Estandarización de la técnica para la obtención de trypomastigotes en células 3T3 a partir de una cepa local de epimastigotes de Tripanosoma cruzi / Acquisition mode pesticides and drugs in intoxicated patients treated in emergency Hospital Clínico Viedma

Objetivo: evaluar el empleo de cultivos celulares para la obtención de tripomastigotes en células 3T3, a partir de una cepa local de epimastigotes de Tripanosoma cruzi. Método: se realizó cultivo in vitro de células 3T3 en medio DMEM-SBF al 10% más penicilina estreptomicina, a 37°C, 95% de humedad y 5% de CO2 al séptimo día, fueron infectados con epimastigotes de T. cruzi cepa TcV, aislados de pacientes con Chagas agudo y cultivados preliminarmente en medios bifásicos como NNN y LIT. Resultados: a los 14 días de infección se observó al parásito en las formas: de amastigotes (forma intracelular), posteriormente la tripomastigote (forma extracelular) que fueron liberados al medio una vez lisadas las células infectadas. Posteriores sub cultivos de células 3T3 con trypomastigotes obtenidos a partir de los epimastigotes mejoran la obtención de T. cruzi. Conclusiones: es posible la obtención de trypomastigotes a partir de una cepa local de epimastigotes recreando el ciclo biológico del parásito in vitro.

Objective: to evaluate the use of cell cultures for the production of trypomastigotes in 3T3 cells, from a local strain of Trypanosoma cruzi epimastigotes. Method: we previously performed growing 3T3 cells in DMEM-10% FBS more penicillin-streptomycin in vitro at 37 °C, 95% humidity and 5% CO2 on the seventh day, they were infected with T. cruzi epimastigotes TcV strain, isolated from patients with acute Chagas and preliminarily grown in biphasic media as NNN and LIT. Results: after 14 days of infection was observed the parasite forms: extracellular) that were released into the infected cells once lysed. Subsequent sub 3T3 cell cultures trypomastigotes obtained from epimastigotes obtaining improved T. cruzi-TcV. Conclusions: it is possible to obtain trypomastigotes from a local strain epimastigotes recreating the life cycle of the parasite in vitro.

Purificación de una fracción de antígenos de tripomastigotes de Trypanosoma cruzi para el diagnóstico de la enfermedad de Chagas / Purification of a trypomastigote antigen fraction from Trypanosoma cruzi for Chagas’ disease diagnosis

Los antígenos excretados/secretados por las formas tripomastigotes de T. cruzi (antígenos TESA) pertenecen a la familia de las transialidasas, las cuales son responsables de la transferencia de ácido siálico exógeno a moléculas aceptadoras en la superficie de los tripomastigotes. En el presente trabajo se purifican varias proteínas de los antígenos TESA utilizando cromatografía de afinidad con resina de sefarosa B4-concanavalina A, con la intención de ser utilizados en el diagnóstico de la enfermedad de Chagas. El buffer de elución contenía una mezcla de α-D-manopiranósido y α-D-glucopiranósido. Se realizó electroforesis unidimensional en gel con poliacrilamida para identificar las bandas purificadas y la prueba de inmunoelectrotransferencia para visualizar las bandas reactivas con el pool de sueros de individuos con infección por T. cruzi. El gel teñido con azul de Coomassie coloidal permitió visualizar 3 bandas de aproximadamente 220, 170, y 20 kDa. La inmunoelectrotransferencia utilizando un pool de sueros positivos, confirmados para la infección por T. cruzi, reveló 5 bandas inmunogénicas de 220, 120, 85, 50 y 32 kDa mientras que el revelado con diaminobenzidina permitió observar las bandas de 220, 120, 85, 50 32 y 20 kDa. Asimismo las bandas purificadas no fueron reconocidas en la inmunoelectrotransferencia por el pool de sueros confirmados como negativos. Estos resultados sugieren el potencial de estas proteínas purificadas de TESA para ser usadas como nueva herramienta para el diagnóstico de la enfermedad de Chagas.

Trypanosoma cruzi excreted/secreted antigens (TESA) belong to the transialidase family, which are responsible for the transfer of exogenous sialic acid to accepting molecules at the trypomastigote surface. In the present study we purified several proteins from TESA antigens using affinity chromatography with sepharose B4-concanavalin A resin, with the purpose of using them for Chagas’ disease diagnosis. The elution buffer contained a mixture of α-D-manopiranosid and α-D-glucopiranosid. A unidimensional electrophoresis in polyacrilamide gel to identify the purified bands, and an immunoelectrotransference test with a pool of sera from T. cruzi infected individuals to visualize the reactive bands were carried out. The colloidal Coomassie blue stained gel allowed visualizing 3 bands of approximately 220, 170 and 20 kDa. The immunoelectrotransference using a pool of positive sera with confirmed T. cruzi infection showed 5 immunogenic 220, 120, 50 and 32 kDa bands, while a developing with diaminobenzidine showed 220, 120, 85, 50, 32 and 20 kDa bands. The purified bands were not recognized in an immunoelectrotransference test when a pool of confirmed negative sera was used. These results suggest the potential of these TESA purified proteins for using them as a new tool for Chagas’ disease diagnosis.

Molecular mechanisms of Trypanosoma cruzi infection by oral route

Frequent reports on outbreaks of acute Chagas' disease by ingestion of food contaminated with parasites from triatomine insects illustrate the importance of this mode of transmission. Studies on oral Trypanosoma cruzi infection in mice have indicated that metacyclic trypomastigotes invade the gastric mucosal epithelium. A key molecule in this process is gp82, a stage-specific surface glycoprotein that binds to both gastric mucin and to target epithelial cells. By triggering Ca2+ signalling, gp82 promotes parasite internalisation. Gp82 is relatively resistant to peptic digestion at acidic pH, thus preserving the properties critical for oral infection. The infection process is also influenced by gp90, a metacyclic stage-specific molecule that negatively regulates the invasion process. T. cruzi strains expressing high gp90 levels invade cells poorly in vitro. However, their infectivity by oral route varies considerably due to varying susceptibilities of different gp90 isoforms to peptic digestion. Parasites expressing pepsin-susceptible gp90 become highly invasive against target cells upon contact with gastric juice. Such is the case of a T. cruzi isolate from an acute case of orally acquired Chagas' disease; the gp90 from this strain is extensively degraded upon short period of parasite permanence in the gastric milieu. If such an exacerbation of infectivity occurs in humans, it may be responsible for the severity of Chagas' disease reported in outbreaks of oral infection.

The utility of anti-trypomastigote lytic antibodies for determining cure of Trypanosoma cruzi infections in treated patients: an overview and perspectives

In previous work, we proposed alternative protocols for following patients with treated Chagas disease and these are reviewed herein. Evidence was provided to support the following: (i) functional anti-trypomastigote antibodies are indicative of ongoing chronic Trypanosoma cruzi infections; (ii) specific antibodies detected by conventional serology (CS) with epimastigote extracts, fixed trypomastigotes or other parasite antigens may circulate years after parasite elimination; (iii) functional antibodies are evidenced by complement-mediated lysis of freshly isolated trypomastigotes, a test which is 100 percent specific, highly sensitive, and the first to revert after T. cruzi elimination and (iv) the parasite target for the lytic antibodies is a glycoprotein of high molecular weight (gp160) anchored at the parasite surface. The complement regulatory protein has been cloned, sequenced and produced as a recombinant protein by other groups and is useful for identifying functional anti-T. cruzi antibodies in ELISA tests, thus dispensing with the need for live trypomastigotes to manage treated patients. If used instead of CS to define cures for Chagas patients, ELISA will avoid unnecessary delays in finding anti-T. cruzi drugs. Other highly sensitive techniques for parasite DNA detection, such as PCR, need to be standardized and included in future protocols for the management of patients with drug-treated Chagas disease.

A century of research: what have we learned about the interaction of Trypanosoma cruzi with host cells?

Since the discovery of Trypanosoma cruzi and the brilliant description of the then-referred to "new tripanosomiasis" by Carlos Chagas 100 years ago, a great deal of scientific effort and curiosity has been devoted to understanding how this parasite invades and colonises mammalian host cells. This is a key step in the survival of the parasite within the vertebrate host, and although much has been learned over this century, differences in strains or isolates used by different laboratories may have led to conclusions that are not as universal as originally interpreted. Molecular genotyping of the CL-Brener clone confirmed a genetic heterogeneity in the parasite that had been detected previously by other techniques, including zymodeme or schizodeme (kDNA) analysis. T. cruzi can be grouped into at least two major phylogenetic lineages: T. cruzi I, mostly associated with the sylvatic cycle and T. cruzi II, linked to human disease; however, a third lineage, T. cruziIII, has also been proposed. Hybrid isolates, such as the CL-Brener clone, which was chosen for sequencing the genome of the parasite (Elias et al. 2005, El Sayed et al. 2005a), have also been identified. The parasite must be able to invade cells in the mammalian host, and many studies have implicated the flagellated trypomastigotes as the main actor in this process. Several surface components of parasites and some of the host cell receptors with which they interact have been described. Herein, we have attempted to identify milestones in the history of understanding T. cruzi- host cell interactions. Different infective forms of T. cruzi have displayed unexpected requirements for the parasite to attach to the host cell, enter it, and translocate between the parasitophorous vacuole to its final cytoplasmic destination. It is noteworthy that some of the mechanisms originally proposed to be broad in function turned out not to be universal, and multiple interactions involving different...