IL-10 participates in the expansion and functional activation of CD8+ T cells during acute infection with Trypanosoma cruzi.

IL-10 is a pleiotropic cytokine with immunoregulatory functions affecting various cell types. In a model of experimental infection with the protozoan Trypanosoma cruzi (T. cruzi), we found increased morbidity and lower parasite control in IL-10 deficient mice (IL-10 KO) compared to wild-type (WT) mice. Despite enhanced Mϕ function and dendritic cell activation, IL-10 KO mice were more susceptible to infection. The kinetics of T cells in spleen and peripheral blood revealed that infected IL-10 KO mice failed to increase the number of spleen and circulating total CD8+ T cells, a phenomenon observed from the second week of infection in WT mice. Total CD8+ T cells from IL-10 KO mice exhibited diminished proliferation, cytotoxic potential and IFN-γ production than their WT counterparts and T. cruzi-specific CD8+ T cells displayed reduced in vivo cytotoxicity. The absence of IL-10 selectively affected expansion, survival, and increased PD-1 expression of CD8+ T cells without altering these same parameters on CD4+ T cells. Increased inhibitory receptors expression and down-modulation of T-bet by CD8+ T cells from IL-10 KO infected mice were compatible with a T cell exhaustion phenotype. Collectively, these findings reveal that during acute infection, IL-10 plays a previously unrecognized stimulatory role on CD8+ T cells, the most relevant lymphocyte population for the control of intracellular T. cruzi stages. A clear knowledge of the underlying mechanisms that drive effector functions of cytotoxic T cells is critical to understand pathogen persistence and rational design of prophylactic strategies against T. cruzi.

Strongyloidiasis Outside Endemic Areas: Long-term Parasitological and Clinical Follow-up After Ivermectin Treatment.

Background: Strongyloides stercoralis affects 30-100 million people worldwide. The first-line therapy is ivermectin. Cure is defined as the absence of larvae by parasitological methods 1 year after treatment. To date, no longitudinal parasitological studies for longer periods of time have been conducted to confirm its cure. Here, we evaluated treatment response in long-term follow-up patients with chronic infection using parasitological and molecular methods for larvae or DNA detection. Methods: A prospective, descriptive, observational study was conducted between January 2009 and September 2015 in Buenos Aires, Argentina. Twenty-one patients with S. stercoralis diagnosis were evaluated 30, 60, and 90 days as well as 1, 2, 3, and/or 4 years after treatment by conventional methods (fresh stool, Ritchie method, agar plate culture), S. stercoralis-specific polymerase chain reaction (PCR) in stool DNA, and eosinophil values. Results: During follow-up, larvae were detected by conventional methods in 14 of 21 patients. This parasitological reactivation was observed starting 30 days posttreatment (dpt) and then at different times since 90 dpt. Eosinophil values decreased (P = .001) 30 days after treatment, but their levels were neither associated with nor predicted these reactivations. However, S. stercoralis DNA was detected by PCR in all patients, both in their first and subsequent stool samples, thus reflecting the poor efficacy of ivermectin at eradicating parasite from host tissues. Asymptomatic eosinophilia was the most frequent clinical form among chronically infected patients. Conclusions: These results suggest that the parasitological cure is unlikely. Strongyloidiasis must be considered a chronic infection and ivermectin administration schedules should be reevaluated.

Dual role of monocyte-derived dendritic cells in Trypanosoma cruzi infection.

Pathogens can cause inflammation when inoculated into the skin. The vector-transmitted protozoan parasite Trypanosoma cruzi induces poor cellular-infiltration and disseminates, causing high mortality in the experimental model. Here, we characterized the inflammatory foci at the parasite inoculation site and secondary lymphoid organs using a murine model. While no macrophages and few neutrophils and monocytes (Mo) were recruited into the skin, T. cruzi infection elicited the mobilization of Ly6C+ Mo to draining lymph nodes and spleen. Over time, this population became enriched in CD11b+ Ly6C+ CD11c+ MHCII+ CD86+ cells resembling inflammatory dendritic cells (DCs). Adoptive transfer of Ly6C+ Mo purified from the bone marrow of CD11c-GFP transgenic mice confirmed the monocytic origin of Ly6C+ DCs found in the spleen of infected animals. Isolated Mo-derived cells not only produced TNF-α and nitric oxide, but also IL-10 and displayed a poor capacity to induce lymphoproliferation. Ablation of Mo-derived cells by 5-fluorouracil confirmed their dual role during infection, limiting the parasite load by inducible nitric oxide synthase-related mechanisms and negatively affecting the development of anti-parasite T-cell response. This study demonstrated that consistent with their antagonistic properties, these cells not only control the parasite spreading but also its persistence in the host.

Trypanosoma cruzi Infection Imparts a Regulatory Program in Dendritic Cells and T Cells via Galectin-1-Dependent Mechanisms.

Galectin-1 (Gal-1), an endogenous glycan-binding protein, is widely distributed at sites of inflammation and microbial invasion. Despite considerable progress regarding the immunoregulatory activity of this lectin, the role of endogenous Gal-1 during acute parasite infections is uncertain. In this study, we show that Gal-1 functions as a negative regulator to limit host-protective immunity following intradermal infection with Trypanosoma cruzi. Concomitant with the upregulation of immune inhibitory mediators, including IL-10, TGF-ß1, IDO, and programmed death ligand 2, T. cruzi infection induced an early increase of Gal-1 expression in vivo. Compared to their wild-type (WT) counterpart, Gal-1-deficient (Lgals1(-/-)) mice exhibited reduced mortality and lower parasite load in muscle tissue. Resistance of Lgals1(-/-) mice to T. cruzi infection was associated with a failure in the activation of Gal-1-driven tolerogenic circuits, otherwise orchestrated by WT dendritic cells, leading to secondary dysfunction in the induction of CD4(+)CD25(+)Foxp3(+) regulatory T cells. This effect was accompanied by an increased number of CD8(+) T cells and higher frequency of IFN-γ-producing CD4(+) T cells in muscle tissues and draining lymph nodes as well as reduced parasite burden in heart and hindlimb skeletal muscle. Moreover, dendritic cells lacking Gal-1 interrupted the Gal-1-mediated tolerogenic circuit and reinforced T cell-dependent anti-parasite immunity when adoptively transferred into WT mice. Thus, endogenous Gal-1 may influence T. cruzi infection by fueling tolerogenic circuits that hinder anti-parasite immunity.

Impairment in natural killer cells editing of immature dendritic cells by infection with a virulent Trypanosoma cruzi population.

Early interactions between natural killer (NK) and dendritic cells (DC) shape the immune response at the frontier of innate and adaptive immunity. Activated NK cells participate in maturation or deletion of DCs that remain immature. We previously demonstrated that infection with a high virulence (HV) population of the protozoan parasite Trypanosoma cruzi downmodulates DC maturation and T-cell activation capacity. Here, we evaluated the role of NK cells in regulating the maturation level of DCs. Shortly after infection with HV T. cruzi, DCs in poor maturation status begin to accumulate in mouse spleen. Although infection induces NK cell cytotoxicity and cytokine production, NK cells from mice infected with HV T. cruzi exhibit reduced ability to lyse and fail to induce maturation of bone marrow-derived immature DCs (iDCs). NK-mediated lysis of iDCs is restored by in vitro blockade of the IL-10 receptor during NK-DC interaction or when NK cells are obtained from T. cruzi-infected IL-10 knockout mice. These results suggest that infection with a virulent T. cruzi strain alters NK cell-mediated regulation of the adaptive immune response induced by DCs. This regulatory circuit where IL-10 appears to participate might lead to parasite persistence but can also limit the induction of a vigorous tissue-damaging T-cell response.

High rate of strongyloidosis infection, out of endemic area, in patients with eosinophilia and without risk of exogenous reinfections.

Strongyloides stercoralis chronic infections are usually asymptomatic and underestimated. We used direct fresh stool examination, Ritchie's method, and agar plate culture for diagnosis in patients with eosinophilia and previous residence in endemic areas. The frequency of strongyloidosis detected among these patients was high: 21 of 42 were positive. Among them, 10 were positive only by agar plate culture. After ivermectin treatment, patients resulted negative for parasitological tests and reduced their eosinophil counts. Half of the submitted patients that were followed 4-12 months after treatment remained negative without eosinophilia, except one who showed an eosinophil ascending curve before reappearance of larvae in stools. The high frequency of strongyloidosis found in this group emphasizes the relevance of including this parasitosis among differential diagnosis in patients with eosinophilia and past risk of S. stercoralis infection to prevent disseminated infections secondary to corticoid therapy.

Trypanosoma cruzi: Immunological predictors of benznidazole efficacy during experimental infection.

C3H/HeN male mice were infected with a lethal population of Trypanosoma cruzi and treated with benznidazole (Bz). Parasitemia, body weight and survival rate were registered during the therapy with significant improvement for T. cruzi-infected Bz-treated animals. Besides, flow cytometry resulted a useful method to discriminate between cured animals from those not cured by monitoring IgG(1) bound to live trypomastigotes levels. At the end of Bz therapy, the LT splenocyte compartment was studied for activation/memory cell surface markers (CD(69)(+) and CD(44)(+)). Cytofluorometric analysis showed that T. cruzi-infected untreated mice increased their activated LT numbers and this effect was completely abolished only in cured mice at the end of Bz administration. The same behavior was observed for the memory LT subpopulation correlating to an effector memory (CD(62L)(-)) displayed by T. cruzi infection. Bz treatment was able to modulate the immunological response by reducing the deleterious effect of the acute phase in all T. cruzi-infected mice.

Identification of novel vaccine candidates for Chagas' disease by immunization with sequential fractions of a trypomastigote cDNA expression library.

The protozoan Trypanosoma cruzi is the etiological agent of Chagas' disease, a major chronic infection in Latin America. Currently, there are neither effective drugs nor vaccines for the treatment or prevention of the disease. Several T. cruzi surface antigens are being tested as vaccines but none of them proved to be completely protective, probably because they represent only a limited repertoire of all the possible T. cruzi target molecules. Taking into account that the trypomastigote stage of the parasite must express genes that allow the parasite to disseminate into the tissues and invade cells, we reasoned that genes preferentially expressed in trypomastigotes represent potential targets for immunization. Here we screened an epimastigote-subtracted trypomastigote cDNA expression library by genetic immunization, in order to find new vaccine candidates for Chagas' disease. After two rounds of immunization and challenge with trypomastigotes, this approach led to the identification of a pool of 28 gene fragments that improved in vivo protection. Sequence analysis of these putative candidates revealed that 19 out of 28 (67.85%) of the genes were hypothetical proteins or unannotated T. cruzi open reading frames, which certainly would not have been identified by other methods of vaccine discovery.

Clusters of hantavirus infection, southern Argentina.

Person-to-person transmission of a hantavirus was first confirmed during a 1996 outbreak of hantavirus pulmonary syndrome in southern Argentina, where Andes virus is endemic. To identify other episodes of secondary transmission, we reviewed reports of 51 cases of hantavirus infection from this region (November 1993-June 2005). Nine clusters involving 20 cases (39.2%) were found. Two patients, who had symptoms 3 weeks after they shared risks for rodent exposure, were considered a cluster. The other 8 clusters each began with an index case, which was almost always fatal, followed 19-40 days later by the illness of at least 1 person who had close and prolonged contact with the index case-patient. Person-to-person transmission was considered the probable source of these 8 clusters. The probability of initiating secondary cases was 41% for patients who died versus 4% for those who survived (p = 0.005). Interpersonal transmission of Andes virus infection should be considered even when rodent exposure cannot be definitively excluded.

Differential expression of a virulence factor, the trans-sialidase, by the main Trypanosoma cruzi phylogenetic lineages.

The clinical outcome of Chagas disease is highly variable, mainly because of the heterogeneity of Trypanosoma cruzi, a parasite for which 2 major phylogenetic groups (I and II) were recently defined. Epidemiological and immunological data indicate that the prevalence of T. cruzi II in patients living in the southern cone of South America correlates with the alterations caused by Chagas disease. We report here that infection with T. cruzi II isolates induces 100% mortality in mice, in contrast to infection with T. cruzi I isolates, in which almost all mice enter the chronic phase even when a 1000-fold higher inoculum is administered. Trypomastigotes from T. cruzi II strains express and shed significantly higher amounts of trans-sialidase than do those from the T. cruzi I lineage. Disorganization of the thymus histoarchitecture associated with the circulating enzyme was observed after infection with T. cruzi II strains, in contrast to transient thymus lesions found in mice infected with T. cruzi I strains. Therefore, trans-sialidase becomes the first T. cruzi virulence factor identified that is differentially expressed by the main parasite groups and that contributes to their contrasting behaviors.

Trypanosoma cruzi infection modulates in vivo expression of major histocompatibility complex class II molecules on antigen-presenting cells and T-cell stimulatory activity of dendritic cells in a strain-dependent manner.

A striking feature of Chagas' disease is the diversity of clinical presentations. Such variability may be due to the heterogeneity among Trypanosoma cruzi isolates or to the host immune response. Employing two strains which differ in their virulence, we investigated the effect of in vivo infection on professional antigen-presenting cells (APC). Acute infection with the virulent RA strain downregulated the expression of major histocompatibility complex (MHC) class II on splenic dendritic cells (DC) and inhibited its induction on peritoneal macrophages and splenic B cells. It also impaired the ability of DC to prime allogeneic T cells and to form homotypic clusters, suggesting a low maturation state of these cells. In contrast, the low-virulence K98 strain maintained the expression of MHC class II on DC or stimulated it on peritoneal macrophages and B cells and preserved DC's T-cell priming capacity and homotypic clustering. DC from RA-infected mice elicited a lower activation of T. cruzi-specific T-cell proliferation than those from K98-infected mice. APC from RA-infected mice that reached the chronic phase of infection restored MHC class II levels to those found in K98-infected mice and upregulated costimulatory molecules expression, suggesting that the immunosuppression caused by this strain is only transient. Taken together, the results indicate that in vivo infection with T. cruzi modulates APC functionality and that this is accomplished in a strain-dependent manner.

Immunogenicity of Trypanosoma cruzi strains determined by neutralization test

Los tripomastigotes (trip) de la cepa CA-I de Trypanosoma Cruzi (cepa escasamente letal para el ratón) presentaron anticuerpos adheridos a sus antígenos de membrana cuando se los obtuvo a los 25-28 días pi y se los procesó a 4-C; a 37-C se negativizaron dentro de los 10-15 min. Estos trip liberados de los complejos antígeno-anticuerpo demostraron ser um blanco adecuado para la detección de anticuerpos neutralizantes; es así como trip - CA-I preincubados con un suero anti-RA de conejo (en el que se había demostrado la existencia de anticuerpos neutralizantes contra la cepa homóloga) indujeron en el ratón un pico de parasitemia muy bajo, de breve duración y aparición tardía. Cuando estos trip se preincubaron con un antisuero homólogo el efecto fue menos marcado produciéndose sólo una demora en la curva de parasitemia para alcanzar a partir de los 34 días pi valores que no diferían significativamente de los registrados en el grupo control . Cuando estos sueros se evaluaron empleando como célula blanco trip-RA y la técnica estandar de neutralización, no se registró reactividad en los sueros anti - CA-I (promedio de sobrevida de ratones inoculados con trip-RA preincubados con suero de conejo: a) normal: 13 ñ 1 días, b anti-CA-I: 14 ñ 1 yc) anti-RA: 20 ñ 1). Para la cepa RA se demostró además que la potencia de la capacidad neutralizante de un suero estaba en función del tamaño de la dosis infectante que se había empleado para producir-lo; estos estudios se realizaron con inmunosueros producidos en ratón, y si bien todos los inmunosueros ensayados fueron reactivos, aquéllos preparados con inóculos de hasta 10**2 trip fueron los más potentes...

Immunogenicity of Trypanosoma cruzi strains determined by neutralization test

Los tripomastigotes (trip) de la cepa CA-I de Trypanosoma Cruzi (cepa escasamente letal para el ratón) presentaron anticuerpos adheridos a sus antígenos de membrana cuando se los obtuvo a los 25-28 días pi y se los procesó a 4-C; a 37-C se negativizaron dentro de los 10-15 min. Estos trip liberados de los complejos antígeno-anticuerpo demostraron ser um blanco adecuado para la detección de anticuerpos neutralizantes; es así como trip - CA-I preincubados con un suero anti-RA de conejo (en el que se había demostrado la existencia de anticuerpos neutralizantes contra la cepa homóloga) indujeron en el ratón un pico de parasitemia muy bajo, de breve duración y aparición tardía. Cuando estos trip se preincubaron con un antisuero homólogo el efecto fue menos marcado produciéndose sólo una demora en la curva de parasitemia para alcanzar a partir de los 34 días pi valores que no diferían significativamente de los registrados en el grupo control . Cuando estos sueros se evaluaron empleando como célula blanco trip-RA y la técnica estandar de neutralización, no se registró reactividad en los sueros anti - CA-I (promedio de sobrevida de ratones inoculados con trip-RA preincubados con suero de conejo: a) normal: 13 ñ 1 días, b anti-CA-I: 14 ñ 1 yc) anti-RA: 20 ñ 1). Para la cepa RA se demostró además que la potencia de la capacidad neutralizante de un suero estaba en función del tamaño de la dosis infectante que se había empleado para producir-lo; estos estudios se realizaron con inmunosueros producidos en ratón, y si bien todos los inmunosueros ensayados fueron reactivos, aquéllos preparados con inóculos de hasta 10**2 trip fueron los más potentes...(AU)

Isolation by Percoll gradient centrifugation and radiolabeling of bloodstream forms of Trypanosoma cruzi

Se diseñó un método para purificar las formas sanguíneas de T. cruzi utilizando un gradiente de Percoll contínuo de 6 cm de longitud (Percoll 60% en "HSA-buffer", preformado por centrifugación a 20.000 g durante 90 min a 4-C en un rotor Sorvall SS 34). El mismo se sembró con 6 x 10**7 elementos (parásitos semipurificados de glóbulos rojos por centrifugación diferencial a partir de sangre desfibrinada) y se centrifugó a 800 g, 15 min. Los parásitos se ubicaron en 1,063 a 1,064 g/ml, existiendo una buena resolución entre ellos y los elementos figurados de la sangre. Los parásitos purificados incorporaron H-uridina y dieron valores del 23% precipitables por TCA. La viabilidad de los mismos fue totalmente preservada según lo evidenciado en los ensayos de infectividad "in vivo"

Isolation by Percoll gradient centrifugation and radiolabeling of bloodstream forms of Trypanosoma cruzi

Se diseñó un método para purificar las formas sanguíneas de T. cruzi utilizando un gradiente de Percoll contínuo de 6 cm de longitud (Percoll 60% en "HSA-buffer", preformado por centrifugación a 20.000 g durante 90 min a 4-C en un rotor Sorvall SS 34). El mismo se sembró con 6 x 10**7 elementos (parásitos semipurificados de glóbulos rojos por centrifugación diferencial a partir de sangre desfibrinada) y se centrifugó a 800 g, 15 min. Los parásitos se ubicaron en 1,063 a 1,064 g/ml, existiendo una buena resolución entre ellos y los elementos figurados de la sangre. Los parásitos purificados incorporaron H-uridina y dieron valores del 23% precipitables por TCA. La viabilidad de los mismos fue totalmente preservada según lo evidenciado en los ensayos de infectividad "in vivo" (AU)

Trypanossoma cruzi: diferencias en el requerimiento de protectores proteicos presentadas por dos cepas argentinas / Trypanosoma cruzi: differences in the requiriment of proteic protectors presented by 2 Argentinian strains

Se estandarizaron los requerimientos de protectores proteícos para los tripomastigotes de las cepas CA-I y RA de Trypanosoma cruzi. Mientras que para la primera fue necesario 5% de suero o albúmina en las soluciones de lavado y/o de suspensión, para la RA se necesitó 1%. La viabilidad de los parásitos fue establecida por la movilidad evidenciada en las preparaciones microscópicas frescas y por inoculación al ratón. Esta diferencia señala una característica más acerca del comportamiento distinto comunicado para la cepa CA-I con relación a la RA

Citocromos en diferentes estadíos, cepas y poblaciones de Trypanosoma cruzi / Cytochromes in different stages, strains and populations of Trypanosoma cruzi

Los espectros diferenciales de muestras "reducida menos oxidada" (Red-Ox) y "reducida. CO menos reducida" (Re. CO-Red) de tripomastigotes metacíclicos del Trypanosoma cruzi (cepa Tulahuen) revelaron la presencia de los citocrómos aa3, b, c555 y o. Espectros concordantes se obtuvieron con la fracción mitocondrial de la forma epimastigote de la misma cepa, después de filtrar las membranas por Sephadex G-50 para excluir hemoproteínas espúreas provenientes del medio de cultivo o hemoproteínas solubles (hemoglobina, peroxidasa, catalasa, etc.), o citocrómos P-420 y P-450. El tratamiento de la fracción mitocondrial cruda con guanidina y colato de Na y la formación de complejos con cianuro, revelaron que el citocrómo o es un constituyente integral de esas membranas. El análisis de los piridinahemocrómos provenientes de los citocrómos mitocondriales demostró la presencia de los hemos A, B y C. Una banda espectral a 625 nm en los espectros de los tripomastigotes, en las membranas mitocondriales, y en los piridina-hemocrómos indicó la presencia de un citocrómo d. Un examen comparativo del contenido en citocrómos o y b de una serie de poblaciones de T. cruzi, provenientes originariamente en su mayoría de pacientes con formas agudas o crónicas de la enfermedad de Chagas, demostró variabilidad en el contenido en citocrómos y falta de correlación de este parámetro con las propiedades biológicas del parásito, en particular con la letalidad para el ratón

Trypanossoma cruzi: diferencias en el requerimiento de protectores proteicos presentadas por dos cepas argentinas / Trypanosoma cruzi: differences in the requiriment of proteic protectors presented by 2 Argentinian strains

Se estandarizaron los requerimientos de protectores proteícos para los tripomastigotes de las cepas CA-I y RA de Trypanosoma cruzi. Mientras que para la primera fue necesario 5% de suero o albúmina en las soluciones de lavado y/o de suspensión, para la RA se necesitó 1%. La viabilidad de los parásitos fue establecida por la movilidad evidenciada en las preparaciones microscópicas frescas y por inoculación al ratón. Esta diferencia señala una característica más acerca del comportamiento distinto comunicado para la cepa CA-I con relación a la RA (AU)