Molecular typing of Strongyloides stercoralis in Latin America, the clinical connection.

This study analysed Strongyloides stercoralis genetic variability based on a 404 bp region of the cox1 gene from Latin-American samples in a clinical context including epidemiological, diagnosis and follow-up variables. A prospective, descriptive, observational study was conducted to evaluate clinical and parasitological evolution after ivermectin treatment of 41 patients infected with S. stercoralis. Reactivation of the disease was defined both by clinical symptoms appearance and/or direct larvae detection 30 days after treatment or later. We described 10 haplotypes organized in two clusters. Most frequent variants were also described in the Asian continent in human (HP24 and HP93) and canine (HP24) samples. Clinical presentation (intestinal, severe, cutaneous and asymptomatic), immunological status and eosinophil count were not associated with specific haplotypes or clusters. Nevertheless, presence of cluster 1 haplotypes during diagnosis increased the risk of reactivation with an odds ratio (OR) of 7.51 [confidence interval (CI) 95% 1.38­44.29, P = 0.026]. In contrast, reactivation probability was 83 times lower if cluster 2 (I152V mutation) was detected (OR = 0.17, CI 95% 0.02­0.80, P = 0.02). This is the first analysis of S. stercoralis cox1 diversity in the clinical context. Determination of clusters during the diagnosis could facilitate and improve the design of follow-up strategies to prevent severe reactivations of this chronic disease.

Early Immune Response Elicited by Different Trypanosoma cruzi Infective Stages.

Trypanosoma cruzi is a protozoan parasite that affects millions of people in Latin America. Infection occurs by vectorial transmission or by transfusion or transplacental route. Immune events occurring immediately after the parasite entrance are poorly explored. Dendritic cells (DCs) are target for the parasite immune evasion mechanisms. Recently, we have demonstrated that two different populations of DCs display variable activation after interaction with the two infective forms of the parasite: metacyclic or blood trypomastigotes (mTp or bTp) in vitro. The skin constitutes a complex network with several populations of antigen-presenting cells. Previously, we have demonstrated T. cruzi conditioning the repertoire of cells recruited into the site of infection. In the present work, we observed that mTp and bTp inoculation displayed differences in cell recruitment to the site of infection and in the activation status of APCs in draining lymph nodes and spleen during acute infection. Animals inoculated with mTp exhibited 100% of survival with no detectable parasitemia, in contrast with those injected with bTp that displayed high mortality and high parasite load. Animals infected with mTp and challenged with a lethal dose of bTp 15 days after primary infection showed no mortality and incremented DC activation in secondary lymphoid organs compared with controls injected only with bTp or non-infected mice. These animals also displayed a smaller number of amastigote nests in cardiac tissue and more CD8 T cells than mice infected with bTp. All the results suggest that both Tp infective stages induce an unequal immune response since the beginning of the infection.

Modulatory Effect of Trypanosoma cruzi Infective Stages in Different Dendritic Cell Populations in vitro.

Trypanosoma cruzi is a protozoan parasite that infects at least 7 million persons in the world (OMS, 2019). In endemic areas, infection normally occurs by vectorial transmission; however, outside, it normally happens by blood and includes congenital transmission. The persistence of T. cruzi during infection suggests the presence of immune evasion mechanisms and the modulation of the anti-parasite response to a profile incapable of eradicating the parasite. Dendritic cells (DCs) are a heterogeneous population of antigen-presenting cells (APCs) that patrol tissues with a key role in mediating the interface between the innate and adaptive immune response. Previous results from our lab and other groups have demonstrated that T. cruzi modulates the functional properties of DCs, in vitro and in vivo. During vectorial transmission, metacyclic (m) trypomastigotes (Tps) eliminated along with the insect feces reach the mucous membranes or injured skin. When transmission occurs by the hematic route, the parasite stage involved in the infection is the circulating or blood (b) Tp. Here, we studied in vitro the effect of both infective mTp and bTp in two different populations of DCs, bone marrow-derived DCs (BMDCs) and XS106, a cell line derived from epidermal DCs. Results demonstrated that the interaction of both Tps imparts a different effect in the functionality of these two populations of DCs, suggesting that the stage of T. cruzi and DC maturation status could define the immune response from the beginning of the ingress of the parasite, conditioning the course of the infection.

Dendritic cells devoid of IL-10 induce protective immunity against the protozoan parasite Trypanosoma cruzi.

In diverse models of microbial infections, protection is improved by immunization with dendritic cells (DC) loaded with whole pathogen lysate. However, pathogens that modulate DC function as a way to evade immunity may represent a challenge for these vaccination strategies. Thus, DC must be instructed in a particular manner to circumvent this issue and drive an effective immune response. Trypanosoma cruzi or its molecules alter DC function and, as we demonstrated, this phenomenon is associated with the parasite-driven stimulation of IL-10 production by DC. Here, we show that DC from IL-10-deficient mice pulsed in vitro with trypomastigote lysate secreted increased amounts of Th1-related cytokines and stimulated higher allogeneic and antigen-specific lymphocyte responses than their wild-type counterparts. In a model of DC-based immunization, these antigen-pulsed IL-10-deficient DC conferred protection against T. cruzi infection to recipient mice. Efficient immunity was associated with enhanced antigen-specific IFN-gamma production and endogenous DC activation. We illustrate for the first time a DC-based vaccination against T. cruzi and evidence the key role of IL-10 produced by sensitizing DC in inhibiting the induction of protection. These results support the rationale for vaccination strategies that timely suppress the effect of specific cytokines secreted by antigen presenting DC.

Trypanosoma cruzi (Chagas' disease agent) reduces HIV-1 replication in human placenta.

BACKGROUND: Several factors determine the risk of HIV mother-to-child transmission (MTCT), such as coinfections in placentas from HIV-1 positive mothers with other pathogens. Chagas' disease is one of the most endemic zoonoses in Latin America, caused by the protozoan Trypanosoma cruzi. The purpose of the study was to determine whether T. cruzi modifies HIV infection of the placenta at the tissue or cellular level. RESULTS: Simple and double infections were carried out on a placental histoculture system (chorionic villi isolated from term placentas from HIV and Chagas negative mothers) and on the choriocarcinoma BeWo cell line. Trypomastigotes of T. cruzi (VD lethal strain), either purified from mouse blood or from Vero cell cultures, 24 h-supernatants of blood and cellular trypomastigotes, and the VSV-G pseudotyped HIV-1 reporter virus were used for the coinfections. Viral transduction was evaluated by quantification of luciferase activity. Coinfection with whole trypomastigotes, either from mouse blood or from cell cultures, decreased viral pseudotype luciferase activity in placental histocultures. Similar results were obtained from BeWo cells. Supernatants of stimulated histocultures were used for the simultaneous determination of 29 cytokines and chemokines with the Luminex technology. In histocultures infected with trypomastigotes, as well as in coinfected tissues, IL-6, IL-8, IP-10 and MCP-1 production was significantly lower than in controls or HIV-1 transducted tissue. A similar decrease was observed in histocultures treated with 24 h-supernatants of blood trypomastigotes, but not in coinfected tissues. CONCLUSION: Our results demonstrated that the presence of an intracellular pathogen, such as T. cruzi, is able to impair HIV-1 transduction in an in vitro system of human placental histoculture. Direct effects of the parasite on cellular structures as well as on cellular/viral proteins essential for HIV-1 replication might influence viral transduction in this model. Nonetheless, additional mechanisms including modulation of cytokines/chemokines at placental level could not be excluded in the inhibition observed. Further experiments need to be conducted in order to elucidate the mechanism(s) involved in this phenomenon. Therefore, coinfection with T. cruzi may have a deleterious effect on HIV-1 transduction and thus could play an important role in viral outcome at the placental level.

Trypanosoma cruzi induces regulatory dendritic cells in vitro.

A main feature of acute infection with Trypanosoma cruzi is the presence of immunological disorders. A previous study demonstrated that acute infection with the virulent RA strain downregulates the expression of major histocompatibility complex class II (MHC-II) on antigen-presenting cells and impairs the T-cell stimulatory capacity of splenic dendritic cells (DC). In the present work, we assessed the ability of trypomastigotes (Tp) to modulate the differentiation stage and functionality of bone marrow-derived DC in vitro. We observed that the Tp stage of T. cruzi failed to activate DC, which preserved their low expression of MHC-II and costimulatory molecules, as well as their endocytic activity. We also show that Tp induced transforming growth factor beta (TGF-beta) secretion by DC and enhanced the gap between interleukin-10 (IL-10) and IL-12p70 production, showing a higher IL-10/IL-12p70 ratio upon lipopolysaccharide (LPS) treatment. In addition, we observed that Tp prevented DC full activation induced by LPS, thereby downregulating their MHC-II surface expression and inhibiting their capacity to stimulate lymphocyte proliferation. In vitro IL-10 neutralization during the differentiation process of DC with Tp+LPS showed a reversion of their inhibitory effect during mixed lymphocyte reaction. In contrast, only simultaneous neutralization of IL-10 and TGF-beta, after DC differentiation, was involved in the partial restitution of lymphocyte proliferation. Since both TGF-beta and IL-10 are immunosuppressive cytokines essential in the modulation of the immune response and important in the induction of tolerance, our results suggest for the first time that Tp are responsible for the generation of regulatory DC in vitro.

Chagas' disease: TCRBV9 over-representation and sequence oligoclonality in the fine specificity of T lymphocytes in target tissues of damage.

Using the same mouse strain and two Trypanosoma cruzi sub-populations (CA-I and RA) it is possible to induce pathology in different target tissues: skeletal muscle (CA-I) or sciatic nerve and spinal cord (RA). On the other hand, T cells are directly involved in tissue injury in a strain-dependent way, resembling the abnormalities of chronic Chagas' disease. In the present work, we examined the TCRBV repertoire and the CDR3 sequence polymorphism of T cells infiltrating spinal cord, sciatic nerve and skeletal muscle in chronically infected mice. The TCRBV9 segment was systematically over-represented in the target tissues for each T. cruzi strain: sciatic nerve and spinal cord in RA and skeletal muscle in CA-I-infected mice. The analysis of CDR3 sequence polymorphism in the same tissues showed a high proportion of identical TCRBV9 clones in RA-infected mice: 66.6% of the TCRBV9 clones found in sciatic nerve and spinal cord expressed one out of four major CDR3 rearrangements. Sequence identity was shared among clones from sciatic nerve and spinal cord, tissues that are also damaged by passive transfer of CD8 + TL. Those observations are consistent with an antigen driven T-cell expansion sequestered at the inflammation site and demonstrate -- for the first time -- the presence of an oligoclonal repertoire in the antigen recognition site of over-represented T cells in nervous system tissues in chronic Chagas' disease.

The circadian system of Trypanosoma cruzi-infected mice.

The effects of Chagas disease on the mammalian circadian system were studied in Trypanosoma cruzi-infected C57-B16J mice. Animals were inoculated with CAI or RA strains of T. cruzi or vehicle, parasitism confirmed by blood specimen visualization and locomotor activity rhythms analyzed by wheel-running recording. RA-strain infected mice exhibited significantly decreased amplitude of circadian rhythms, both under light-dark and constant dark conditions, probably due to motor deficiencies. CAI-treated animals showed normal locomotor activity rhythms. However, in these mice, reentrainment to a 6h phase shift of the LD cycle took significantly longer than controls, and application of 15min light pulses in DD produced smaller phase delays of the rhythms. All groups exhibited light-induced Fos expression in the suprachiasmatic nuclei. We conclude that the main effect of T. cruzi infection on the circadian system is an impairment of the motor output from the clock toward controlled rhythms, together with an effect on circadian visual sensitivity.

Trypanosoma cruzi: effect of parasite subpopulation on murine pregnancy outcome.

C3H/HeN female mice infected with distinct Trypanosoma cruzi subpopulations (RA strain [pantropic/reticulotropic] and K98 clone of the CA-I strain [myotropic]) show differences both in inflammatory compromise of the genital tract and in the outcome of pregnancy. The group of mice infected with the K98 clone show lymphomononuclear infiltrates in pelvian fat and in uterus interstitium, coexisting with the presence of T. cruzi DNA, and show moderate oophoritis, perioophoritis, and vasculitis. However, neither parasite DNA nor inflammatory foci were detected in the uterus, and only mild oophoritis was observed among RA-infected mice at mating time. Independently from the parasite subpopulation, females developed estrous 30 days postinoculation (PI), and at the same time, parasite counts were similar for K98 and for RA-infected mice. However, fertility was significantly diminished in K98-infected females. On day 14 of gestation, fetal resorptions increased in this group and cannot be attributed to hormonal disbalance because similar serum progesterone levels were found in all groups. At this time (44 days PI), parasitemia was higher in K98- than in RA-infected mice. However, resorptions were not triggered by massive infection because polymerase chain reaction failed to prove parasite DNA in resorbing fetuses. In contrast with K98 females, RA-infected mice delivered T. cruzi-infected newborns.

Characterization of Biomphalaria orbignyi, Biomphalaria peregrina and Biomphalaria oligoza by polymerase chain reaction and restriction enzyme digestion of the internal transcribed spacer region of the RNA ribosomal gene

The correct identification of Biomphalaria oligoza, B. orbignyi and B. peregrina species is difficult due to the morphological similarities among them. B. peregrina is widely distributed in South America and is considered a potential intermediate host of Schistosoma mansoni. We have reported the use of the polymerase chain reaction and restriction fragment length polymorphism analysis of the internal transcribed spacer region of the ribosomal DNA for the molecular identification of these snails. The snails were obtained from different localities of Argentina, Brazil and Uruguay. The restriction patterns obtained with MvaI enzyme presented the best profile to identify the three species. The profiles obtained with all enzymes were used to estimate genetic similarities among B. oligoza, B. peregrina and B. orbignyi. This is also the first report of B. orbignyi in Uruguay

Enfermedad de Chagas y transfusión sanguinea: actividad tripanocida del clorhidrato de maprotilina y del violeta de genciana / Chagas' disease and blood transfusion: trypanocidal activity of maprotiline hydrochloride and gentian violet

Se evaluó por inoculación en ratón la actividad tripanocida del Clorhidrati de Maprotilina comparativamente con la presentada por el Violeta de Genciana cuando estos compuestos se adicionaron a muestras de sangre conteniendo bajas concentraciones de parásitos. Ambas drogas presentaon actividad tripanocida cuando se las utilizó en concentraciónes del orden 10**-3M. Sin embargo, aun empleando esta concentración pudo detectarse esporádicamente infección en alguno de los animales inyectados con muestras de sangre conteniendo 10 o 100 tripomastigotes, despues de haber sido incubadas 24 h a 4-C con uno de ambos compuestos. Debido a la baja solubilidad del Clorhidrato de Maprotilina el presente estudio se realizó con muestras de sangre diluidas al medio siendo imposible evitar esta condición para la concentración 10**-3M de este compuesto. Estos resultados descartan el uso de clorhidrato de Maprotilina en bancos de sangre y previenen sobre la posibilidad eventual de transmitir infección por Trypanosoma cruzi aun con sangre tratada con Violeta de Genciana. De los 3 métodos utilizados para evaluar viabilidad parasitaria remanente en las muestras de sangre químicamente tratadas, el microhematocrito fue el más sensible

Enfermedad de Chagas y transfusión sanguinea: actividad tripanocida del clorhidrato de maprotilina y del violeta de genciana / Chagas disease and blood transfusion: trypanocidal activity of maprotiline hydrochloride and gentian violet

Se evaluó por inoculación en ratón la actividad tripanocida del Clorhidrati de Maprotilina comparativamente con la presentada por el Violeta de Genciana cuando estos compuestos se adicionaron a muestras de sangre conteniendo bajas concentraciones de parásitos. Ambas drogas presentaon actividad tripanocida cuando se las utilizó en concentraciónes del orden 10**-3M. Sin embargo, aun empleando esta concentración pudo detectarse esporádicamente infección en alguno de los animales inyectados con muestras de sangre conteniendo 10 o 100 tripomastigotes, despues de haber sido incubadas 24 h a 4-C con uno de ambos compuestos. Debido a la baja solubilidad del Clorhidrato de Maprotilina el presente estudio se realizó con muestras de sangre diluidas al medio siendo imposible evitar esta condición para la concentración 10**-3M de este compuesto. Estos resultados descartan el uso de clorhidrato de Maprotilina en bancos de sangre y previenen sobre la posibilidad eventual de transmitir infección por Trypanosoma cruzi aun con sangre tratada con Violeta de Genciana. De los 3 métodos utilizados para evaluar viabilidad parasitaria remanente en las muestras de sangre químicamente tratadas, el microhematocrito fue el más sensible (AU)

Caracterización inmunobiologica de una cepa presuntamente atipica de Trypanosoma cruzi / Immunobiological characterization of a presumed atypical straim of Trypanosoma cruzi

Se analisaron para la cepa LP de Trypanosoma cruzi algunas características biológicas e inmunológicas y se las comparó con las presentadas por cepas de referencia, RA y CA-I. La cepa LP produjo niveles de parasistemia próximoss a los inducidos por la RA pero, asemejándose a la cepa CA-I, la elevada parasitemia fue persistente y los ratones sobrevivieron a la infección aguda; sin embargo, alrededor de 40-45 días pi los animales inoculados con LP presentaron una caída en crisis de los parásitos circulantes. Dosis supresoras de infurtimox retardaron la positivación de la parasitemia en los 3 casos y los valores máximos alcanzados fueron una unidad logarítmica menor comparado con los controles sin tratamiento. La respuesta inmune estimulada por la cepa LP en conejo y en ratón fue positiva para las pruebas serológicas convencionales (IFA y AD) al igual que lo ocurrido con las otras 2 cepas. En el caso de la IFA todos los inmunosueros fueron reactivos con cualquiera de los 4 antígenos utilizados (epimastigotes de LP, RA, CA-I y Tulahuén). La cepa LP también estimuló la formación de anticuerpos capaces de interactuar ccon la forma circulante del parásito modificando su virulencia o produciendo su lisis; estos anticuerpos se detectaron tardíamente, lo que podría justificar la prolongada parasitemia presentada por los ratones inoculados con LP. por los resultados aquí obtenidos el comportamiento atípico comunicado previamente para esta cepa dependería de la asociación hospedeiro-parásito más que de características propias de la población parasitaria

Caracterización inmunobiologica de una cepa presuntamente atipica de Trypanosoma cruzi / Immunobiological characterization of a presumed atypical straim of Trypanosoma cruzi

Se analisaron para la cepa LP de Trypanosoma cruzi algunas características biológicas e inmunológicas y se las comparó con las presentadas por cepas de referencia, RA y CA-I. La cepa LP produjo niveles de parasistemia próximoss a los inducidos por la RA pero, asemejándose a la cepa CA-I, la elevada parasitemia fue persistente y los ratones sobrevivieron a la infección aguda; sin embargo, alrededor de 40-45 días pi los animales inoculados con LP presentaron una caída en crisis de los parásitos circulantes. Dosis supresoras de infurtimox retardaron la positivación de la parasitemia en los 3 casos y los valores máximos alcanzados fueron una unidad logarítmica menor comparado con los controles sin tratamiento. La respuesta inmune estimulada por la cepa LP en conejo y en ratón fue positiva para las pruebas serológicas convencionales (IFA y AD) al igual que lo ocurrido con las otras 2 cepas. En el caso de la IFA todos los inmunosueros fueron reactivos con cualquiera de los 4 antígenos utilizados (epimastigotes de LP, RA, CA-I y Tulahuén). La cepa LP también estimuló la formación de anticuerpos capaces de interactuar ccon la forma circulante del parásito modificando su virulencia o produciendo su lisis; estos anticuerpos se detectaron tardíamente, lo que podría justificar la prolongada parasitemia presentada por los ratones inoculados con LP. por los resultados aquí obtenidos el comportamiento atípico comunicado previamente para esta cepa dependería de la asociación hospedeiro-parásito más que de características propias de la población parasitaria (AU)