Producción y purificación de anticuerpos (IgY) a partir de huevos de gallinas inmunizadas con epimastigotas de trypanosoma cruzi / Production and purification of IgY antibodies from eggs laid by hens inmunized with trypanosoma cruzi epimastigotes

No obstante las ventajas de producir anticuerpos en gallinas, no se ha reportado inducción y purificación de anticuerpos contra estadios de Trypanosoma cruzi en el modelo aviario. La inducción de anticuerpos anti-estadios de T. cruzi en gallina puede garantizar la producción de reactivos útiles en diagnóstico y terapéutica de la enfermedad de Chagas. Aquí reportamos la obtención y rápido aislamiento de IGY procedente de yema de huevos de gallinas inmunizadas contra epimastigotas de T. cruzi a los 7, 22 y 61 días post-inmunización. Se compararon tres estrategias de purificación de IgY: el método de la dilución acuosa, el método del polietilén glicol (PEG) y el método del cloroformo-PEG, respecto a un estuche comercial. El método del cloroformo-PEG dio un rendimiento de 6,4 mg/mL de proteínas de yema de huevo de 61 días con una sensibilidad tal que 7 ng de IgY detectaron 1 µg de antígeno (ELISA). Por Western blot, 5 µg/mL de IgY revelaron 11 antígenos diferentes en 8 µg de proteína total de epimastigotas. El análisis por SDS-PAGE demostró que las IgY extraídas contienen dos bandas proteícas dominantes de 70/57 y 37/35 kDa y dos tenues de 81 y 41 kDa, las cuales pueden ser eliminadas por re-precipitación con PEG. Para la extracción de IgY el método del cloroformo-polietilén glicol dio la mejor combinación por facilidad de ejecución y bajo costo. Si bien rindió 50 por ciento menos que el estuche comercial bajo las mismas condiciones, la sensibilidad de los anticuerpos fue 4 veces mayor. Estos resultados evidencian la factibilidad del modelo aviario para producir anticuerpos contra estadios de T. cruzi y muestran la experticia para purificarlos usando una tecnología local de bajo costo

Morphological comparison of axenic amastigogenesis of trypomastigotes and metacyclic forms of Trypanosoma cruzi

Amastigogenesis occurs first when metacyclic trypomastigotes from triatomine urine differentiate into amastigotes inside mammalian host cells and a secondary process when tissue-derived trypomastigotes invade new cells and differentiate newly to amastigotes. Using scanning electron microscopy, we compared the morphological patterns manifested by trypomastigotes and metacyclic forms of Trypanosoma cruzi during their axenic-transformation to amastigotes in acidic medium at 37ºC. We show here that in culture MEMTAU medium, secondary and primary axenic amastigogenesis display different morphologies. As already described, we also observed a high differentiation rate of trypomastigotes into amastigotes. Conversely, the transformation rate of in vitro-induced-metacyclic trypomastigotes to amastigotes was significantly slower and displayed distinct patterns of transformation that seem environment-dependent. Morphological comparisons of extracelullar and intracellular amastigotes showed marked similarities, albeit some differences were also detected. SDS-PAGE analyses of protein and glycoprotein from primary and axenic extracelullar amastigotes showed similarities in glycopeptide profiles, but variations between their proteins demonstrated differences in their respective macromolecular constitutions. The data indicate that primary and axenic secondary amastigogenesis of T. cruzi may be the result of different developmental processes and suggest that the respective intracellular mechanisms driving amastigogenesis may not be the same

Production of amastigotes from metacyclic trypomastigotes of Trypanosoma cruzi

Attempts to recreate all the developmental stages of Trypanosoma cruzi in vitro have thus far been met with partial success. It is possible, for instance, to produce trypomastigotes in tissue culture and to obtain metacyclic trypomastigotes in axenic conditions. Even though T. cruzi amastigotes are known to differentiate from trypomastigotes and metacyclic trypomastigotes, it has only been possible to generate amastigotes in vitro from the tissue-culture-derived trypomastigotes. The factors and culture conditions required to trigger the transformation of metacyclic trypomastigotes into amastigotes are as yet undetermined. We show here that pre-incubation of metacyclic trypomastigotes in culture (MEMTAU) medium at 37ºC for 48 h is sufficient to commit the parasites to the transformation process. After 72 h of incubation in fresh MEMTAU medium, 90 percent of the metacyclic parasites differentiate into forms that are morphologically indistinguishable from normal amastigotes. SDS-PAGE, Western blot and PAABS analyses indicate that the transformation of axenic metacyclic trypomastigotes to amastigotes is associated with protein, glycoprotein and antigenic modifications. These data suggest that (a) T. cruzi amastigotes can be obtained axenically in large amounts from metacyclic trypomastigotes, and (b) the amastigotes thus obtained are morphological, biological and antigenically similar to intracellular amastigotes. Consequently, this experimental system may facilitate a direct, in vitro assessment of the mechanisms that enable T. cruzi metacyclic trypomastigotes to transform into amastigotes in the cells of mammalian hosts

Separación de polipéptidos antigénicos de trypanosoma cruzi usando soluciones de pH diferentes / Separation of antigenic polypeptides of tripanosoma cruzi using defferential solubility in different pHs

Fracciones antigénicas útiles para el diagnóstico de la enfermedad de Chagas son obtenidas utilizando metodologías costosas, por eso es necesario desarrollar tecnologías que reduzcan los costos de producción. Previamente mostramos que los polipéptidos de trypanosoma cruzi se solubilizan diferencialmente en soluciones con valores de pH diferentes. El Propósito en este estudio fue obtener fracciones enriquecidas en polipéptidos de T. cruzi antigénicamente funcionales en base a su solubilidad diferencial. Los perfiles polipeptídicos de los epimastigotas fueron identicados por electroforesis en geles de poliacrilamida con dodecil sulfato de sodio (SDS-PAGE), coloreados por el método de la plata metálica. La antigenicidad de los polipéptidos se estudió mediante Western Bot usando un suero específico anti-epimastigotas. Se encontró que: a) la solubilidad de los polipéptidos no fue afectada por cambios en la fuerza iónica entre 0 y 250 mM NaCl; b) todos los polipéptidos se precipitaron a pH 4,0 mientras que el incremento secuencial del pH de la solución de resuspensión produjo un enriquecimiento diferencial de polipéptidos de mayor peso molecular; c) las fracciones enriquecidas, procedentes de soluciones con valores pH menores que 7,0, mostraron una fuerte actividad proteolítica la cual fue inhibida a pH alcalino y; d) los polipéptidos conservaron su antigenicidad. Estos resultados muestran una metodología sencilla y relativamente económica para producir fracciones antigénicas. Están en progreso experimentos para reducir la actividad proteolítica y obtener polipéptidos útiles para el diagnóstico de la enfermedad de Chagas

Trypanosoma cruzi: maintenance in culture modify gene and antigenic expression of metacyclic trypomastigotes

In this study we examined whether the maintenance of Trypanosoma cruzi by long-time in axenic culture produces changes in gene expression and antigenic profiles. The studies were made with a Dm30L-clone from a low-virulent strain and a non-cloned virulent EP-strain of T. cruzi. Both parasites were maintained, for at least seven years, by successive alternate passage triatomine/mouse (triatomine condition), or by serial passage in axenic medium (culture condition). The comparison of the [35S]methionine metabolic labeling products of virulent and non-virulent parasites by 2D-SDS-PAGE, clearly indicates that the expression of metacyclic trypomastigotes (but not of epimastigotes) proteins have been altered by laboratory maintenance conditions. Western blot analysis of EP and Dm30L-epimastigotes using a serum anti-epimastigotes revealed that although most of antigens are conserved, four antigens are characteristics of triatomine condition parasites and three other are characteristics of culture condition parasites. Anti-metacyclics serum revealed significative differences in EP- and Dm30L-metacyclic trypomastigotes from triatomine condition. However, avirulent metacyclic forms were antigenically very similar. These results suggest that besides a possible selection of avirulent subpopulation from T. cruzi strains genetically heterogeneous when maintained by long time in axenic culture, changes in virulence might be due to post-translational modifications of antigens induced by the absence of the natural alternability (vertebrate-invertebrate) in the life-cycle of T. cruzi.

Trypanosoma cruzi: metacyclogenesis in vitro - I. Changes in the properties of metacyclic trypomastigotes maintained in the laboratory by different methods

In this work we have studied the modifications in the biological properties of Trypanosoma cruzi when the parasite is maintained for a long time in axenic culture. The studies were done with a clone from an avirulent strain (Dm30L) and a non-cloned virulent strain (EP) of T. cruzi. Both parasiteswere maintained, for at least three years, by successive triatomine/mouse alternate passage (control condition), or by serial passage in axenic medium (culture condition), or only in the mouse (mouse condition). The comparison between parasites of culture and control condition showed that metacyclogenesis capacity was reduced in the former and that the resulting metacyclics displayed an attenuatedvirulence. In order to compare the virulence of metacyclics from the urine of the insect vector, Rhodnius prolixus were infected by artificial feeding with parasites of the control or culture condition. After three triatomine/triatomine passages, there was observed an almost identical biological behavior for these parasites, hence indicating that the maintenance of T. cruzi for a long time in axenic culture affects the differentiation capacity and the virulence of the parasite. Additionally, it was demonstrated that it is possible to maintain T. cruzi exclusively through passages in the invertebrate host

Biological properties of Trypanosoma cruzi metacyclic trypomastigotes: usefulness for studying Chagas' disease (brief report)

We have investigated changes in the biological properties of metacyclic trypomastigotes obtained from various sources, kept in the laboratory under diverse conditions and subjected to different procedures. Results demonstrate the great capacity of adaptation of Trypanosoma cruzi to changeable environments. The usefulness of different chemically defined media and consecutive passages through various hosts emphasize the importance of mimicking the life cycle of the parasite

Biological aspects of the DM28C clone of Trypanosoma cruzi after metacylogenesis in chemically defined media

A caracterizaçäo biológica do clone Dm 28c de Trypanosoma cruzi em termos do seu crescimento em meio LIT, ciclo celular, infectividade para camundongos e interaçäo com células fagocíticas profissionais e näo-profissionais, mostra que o mesmo comporta-se como um fiel representante da espécie T. cruzi. As propriedades biológicas deste clone miotrópico näo mudam de acordo com a proveniência das formas tripomastigotas (i. e., de triatomíneos, de camundongos infectados, de cultura celular ou dos meios quimicamente definidos TAUP e TAU3AAG). Ainda mais, formas tripomastigotas metacíclicas do clone Dm 28c derivado do meio TAU3AAG apresentam um alto grau de infectividade para fibroblastos e células de músculo. Experimentos de ligaçäo de ferritina cationizada à superfície de tripomastigotas, mostram a existência de acúmulos ("caps") de ferritina em regiöes próximas ao cinetoplasto, todavia a natureza e o papel destes sítios aniônicos resta a ser determinado. Os resultados indicam que tripomastigotas metacíclicos do clone Dm 28c, obtidos em condiçöes quimicamente definidas, reproduzem o comportamento biológico de T.cruzi, tornando este sistema bastante apropriado para o estudo da interaçäo célula-parasito e para o isolamento de macromoléculas relevantes