Inactive trans-sialidase expression in iTS-null Trypanosoma cruzi generates virulent trypomastigotes

Disclosing virulence factors from pathogens is required to better understand the pathogenic mechanisms involved in their interaction with the host. In the case of Trypanosoma cruzi several molecules are associated with virulence. Among them, the trans-sialidase (TS) has arisen as one of particular relevance due to its effect on the immune system and involvement in the interaction/invasion of the host cells. The presence of conserved genes encoding for an inactive TS (iTS) isoform is puzzlingly restricted to the genome of parasites from the Discrete Typing Units TcII, TcV, and TcVI, which include highly virulent strains. Previous in vitro results using recombinant iTS support that this isoform could play a different or complementary pathogenic role to that of the enzymatically active protein. However, direct evidence involving iTS in in vivo pathogenesis and invasion is still lacking. Here we faced this challenge by transfecting iTS-null parasites with a recombinant gene that allowed us to follow its expression and association with pathological events. We found that iTS expression improves parasite invasion of host cells and increases their in vivo virulence for mice as shown by histopathologic findings in heart and skeletal muscle.Fil: Pascuale, Carla Antonela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Burgos, Juan Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Postan, Miriam. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; ArgentinaFil: Lantos, Andrés Bernardo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Bertelli, Adriano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Campetella, Oscar Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Leguizamon, Maria Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

Immunomodulatory and anti-fibrotic effects of ganglioside therapy on the cardiac chronic form of experimental Trypanosoma cruzi infection

Heart failure and sudden death are the most common causes of death in patients with Chagas' disease. The main drug available for Chagas treatment is benznidazole, which eradicates Trypanosoma cruzi parasites during the acute stage of infection. However, its effectiveness during the chronic phase remains unclear. Ganglioside GM1 administration in chronically infected patients resulted to be an effective treatment for the cardiac manifestations of Chagas' disease. However, the precise mechanisms of GM1-induced improvement during chronic T. cruzi infection still remain unknown. The aim of the present study was to evaluate the potential benefits of ganglioside GM1 treatment during the chronic stage of murine chagasic infection, analyzing its influence on myocardial pathology as well as its immunomodulatory effects. The results obtained showed that GM1 therapy diminished the extent of myocardial fibrosis induced by T. cruzi in chronically infected mice. In addition, GM1 treatment resulted in a significant reduction in the myocardial expression of the fibrogenic cytokine TGF-β as well as the proinflammatory cytokines and chemokines IFN-γ, TNF-α and CCL5/RANTES. Our experimental data indicate that GM1 could be a promising mmunomodulatory agent with capacity to limit the inflammatory process leading to myocardial tissue damage in chronic chagasic patients.Fil: Cutrullis, Romina Andrea. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Parasitología y Chagas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Poklépovich Caride, Tomás Javier. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Parasitología y Chagas; ArgentinaFil: Postan, Miriam. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Parasitología y Chagas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Freilij, Hector León. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Parasitología y Chagas; ArgentinaFil: Petray, Patricia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Parasitología y Chagas; Argentin

Circulating T Follicular Helper Cell Abnormalities Associated to Different Clinical Forms of Chronic Chagas Disease

Multiple perturbations of the immune response affecting a range of cells have been reported in Trypanosoma cruzi-infected individuals and associated to clinical manifestations of chronic Chagas disease. There is a paucity of knowledge about the role of T follicular helper (Tfh) cells in this infection. Here, we sought to characterize circulating Tfh (cTfh) cells in chronic Chagas disease patients and to identify potential associations with disease severity in humans. cTfh cells were characterized by flow cytometry in freshly isolated PBMCs from 7 T. cruzi-infected asymptomatic patients (ASYMP), 5 patients with chronic chagasic dilated cardiomyopathy (CCC) and 8 healthy controls, using antibodies against chemokine receptors CXCR5, CXCR3, CCR6, and CCR7. Our results showed significant expansion of CD4+CD45RO+CXCR5+CCR6+ cells in ASYMP and CCC patients, along with a contraction of CD4+CD45RO+CXCR5+CXCR3-CCR6- (cTfh2) cells. ASYMP patients further exhibited decreased CD4+CD45RO+CXCR5+CXCR3+CCR6- (cTfh1) cells and expanded CD4+CD45RO+CXCR5+CXCR3-CCR6+ (cTfh17) cells while CCC patients exhibited significantly increased frequencies of CD4+CD45RO+CXCR5+CCR7+ cells. Linear regression analysis revealed a positive trend of CD4+CD45RO+CXCR5+CXCR3+CCR6+ (cTfh1/17) cells and negative trends of cTfh1 and cTfh2 cells as disease was more severe. There was no correlation between the frequencies of cTfh cells and circulating CD19+IgD-IgG+ cells or serum levels of T. cruzi-specific IgG. These results demonstrate that the cTfh compartment of humans chronically infected with T. cruzi comprises expanded CCR6-expressing cells and reduced cTfh2 cells. The association of discrete phenotypic changes in cTfh subsets with different clinical forms suggests the potential contribution of T follicular helper cells to Chagas heart disease progression.Fil: Quebrada Palacio, Luz Piedad. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; ArgentinaFil: Fernández, Esteban Rodrigo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; ArgentinaFil: Hernandez Vasquez, Yolanda Maria. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; ArgentinaFil: Petray, Patricia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Postan, Miriam. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán". Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben"; Argentin

Anti-inflammatory Role of Galectin-8 During Trypanosoma cruzi Chronic Infection

Galectins are animal lectins with high affinity for β-galactosides that drive the immune response through several mechanisms. In particular, the role of galectin-8 (Gal-8) in inflammation remains controversial. To analyze its role in a chronic inflammatory environment, we studied a murine model of Trypanosoma cruzi infection. The parasite induces alterations that lead to the development of chronic cardiomyopathy and/or megaviscera in 30% of infected patients. The strong cardiac inflammation along with fibrosis leads to cardiomyopathy, the most relevant consequence of Chagas disease. By analyzing infected wild-type (iWT) and Gal-8-deficient (iGal-8KO) C57BL/6J mice at the chronic phase (4–5 months post-infection), we observed that the lack of Gal-8 favored a generalized increase in heart, skeletal muscle, and liver inflammation associated with extensive fibrosis, unrelated to tissue parasite loads. Remarkably, increased frequencies of neutrophils and macrophages were observed within cardiac iGal-8KO tissue by flow cytometry. It has been proposed that Gal-8, as well as other galectins, induces the surface expression of the inner molecule phosphatidylserine on activated neutrophils, which serves as an “eat-me” signal for macrophages, favoring viable neutrophil removal and tissue injury protection, a process known as preaparesis. We found that the increased neutrophil rates could be associated with the absence of Gal-8-dependent preaparesis, leading to a diminished neutrophil-clearing capability in macrophages. Thus, our results support that Gal-8 exerts an anti-inflammatory role in chronic T. cruzi infection.Fil: Bertelli, Adriano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Sanmarco, Liliana Maria. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Pascuale, Carla Antonela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Postan, Miriam. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán". Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Aoki, Maria del Pilar. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Leguizamon, Maria Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

CD8(+) T-Cell Responses to Trypanosoma cruzi Are Highly Focused on Strain-Variant trans-Sialidase Epitopes

CD8(+) T cells are crucial for control of a number of medically important protozoan parasites, including Trypanosoma cruzi, the agent of human Chagas disease. Yet, in contrast to the wealth of information from viral and bacterial infections, little is known about the antigen specificity or the general development of effector and memory T-cell responses in hosts infected with protozoans. In this study we report on a wide-scale screen for the dominant parasite peptides recognized by CD8(+) T cells in T. cruzi–infected mice and humans. This analysis demonstrates that in both hosts the CD8(+) T-cell response is highly focused on epitopes encoded by members of the large trans-sialidase family of genes. Responses to a restricted set of immunodominant peptides were especially pronounced in T. cruzi–infected mice, with more than 30% of the CD8(+) T-cell response at the peak of infection specific for two major groups of trans-sialidase peptides. Experimental models also demonstrated that the dominance patterns vary depending on the infective strain of T. cruzi, suggesting that immune evasion may be occurring at a population rather than single-parasite level

High Throughput Selection of Effective Serodiagnostics for Trypanosoma cruzi infection

The diagnosis of Trypanosoma cruzi infection (the cause of human Chagas disease) is difficult because the symptoms of the infection are often absent or non-specific, and because the parasites themselves are usually below the level of detection in the infected subjects. Therefore, diagnosis generally depends on the measurement of T. cruzi–specific antibodies produced in response to the infection. However, current methods to detect anti–T. cruzi antibodies are relatively poor. In this study, we have conducted a broad screen of >400 T. cruzi proteins to identify those proteins which are best able to detect anti–T. cruzi antibodies. Using a set of proteins selected by this screen, we were able to detect 100% of >100 confirmed positive human cases of T. cruzi infection, as well as suspect cases that were negative using existing tests. This protein panel was also able to detect apparent changes in infection status following drug treatment of individuals with chronic T. cruzi infection. The results of this study should allow for significant improvements in the detection of T. cruzi infection and better screening methods to avoid blood transfusion–related transmission of the infection, and offer a crucial tool for determining the success or failure of drug treatment and other intervention strategies to limit the impact of Chagas disease

Myocardial cell response to Trypanosoma cruzi infection

Fil: Postan, Mirian. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Arnaiz, María Rosa. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Fichera, Laura E. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.El objetivo de este estudio es determinar la capacidad de respuesta de la célula muscular cardíaca a la infección por Trypanosoma cruzi. Se investigó el rol de la multiplicación de las células musculares en la remodelación cardíaca, y su capacidad de producción de óxido nítrico y control del parasitismo intracelular. Para ello, se determinó la presencia del antígeno nuclear de proliferación celular (PCNA) en los miocitos cardíacos de ratas Wistar infectadas experimentalmente con T. cruzi, determinándose un aumento significativo del número de núcleos PCNA+ en los animales infectados agudos y crónicos. La capacidad de control del parasitismo intracelular y de producción de óxido nítrico fueron evaluados en cultivos primarios de miocitos cardíacos de ratas neonatas, estimulados con citoquinas in vitro. El análisis del parasitismo mostró que el número de miocitos cardíacos con amastigotes intracelulares fue menor en los cultivos estimulados con IL-1β, TNF-α e IFN-γ. La producción de óxido nítrico fue mayor en los sobrenadantes de los cultivos celulares estimulados con citoquinas, tanto en los infectados como en los controles. Estos datos demuestran que la célula muscular cardíaca participa activamente en la respuesta del organismo durante la infección con T. cruzi. The aim of this study is to establish the response of cardiac myocytes to the infection with Trypanosoma cruzi. The role of myocardial cell proliferation on heart remodelation and the ability of these cells to produce nitric oxide and control intracellular parasite growth during T. cruzi infection were evaluated. The presence of proliferating cell nuclear antigen (PCNA) was determined in myocardial cells of Wistar rats infected with T. cruzi, resulting in a significant increase of PCNA+ labelling in all stages of disease. The ability of myocardial cells to control growth of intracellular parasites and the production of nitric oxide were evaluated in cultures of cardiac myocytes obtained from neonatal rats. Different combinations of cytokines were added to culture media. The number of cardiac cells displaying intracellular amastigotes was lower in cultures supplemented with IL-1β, TNF-α and IFN-γ than with other cytokine combinations and controls. The addition of cytokines resulted also in an increase of nitric oxide production in both infected and non-infected controls. These results demonstrate that myocardial cells participate actively in the response of the heart to the infection with T. cruzi

Cardiac myocyte hypertrophy and proliferating cell nuclear antigen expression in Wistar rats infected with Trypanosoma cruzi

Fil: Arnaiz, María Rosa. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Fichera, Laura Edith. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Postan, Miriam. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Chagasic cardiomyopathy is a major life-threatening complication of Trypanosoma cruzi infection in human beings. This study focuses on the hypertrophic and hyperplastic mechanisms underlying the structural changes of the heart during experimental infection. Proliferating cell nuclear antigen (PCNA) expression, transversal diameter, nuclear area, and number of nuclei per unit volume were determined in the ventricular myocytes of T. cruzi-infected Wistar rats. PCNA expression was enhanced throughout the inflamed myocardium and in the spared areas of the left ventricular wall and the septum. Myocyte width increased from 26 to 75% at the inflammation-free myocardium (P 10-36%, P 0.2-32%, P < 0.03, respectively) and decreased at the inflamed areas of the left ventricular wall (10-22%. P < 0.02) with respect to the controls. The number of nuclei per unit volume decreased at the inflamed myocardium regardless of topographical location (36-65%) with respect to the controls (P < 0.0001) and in the inflammation-free myocardium of the right ventricle and the septum (<21-37%, P < 0.002 and <8-39%, P < 0.002, respectively). These results show that the heart responds to T. cruzi infection with DNA repair and cell multiplication in the inflamed sites and with hypertrophy of the unaffected myocardium

Myocardial cell response to Trypanosoma cruzi infection

Fil: Postan, Mirian. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Arnaiz, María Rosa. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Fichera, Laura E. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.El objetivo de este estudio es determinar la capacidad de respuesta de la célula muscular cardíaca a la infección por Trypanosoma cruzi. Se investigó el rol de la multiplicación de las células musculares en la remodelación cardíaca, y su capacidad de producción de óxido nítrico y control del parasitismo intracelular. Para ello, se determinó la presencia del antígeno nuclear de proliferación celular (PCNA) en los miocitos cardíacos de ratas Wistar infectadas experimentalmente con T. cruzi, determinándose un aumento significativo del número de núcleos PCNA+ en los animales infectados agudos y crónicos. La capacidad de control del parasitismo intracelular y de producción de óxido nítrico fueron evaluados en cultivos primarios de miocitos cardíacos de ratas neonatas, estimulados con citoquinas in vitro. El análisis del parasitismo mostró que el número de miocitos cardíacos con amastigotes intracelulares fue menor en los cultivos estimulados con IL-1β, TNF-α e IFN-γ. La producción de óxido nítrico fue mayor en los sobrenadantes de los cultivos celulares estimulados con citoquinas, tanto en los infectados como en los controles. Estos datos demuestran que la célula muscular cardíaca participa activamente en la respuesta del organismo durante la infección con T. cruzi. The aim of this study is to establish the response of cardiac myocytes to the infection with Trypanosoma cruzi. The role of myocardial cell proliferation on heart remodelation and the ability of these cells to produce nitric oxide and control intracellular parasite growth during T. cruzi infection were evaluated. The presence of proliferating cell nuclear antigen (PCNA) was determined in myocardial cells of Wistar rats infected with T. cruzi, resulting in a significant increase of PCNA+ labelling in all stages of disease. The ability of myocardial cells to control growth of intracellular parasites and the production of nitric oxide were evaluated in cultures of cardiac myocytes obtained from neonatal rats. Different combinations of cytokines were added to culture media. The number of cardiac cells displaying intracellular amastigotes was lower in cultures supplemented with IL-1β, TNF-α and IFN-γ than with other cytokine combinations and controls. The addition of cytokines resulted also in an increase of nitric oxide production in both infected and non-infected controls. These results demonstrate that myocardial cells participate actively in the response of the heart to the infection with T. cruzi

Effects of artesunate against Trypanosma cruzi

Therapy against Trypanosma cruzi relies on only two chemically relatednitro-derivative drugs, benznidazole and nifurtimox, both limited by poorefficacy and toxicity. It is suspected that with prolonged usage of these drugs, resistant parasites will be selected, which results in risk for treatment failureover the time. Herein, we studied the in vitro activity of artesunate, the mosteffective drug to treat severe P. falciparum and chloroquine-resistant P. vivax, on three strains of T. cruzi originated in different regions of Latin America(Argentina, Nicaragua and Brazil). The results of these assays showed thatartesunate inhibits multiplication of epimastigotes (IC50 = 50, 6.10 and 23 µM,respectively) and intracellular amastigotes (IC50 = 15, 0.12 and 6.90 µM,respectively), indicating that it represents a potent anti-T. cruzi compound interms of inhibiting parasite multiplication in vitro. We then tested the effectof artesunate in Balb/c mice infected with Brazil strain and found that it failedto cure the infection, suggesting that the drug may be unsuitable for in vivotreatment. When infected mice were treated with high doses AS + BZ, the outcomeof infection was similar to that observed in mice treated with BZ alone.Nevertheless, understanding of structure-activity relationship of artesunatemight lead to the development of new and effective drugs against T. cruzi.Fil: Olivera, Gabriela Carina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Postan, Miriam. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: González, Mariela Natacha. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin