and methods. The amplification, cloning and sequencing of the h2a gene intergenic region was undertaken for the Tre and 5048 [KP1(-)] strains for comparison with the Choachí [KP1(+)] strain. These sequences, along with those previously reported for the KP1 (+) and KP1 (-) H14 and C23 strains, were used to reconstruct phylogenetic trees based on the “neighborjoining”, maximum parsimony and maximum likelihood methods. The Y strain of Trypanosoma cruzi was chosen as the outgroup. Results. Intra-specific heterogeneity was observed in the size of the gene region under study, supported by bootstarp values of 85% (neighbor-joining), 66% (maximum parsimony) and 57% (maximum likelihood). The KP1(-) strains were grouped apart, clearly differentiated from the KP1(+) strains. The latter demonstrated a higher intra-specific heterogeneity, both in sequence length and composition. In addition, a closer phylogenetic relationship between T. rangeli and T. cruzi was found to be more closely related to one another than to T. rangeli and Trypanosoma brucei. Conclusion. Phylogenetic analyses of analyzed strains based on the intergenic region of the h2a genes supported the T. rangeli grouping in two major subpopulations known as KP1(+) and KP1(-) strains. However, a higher number of strains are needed to confirm this finding.

Detection of Trypanosoma cruzi and Trypanosoma rangeli infection in triatomine vectors by amplification of the histone H2A/SIRE and the sno-RNA-C11 genes.

Trypanosoma rangeli is non pathogenic for humans but of important medical and epidemiological interest because it shares vertebrate hosts, insect vectors, reservoirs and geographic areas with T. cruzi, the etiological agent of Chagas disease. Therefore, in this work, we set up two PCR reactions, TcH2AF/R and TrFR2, to distinguish T. cruzi from T. rangeli in mixed infections of vectors based on amplification of the histone H2A/SIRE and the small nucleolar RNA Cl1 genes, respectively. Both PCRs were able to appropriately detect all T. cruzi or T. rangeli experimentally infected-triatomines, as well as the S35/S36 PCR which amplifies the variable region of minicircle kDNA of T. cruzi. In mixed infections, whereas T. cruzi DNA was amplified in 100% of samples with TcH2AF/R and S35/S36 PCRs, T. rangeli was detected in 71% with TrF/R2 and in 6% with S35/S36. In a group of Rhodnius colombiensis collected from Coyaima (Colombia), T. cruzi was identified in 100% with both PCRs and T. rangeli in 14% with TrF/R2 and 10% with S35/S36 PCR. These results show that TcH2AF/R and TrF/R2 PCRs which are capable of recognizing all T. cruzi and T. rangeli strains and lineages could be useful for diagnosis as well as for epidemiological field studies of T. cruzi and T. rangeli vector infections.

Detection of Trypanosoma cruzi and Trypanosoma rangeli infection in triatomine vectors by amplification of the histone H2A/SIRE and the sno-RNA-C11 genes

Trypanosoma rangeli is non pathogenic for humans but of important medical and epidemiological interest because it shares vertebrate hosts, insect vectors, reservoirs and geographic areas with T. cruzi, the etiological agent of Chagas disease. Therefore, in this work, we set up two PCR reactions, TcH2AF/R and TrFR2, to distinguish T. cruzi from T. rangeli in mixed infections of vectors based on amplification of the histone H2A/SIRE and the small nucleolar RNA Cl1 genes, respectively. Both PCRs were able to appropriately detect all T. cruzi or T. rangeli experimentally infected-triatomines, as well as the S35/S36 PCR which amplifies the variable region of minicircle kDNA of T. cruzi. In mixed infections, whereas T. cruzi DNA was amplified in 100 percent of samples with TcH2AF/R and S35/S36 PCRs, T. rangeli was detected in 71 percent with TrF/R2 and in 6 percent with S35/S36. In a group of Rhodnius colombiensis collected from Coyaima (Colombia), T. cruzi was identified in 100 percent with both PCRs and T. rangeli in 14 percent with TrF/R2 and 10 percent with S35/S36 PCR. These results show that TcH2AF/R and TrF/R2 PCRs which are capable of recognizing all T. cruzi and T. rangeli strains and lineages could be useful for diagnosis as well as for epidemiological field studies of T. cruzi and T. rangeli vector infections.

Embora o Trypanosoma rangeli não seja patogênico para o homem, sua importância médica e epidemiológica reside no fato de compartilhar vetores, reservatórios e áreas geográficas com o Trypanosoma cruzi, agente causal da Doença de Chagas. Neste estudo, para distinguir T. cruzi de T. rangeli em vetores com infecções mistas, se utilizaram duas amplificações de PCR; TcH2AF/R para o gen da histona H2A/SIRE e TrFR2, para um gen repetitivo de ARN nucleolar Cl1 (sno-RNA-Cl1). Assim como a PCR S35/S36, ambas as reações foram capazes de detectar corretamente a presença de T. cruzi ou T. rangeli em triatomíneos infectados experimentalmente. Nas infecções mistas, o ADN de T. cruzi foi amplificado em 100 por cento das amostras quando se utilizaram TcH2AF/R e S35/S36, enquanto T. rangeli foi detectado em 71 por cento delas com os iniciadores TrF/R2 e em 6 por cento, com S35/S36. Adicionalmente, em um grupo de Rhodnius colombiensis coletados na região de Coyaima (Tolima), T. cruzi foi identificado em 100 por cento com ambas PCRs e T. rangeli em 14 por cento delas com os iniciadores TrF/R2 e em 10 por cento, com S35/S36. Estes resultados mostram que as reações de PCR TcH2AF/R e TrF/R2, capazes de reconhecer todas as cepas e linhagens de T. cruzi e T. rangeli, podem ser úteis no diagnóstico e também nos estudos epidemiológicos do campo com vetores infectados pelo T. cruzi e T. rangeli.