RESUMEN
BACKGROUND: To deeply understand the role of antibodies in the context of Trypanosoma cruzi infection, we decided to characterize A2R1, a parasite antibody selected from single-chain variable fragment (scFv) phage display libraries constructed from B cells of chronic Chagas heart disease patients. METHODS: Immunoblot, ELISA, cytometry, immunofluorescence and immunohistochemical assays were used to characterize A2R1 reactivity. To identify the antibody target, we performed an immunoprecipitation and two-dimensional electrophoresis coupled to mass spectrometry and confirmed A2R1 specific interaction by producing the antigen in different expression systems. Based on these data, we carried out a comparative in silico analysis of the protein target´s orthologues, focusing mainly on post-translational modifications. FINDINGS: A2R1 recognizes a parasite protein of ~50 kDa present in all life cycle stages of T. cruzi, as well as in other members of the kinetoplastid family, showing a defined immunofluorescence labeling pattern consistent with the cytoskeleton. A2R1 binds to tubulin, but this interaction relies on its post-translational modifications. Interestingly, this antibody also targets mammalian tubulin only present in brain, staining in and around cell bodies of the human peripheral and central nervous system. INTERPRETATION: Our findings demonstrate for the first time the existence of a human antibody against T. cruzi tubulin capable of cross-reacting with a human neural protein. This work re-emphasizes the role of molecular mimicry between host and parasitic antigens in the development of pathological manifestations of T. cruzi infection.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Antiprotozoarios/farmacología , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Proteínas Recombinantes de Fusión/farmacología , Trypanosoma cruzi/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antiprotozoarios/inmunología , Anticuerpos Antiprotozoarios/uso terapéutico , Especificidad de Anticuerpos/inmunología , Antígenos de Protozoos/inmunología , Línea Celular , Clonación Molecular , Reacciones Cruzadas/inmunología , Desarrollo de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Inmunoprecipitación , Espectrometría de Masas , Ratones , Imitación Molecular , Ratas , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/uso terapéutico , Análisis de Secuencia de ADN , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/farmacología , Anticuerpos de Cadena Única/uso terapéuticoRESUMEN
Trypanosoma cruzi is a flagellate protozoan pathogen that causes Chagas disease. Currently there is no preventive treatment and the efficiency of the two drugs available is limited to the acute phase. Therefore, there is an unmet need for innovative tools to block transmission in endemic areas. In this study, we engineered a novel recombinant molecule able to adhere to the T. cruzi surface, termed scFv-10D8, that consists of a single-chain variable fragment (scFv) derived from mAb-10D8 that targets gp35/50. The synthetic gene encoding scFv-10D8 was cloned and fused to a 6×His tag and expressed in a prokaryotic expression system. Total periplasmic or 6xHis tag affinity-purified fractions of scFv-10D8 retained the capacity to bind to gp35/50, as shown by Western blot analyses. Pre-incubation of metacyclic trypomastigotes with scFv-10D8 showed a remarkable reduction in cell invasion capacity. Our results suggest that scFv-10D8 can be used in a paratransgenic approach to target parasites in insect vectors, avoiding dissemination of infective forms. Such advances in the development of this functional molecule will surely prompt the improvement of alternative strategies to control Chagas disease by targeting mammalian host stages.
Asunto(s)
Antígenos de Protozoos/inmunología , Ingeniería de Proteínas/métodos , Anticuerpos de Cadena Única/genética , Trypanosoma cruzi/inmunología , Anticuerpos Antiprotozoarios/genética , Anticuerpos Antiprotozoarios/farmacología , Línea Celular , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Enfermedad de Chagas/prevención & control , Células HeLa , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Anticuerpos de Cadena Única/farmacología , Trypanosoma cruzi/efectos de los fármacosRESUMEN
The aim of this study was to evaluate the therapeutic efficacy of specific avian polyclonal antibodies (IgY) against Trypanosoma cruzi and their interaction with ecto-enzymes of the purinergic system (NTPDase and adenosine deaminase (ADA) activities) in splenic lymphocytes. For this, mice were divided into six groups: three non-infected (A, B, and C) and three infected (D, E, and F). The groups A and D were composed by negative and positive controls, respectively; while the groups B and E were treated prophylactically with IgY (50 mg/kg), and the groups C and F were treated therapeutically with IgY (50 mg/kg). Treatment with IgY reduced parasitemia on day 6 post-infection (PI) compared to the infected control group, but it was similar on day 8 PI. Moreover, infected and treated animals (the groups E and F) did not show neither amastigotes in the cardiac tissue nor cardiac lesions when compared to the positive control group (the group D). The E-NTPDase (ATP and ADP as substrate) and ADA activities in splenic lymphocytes increased significantly in the positive control group (the group D) compared to the negative control group (the group A). The therapeutic treatment of IgY (the group F) was able to prevent the increase of E-NTPDase and E-ADA activities compared to the positive control group (the group D), but this finding was not observed in animals that received the prophylactic treatment (the group E). The therapeutic treatment of IgY may be considered an interesting approach to improve the immune response of mice experimentally infected by T. cruzi.
Asunto(s)
Adenosina Desaminasa , Anticuerpos Antiprotozoarios/farmacología , Proteínas Aviares/farmacología , Enfermedad de Chagas , Inmunoglobulinas/farmacología , Proteínas Protozoarias , Bazo , Trypanosoma cruzi , Adenosina Desaminasa/inmunología , Adenosina Desaminasa/metabolismo , Animales , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/enzimología , Enfermedad de Chagas/inmunología , Pollos , Femenino , Linfocitos/enzimología , Linfocitos/inmunología , Linfocitos/patología , Ratones , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Bazo/enzimología , Bazo/inmunología , Bazo/parasitología , Bazo/fisiología , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/inmunologíaRESUMEN
The ribosomal P proteins are located on the stalk of the ribosomal large subunit and play a critical role during the elongation step of protein synthesis. The single chain recombinant antibody C5 (scFv C5) directed against the C-terminal region of the Trypanosoma cruzi P2ß protein (TcP2ß) recognizes the conserved C-terminal end of all T. cruzi ribosomal P proteins. Although this region is highly conserved among different species, surface plasmon resonance analysis showed that the scFv C5 possesses very low affinity for the corresponding mammalian epitope, despite having only one single amino-acid change. Crystallographic analysis, in silico modelization and NMR assays support the analysis, increasing our understanding on the structural basis of epitope specificity. In vitro protein synthesis experiments showed that scFv C5 was able to specifically block translation by T. cruzi and Crithidia fasciculata ribosomes, but virtually had no effect on Rattus norvegicus ribosomes. Therefore, we used the scFv C5 coding sequence to make inducible intrabodies in Trypanosoma brucei. Transgenic parasites showed a strong decrease in their growth rate after induction. These results strengthen the importance of the P protein C terminal regions for ribosomal translation activity and suggest that trypanosomatid ribosomal P proteins could be a possible target for selective therapeutic agents that could be derived from structural analysis of the scFv C5 antibody paratope.
Asunto(s)
Anticuerpos Antiprotozoarios/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Protozoarias/biosíntesis , Proteínas Ribosómicas/antagonistas & inhibidores , Anticuerpos de Cadena Única/farmacología , Trypanosoma cruzi/metabolismo , Anticuerpos Antiprotozoarios/química , Anticuerpos Antiprotozoarios/genética , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/metabolismo , Epítopos/química , Epítopos/inmunología , Expresión Génica , Humanos , Modelos Moleculares , Filogenia , Unión Proteica/efectos de los fármacos , Conformación Proteica , Proteínas Protozoarias/clasificación , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Proteínas Ribosómicas/biosíntesis , Proteínas Ribosómicas/clasificación , Proteínas Ribosómicas/inmunología , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genética , Trypanosoma cruzi/genética , Trypanosoma cruzi/inmunologíaRESUMEN
La enfermedad de Chagas o tripanosomiasis sudamericana, es una parasitosis crónica causada por el protozoario flagelado Tripanosoma cruzi. Este agente puede ser transmitido de la madre al hijo por vía transplacentaria durante la fase aguda y/o crónica de la infección materna. Objetivo: Evaluar la seroprevalencia de la enfermedad de Chagas en gestantes y en niños de madres seropositivas a T. cruzi y su relación con algunos factores condicionantes en el Municipio Roscio del estado Guárico. Métodos: Se realizó un estudio de tipo transversal, estudiando la enfermedad de Chagas en gestantes y en niños (as) nacidos de madres reactivas al T. cruzi. La muestra estuvo conformado por una población representada por todas las gestantes (925) que acudieron al primer control de embarazo en la red primaria de atención, y otra, por los hijos (as) (2) nacidos de madres seropositivas a T. cruzi. Resultados: Se detectaron dos gestantes reactivas utilizando Anticuerpos IgG, obteniéndose una seroprevalencia de 0,22 por ciento. Los dos hijos nacidos de estas madres reactivas resultaron No Reactivos mediante la técnica parasitológica directa (Microhematocrito), confirmando el diagnóstico con ELISA y Hemaglutinación Indirecta.
Asunto(s)
Femenino , Embarazo , Recién Nacido , Enfermedad de Chagas/congénito , Enfermedad de Chagas/transmisión , Estudios Seroepidemiológicos , Trypanosoma cruzi/genética , Anticuerpos Antiprotozoarios/farmacología , Terapia Conductista , Epidemiología , Transmisión Vertical de Enfermedad InfecciosaRESUMEN
Phagocytic removal of apoptotic lymphocytes exacerbates replication of Trypanosoma cruzi in macrophages. We investigated the presence of Ab against apoptotic lymphocytes in T. cruzi infection and the role of these Ab in parasite replication. Both control and chagasic serum contained IgG Ab that opsonized apoptotic lymphocytes. Treatment of apoptotic lymphocytes with purified IgG from chagasic, but not control serum, reduced T. cruzi replication in macrophages. The protective effect of chagasic IgG depended on Fcgamma receptors, as demonstrated by the requirement for the intact Fc portion of IgG, and the effect could be abrogated by treating macrophages with an anti-CD16/CD32 Fab fragment. Chagasic IgG displayed increased reactivity against a subset of apoptotic cell Ag, as measured by flow cytometry and immunoblot analyses. Apoptotic lymphocytes treated with chagasic IgG, but not control IgG, increased production of TNF-alpha, while decreasing production of TGF-beta1 by infected macrophages. Increased control of parasite replication required TNF-alpha production. Previous immunization with apoptotic cells or injection of apoptotic cells opsonized with chagasic IgG reduced parasitemia in infected mice. These results indicate that Ab raised against apoptotic cells could play a protective role in control of T. cruzi replication by macrophages.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Enfermedad de Chagas/inmunología , Linfocitos/inmunología , Macrófagos/inmunología , Trypanosoma cruzi/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Traslado Adoptivo , Animales , Anticuerpos Antiprotozoarios/farmacología , Apoptosis , Células Cultivadas , Enfermedad de Chagas/parasitología , Enfermedad de Chagas/terapia , Técnicas de Cocultivo , Citometría de Flujo , Immunoblotting , Inmunoglobulina G/inmunología , Inmunoglobulina G/farmacología , Linfocitos/citología , Linfocitos/efectos de los fármacos , Macrófagos/citología , Macrófagos/parasitología , Masculino , Ratones , Ratones Endogámicos BALB C , Parasitemia/inmunología , Parasitemia/parasitología , Parasitemia/terapia , Fagocitosis , Factor de Crecimiento Transformador beta1/metabolismo , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
Trypanosoma evansi and Trypanosoma vivax have shown a very high immunological cross-reactivity. Anti-T. vivax antibodies were used to monitor changes in the T. evansi intracellular Ca2+ concentration ([Ca2+]i) by fluorometric ratio imaging from single parasites. A short-time exposure of T. evansi parasites to sera from T. vivax-infected bovines induced an increase in [Ca2+]i, which generated their complete lysis. The parasite [Ca2+]i boost was reduced but not eliminated in the absence of extracellular Ca2+ or following serum decomplementation. Decomplemented anti-T. evansi VSG antibodies also produced an increase in the parasite [Ca2+]i, in the presence of extracellular Ca2+. Furthermore, this Ca2+ signal was reduced following blockage with Ni2+ or in the absence of extracellular Ca2+, suggesting that this response was a combination of an influx of Ca2+ throughout membrane channels and a release of this ion from intracellular stores. The observed Ca2+ signal was specific since (i) it was completely eliminated following pre-incubation of the anti-VSG antibodies with the purified soluble VSG, and (ii) affinity-purified anti-VSG antibodies also generated an increase in [Ca2+]i by measurements on single cells or parasite populations. We also showed that an increase of the T. evansi [Ca2+]i by the calcium A-23187 ionophore led to VSG release from the parasite surface. In addition, in vivo immunofluorescence labelling revealed that anti-VSG antibodies induced the formation of raft patches of VSG on the parasite surface. This is the first study to identify a ligand that is coupled to calcium flux in salivarian trypanosomes.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Anticuerpos Antiprotozoarios/farmacología , Señalización del Calcio/efectos de los fármacos , Trypanosoma vivax/inmunología , Trypanosoma/inmunología , Tripanosomiasis Bovina/inmunología , Glicoproteínas Variantes de Superficie de Trypanosoma/inmunología , Animales , Especificidad de Anticuerpos , Antígenos de Protozoos/inmunología , Calcio/metabolismo , Bovinos , Proteínas del Sistema Complemento , Sueros Inmunes , Trypanosoma/clasificación , Trypanosoma/metabolismo , Trypanosoma vivax/metabolismo , Trypanosoma vivax/patogenicidad , Tripanosomiasis Bovina/parasitología , Glicoproteínas Variantes de Superficie de Trypanosoma/aislamiento & purificaciónRESUMEN
To date, there are no vaccines against Leishmania, and chemotherapy remains the mainstay for the control of leishmaniasis. The drugs of choice used for leishmaniasis therapy are significantly toxic, expensive and with a growing frequency of refractory infections. Because of these limitations, a combination therapy is the better hope. This work demonstrates that the essential oil from Chenopodium ambrosioides shows a synergic activity after incubation in conjunction with pentamidine against promastigotes of Leishmania amazonensis. However, an indifferent effect has been found for combinations of meglumine antimoniate or amphotericin B and the essential oil(AU)
Asunto(s)
Animales , Ratones , Anticuerpos Antiprotozoarios/farmacología , Chenopodium ambrosioides/química , Aceites Volátiles/farmacología , Pruebas de Sensibilidad Parasitaria , Aceites de Plantas/farmacocinética , Pentamidina/farmacologíaRESUMEN
Trichomonas vaginalis infects the epithelium of the genital tract. The mechanism by which it invades the tissue leading to the disease is not thoroughly understood. However, results of several studies seem to agree that parasite adhesion to epithelium cells is the initial step leading to infection in women. T. vaginalis is associated with high levels of proteolytic activity. The role of some of these proteinases in the development of infection has been demonstrated. The current study establishes the role of a 62 kDa excretion-secretion proteinase in parasite cytoadherence. Monoclonal antibodies (MAbs) against this enzyme were tested for their ability to inhibit this process. Three stable hybrid producers of IgG(1)class MAbs (4D8, 1A8, 3C11) against the 62 kDa proteinase were obtained. Two of them (4D8 and 1A8) showed parasite recognition by immunofluorescence. Parasite cytoadherence to a monolayer of HeLa cells was inhibited by the 4D8, 1A8 and 3C11 antibodies. MAb 4D8 administered 24 h before a challenge with T. vaginalis by the intraperitoneal route was able to protect the majority of mice. Nitric oxide levels in the serum of animals inoculated with MAb 4D8 and challenged with the parasite were significantly different from those recorded in mice treated with an unrelated MAb. These studies show that an appropriate antibody against 62 kDa proteinase can help the host resist a challenge by the intraperitoneal route with T. vaginalis.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Antiprotozoarios/farmacología , Endopeptidasas/inmunología , Trichomonas vaginalis/enzimología , Trichomonas vaginalis/inmunología , Animales , Adhesión Celular , Endopeptidasas/química , Endopeptidasas/aislamiento & purificación , Células Epiteliales/inmunología , Células Epiteliales/parasitología , Femenino , Células HeLa , Humanos , Inmunización Pasiva , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Vaginitis por Trichomonas/inmunología , Vaginitis por Trichomonas/parasitología , Vaginitis por Trichomonas/prevención & control , Trichomonas vaginalis/patogenicidadRESUMEN
It has been proposed that anti-myocardial antibodies (Ab) against neurotransmitter (NT) receptors are involved in the immunopathology of chronic Chagas' heart disease. We demonstrated that an anti-Trypanosoma cruzi monoclonal Ab (mAb), CAK20.12, binds to murine cardiac beta-adrenergic and muscarinic acetyl choline (mACh) receptors eliciting abnormal physiological responses on normal heart. No cross-linking requirement for mAb actions was demonstrated using Fab fragment derived from CAK20.12. mAb binding to synthetic peptides from the second extracellular loop of both beta1-adrenergic and mACh receptors, demonstrated by ELISA, identified the region of NT receptors involved. Cross-reactivity between these peptides and T. cruzi antigen was confirmed by binding inhibition assays. These results support the existence of cross-reactivity due to molecular mimicry between a parasite antigen and the major antigenic epitopes present on both beta1-adrenergic and M2-ACh receptors. Its possible relationship with cardiac dysfunction during chronic stage of Chagas' disease is also discussed.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Antiprotozoarios/farmacología , Contracción Miocárdica/efectos de los fármacos , Pindolol/análogos & derivados , Receptor Muscarínico M2/inmunología , Receptores Adrenérgicos beta 1/inmunología , Trypanosoma cruzi/inmunología , Antagonistas Adrenérgicos beta , Análisis de Varianza , Animales , Unión Competitiva/efectos de los fármacos , Unión Competitiva/fisiología , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos/metabolismo , Epítopos/farmacología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Técnicas In Vitro , Isótopos de Yodo/farmacocinética , Ratones , Ratones Endogámicos BALB C , Antagonistas Muscarínicos/farmacocinética , Contracción Miocárdica/fisiología , Pindolol/farmacocinética , Quinuclidinil Bencilato/farmacocinética , Radioinmunoensayo/métodos , Ensayo de Unión Radioligante/métodos , Receptor Muscarínico M2/química , Receptores Adrenérgicos beta 1/química , Volumetría/métodos , Trypanosoma cruzi/químicaRESUMEN
In this work it was shown that the infectivity of trypomastigote forms of Trypanosoma cruzi was affected upon the interaction with the Monoclonal Antibody (McAb) 2E9, which was raised against a glycoconjugated fraction of membranes of epimastigotes (Tulahuen strain). Characterization of the epitope recognized by this McAb, as well as its effects on complement mediated lysis and host cell invasion are reported. Immunocytochemical analysis showed that the McAb was reactive with two macromolecules (41-58 kDa) present on Trypanosoma cruzi epimastigotes (Tulahuen and Y strain), while it recognized several trypomastigotes macromolecules, showing a more intense reactivity with a band of 80 kDa. By indirect immunofluorescence, it was found there were subpopulations of blood and tissue culture derived trypomastigotes which attach the antibody to varying degrees. Studies using chemical or enzymatically treated antigens suggested that the McAb 2E9 was directed against carbohydrate epitopes, which were identified as being--galactosyl residues. In addition, preliminary results are shown, suggesting that the epitope recognized by the McAb 2E9 is involved in adhesion/or internalization of trypomastigotes.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Antiprotozoarios/farmacología , Glicoconjugados/inmunología , Trypanosoma cruzi/crecimiento & desarrollo , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antiprotozoarios/inmunología , Especificidad de Anticuerpos , Antígenos de Protozoos/inmunología , Carbohidratos/inmunología , Línea Celular , Enfermedad de Chagas/prevención & control , Epítopos/inmunología , Eritrocitos/parasitología , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Parasitemia/prevención & control , Proteínas Protozoarias/inmunología , Porcinos , Trypanosoma cruzi/inmunologíaRESUMEN
We have already demonstrated the presence of antibodies in the sera of chagasic patients with the ability to interact with neurotransmitter receptors triggering several intracellular pathways of transduction signals. Here we show that, chagasic IgG induced protein kinase C (PKC) translocation to rat cardiac membranes and this effect was inhibited by muscarinic cholinergic blockers atropine and AF-DX 116 pointing to the participation of M2 receptors in this effect. It was also able to stimulate nitric oxide synthase (NOS) activity and this action was blunted by phospholipase C (PLC) and PKC inhibitors indicating that the production of nitric oxide (NO) would be the consequence of the cascade of enzymatic pathways triggered by mAChR activation. PKC and NOS activities were involved in chagasic IgG negative inotropic actions on rat isolated myocardium as its effects were blunted by staurosporine and L-N-monomethyl arginine. Furthermore, low concentrations of chagasic IgG inhibited the cardiac mechanical action of carbachol in a non-competitive manner. These data suggested that PKC activation in myocardium by chagasic IgG would be involved in its physiological actions by modulating NOS activity. The participation of PKC-mediated phosphorylation of mAChR leading to receptor desensitization as one of the causes of dysautonomia is also discussed.