Trypanosoma cruzi: in vitro activity of Epoxy-alpha-Lap, a derivative of alpha-lapachone, on trypomastigote and amastigote forms.

Chagas disease is an endemic parasitic infection caused by Trypanosomacruzi that affects 18-20 million people in Central and South America. Recently we described the Epoxy-alpha-Lap, an oxyran derivative of alpha-lapachone, which presents a low toxicity profile and a high inhibitory activity against T.cruzi epimastigotes forms, the non-infective form of this parasite. In this work we described the trypanocidal effects of Epoxy-alpha-Lap on extracellular (trypomastigote) and intracellular (amastigote) infective forms of two T. cruzi strains (Y and Colombian) known by their different infective profile. Our results showed that Epoxy-alpha-Lap is lethal to trypomastigote Y and Colombian strains (97% and 84%, respectively). Interestingly, Epoxy-alpha-Lap also showed a trypanocidal effect in human macrophage infected with T. cruzi Y (85.6%) and Colombian (71.9%) strains amastigote forms. Similar effects were observed on T. cruzi amastigote infected Vero cells (96.4% and 95.0%, respectively). Our results pointed Epoxy-alpha-Lap as a potential candidate for Chagas disease chemotherapy since it presents trypanocidal activity on all T. cruzi forms with low) toxicity profile.

The main proteinases in Dermatobia hominis second and third instars larvae are serine-proteinases.

We performed a combination of proteinase assay, either in solution or immobilized in sodium dodecyl sulfate-polyacrylamide gel copolymerized with gelatin, to detect and quantify proteinases of Dermatobia hominis second (L2) and third (L3) instar larvae. In the quantitative assay, we examined proteinase activity by hydrolysis of a panel of peptide bonds specific for the main proteinase classes. We verified that the pGlu-Phe-Leu p-nitroanilide substrate was hydrolyzed by crude extracts of L2 (3.0+/-0.2 nmol h(-1)mg of protein(-1)) and L3 (7.7+/-0.1 nmol h(-1)mg of protein(-1)) and that both activities were partially inhibited by trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane, 15% and 3%, respectively. Also, we demonstrated that the Nalpha-p-Tosyl-l-Arg methyl ester substrate was hydrolyzed by crude extracts of L2 (117+/-24 nmol h(-1)mg of protein(-1)) and L3 (111+/-10 nmol h(-1)mg of protein(-1)), suggesting a predominance of esterase activity in the crude larval preparation. Interestingly, the specific activity of serine-proteinases was totally inhibited by phenylmethylsulphonyl fluoride in the L3 crude extract, while only 10% of this enzyme class activity was inhibited in the L2 crude extract. The results of the qualitative assays with substrate gels suggested that L2 and L3 larvae express serine-proteinases with similar (13 and 22 kDa) and distinct (50 kDa in L2 and 30 kDa in L3) relative molecular masses. These findings contribute to the biochemical characterization of D. hominis L2 and L3 larvae.

Trypanosoma cruzi infection: a continuous invader-host cell cross talk with participation of extracellular matrix and adhesion and chemoattractant molecules

Several lines of evidence have shown that Trypanosoma cruzi interacts with host extracellular matrix (ECM) components producing breakdown products that play an important role in parasite mobilization and infectivity. Parasite-released antigens also modulate ECM expression that could participate in cell-cell and/or cell-parasite interactions. Increased expression of ECM components has been described in the cardiac tissue of chronic chagasic patients and diverse target tissues including heart, thymus, central nervous system and skeletal muscle of experimentally T. cruzi-infected mice. ECM components may adsorb parasite antigens and cytokines that could contribute to the establishment and perpetuation of inflammation. Furthermore, T. cruzi-infected mammalian cells produce cytokines and chemokines that not only participate in the control of parasitism but also contribute to the establishment of chronic inflammatory lesions in several target tissues and most frequently lead to severe myocarditis. T. cruzi-driven cytokines and chemokines may also modulate VCAM-1 and ICAM-1 adhesion molecules on endothelial cells of target tissues and play a key role in cell recruitment, especially of activated VLA-4+LFA-1+CD8+ T lymphocytes, resulting in a predominance of this cell population in the inflamed heart, central nervous system and skeletal muscle. The VLA-4+-invading cells are surrounded by a fine network of fibronectin that could contribute to cell anchorage, activation and effector functions. Since persistent "danger signals" triggered by the parasite and its antigens are required for the establishment of inflammation and ECM alterations, therapeutic interventions that control parasitism and selectively modulate cell migration improve ECM abnormalities, paving the way for the development of new therapeutic strategies improving the prognosis of T. cruzi-infected individuals

Trypanosoma cruzi infection: a continuous invader-host cell cross talk with participation of extracellular matrix and adhesion and chemoattractant molecules.

Several lines of evidence have shown that Trypanosoma cruzi interacts with host extracellular matrix (ECM) components producing breakdown products that play an important role in parasite mobilization and infectivity. Parasite-released antigens also modulate ECM expression that could participate in cell-cell and/or cell-parasite interactions. Increased expression of ECM components has been described in the cardiac tissue of chronic chagasic patients and diverse target tissues including heart, thymus, central nervous system and skeletal muscle of experimentally T. cruzi-infected mice. ECM components may adsorb parasite antigens and cytokines that could contribute to the establishment and perpetuation of inflammation. Furthermore, T. cruzi-infected mammalian cells produce cytokines and chemokines that not only participate in the control of parasitism but also contribute to the establishment of chronic inflammatory lesions in several target tissues and most frequently lead to severe myocarditis. T. cruzi-driven cytokines and chemokines may also modulate VCAM-1 and ICAM-1 adhesion molecules on endothelial cells of target tissues and play a key role in cell recruitment, especially of activated VLA-4+LFA-1+CD8+ T lymphocytes, resulting in a predominance of this cell population in the inflamed heart, central nervous system and skeletal muscle. The VLA-4+-invading cells are surrounded by a fine network of fibronectin that could contribute to cell anchorage, activation and effector functions. Since persistent "danger signals" triggered by the parasite and its antigens are required for the establishment of inflammation and ECM alterations, therapeutic interventions that control parasitism and selectively modulate cell migration improve ECM abnormalities, paving the way for the development of new therapeutic strategies improving the prognosis of T. cruzi-infected individuals.

Effect of Trypanosoma cruzi released antigens binding to non-infected cells on anti-parasite antibody recognition and expression of extracellular matrix components.

It has been proposed that antigens released by Trypanosoma cruzi sensitize vertebrate cells leading to their destruction by the immune response raised against the parasite. Here, we characterized antigens released by trypomastigotes of T. cruzi that bind to non-infected cells and investigated biological consequences of this adsorption. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of antigens released by [(35)S]-methionine-labeled parasites revealed the presence of polypeptides mainly ranging from 85 to 170 kDa that were specifically recognized by sera from chronically T. cruzi infected rabbits. Polypeptides of 85-110 and 160-170 kDa bound to non-infected epithelial, fibroblast and muscle mammalian cell lines, which thus became targets for anti-T. cruzi antibody binding. Cysteine-proteinase, but not trans-sialidase, was detected among the cell-bound antigens, and purified cysteine-proteinase was adsorbed to non-infected cells. Immunoelectron microscopic studies showed that parasite antigens were mainly released as membrane vesicles that adhered to membrane microvilli and were internalized by mammalian cells. We provide evidence that adsorption of parasite antigens induced an increase in expression of extracellular matrix (ECM) components (fibronectin, laminin and type I collagen) by sensitized cells. Thus, our data reinforce the idea that in vivo T. cruzi released antigens might be involved in the establishment of inflammation, sensitizing non-infected host cells and triggering an immune response against parasite antigens. Further, our data showed that antigen sensitization modulates biological cell functions as ECM expression that could mediate cell-cell or parasite-host cell interactions, contributing to the establishment of inflammation.

Saliva ELISA: a method for the diagnosis of chronic Chagas disease in endemic areas.

An indirect enzyme linked immunosorbent assay (ELISA) was applied to saliva to detect chronic infection by Trypanosoma cruzi in humans. Saliva samples from 114 Chagas' disease chronically infected individuals, characterized by three serological tests and clinical evaluation and from 100 healthy controls were tested for T. cruzi specific IgG antibodies. At dilution of 1 in 2, specific antibodies were detected in saliva samples from 103 of 114 samples from infected patients and 5 of 100 controls (sensitivity 90.4%, specificity 95%). There was no significant correlation between the antibody titre and cardiac or gastrointestinal tract disease. This assay possesses some advantages over other methods as saliva collection is non-invasive, requires no special equipment and whole saliva gave reproducible results. Although serology remains the gold standard for T. cruzi infection, these results suggest that T. cruzi specific salivary antibody detection may provide a screening diagnostic test and contribute to epidemiological studies of chronic trypanosomiasis infection in endemic areas.

Preparative isolation of the lectin jacalin by anion-exchange high-performance liquid chromatography.

The lectin jacalin from Artocarpus integrifolia was purified to homogeneity in a single step by preparative anion-exchange high-performance liquid chromatography (HPLC). Selection of the optimum chromatographic parameters in gradient elution allowed a rapid procedure to be obtained for the qualitative and quantitative isolation of the most important alpha- and alpha'-jacalin components. A recovery of 27-33% was obtained from a total soluble extract using a polyacrylate-DEAE HPLC column. The identities of the two isolated polypeptides were established by N-terminal amino acid sequence analysis and from the IgA1 binding lectin activity.

A glass wool-based method for purifying Trypanosoma cruzi trypomastigotes and identification of an epimastigote-specific glass-adherent surface peptide.

Glass wool, hydrophilic cotton wool, non-electrically charged BIO-GEL P2 and common tissue paper columns were used to purify trypomastigotes from a mixed Trypanosoma cruzi population grown in axenic culture medium. With all these columns, highly purified (up to 98%) trypomastigote preparations were obtained. Trypomastigote yields from cotton wool, BIO-GEL P2 and common tissue paper columns were not as high as from glass wool columns, from which yields varied from 69 to 80%. Purification on glass wool did not affect trypomastigote infectivity or virulence. Dead trypomastigotes could not be purified on glass wool columns. A glass-adherent amphiphilic peptide of 45 kDa, present in the cell membrane, was isolated from epimastigote but not from trypomastigote preparations.

Isolation and immunological analysis of Trypanosoma cruzi glycolipids.

A water-soluble glycolipidic fraction from Trypanosoma cruzi was isolated using a mixture of hexane and isopropanol. Analysis by SDS-polyacrylamide gel electrophoresis, after staining by silver and Sudan Black B, showed that the fraction contained one band with a relatively high mobility. Its reactivity and specificity with human chagasic sera and T. cruzi infected mouse sera or with sera from patients with several other pathologies was determined by an immunoradiometric assay. The glycolipid-based radioimmunoassay for the detection of T. cruzi antigens provided a sensitive measure of its activity. However, cross-reactivity with several sera from patients with visceral and cutaneous leishmaniasis was detected.

Trypanosoma cruzi: identification of specific epimastigote antigens by human immune sera

Antígenos solúveis de epimastigotas de Trypanosoma cruzi foram analisados por "imunoblot" a fim de verificar sua reatividade com soros de pacientes com doença de Chagas. Além disso, soro de pacientes com leishmaniose visceral (LVA) e tegumentar americana (LTA) foram também analisados com o objetivo de se identificar oa antígenos de reaçäo cruzada com o Trypanosoma cruzi. Pelo menos 28 polipeptídeos, com pesos moleculares variando de 14 a 113 kDa foram identificados com soros de pacientes com doença de Chagas. Uma intensa reatividade cruzada foi observada quando foram utilizados soros de pacientes com leishmaniose visceral, enquanto que uma fraca reaçäo cruzada foi observada com soros de pacientes portadores de leishmaniose tegumentar. Por outro lado, pelo menos 10 polipeptídeos puderam ser identificados apresentando reaçäo específica com soros de pacientes chagásicos. Entre estes, os polipeptídeos de pesos moleculares de 46 kDa e 25 kDa que reagiram com todos esses soros e säo potencialmente bons candidatos a antígenos específicos no diagnósticos sorológico da doença de Chagas

Trypanosoma cruzi: identification of specific epimastigote antigens by human immune sera.

Soluble antigens from epimastigotes of Trypanosoma cruzi were analyzed by western blot in terms of their reactivity with sera from patients with Chagas' disease. In addition, sera from patients with visceral (AVL) and tegumental leishmaniasis (ATL) were also tested in order to identify cross-reactivities with Trypanosoma cruzi antigens. Twenty eight polypeptides with molecular weights ranging from 14 kDa to 113 kDa were identified with sera from Chagas' disease patients. An extensive cross-reactivity was observed when sera from human visceral leishmaniasis were used, while only a slight cross-reaction was observed with sera from tegumental leishmaniasis. On the other hand, 10 polypeptides specifically reacting with sera from Chagas' disease patients were identified. Among them, the antigens with molecular weights of 46 kDa and 25 kDa reacted with all sera tested and may be good candidates for specific immunodiagnosis of Chagas' disease.

Characterization of plasma menbrane polypeptides of trypanosoma from bats

As proteínas de superfície de Trypanosoma dionisii, Trypanosoma vespertilionis e Trypanosoma sp. (M238) foram radiodinados e sua distribuiçäo na fase rica em detergente (DRP) e fase pobre em detergente (DPP) foram estudadas pela técnica de separaçäo de fases com Triton X-114 e por eletroforese em gel e policrilamida em presença de dodecil sulfato de sódio (SDS-PAGE). Foram observadas diferenças significativas nas proteínas presentes na DRP quando a três espécies de tripanosomas foram comparadas. Duas bandas com 88 e 70 KDa foram observadas em T. sp. (M238) e näo foram detectadas em T. dionisii e t. vespertilionis. Três polípeptídeos com 96, 77 e 60 KDa foram identificados na fase DRP de T. vespertilionis. Três bandas com 84, 72 e 60 KDa foram visualizadas na fase DRP de T. dionisii. Dois polipeptídeos com 34-36 KDA presentes na fase DPP, foram observados nas três espécies de tripanosomas analisadas. Nossas observaçöes mostraram que T. sp. (M238) possui polipeptídeos de superfície característicos, que näo säo encontrados em T. dionisii e T. vespertilionis

Characterization of plasma membrane polypeptides of Trypanosoma from bats.

Cell surface proteins of Trypanosoma dionisii, Trypanosoma vespertilionis and Trypanosoma sp. (M238) were radiodinated and their distribution both in the detergent-poor (DPP) and detergent-enriched phase (DRP) was studied using a phase separation technique in Triton X-114, as well as polyacrylamide gel electrophoresis in sodium dodecyl sulphate (SDS-PAGE). Significant differences were observed in the proteins present in the DRP when the three species of trypanosoma were compared. Two major bands with 88 and 70 KDa were observed in T. sp. (M238) but were not detectable in T. dionisii and T. vespertilionis. Three polypeptides with 96, 77 and 60 KDa were identified in the DRP of T. vespertilionis. Three major bands with 84, 72 and 60 KDa were observed in the DRP of T. dionisii. Two polypeptides with 34-36 KDa present in the DPP, were observed in the three Trypanosome species analyzed. Our observations show that T. sp. (M238) has characteristic surface polypeptides not found in T. dionisii and T. vespertilionis.

A study on the amphiphilic proteins of three Trypanosoma cruzi populations

Amphiphilic proteins were partially purified from culture-derived metacyclic trypomastigotes of the CL and Colombian strains and of the Dm 28c clone of T. cruzi by the use of Triton X-114. These proteins were subjected to one-and two-dimensional polyacrylamide electrophoresis in the presence of sodium dodecyl sulphate. Relatively simple protein profiles with only 5 to 6 major bands were obtained. The CL and Colombian strains produced at least one additional major protein band (86 kDa) compared to the Dm 28c proteins. Trypomastigote amphiphilic proteins displayed both electrophoretic mobilities and isoelectric points identical to those of two polypeptides precipitated by a rabbit antiserum which recognized metacyclic trypomastigote-specific surface antigens. The partial purification of the T. cruzi amphiphilic proteins with Triton X-114 may provide a simple preparative step for the study of these differentiation-related polypeptides, as well as for the study of strain-specific (glyco)proteins and of their possible biological role

A study on the amphiphilic proteins of three Trypanosoma cruzi populations.

1. Amphiphilic proteins were partially purified from culture-derived metacyclic trypomastigotes of the CL and Colombian strains and of the Dm 28c clone of T. cruzi by the use of Triton X-114. These proteins were subjected to one- and two-dimensional polyacrylamide electrophoresis in the presence of sodium dodecyl sulphate. 2. Relatively simple protein profiles with only 5 to 6 major bands were obtained. The CL and Colombian strains produced at least one additional major protein band (86 kDa) compared to the Dm 28c proteins. 3. Trypomastigote amphiphilic proteins displayed both electrophoretic mobilities and isoelectric points identical to those of two polypeptides precipitated by a rabbit antiserum which recognized metacyclic trypomastigote-specific surface antigens. 4. The partial purification of the T. cruzi amphiphilic proteins with Triton X-114 may provide a simple preparative step for the study of these differentiation-related polypeptides, as well as for the study of strain-specific (glyco)proteins and of their possible biological role.

Interactions between Leishmania mexicana mexicana promastigotes and amastigotes and murine peritoneal macrophages in vitro.

Celulas peritoneais de camundongos cultivadas in vitro foram infectadas com inoculos identicos de amastigotas (AM) e de promastigotas, respectivamente, virulentas (VP), avirulentas (AVP) e fixadas (FP) de uma mesma cepa de Leishmania m. mexicana.As monocamadas coradas com May-Grunwald-Giemsa foram examinadas em diferentes intervalos de tempo. Ao nivel de 3a. hora pos-infeccao o numero de macrofagos contendo parasitas variou entre 60,5% (VP) e 84% (AM) nas monocamadas expostas aos parasitas viaveis, comparados com 6,5% para aquelas expostas aos parasitas fixados.Entretanto, nesse tempo de interacao, houve alteracoes degenerativas parasitarias e o numero de macrofagos contendo parasitas intatos variou significantemente entre as celulas infectadas com AM (84%) e aquelas infectadas com VP (42%) ou AVP (40%). O numero medio de parasitas intatos/macrofago tambem diferiu significantemente entre as celulas infectadas com AM e aquelas infectadas com promastigotas viaveis ou fixadas. A analise quantitativa de parasitas intatos e degenerados indicou uma multiplicacao e destruicao parasitaria nas monocamadas infectadas com VP e sobrevivencia e multiplicacao parasitarias naquelas infectadas com AM. Em contraste, nao houve evidencia de multiplicacao de parasitas nas celulas infectadas com AVP. Exses resultados sugerem que os eventos cruciais que determinam a evolucao da infeccao ocorrem durante as primeiras 24 horas da interacao parasito-hospedeiro. Esses eventos sao aparentemente influenciados nao somente pela cepa do parasita ou do hospedeiro, mas tambem por variacoes ambientais induzidas em uma dada cepa parasitaria

Macrophage-trypanosome infection. Dead and live cell scoring.

Descreve-se uma tecnica, usando azul de tripano e posterior fixacao, que permite visualizacao perfeita de T. cruzi no interior de macrofagos e ao mesmo tempo a distincao entre celulas hospedeiras vivas e mortas