RESUMO
Bone marrow mesenchymal stromal cells (BM-MSCs) with regenerative potential have been identified in heart. Whether these cells become new cardiac lineage cells by phenomena of transdifferentiation or fusion is also being investigated. Although, these mechanisms give cardiomyocytes, it has to be considered that MSCs transplantation could carry out ossification and calcification processes. An alternative might be the use of myocytes; however, the problem is the arrythmia. For those reasons, is that we investigated how to obtain cardiomyocyte-like cells from human MSCs (hMSCs). The aim of the present work was to evaluate a nuclear reprogramming of the hMSCs by a neonatal rat cardiomyocytes extract (EX) using Streptolysin O (SLO) treatment. hMSCs treated with 57.5ng/ml SLO presented ball-like, stick-like and myotube-like morphology. In the absence of cardiomyogenic stimuli, hMSCs expressed markers of cardiac phenotype-like sarcomeric alpha-actinin, connexin-43 and GATA-4. However, when hMSCs were treated with SLO+EX or 10 microM of 5-azacytidine (5-AZA), the expression of these markers were significantly increased and furthermore, expressed SERCA-2, cardiac Troponin I, beta-MyHC, desmin, MLC-2a and MLC-2v thus showing the phenotype of mature cardiomyocytes. PCR analysis showed that cardiomyocyte-related genes, such as beta1-adrenergic receptor (beta1-AR), MLC-2a and cardiac Troponin T, were expressed after SLO+EX treatment like with 5-AZA. We concluded that the extract of neonatal rat cardiomyocytes could promote a nuclear modification of hMSCs to cardiomyogenic-like cells differentiation. Since the 5-AZA treatment appears to be genotoxic and taking into account the obtained results, the nuclear reprogramming by cell extract may be an approach leading to the identification of soluble factors that drives the reprogramming.
Assuntos
Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Miócitos Cardíacos/metabolismo , Adolescente , Adulto , Animais , Azacitidina/farmacologia , Células da Medula Óssea/metabolismo , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Criança , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Miócitos Cardíacos/citologia , RatosRESUMO
Heart transplantation (HTx) is a useful therapy for end-stage Chagas cardiomyopathy; however, Chagas reactivation remains a mayor complication. Parasitological methods offer poor diagnostic sensitivity, and use of more sensitive tools such as the Polymerase chain reaction (PCR) is usually necessary. In the present study, reactivation incidence and PCR usefulness for early reactivation diagnosis, as well as for treatment response evaluation during follow-up, were analyzed using Strout parasite detection test, in 10 of 222 consecutive HTx patients suffering Chagas cardiomyopathy. PCR strategies targeted to minicircle sequences (kDNA, detection limit 1 parasite/ 10 mL blood) and miniexon genes (SL-DNA, 200 parasite/10 mL) were performed to compare parasite burdens between samples. No patients received prophylactic antiprotozoal therapy (benznidazole). Five patients (50%) exhibited clinical reactivation within a mean period of 71.6 days; positive Strout results were observed in most cases presenting clinical manifestations. kDNA-PCR was positive 38-85 days before reactivation, whereas SLDNA-PCR became positive only 7-21 days later, revealing post-HTx parasitic load enhancement present prior to clinical reactivation development. Reactivations were successfully treated with benznidazole and generated negative PCR results. Results observed in this study indicate the value of PCR testing for an early diagnosis of Chagas reactivation as well as for monitoring treatment efficacy.
Assuntos
Cardiomiopatia Chagásica/patologia , Cardiomiopatia Chagásica/cirurgia , Doença de Chagas/diagnóstico , Transplante de Coração , Adulto , Animais , Cardiomiopatia Chagásica/diagnóstico , Feminino , Seguimentos , Humanos , Imunossupressores/classificação , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Recidiva , Trypanosoma cruzi/genética , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
Chronic Chagas heart disease (cChHD), a chronic manifestation of the Trypanosoma cruzi infection, is characterized by high antibody levels against the C-terminal region of the ribosomal P proteins (i.e. peptide R13, EEEDDDMGFGLFD) which bears similarity with the second extracellular loop of beta1-adrenergic receptor (beta1-AR, peptide H26R HWWRAESDEARRCYNDPKCCDFVTNR). Because it has not been demonstrated clearly that IgGs from cChHD patients bind to native human beta1-AR, the aim of this study was to investigate further the physical interaction between cChHD IgGs and the human beta1-AR. Immunofluorescence assays demonstrated the binding of these antibodies to the receptor expressed on stably transfected cells, together with a beta1-AR agonist-like effect. In addition, immunoadsorption of the serum samples from cChHD patients with a commercially available matrix, containing peptides representing the first and the second extracellular loop of the beta1-AR, completely abolished reactivity against the H26R peptide and the physiological response to the receptor. The follow-up of this specificity after in vitro immunoadsorption procedures suggests that this treatment might be used to diminish significantly the serum levels of anti-beta1-AR antibodies in patients with Chagas heart disease.
Assuntos
Anticorpos Antiprotozoários/metabolismo , Autoanticorpos/metabolismo , Doença de Chagas/imunologia , Receptores Adrenérgicos beta 1/imunologia , Animais , Células CHO , Células COS , Chlorocebus aethiops , Doença Crônica , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Imunoglobulina G/metabolismo , Técnicas de Imunoadsorção , Fragmentos de Peptídeos , Transfecção , Trypanosoma cruzi/imunologiaRESUMO
Flagellates indistinguishable from Trypanosoma cruzi were detected by microscopy in faecal samples of 2/110 Triatoma guasayana and 2/283 Triatoma garciabesi captured in a rural area of northwestern Argentina. Inoculation of faecal homogenates to mice followed by xenodiagnosis, haemoculture, histopathology and culture from cardiac homogenates, and PCR based on T. cruzi minicircle and nuclear sequences failed to detect T. cruzi infection, pointing to another trypanosomatidean. A PCR strategy targeted to the D7 domain of 24salpha ribosomal DNA genes amplified a 250 bp sequence from one T. guasayana and one T. garciabesi faecal lysate. Sequence analysis revealed 100% identity with 24salpha rDNA amplicons from Blastocrithidia triatomae obtained from faeces of reared Triatoma infestans bugs. Phylogenetic analysis clustered this sequence with C. fasciculata and L. major, separated from the Trypanosoma branch (bootstrap: 968/1000), in concordance with a Neighbour-joining dendrogram based on 18s rDNA sequences. This PCR procedure provides a rapid sensitive tool for differential diagnosis of morphologically similar trypanosomatids in field surveys of Chagas disease vectors and laboratory-reared triatomines used for xenodiagnosis.
Assuntos
Insetos Vetores/parasitologia , Triatoma/parasitologia , Trypanosoma cruzi/isolamento & purificação , Trypanosomatina/isolamento & purificação , Animais , Argentina , Sequência de Bases , DNA de Cinetoplasto/química , DNA de Cinetoplasto/genética , Fezes/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , RNA Ribossômico/química , RNA Ribossômico/genética , População Rural , Alinhamento de Sequência , Análise de Sequência de DNA , Trypanosoma cruzi/classificação , Trypanosoma cruzi/genética , Trypanosomatina/classificação , Trypanosomatina/genética , XenodiagnósticoRESUMO
Antibodies from patients with Chagas heart disease and monoclonal antibodies (or mAb) to the carboxy-terminal end (B cell epitope R13) of the ribosomal P2beta protein of Trypanosoma cruzi (TcP2beta) cross-react with the beta1 adrenergic receptor (beta1-AR). Two single-chain Fv fragments (scFv) C5 and B7 derived from the variable regions of the anti-R13 mAb 17.2 were expressed. scFv C5 was a dimer and bound to TcP2beta with an affinity of K(d) = 8 nM, whereas scFv B7 was monomeric and had less affinity than scFv C5 for TcP2beta, K(d) = 46 nM. The affinity constant of scFv C5 to the second extracellular loop of the human beta1-AR was of 10 microM. Moreover, scFv C5 induced an increase in cAMP levels of CHO-K cells transfected with the human beta1-AR; scFv B7 had no effect but blocked isoproterenol stimulation. The agonist-like activity of scFv C5 and the antagonist activity of scFv B7 were both confirmed in vivo on heart beating frequency after their passive transfer to mice. Molecular modeling of the variable region of mAb 17.2 indicated which amino acids were likely to be involved in recognizing both peptide EDDDMGFGLF, derived from the R13 epitope of TcP2beta, and peptide ESDEARRCYN from the second extracellular loop of the human beta1-AR. It is plausible that the recently described cross-reaction of mAb 17.2 with rhodopsin can also be explained by this model. The physiological effects of this type of anti-T. cruzi antibodies may increase the liability of patients with Chagas disease.
Assuntos
Anticorpos Antiprotozoários/imunologia , Doença de Chagas/imunologia , Fosfoproteínas/imunologia , Proteínas de Protozoários/imunologia , Receptores Adrenérgicos beta 1/imunologia , Proteínas Ribossômicas/imunologia , Trypanosoma cruzi/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Cricetinae , Reações Cruzadas , Primers do DNA , Frequência Cardíaca , Humanos , Fragmentos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Reação em Cadeia da Polimerase , Proteínas de Protozoários/genética , RatosRESUMO
Long-term variations in the dynamics and intensity of sylvatic transmission of Trypanosoma cruzi were investigated around eight rural villages in the semiarid Argentine Chaco in 2002-2004 and compared to data collected locally in 1984-1991. Of 501 wild mammals from 13 identified species examined by xenodiagnosis, only 3 (7.9%) of 38 Didelphis albiventris opossums and 1 (1.1%) of 91 Conepatus chinga skunks were infected by T. cruzi. The period prevalence in opossums was four-fold lower in 2002-2004 than in 1984-1991 (32-36%). The infection prevalence of skunks also decreased five-fold from 4.1-5.6% in 1984-1991 to 1.1% in 2002-2004. Infection in opossums increased with age and from summer to spring in both study periods. The force of infection per 100 opossum-months after weaning declined more than six-fold from 8.2 in 1988-1991 to 1.2 in 2002-2004. Opossums were mainly infected by T. cruzi lineage I and secondarily by lineage IId in 1984-1991, and only by T. cruzi I in 2002-2004; skunks were infected by T. cruzi IId in 1984-1991 and by IIc in 2002-2004. The striking decline of T. cruzi infection in opossums and skunks occurred in parallel to community-wide insecticide spraying followed by selective sprays leading to very low densities of infected Triatoma infestans in domestic and peridomestic habitats since 1992; to massive deforestation around one of the villages or selective extraction of older trees, and apparent reductions in opossum abundance jointly with increases in foxes and skunks. These factors may underlie the dramatic decrease of T. cruzi infection in wild reservoir hosts.
Assuntos
Doenças dos Animais/epidemiologia , Doença de Chagas/veterinária , Conservação dos Recursos Naturais , Mamíferos/parasitologia , Árvores , Trypanosoma cruzi/isolamento & purificação , Doenças dos Animais/parasitologia , Animais , Argentina , Doença de Chagas/epidemiologiaRESUMO
This study applied improved DNA extraction and polymerase chain reaction strategies for screening and identification of Trypanosoma cruzi lineages directly from faeces of triatomines collected in a well-defined rural area in northwestern Argentina. Amplification of the variable regions of the kinetoplastid minicircle genome (kDNA-PCR) was performed in faecal lysates from 33 microscope (MO)-positive and 93 MO-negative Triatoma infestans, 2 MO-positive and 38 MO-negative Triatoma guasayana and 2 MO-positive and 73 MO-negative Triatoma garciabesi. kDNA-PCR detected T. cruzi in 91% MO-positive and 7.5% MO-negative T. infestans, which were confirmed by amplification of the minicircle conserved region. In contrast, kDNA-PCR was negative in all faecal samples from the other triatomine species. A panel of PCR-based genomic markers (intergenic region of spliced-leader DNA, 24Salpha and 18S rRNA genes and A10 sequence) was implemented to identify the parasite lineages directly in DNA lysates from faeces and culture isolates from 28 infected specimens. Two were found to be infected with TCI, 24 with TCIIe, 1 with TCIId and 1 revealed a mixed TCI+TCII infection in the faecal sample whose corresponding culture only showed TCII, providing evidence of the advantages of direct typing of biological samples. This study provides an upgrade in the current diagnosis and lineage identification of T. cruzi in field-collected triatomines and shows T. cruziII strains as predominant in the region.
Assuntos
DNA de Cinetoplasto/análise , Fezes/parasitologia , Insetos Vetores/parasitologia , Reação em Cadeia da Polimerase/métodos , Triatoma/parasitologia , Trypanosoma cruzi/classificação , Trypanosoma cruzi/isolamento & purificação , Animais , Argentina , Doença de Chagas/diagnóstico , Doença de Chagas/parasitologia , DNA de Cinetoplasto/isolamento & purificação , Amplificação de Genes , Humanos , Filogenia , Trypanosoma cruzi/genéticaRESUMO
BALB/c mice immunized with recombinant Trypanosoma cruzi ribosomal P2beta protein (TcP2beta) develop a strong and specific antibody response against its 13 residue-long C-terminal epitope (peptide R13: EEEDDDMGFGLFD) that has a concomitant beta1-adrenergic stimulating activity. However, other animals that undergo similar immunizations seem tolerant to this epitope. To evaluate further the antibody response against the ribosomal P proteins, 25 BALB/c and 25 Swiss mice were immunized with TcP2beta. From the 50 animals, 31 developed a positive anti-R13 response, whereas 19 were non-responsive. From the 31 anti-R13 positive mice, 25 had anti-R13 antibodies that recognized the discontinuous motif ExDDxGF, and their presence correlated with the recording of supraventricular tachycardia. The other six had anti-R13 antibodies but with a normal electrocardiographic recording. These anti-R13 antibodies recognized the motif DDxGF shared by mammals and T. cruzi and proved to be a true anti-P autoantibody because they were similar to those elicited in Swiss, but not in BALB/c mice, by immunization with the C-terminal portion of the mouse ribosomal P protein. Our results show that the recognition of the glutamic acid in position 3 of peptide R13 defines the ability of anti-R13 antibodies to react with the motif AESDE of the second extracellular loop of the beta1-adrenergic receptor, setting the molecular basis for their pathogenic beta1 adrenoceptor stimulating activity.
Assuntos
Anticorpos Antiprotozoários/imunologia , Epitopos/imunologia , Proteínas de Protozoários/imunologia , Receptores Adrenérgicos beta 1/imunologia , Proteínas Ribossômicas/imunologia , Trypanosoma cruzi/imunologia , Animais , Autoanticorpos/imunologia , Autoimunidade/imunologia , Doença de Chagas/imunologia , Doença de Chagas/fisiopatologia , Eletrocardiografia , Mapeamento de Epitopos/métodos , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/imunologiaRESUMO
High levels of antibodies against the C-terminus of the Trypanosoma cruzi TcP2 beta ribosomal protein, defined by the peptide EEEDDDMGFGLFD, named R13, have been measured in sera from patients with chronic Chagas' Heart Disease (cChHD). These antibodies also recognize an epitope on the second extracellular loop of the beta 1-adrenergic receptor, inducing a functional response on cardiomyocytes. The aim of this study was to gain novel insights into the structural basis of this cross-reactivity as well as to evaluate the origin of anti-M2- cholinergic receptor antibodies, which are also commonly found in cChHD patients. To address these questions we immunopurified anti-R13 antibodies and studied the structural requirements of epitope recognition. Results showed that the immunopurified antibodies recognized a conformation of R13 in which the third Glu residue was essential for binding, explaining their low affinity for the mammalian homologue (peptide H13: EESDDDMGFGLFD). Alanine mutation scanning showed individual variations in epitope recognition in each of the studied patients. The importance of a negatively charged residue at position 3 for the recognition of anti-R13 antibodies was further confirmed by competition experiments using a Ser3-phosphorylated H13 analogue, which had 10 times more affinity for the anti-R13 antibody than the native H13 peptide. Moreover, anti-R13 antibodies stimulated either the beta 1-adrenergic or the M2-cholinergic receptor, in strict agreement with the functional properties of the IgG fractions from which they derived, demonstrating that the same parasite antigen may generate antibody specificities with different functional properties. This may be a clue to explain the high variability of electrophysiological disturbances found in cChHD.
Assuntos
Anticorpos Antiprotozoários/sangue , Doença de Chagas/imunologia , Miócitos Cardíacos/imunologia , Proteínas Ribossômicas/imunologia , Trypanosoma cruzi/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Células Cultivadas , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática/métodos , Mapeamento de Epitopos , Epitopos/imunologia , Humanos , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos beta/imunologia , Receptores Colinérgicos/imunologiaRESUMO
Sera from patients with chronic Chagas heart disease recognize the carboxyl-terminal regions of the Trypanosoma cruzi ribosomal P proteins defined by B cell epitopes P013 (EDDDDDFGMGALF) and R13 (EEEDDDMGFGLFD) corresponding to the T. cruzi ribosomal P0 (TcP0) and P2beta (TcP2beta) proteins, respectively. It has been hypothesized that both epitopes may induce antibodies that cross-react and stimulate the beta1-adrenoreceptor. However, no proof as to their pathogenicity has been obtained. We investigated the consequences of immunizing mice with either TcP0 or TcP2beta proteins. Of 24 immunized animals, 16 generated antibodies against the carboxyl-terminal end of the corresponding protein, 13 of which showed an altered ECG (P<0.001, 81%). Immunization with TcP0 induced anti-P013 antibodies that bind to and stimulate cardiac G-protein-coupled receptors and are linked to the induction of supraventricular arrhythmia, repolarization, and conduction abnormalities as monitored by serial electrocardiographic analysis. In contrast, immunization with TcP2beta generated anti-R13 antibodies with an exclusive beta1-adrenergic-stimulating activity whose appearance strictly correlated with the recording of supraventricular tachycardia and death. These findings demonstrate that anti-P antibodies are arrhythmogenic in the setting of a normal heart, since no inflammatory lesions or fibrosis were evident to light microscopic examination.
Assuntos
Anticorpos Antiprotozoários/imunologia , Proteínas de Protozoários , Proteínas Ribossômicas/imunologia , Trypanosoma cruzi/imunologia , Alanina/genética , Sequência de Aminoácidos , Animais , Formação de Anticorpos , Células COS , Proteínas de Transporte/genética , Cardiomiopatia Chagásica/sangue , Cardiomiopatia Chagásica/imunologia , Cardiomiopatia Chagásica/fisiopatologia , Clonagem Molecular , Eletrocardiografia , Mapeamento de Epitopos , Glutationa Transferase/genética , Frequência Cardíaca/fisiologia , Humanos , Imunização , Imunoglobulina G/sangue , Proteínas Ligantes de Maltose , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutagênese , Miocárdio/citologia , Miocárdio/imunologia , Ratos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Trypanosoma cruzi/genéticaRESUMO
OBJECTIVE: To determine the safety and immunogenicity of varicella vaccine in children with human immunodeficiency virus (HIV) infection. Children (n = 41) who were mildly affected by HIV (Centers for Disease Control and Prevention stage N1 or A1) and had no history or serum antibody indicative of prior varicella infection were immunized with two doses of live attenuated varicella vaccine. RESULTS: A minority of the vaccine recipients had mild local or systemic reactions. Vaccination had no effect on the clinical stage of HIV or the HIV RNA plasma load. CD4 cell percentage and CD4 cell count were marginally decreased at week 4 after the first vaccination; this effect was no longer present at week 8 after vaccination. Two months after the second dose of vaccine, 60% of vaccine recipients had anti-varicella antibody in their serum, and 83% had a positive lymphocyte proliferation assay response to varicella antigen. CONCLUSION: On the basis of its safety and immunogenicity, varicella vaccine should be considered in the childhood vaccines given to mildly affected HIV-infected children.
Assuntos
Anticorpos Antivirais/sangue , Vacina contra Varicela/efeitos adversos , Varicela/imunologia , Infecções por HIV/imunologia , Contagem de Linfócito CD4 , Vacina contra Varicela/imunologia , Criança , Pré-Escolar , Humanos , Lactente , Estudos Multicêntricos como Assunto , Carga ViralRESUMO
Monoclonal antibodies were raised against a recombinant ribosomal P2beta protein of Trypanosoma cruzi. One of these reacted with the C terminus of this protein (peptide R13, EEEDDDMGFGLFD) and epitope mapping confirmed that this epitope was the same as the one defined by the serum of immunized mice, and similar to the previously described chronic Chagas' heart disease (cChHD) anti-P epitope. Western blotting showed that the monoclonal antibody recognized the parasite ribosomal P proteins, as well as the human ribosomal P proteins. Electron microscopy showed that it stained different structures in parasite and human cells. Interestingly, surface plasmon resonance measurements indicated that the affinity for the parasite ribosomal P protein epitope (R13) was five times higher than for its human counterpart (peptide H13, EESDDDMGFGLFD). Since the human epitope contained an acidic region (EESDD) similar to the AESDE peptide recognized by cChHD patients in the second extra-cellular loop of the human beta1-adrenergic receptor, the biological activity of the antibody was assessed on neonatal rat cardiomyocytes in culture. The monoclonal antibody had an agonist-like effect. These results, together with the fact that the monoclonal reacted in Western blots with the different isoforms of the heart beta1-adrenergic receptor, confirm the possible pathogenic role of antibodies against the parasite ribosomal P protein based on their cross-reaction with the human beta1-adrenergic receptor.
Assuntos
Anticorpos Antiprotozoários/imunologia , Autoanticorpos/imunologia , Proteínas de Protozoários/imunologia , Receptores Adrenérgicos beta 1/imunologia , Proteínas Ribossômicas/imunologia , Trypanosoma cruzi/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Células Cultivadas , Cardiomiopatia Chagásica/imunologia , Reações Cruzadas , Células HeLa , Humanos , Epitopos Imunodominantes/imunologia , Miocárdio/imunologia , Coelhos , Ratos , Ratos Wistar , Trypanosoma cruzi/ultraestruturaRESUMO
The P0 protein is part of the ribosomal eukaryotic stalk, which is an elongated lateral protuberance of the large ribosomal subunit involved in the translocation step of protein synthesis. P0 is the minimal portion of the stalk that is able to support accurate protein synthesis. The P0 C-terminal peptide is highly antigenic and a major target of the antibody response in patients with systemic lupus erythematosus and patients suffering chronic heart disease produced by the Trypanosoma cruzi parasite. The T. cruzi P0 (TcP0) protein was cloned into the pRSET A vector and expressed in Escherichia coli fused to a His-tag. The identity of the protein was confirmed by immunoblotting. Due to the formation of inclusion bodies the protein was purified using the following steps: (i) differential centrifugation to separate the inclusion bodies from soluble proteins and (ii) affinity chromatography under denaturing conditions. TcP0 showed high tendency to aggregation during refolding assays. However, TcP0 could be efficiently folded in the presence of a low concentration of SDS. The folding of the protein was confirmed using urea gradient electrophoresis, limited proteolysis, circular dichroism, and tryptophan fluorescence. Native electrophoresis showed that the folded TcP0 (and not a folding intermediate) was the cause of aggregation in the absence of SDS. The protocol described here permitted us to obtain large amounts (up to 30 mg per culture liter) of pure and folded TcP0, a very hydrophobic protein with a high tendency to aggregation.
Assuntos
Fosfoproteínas/química , Fosfoproteínas/genética , Dobramento de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Trypanosoma cruzi/química , Animais , Dicroísmo Circular , Vetores Genéticos , Substâncias Macromoleculares , Fosfoproteínas/biossíntese , Conformação Proteica , Desnaturação Proteica , Estrutura Secundária de Proteína , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/isolamento & purificaçãoRESUMO
Trypanosoma cruzi ribosomes from epimastigote forms were purified as determined by electron microscopy and isoelectrofocusing was used to analyse this purified ribosome fraction. Silver stained gels revealed that acidic proteins are present in at least 10 different isoforms, in accord with previous cloning studies. To detect phosphorylation, in vitro phosphorylation assays using the recombinant protein TcP2beta-mbp were carried out. The results showed that T. cruzi cytosolic fraction possesses protein kinase activity able to phosphorylate the recombinant protein. Purified ribosomes contain protein kinases that could also phosphorylate the recombinant protein TcP2beta-mbp. Labelling parasites with [(32)Pi] in a phosphate free medium demonstrated that ribosome proteins, recognised with a specific mouse antiserum against recombinant TcP2beta proteins, are phosphorylated in vivo. All these results suggest that in vivo phosphorylation of ribosome TcP2beta proteins are mediated by protein kinase(s) not yet identified.
Assuntos
Proteínas de Protozoários , Proteínas Ribossômicas/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , Microscopia Eletrônica , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/química , Ribossomos/química , Ribossomos/metabolismoRESUMO
BACKGROUND: The relationship between anti-beta-adrenergic (anti-betaR) and anti-M(2)-cholinergic (anti-M2R) receptor antibodies (Abs) and cardiac arrhythmias and their biochemical effects have not been systematically investigated. METHODS AND RESULTS: We studied 41 patients, 28 with ventricular arrhythmias (primary or due to Chagas' heart disease or idiopathic dilated cardiomyopathy; group I), 13 with sinus node dysfunction (primary or caused by Chagas' heart disease; group II), and 10 healthy controls (group III). The chronotropic effects of the IgG and immunopurified anti-beta(1)RAbs or anti-M2RAbs were assessed on cultured cardiomyocytes before and after exposure to atropine and propranolol. The biochemical effects of the IgG from 9 patients from group I, 6 from group II, and 6 controls were evaluated on COS7 cells transfected with genes encoding for beta(1),beta(2)-adrenergic receptors (cAMP increment) or M(2)-cholinergic receptors (phosphatidylinositol increment). The IgG from group I patients exerted a positive chronotropic action, with a high prevalence of anti-betaRAbs (75%) and low prevalence of anti-M2RAbs (10.7%) and induced a clear-cut and long-lasting increment in cAMP. The IgG from group II patients depressed chronotropism, with a high prevalence of anti-M2RAbs (76.9%) and low prevalence of anti-betaRAbs (15.4%) and evoked a marked augmentation of phosphatidylinositol. CONCLUSIONS: Our results demonstrate a strong correlation between anti-betaRAbs and ventricular arrhythmias and anti-M2RAbs and sinus node dysfunction. Anti-betaRAbs increase and anti-M2RAbs inhibit cAMP production. These findings offer new insight into the etiology and pathophysiology of cardiac arrhythmias, with therapeutic implications.
Assuntos
Arritmia Sinusal/imunologia , Arritmias Cardíacas/imunologia , Autoanticorpos/imunologia , Receptores Adrenérgicos beta/imunologia , Receptores Colinérgicos/imunologia , Adulto , Idoso , Sequência de Aminoácidos , Animais , Arritmia Sinusal/complicações , Arritmias Cardíacas/complicações , Atropina/farmacologia , Autoanticorpos/análise , Células COS , Cardiomiopatia Dilatada/complicações , Cardiomiopatia Dilatada/imunologia , Cardiomiopatia Chagásica/complicações , Cardiomiopatia Chagásica/imunologia , AMP Cíclico/metabolismo , Eletrocardiografia , Feminino , Frequência Cardíaca/efeitos dos fármacos , Humanos , Imunoglobulina G/análise , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fosfatidilinositóis/metabolismo , Propranolol/farmacologia , Receptores Adrenérgicos beta/química , Receptores Adrenérgicos beta/genética , Receptores Colinérgicos/química , Receptores Colinérgicos/genética , Sistemas do Segundo Mensageiro/efeitos dos fármacosAssuntos
Proteínas de Transporte/genética , Genes de Protozoários , Proteínas de Filamentos Intermediários/genética , Proteínas de Protozoários/genética , RNA Helicases/genética , Trypanosoma cruzi/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Northern Blotting , Southern Blotting , Proteínas de Transporte/química , Mapeamento Cromossômico , RNA Helicases DEAD-box , Etiquetas de Sequências Expressas , Proteínas de Filamentos Intermediários/química , Sequências Repetitivas Dispersas , Dados de Sequência Molecular , Fases de Leitura Aberta , Proteínas de Protozoários/química , RNA Helicases/química , Análise de Sequência de DNA , Transcrição Gênica , Trypanosoma cruzi/química , Trypanosoma cruzi/enzimologiaRESUMO
Heart transplantation is contraindicated as an effective treatment for end-stage Chagas' heart disease because of post-operative recurrence of Trypanosoma cruzi infection and reactivation of disease after immunosupression. In a follow-up study of a heart transplanted patient with Chagas' disease, we prospectively evaluated the usefulness of the polymerase chain reaction (PCR) for early diagnosis of reactivation. We monitored post-operative recurrence of Trypanosoma cruzi infection with microscopic observation of the parasite in peripheral blood (Strout's method), endomyocardial biopsies (EMBs), skin lesions, and 2 PCR assays, based on the amplification of specific T cruzi kinetoplastid and nuclear DNA sequences. During follow-up, parasite DNA was amplified in blood samples and EMB sections 41 days before we observed patent parasitemia and cutaneous manifestations of reactivation, proving that PCR is much more sensitive than direct microscopic observation for early diagnosis of disease reactivation in heart-transplanted Chagas' disease patients.
Assuntos
Cardiomiopatia Chagásica/cirurgia , Transplante de Coração , Reação em Cadeia da Polimerase , Complicações Pós-Operatórias/diagnóstico , Trypanosoma cruzi/isolamento & purificação , Animais , Biópsia , Cardiomiopatia Chagásica/sangue , Cardiomiopatia Chagásica/diagnóstico , Endocárdio/patologia , Feminino , Seguimentos , Rejeição de Enxerto/sangue , Rejeição de Enxerto/diagnóstico , Humanos , Pessoa de Meia-Idade , Miocárdio/patologia , Complicações Pós-Operatórias/sangue , Valor Preditivo dos Testes , Estudos Prospectivos , RecidivaRESUMO
The short interspersed repetitive element (SIRE) of Trypanosoma cruzi was first detected when comparing the sequences of loci that encode the TcP2beta genes. It is present in about 1,500-3,000 copies per genome, depending on the strain, and it is distributed in all chromosomes. An initial analysis of SIRE sequences from 21 genomic fragments allowed us to derive a consensus nucleotide sequence and structure for the element, consisting of three regions (I, II, and III) each harboring distinctive features. Analysis of 158 transcribed SIREs demonstrates that the consensus is highly conserved. The sequences of 51 cDNAs show that SIRE is included in the 3' end of several mRNAs, always transcribed from the sense strand, contributing the polyadenylation site in 63% of the cases. This study led to the characterization of VIPER (vestigial interposed retroelement), a 2,326-bp-long unusual retroelement. VIPER's 5' end is formed by the first 182 bp of SIRE, whereas its 3' end is formed by the last 220 bp of the element. Both SIRE moieties are connected by a 1,924-bp-long fragment that carries a unique ORF encoding a complete reverse transcriptase-RNase H gene whose 15 C-terminal amino acids derive from codons specified by SIRE's region II. The amino acid sequence of VIPER's reverse transcriptase-RNase H shares significant homology to that of long terminal repeat retrotransposons. The fact that SIRE and VIPER sequences are found only in the T. cruzi genome may be of relevance for studies concerning the evolution and the genome flexibility of this protozoan parasite.
Assuntos
Proteínas de Protozoários/genética , Retroelementos , Proteínas Ribossômicas/genética , Elementos Nucleotídeos Curtos e Dispersos , Sequências Repetidas Terminais , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Consenso , DNA de Protozoário , Expressão Gênica , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
As part of the Trypanosoma cruzi Genome Initiative, we have mapped a large portion of the chromosomal bands XVI (2.3 Mb) and XVII (2.6 Mb) containing the highly repetitive and immunodominant antigenic gene families h49 and jl8. Restriction mapping of the isolated chromosomal bands and hybridization with chromosome specific gene probes showed that genes h49 and jl8 are located in a pair of size-polymorphic homologous chromosomes. To construct the integrated map of the chromosomes harboring the h49 and jl8 loci, we used YAC, cosmid, and lambda phage overlapping clones, and long range restriction analysis using a variety of probes (i.e., known gene sequences, ESTs, polymorphic repetitive sequences, anonymous sequences, STSs generated from the YAC ends). The total length covered by the YAC contig was approximately 670 kb, and its map agreed and was complementary to the one obtained by long-range restriction fragment analysis. Average genetic marker spacing in a 105 kb region around h49 and jl8 genes was estimated to be 6.2 kb/marker. We have detected some polymorphism in the H49/JL8 antigens-encoding chromosomes, affecting also the coding regions. The physical map of this region, together with the isolation of specific chromosome markers, will contribute in the global effort to sequence the nuclear genome of this parasite.
Assuntos
Antígenos de Protozoários/genética , Mapeamento Físico do Cromossomo , Trypanosoma cruzi/genética , Animais , Bandeamento Cromossômico , Cromossomos Artificiais de Levedura/genética , Mapeamento de Sequências Contíguas , Sondas de DNA/genética , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Genes de Protozoários/genética , Humanos , Epitopos Imunodominantes/genética , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
The short interspersed repetitive element (SIRE) of the nuclear genome of Trypanosoma cruzi was first detected when comparing the sequences of loci that encode the TcP2beta genes. The present study was designed to assess its distribution and organization in the nuclear genome of the parasite. Southern blots of genomic DNA from different strains demonstrated that each one possesses a defined and characteristic pattern of SIRE distribution. The conservation of the SIRE sequence in T. cruzi strains allowed the development of a rapid inter-SIRE PCR reaction that yields strain-specific amplicon profiles. In the T. cruzi CL Brener clone, we found 1500 copies of the element distributed in all chromosomes. 16 genomic fragments containing SIRE (SZs) were isolated and characterized. In fragments SZ10, SZ12 and SZ31, SIRE was linked to TcRel, a novel repeated sequence that constitutes the 3' end of vp85 genes. SIRE was also linked to an unknown open reading frame in fragments SZ14 and SZ23 which might be related to the subtelomeric regions of T. cruzi chromosomes. Further sequencing of SZ fragments revealed that SIRE was also linked to protein coding genes that have not yet been described in kinetoplastids such as the one coding for PRP22 helicase and a thimet oligopeptidase. To allow the rapid-generation genetic markers associated with SIRE, we developed a SIRE-bubble PCR reaction that provided several such markers for the construction of the physical map of chromosome XVI. The results herein demonstrate that SIRE-associated sites (SAS) may be of great help in physical mapping and interpretation of T. cruzi genomic sequence data.