Synthesis of epoxypropyloxy chromones and allyl hydroxy chromones and in vitro-in vivo activity on protoscoleces of Echinococcus multilocularis (Cestoda).

Derivatives with epoxypropyloxy, allyloxy and allyl hydroxychromone structures were synthesized and tested against protoscoleces of Echinococcus multilocularis. Compounds IV-IX were tested in vitro and compound VII proved the most active of them with, at 0.1 mmol.L-1, 98% dead protoscoleces in open vesicles after 24 hours and 80% in closed vesicles within 96 hours. Compound VII was tested in vivo on Meriones unguiculatus infested by Echinococcus multilocularis, but was found to be rather inactive.

Synthesis and in vitro activity of alkylaminoalkyloxy chromones on protoscoleces of Echinococcus multilocularis (Cestoda).

Twelve derivatives with alkylaminoalkyloxy chromone structures were synthesized and tested upon protoscoleces of Echinococcus multilocularis metacestode kept alive in vitro. Assays were performed with protoscoleces attached to the germinal layer in open and in closed vesicles. Compounds IVb and IIIe at the concentration of 0.1 mmol.L-1 killed 50% of the protoscoleces in open vesicles in 48 hours and compound IVb killed 100% of the protoscoleces in open vesicles within 96 hours at the same concentration. In closed vesicles after four days compound IVb killed all protoscoleces, compounds IIIe and IVd half of them, whereas in the controls all protoscoleces were alive. Trifluoperazine (TFP) was the reference compound; none of the new compounds showed better activity than TFP.

Polyclonal cell activity of a repeat peptide derived from the sequence of an 85-kilodalton surface protein of Trypanosoma cruzi trypomastigotes.

Some in vitro and in vivo biological activities of an octadecapeptide derived from an 85-kDa surface protein of Trypanosoma cruzi trypomastigote were studied. The peptide coupled to a carrier protein induced the proliferative response of lymph node cells from mice immunized with various antigens. Moreover, sera from mice immunized with the coupled peptide were found to contain antibodies against a number of self and nonself antigens: fibronectin, bovine serum albumin, myosin, tetanus toxoid, ovalbumin, keyhole limpet hemocyanin, and DNA. These results are discussed in the context of Chagas' disease immunopathology.

Potential anti-echinococcal activity of alkylaminoethers.

Trans 2-phenoxy cyclohexanol ethers (IA, IIA, IIIA, IVA, VA, and VIA), the cyclohexanol analog (IB) and one coumarinic compound (IC) were obtained and their activity against Echinococcus multilocularis metacestodes was studied and compared with that of trifluoperazine (TFP). All of these compounds are analogous to IA and belong to three classes. Class A comprises trans 2-phenoxycyclohexanol aminoethers whose alkylaminoether group varies; compound VIA bears one more methylene in its aminoether group than does compound IA. Class B consists of one compound exhibiting no phenoxy function. Class C comprises one coumarinic analog. In vitro assays were performed using metacestodes whose protoscoleces were attached to the germinal layer in open and in closed vesicles. Compounds IA and IIA exhibited the highest activity, but it was lower than that displayed by TFP under the same conditions. Compound IA was tested in an in vivo assay in jirds (50 mg/kg/daily beginning at 80 days p.i.); it produced results that were analogous to those obtained using TFP without inducing the neuroleptic effect associated with the latter. After 40-90 days' treatment, the percentage of diminution in the entire parasitic mass in the jirds that survived minimal treatment (71%) was about 41% as compared with that in untreated jirds. Histologic examination of the parasites in treated jirds revealed numerous dead protoscoleces and some parasitic dedifferentiated cells. This parasitic response may indicate that in alveolar echinococcosis, these drugs exhibit only a parasitostatic effect.

Trypanosoma cruzi: a carbohydrate epitope defined by a monoclonal antibody as a possible marker of the acute phase of human Chagas' disease.

An IgM monoclonal antibody (MAb) against a carbohydrate epitope present in Trypanosoma cruzi trypomastigote excretory-secretory antigens and expressed by different developmental stages of the parasite (epimastigote, trypomastigote and intracellular amastigote) was linked to a solid phase matrix and used as an antigen-capture antibody. Human serum complexes containing the epitope were then detected by using specific secondary antibodies against human immunoglobulin isotypes. Results of detection of IgM, IgG, and IgA serum complexes (SC) containing a T. cruzi polypeptide epitope showed that SC could be detected in 69% of the 13 Chagasic acute phase sera studied with IgG, in 84% with IgM, and in 75% with IgA. Only 16% (IgG-SC), 8% (IgM-SC), and 10% (IgA-SC) of chronic sera from 50 patients were positive. No patients with toxoplasmosis or rheumatoid factor were positive. Of the 11 leishmaniasis sera studied, four had IgG-SC, two had IgA-SC, and five had IgM-SC. Of the eight Yanomamo Indians infected by Onchocerca volvulus, three were found to have IgG-SC, two had IgM-SC, and two had IgA-SC. Thirteen sera from healthy individuals living in an endemic area were also studied. One subject had IgG IgM and IgA-SC. The results presented in this study show for the first time, the specific detection of IgM, IgG, and IgA immune complexes using a MAb against T. cruzi. The presence of the epitope in association with IgM antibodies in sera from patients with the acute phase of the disease suggests that this antigen(s) carrying the epitope that reacts with the MAb could be a marker(s) of active infection. In addition, the specificity of the serum complex capture assay allowed the detection of Chagas' disease in two different endemic areas (Argentina and Venezuela).

Evaluation of a competitive antibody enzyme immunoassay for specific diagnosis of Chagas' disease.

A competitive enzyme immunoassay based on the use of a monoclonal antibody (MAb) specific for "component 5" of Trypanosoma cruzi was evaluated. The antigenicity and immunogenicity of this component has been observed in natural and experimental infections. The studies were conducted in an area of Bolivia where mixed infections with Leishmania braziliensis are frequent and present a problem in the accurate diagnosis of T. cruzi infections. The specificity and sensitivity of this assay as compared to the indirect immunofluorescence and ELISA tests were demonstrated. The present test has proved to be more specific than the immunofluorescence and ELISA tests.

Trypanosoma cruzi: circulating polysaccharide factors excreted in vitro and in vivo.

An antigen factor (EF), thermostable and soluble in trichloroacetic acid was detected in the supernatant fluid of epimastigote cultures of Trypanosoma cruzi and in the sera of patients with acute Chagas disease. An hyperimmune antiserum to this antigenic factor was obtained in rabbits. The EF was revealed on the fibroblast surface membranes of rats infected with trypomastigotes, using the indirect immunofluorescence technique. The presence of EF in the sera of patients with acute Chagas disease as well as in the supernatant of epimastigotes culture at logarithmic phase, leads to its association with a process of parasite proliferation. Being EF a component of the parasite, its origin both in vitro and in vivo could be the result of an excretion-secretion of parasite or simply a result of the parasite's death. It can be postulated that the same as in other protozoic infection, EF could be used by T. cruzi in the process of cell penetration.

Trypanosoma cruzi: circulating polysaccharide factors excreted in vitro and in vivo

Se detectó un factor antigénico, temoestable y soluble en ácido tricloroacético (TCA) en el sobrenadante de cultivo de epimatigotes de Trypanosoma cruzi en fase logarítmica y en los sueros de pacientes con enfermedad de Chagas aguda. Este antígeno fue revelado en la mambrana de superficie de fibroblastos de ratas cuando fueron infectados con trypomastigotes, utilizando la técnica de inmunofluorescencia indirecta. El sobrenadante de cultivo de epimastigotes en medio GLSH libre de células a 28-C fue acidificado a pH5, calentado a ebullición 30 min y luego precipitado con TCA, centrifugado y el sobrenadante tratado con éter de petróleo. La solución extraída fue dializada, concentrada, liofilizada y purificada por cromatografía de afinidad. El eluido fue liofilizado y este material fue denominado factor antígeno excretado (EF). Un antisuero hiperinmune contra estos factores antígenos fue obtenido en conejos. El 76,6% de los sueros correspondientes a pacientes con Chagas agudo demostraron poseer el EF por técnica de inmunodifusión doble (Tabla 1). Monocapas de fibroblastos de ratas infectados previamente con 3 ml de trypomastigotes en una concentración de 5 x 10**6 células/ml sometidas a una inmunofluorescencia indirecta con el antisuero hiperinmune demostraron la presencia del EF en las membranas celulares de fibroblastos infectados y no infectados..

Trypanosoma cruzi: circulating polysaccharide factors excreted in vitro and in vivo.

An antigen factor (EF), thermostable and soluble in trichloroacetic acid was detected in the supernatant fluid of epimastigote cultures of Trypanosoma cruzi and in the sera of patients with acute Chagas disease. An hyperimmune antiserum to this antigenic factor was obtained in rabbits. The EF was revealed on the fibroblast surface membranes of rats infected with trypomastigotes, using the indirect immunofluorescence technique. The presence of EF in the sera of patients with acute Chagas disease as well as in the supernatant of epimastigotes culture at logarithmic phase, leads to its association with a process of parasite proliferation. Being EF a component of the parasite, its origin both in vitro and in vivo could be the result of an excretion-secretion of parasite or simply a result of the parasites death. It can be postulated that the same as in other protozoic infection, EF could be used by T. cruzi in the process of cell penetration.

Trypanosoma cruzi: circulating polysaccharide factors excreted in vitro and in vivo

Se detectó un factor antigénico, temoestable y soluble en ácido tricloroacético (TCA) en el sobrenadante de cultivo de epimatigotes de Trypanosoma cruzi en fase logarítmica y en los sueros de pacientes con enfermedad de Chagas aguda. Este antígeno fue revelado en la mambrana de superficie de fibroblastos de ratas cuando fueron infectados con trypomastigotes, utilizando la técnica de inmunofluorescencia indirecta. El sobrenadante de cultivo de epimastigotes en medio GLSH libre de células a 28-C fue acidificado a pH5, calentado a ebullición 30 min y luego precipitado con TCA, centrifugado y el sobrenadante tratado con éter de petróleo. La solución extraída fue dializada, concentrada, liofilizada y purificada por cromatografía de afinidad. El eluido fue liofilizado y este material fue denominado factor antígeno excretado (EF). Un antisuero hiperinmune contra estos factores antígenos fue obtenido en conejos. El 76,6% de los sueros correspondientes a pacientes con Chagas agudo demostraron poseer el EF por técnica de inmunodifusión doble (Tabla 1). Monocapas de fibroblastos de ratas infectados previamente con 3 ml de trypomastigotes en una concentración de 5 x 10**6 células/ml sometidas a una inmunofluorescencia indirecta con el antisuero hiperinmune demostraron la presencia del EF en las membranas celulares de fibroblastos infectados y no infectados..

Identification and isolation of Trypanosoma cruzi trypomastigote collagen-binding proteins: possible role in cell-parasite interaction.

We have shown here that collagen type I bound efficiently to the trypomastigote surface. In addition, monoclonal and polyclonal antibodies against collagen types I and III inhibited the infection of fibroblasts by the parasite. These results suggested the presence of collagen-binding protein(s) on the parasite surface. This protein was identified from trypomastigote surface antigens using affinity chromatography on a Gelatin Ultrogel column (denatured form of collagen). These collagen-binding proteins were revealed as a low-affinity gelatin binding protein (LAG Bp) of 98 kDa, and a high-affinity binding protein (HAG Bp) of 58 and 68 kDa under non-reducing and reducing conditions respectively. In addition, HAG Bp and LAG Bp bound to collagen type I. The 58/68 kDa protein was purified to homogeneity on a wheat germ agglutinin Sepharose column. A polyclonal antibody to this glycoprotein, as well as a monoclonal antibody (McAb) 155D3 produced against the HAG Bp, immunoprecipitated two parasite surface antigens of 160 and 58 kDa under non-reducing conditions which migrated at a position of 80-85 and 68 kDa when reduced. However, only the 80-85 kDa component could be precipitated from [35S] methionine-labelled trypomastigote antigens under reducing conditions. The antibodies to the 58/68 kDa glycoprotein as well as McAb 155D3 diminished the invasion of fibroblasts by parasites. Taken together these results suggest that the same receptor binds fibronectin and/or collagen and that both the 80-85 and 58/68 kDa glycoproteins form part of the same receptor. These trypomastigote surface molecules may interact with the host cell fibronectin and/or collagen during the initial phase of parasite-cell recognition.

Specific immunodiagnosis of Chagas disease: immunodiffusion test using a specific serum anti-Trypanosoma cruzi component 5.

A micro double diffusion test (MD), allowing the identification of precipitation brand 5 by identity reaction, using a rabbit specific anti-component 5 serum, was evaluated for the immunological diagnosis of Chagas' disease. The previous studies on the Trypanosoma cruzi specificity of component 5[g] were completed, showing it to be absent in Leishmania brazilienis, but present in different strains of T. cruzi. 200 sera from Bolivian patients were studied. (88 with a positive xenodiagnosis, 45 with mucocutaneous leishmaniasis but without Chagas' disease, and 67 controls). Band 5 was found in 74 (84.1%) of the sera with positive xenodiagnosis but was never found either in the leishmaniasis or in the control groups. MD, allowing an easy detection of T. cruzi specific band 5, cheap and simple to perform, can be recommended in association with other serological tests, when highly specific immunodiagnosis of Chagas' disease is required.

Studies on anti-component 5 antibodies in animals infected with Trypanosoma cruzi

Foi usado um teste imunoenzimático competitivo para investigar a presença de anticorpos anticomponente 5 nos soros de sariguês, cäes, coelhos e ratos infectados com o Trypanosoma cruzi. Neste teste, foi utilizado um anticorpo monoclonal contra o antígeno 5 que é específico do T. cruzi. Também foram testados os soros de 51 pacientes venezuelanos com doença de Chagas. Apesar desses anticorpos näo serem detectados nos soros de cäes, ratos e sariguês infectados com o T. cruzi, alguns soros de coelhos infectados apresentaram resultados positivos porém em níveis próximos aos do limite de positividade do teste. Esses achados surgerem que a resposta imune em animais naturalmente ou experimentalmente infectados com o T. cruzi é diferente daquela que é observada na doença de Chagas humana

Complejos inmunes circulantes en los distintos estados evolutivos de la enfermedad de chagas / Circulating immune complexes in different developmental stages of Chagas disease

En este trabajo se demuestra un aumento de los CIC en las fases agudas y crónica de la enfermedad de Chagas. Los complejos inmunes circulantes (CIC) fueron cuantificados por test del Clq marcado con 125I unido a los sistemas antígeno-anticuerpo y precipitados con polietilenglicol al 3% en los diferentes estados evolutivos de la patología. Las muestras de sangre fueron obtenidas de 31 pacientes con infección aguda y 10 individuos normales habitantes de la misma zona y edad similar; el grupo de pacientes con infección crónica estuvo integrado por 23 enfermos con cardiopatía demostrada por 23 enfermos con cardiopatía demostrada y 25 pacientes que no presentaron ningún síntoma y sus exámenes periódicos fueron siempre normales. La población control de este grupo fue constituida por 10 individuos normales con características etarias y de residencia similar. Todos los pacientes estudiados pertenecían a las provincias de Santa Fé y Santiago del Estero, República Argentina. Se demostró un incremento significativo de los CIC en la población de agudos (p<0,001) y en los infectados crónicos considerados en conjunto (p<0,05). Sin embargo, cuando los infectados crónicos cardiópatas fueron comparados con la población normal, no se obtuvieron diferencias significativas en los niveles de CIC (p<0,35); mientras que lo contrario sucedió cuando se comparó el grupo de infectados crónicos sin cardiopatía con los controles (p<0,01). Parecería importante la obtención de estos resultados diferenciado la población crónica en función de sus signos y síntomas clínicos, tal como lo efectuado en este trabajo. Distintos autores le han aribuido a los CIC un posible rol en la regulación de la respuesta inmune. Su demonstración en las distintas fases evolutivas de enfermedad de Chagas constituye un hecho a considerar. La demostración de sus aumentos en la poblacicón crónica sin alteraciones patológicas podría incentivar el estudio de los CIC como signo inmunológico a tener en cuenta en el curso de la evolución de infección a enfermedad. Mientras que en infectados agudos los altos niveles de los CIC inducen, además, a estudiarlos como causa inmunológica de algunos signos presentados por estos pacientes en el examen clínico (AU)

Complejos inmunes circulantes en los distintos estados evolutivos de la enfermedad de chagas / Circulating immune complexes in different developmental stages of Chagas' disease

En este trabajo se demuestra un aumento de los CIC en las fases agudas y crónica de la enfermedad de Chagas. Los complejos inmunes circulantes (CIC) fueron cuantificados por test del Clq marcado con 125I unido a los sistemas antígeno-anticuerpo y precipitados con polietilenglicol al 3% en los diferentes estados evolutivos de la patología. Las muestras de sangre fueron obtenidas de 31 pacientes con infección aguda y 10 individuos normales habitantes de la misma zona y edad similar; el grupo de pacientes con infección crónica estuvo integrado por 23 enfermos con cardiopatía demostrada por 23 enfermos con cardiopatía demostrada y 25 pacientes que no presentaron ningún síntoma y sus exámenes periódicos fueron siempre normales. La población control de este grupo fue constituida por 10 individuos normales con características etarias y de residencia similar. Todos los pacientes estudiados pertenecían a las provincias de Santa Fé y Santiago del Estero, República Argentina. Se demostró un incremento significativo de los CIC en la población de agudos (p<0,001) y en los infectados crónicos considerados en conjunto (p<0,05). Sin embargo, cuando los infectados crónicos cardiópatas fueron comparados con la población normal, no se obtuvieron diferencias significativas en los niveles de CIC (p<0,35); mientras que lo contrario sucedió cuando se comparó el grupo de infectados crónicos sin cardiopatía con los controles (p<0,01). Parecería importante la obtención de estos resultados diferenciado la población crónica en función de sus signos y síntomas clínicos, tal como lo efectuado en este trabajo. Distintos autores le han aribuido a los CIC un posible rol en la regulación de la respuesta inmune. Su demonstración en las distintas fases evolutivas de enfermedad de Chagas constituye un hecho a considerar. La demostración de sus aumentos en la poblacicón crónica sin alteraciones patológicas podría incentivar el estudio de los CIC como signo inmunológico a tener en cuenta en el curso de la evolución de infección a enfermedad. Mientras que en infectados agudos los altos niveles de los CIC inducen, además, a estudiarlos como causa inmunológica de algunos signos presentados por estos pacientes en el examen clínico