RESUMO
Vascular endothelial growth factor (VEGF) gene transfer-mediated angiogenesis has been proposed for peripheral artery disease. However, protocols using single administration have shown little benefit. Given that the transient nature of VEGF gene expression provokes instability of neovasculature, we hypothesized that repeated administration would provide efficient tissue protection. We thus compared single vs repeated transfection in a rabbit model of hindlimb ischemia by injecting a plasmid encoding human VEGF165 (pVEGF165) at 7 (GI, n=10) or 7 and 21 (GII, n=10) days after surgery. Placebo animals (GIII, n=10) received empty plasmid. Fifty days after surgery, single and repeated administration similarly increased saphenous peak flow velocity and quantity of angiographically visible collaterals. However, microvasculature increased only with repeated transfection: capillary density was 49.4+/-15.4 capillaries per 100 myocytes in GI, 84.6+/-14.7 in GII (P<0.01 vs GI and GIII) and 49.3+/-13.6 in GIII, and arteriolar density was 1.9+/-0.6 arterioles per mm2 in GI, 3.0+/-0.9 in GII (P<0.01 vs GI and GIII) and 1.5+/-0.6 in GIII. Muscle lesions were reduced only within repeated transfection. With single administration, gene expression peaked at 7 days and declined rapidly, but with repeated administration, it remained positive at 50 days. At 90 days of repeated transfection (additional animals), gene expression decreased significantly, but neovessel densities did not. Thus, repeated, but not single, VEGF gene transfection resulted in increased microvasculature, which, in turn, afforded effective protection against ischemic muscle damage.
Assuntos
Terapia Genética/métodos , Isquemia/terapia , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Doenças Vasculares Periféricas/terapia , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Modelos Animais de Doenças , Expressão Gênica/fisiologia , Técnicas de Transferência de Genes , Membro Posterior/irrigação sanguínea , Humanos , Injeções Intramusculares , Isquemia/etiologia , Microvasos/diagnóstico por imagem , Doenças Vasculares Periféricas/complicações , Plasmídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Coelhos , Radiografia , Fluxo Sanguíneo Regional/fisiologia , Fatores de Tempo , Transfecção , Transgenes , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
We have recently reported that in pigs with chronic myocardial ischemia heart transfection with a plasmid encoding the 165 isoform of human vascular endothelial growth factor (pVEGF165) induces an increase in the mitotic index of adult cardiomyocytes and cardiomyocyte hyperplasia. On these bases we hypothesized that VEGF gene transfer could also modify the evolution of experimental myocardial infarct. In adult sheep pVEGF165 (3.8 mg, n=7) or empty plasmid (n=7) was injected intramyocardially 1 h after coronary artery ligation. After 15 days infarct area was 11.3+/-1.3% of the left ventricle in the VEGF group and 18.2+/-2.1% in the empty plasmid group (P<0.02). The mechanisms involved in infarct size reduction (assessed in additional sheep at 7 and 10 days after infarction) included an increase in early angiogenesis and arteriogenesis, a decrease in peri-infarct fibrosis, a decrease in myofibroblast proliferation, enhanced cardiomyoblast proliferation and mitosis of adult cardiomyocytes with occasional cytokinesis. Resting myocardial perfusion (99mTc-sestamibi SPECT) was higher in VEGF-treated group than in empty plasmid group 15 days after myocardial infarction. We conclude that plasmid-mediated VEGF gene transfer reduces myocardial infarct size by a combination of effects including neovascular proliferation, modification of fibrosis and cardiomyocyte regeneration.
Assuntos
Terapia Genética/métodos , Infarto do Miocárdio/terapia , Miocárdio/metabolismo , Plasmídeos/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Fibrose , Injeções , Masculino , Mitose , Modelos Animais , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/patologia , Miócitos Cardíacos/patologia , Neovascularização Fisiológica , Regeneração , Ovinos , Tomografia Computadorizada de Emissão de Fóton Único , Transfecção/métodosRESUMO
Replacement of the cell loss occurring after acute myocardial infarction has been proposed as a potential treatment to prevent heart remodeling and failure. On account that cardiomyocytes express VEGF receptors and that VEGF triggers mitogen-activated protein kinases, we investigated if VEGF gene transfer may induce cardiomyocyte replication. In a pig model of chronic myocardial ischemia achieved by Ameroid occlusion of the left circumflex coronary artery, we observed that direct intramyocardial injection of a plasmid encoding human VEGF(165) induced a several-fold increase in cardiomyocyte mitotic index and in the number of cardiomyocyte nuclei per unit volume as compared with pigs receiving plasmid devoid of gene. Despite images of conventional cytokinesis were not observed, the fact that caryokinesis is an obligatory step for cell division suggests that our finding may contribute to the issue of heart regeneration and may potentially widen the therapeutic spectrum of VEGF gene transfer.
Assuntos
Fatores de Crescimento Endotelial/genética , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Peptídeos e Proteínas de Sinalização Intercelular/genética , Linfocinas/genética , Isquemia Miocárdica/terapia , Miócitos Cardíacos/patologia , Animais , Células Cultivadas , Fatores de Crescimento Endotelial/análise , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/análise , Linfocinas/análise , Microscopia de Fluorescência , Mitose , Modelos Animais , Miócitos Cardíacos/metabolismo , Distribuição Aleatória , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sus scrofa , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Trypanosoma cruzi must invade mammalian host cells to replicate and complete its life cycle. Almost all nucleated mammalian cells can be invaded by the parasite following a receptor-ligand recognition as an early prerequisite. In this work, we describe a 67-kDa lectin-like glycoprotein that binds to desialylated human erythrocyte membranes in a galactose-dependent way. This protein is present on the parasite surface in both infective and non-infective stages of T. cruzi. More interestingly, we demonstrate by lectin-immuno-histochemistry assays that the 67kDa protein is involved in the recognition of host-cell receptors in mouse cardiac tissue and human cardiac aortic endothelium and mammary artery tissue. Moreover, antibodies against the 67kDa glycoprotein inhibit in vitro host-cell invasion by 63%. These data suggest that the 67kDa glycoprotein in vivo is needed for host-cell invasion by T. cruzi.
Assuntos
Proteínas de Ligação ao Cálcio , Membrana Eritrocítica/metabolismo , Proteínas de Helminto/isolamento & purificação , Proteínas de Transporte de Monossacarídeos/isolamento & purificação , Proteínas Periplásmicas de Ligação , Trypanosoma cruzi/fisiologia , Animais , Western Blotting , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Endotélio Vascular/metabolismo , Endotélio Vascular/parasitologia , Membrana Eritrocítica/parasitologia , Imunofluorescência , Galactose/metabolismo , Coração/parasitologia , Proteínas de Helminto/imunologia , Proteínas de Helminto/fisiologia , Humanos , Soros Imunes/imunologia , Imuno-Histoquímica , Lectinas , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Transporte de Monossacarídeos/imunologia , Proteínas de Transporte de Monossacarídeos/fisiologia , Coelhos , Trypanosoma cruzi/químicaRESUMO
Studies carried out during the last decades provided evidence in support of an autoimmune pathogenesis for chronic chagasic myocarditis. This opinion was based on 1) the demonstration of molecular mimicry between parasite and host antigens, 2) the appearance of autoantibodies recognizing heart epitopes during the chronic phase of infection, 3) the induction of myocarditis and electrocardiographic alterations in animals immunized with whole parasites, parasite fragments or with biochemically-defined antigens, 4) the isolation from the heart of inflammatory infiltrates of B cells elaborating antibodies against myocardial antigens and 5) or of T cell clones reacting with heart epitopes and 6) induction of heart and nervous tissue alterations by transfer of lymphocytes from infected animals into naive syngeneic hosts. However, the characteristics of the inflammatory infiltrate in human myocarditis, displaying a wide variety of cells, many of them not involved in autoreactivity, such as the presence of giant cell granulomas and abundant eosinophils, as well as its focality and asynchrony, and the frequent association with pericarditis, casts doubts about the possibility of autoimmunity being responsible for the perpetuation of the myocarditis. This is supported by the recent observation that treatment of asymptomatic patients with trypanocidal drugs prevents the development of cardiopathy and that parasite components, either antigens or genomic fragments, are present at the site of the inflammatory lesions. On the basis of this new evidence, other alternative pathogenetic mechanisms should be sought to explain the appearance of a polymorphic long-lasting myocarditis that needs the presence of tiny fragments of parasites to develop. In addition to the well known immunological pathogenesis, the link between such a small amount of parasite components, below the level of microscopic detection, and the induction of such an extensive inflammatory infiltrate, represents interesting avenues for research in the near future.
Assuntos
Doenças Autoimunes/imunologia , Cardiomiopatia Chagásica/imunologia , Adulto , Linfócitos B/imunologia , Doença Crônica , Feminino , Humanos , Imunidade Celular , Linfócitos T/imunologiaRESUMO
Molecular expression cloning techniques revealed that patients with severe chronic Chagas heart disease showed a strong humoral response against the cloned C-terminal portion of the Trypanosoma cruzi ribosomal P2beta protein, previously named JL5. The main linear epitope of this polypeptide was mapped to the 13 C-terminal amino acid sequence EEEDDDMGFGLFD (named R13), which is almost identical to the mammalian ribosomal P consensus sequence EESDDDMGFGLFD (named H13). Enzyme-linked immunosorbent assay measurements demonstrated that sera from patients with chronic Chagas heart disease presented a very specific anti-P humoral response with high anti-R13, but low H13 antibody levels. We attempted to develop an animal model that would reproduce, at least partially, two features of the human infection: (1) the serological pattern of the anti-P response, and (2) specific cardiac symptoms. To this effect, mice were immunized with T. cruzi P2beta recombinant protein. Immunization reproduced the typical anti-P antibody profile defined for chronic infections, but did not induce cardiac inflammatory lesions. However, it altered significantly the electrocardiograms of immunized mice. It is suggested that this assay represents a functional test for assessing the biological activity of antibodies against T. cruzi ribosomal P protein on cardiac muscle.
Assuntos
Eletrocardiografia , Coração/fisiologia , Proteínas de Protozoários , Proteínas Ribossômicas/imunologia , Trypanosoma cruzi/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Autoanticorpos/sangue , Proteínas de Transporte/genética , Humanos , Imunização , Proteínas Ligantes de Maltose , Camundongos , Camundongos Endogâmicos BALB C , Miocárdio/imunologia , Proteínas Recombinantes de Fusão/imunologiaRESUMO
OBJECTIVE: To characterize biochemically and isolate the skeletal and heart muscle cell epitope recognized by the autoantibodies present in the serum of chronically infected Trypanosoma cruzi patients. Secondly, to use that epitope in an immunoenzymatic assay for determining differences in antibody titre among Chagas' and other protozoan and heart diseases and between asymptomatic and cardiopathic chagasic patients. DESIGN: Isolated human skeletal and heart muscle cells were treated with organic solvents, pronase, neuraminidase and sodium metaperiodate before immunofluorescence assay. Glycolipids were extracted from human skeletal muscle for ELISA. PATIENTS: Sera were collected from 155 patients with positive serology for T cruzi infection; 44 healthy blood bank donors; and from patients after heart transplantation (16 patients), during the first month after cardiac infarction (eight) or cardiotomy (10), dilated myocardiopathy (21), leishmaniasis (12), acute toxoplasmosis (four) and hyperthyroid ophthalmopathy (five). MAIN RESULTS: Immunofluorescence assay revealed that the chagasic sera recognized epitopes that appeared to be glycolipid in nature. ELISA showed that the chagasic sera contained a higher titre of antiskeletal muscle glycolipid antibodies than the control sera and that, in the chagasic population, antibody titre was significantly higher in patients with heart failure than in asymptomatic subjects or in those presenting only electrocardiographic abnormalities. CONCLUSIONS: The skeletal and heart muscle epitope recognized by antibodies present in the sera of chagasic patients has the characteristics of a glycolipid. ELISA with glycolipids extracted from human skeletal muscle indicated that chagasic patients presented a higher antibody titre and that patients with heart failure showed a titre significantly higher than those who were asymptomatic or with electrocardiographic abnormalities, suggesting that those antibodies could be immunological markers and even predictors of heart failure in Chagas' disease.
Assuntos
Autoanticorpos/sangue , Cardiomiopatia Chagásica/imunologia , Glicolipídeos/imunologia , Músculo Esquelético/imunologia , Miocárdio/imunologia , Cardiomiopatia Dilatada/imunologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Técnicas In Vitro , Leishmaniose/imunologia , Infarto do Miocárdio/imunologia , Toxoplasmose/imunologiaRESUMO
Chagasic myocarditis is associated with the appearance of circulating antiheart autoantibodies. In order to find out if there was local synthesis of those antibodies we investigated, by means of a solid immunoenzymatic technique, the presence of cells secreting antibody (ASC) against syngeneic soluble heart antigens in the mononuclear cell (MNC) population isolated from the hearts of mice chronically infected with Trypanosoma cruzi. In seven animals the number of ASC ranged between 300 and 2080 per 10(6) MNC. A similar number of cells was observed when the assay was carried out with T. cruzi-soluble antigens. When the ASC were enumerated in an assay simultaneously with both antigens, their number doubled that found in the single antigen assay, suggesting that there was no cross-reactivity between the heart and the parasite antigens. These results indicate that some of the cells in the inflammatory infiltrate of chronic chagasic myocarditis synthesize IgG autoantibodies against heart antigens, a phenomenon which may lead to a local concentration of antibody large enough to induce tissue damage.
Assuntos
Autoanticorpos/biossíntese , Cardiomiopatia Chagásica/imunologia , Miocárdio/imunologia , Animais , Doença Crônica , Feminino , Camundongos , Camundongos Endogâmicos BALB CAssuntos
Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Doença de Chagas/imunologia , Músculos/imunologia , Miocárdio/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Autoantígenos/imunologia , Células Cultivadas , Cardiomiopatia Chagásica/imunologia , Cardiomiopatia Chagásica/patologia , Epitopos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Camundongos , Camundongos Endogâmicos/imunologia , Miocardite/imunologia , Miocardite/patologia , Miocárdio/citologia , Trypanosoma cruzi/imunologiaRESUMO
Human platelets in the presence of sera from humans with chronic Chagas' disease display cytotoxic activity against Trypanosoma cruzi (T. cruzi) trypomastigotes. Adsorption of IgE from those sera decrease the cytotoxic effect and IgG purified from the same sera revealed a cytotoxic action similar to the whole sera. Morphological studies suggest that chagasic serum promotes adhesion between parasites and platelets. On the basis of these results it is postulated that platelets may represent a defensive mechanism in South American trypanosomiasis by killing circulating forms of the parasite and that both IgG and IgE are relevant for that action.
RESUMO
The existence of antigens shared in common by T. cruzi and heart muscle cells is suggested by the presence of antibodies binding to the parasite surface in the serum of mice with autoimmune myocarditis induced by immunization with syngenic heart antigens.
Assuntos
Anticorpos Antiprotozoários/sangue , Doenças Autoimunes/imunologia , Miocardite/imunologia , Trypanosoma cruzi/imunologia , Animais , Antígenos de Protozoários/imunologia , Epitopos/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Miocardite/patologia , Miocárdio/imunologia , Miocárdio/patologiaRESUMO
In an experimental model, T. cruzi infected mice develop a chronic myocarditis with mononuclear infiltrates rich in CD4 lymphocytes and macrophages with scarce B and CD8 lymphocytes. The infected mice present in the spleen lymphocytes cytotoxic for adult syngeneic cardiocytes and in their sera antibodies able to induce antibody-dependent cytotoxicity (ADCC) against adult cardiocytes in a syngeneic system with spleen lymphocytes as effector cells. Myocarditis with electrocardiographic alterations was induced in naive recipients by transfer of lymphocytes from chagasic syngeneic mice. Immunization with subcellular fractions of T. cruzi led to a chronic myocarditis similar to that observed in infected animals; mice with autoimmune myocarditis induced by immunization with heart antigens developed antibodies which bind to T. cruzi epitopes. Glucocorticoids administered with trypanocidal drugs dramatically reduce the electrocardiographic alterations and to a lesser extent the myocarditis. Parasitological "cure" by treatment with trypanocidal drugs led to loss of the premunition state associated with the disappearance of antibodies able to induce complement-mediated lysis or ADCC against circulating forms of T. cruzi. However, both resistance against a challenge with a small number of parasites and cell-mediated anti T. cruzi immunity persisted. Our studies suggest that more than a single immunopathological mechanism is involved since anti-heart T cell cytotoxicity delayed type hypersensitivity and antiheart ADCC have been observed in chronically infected mice; this could be due to the existence of epitopes shared by the heart and the parasite.
Assuntos
Cardiomiopatia Chagásica/imunologia , Animais , Anticorpos Antiprotozoários/análise , Formação de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD4/imunologia , Cardiomiopatia Chagásica/tratamento farmacológico , Cardiomiopatia Chagásica/patologia , Imunidade Celular , Imunossupressores/uso terapêutico , Dose Letal Mediana , Camundongos , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/imunologiaRESUMO
In an experimental model, T. cruzi infected mice develop a chronic myocarditis with mononuclear infiltrates rich in CD4 lymphocytes and macrophages with scarce B and CD8 lymphocytes. The infected mice present in the spleen lymphocytes cytotoxic for adult syngeneic cardiocytes and in their sera antibodies able to induce antibody-dependent cytotoxicity (ADCC) against adult cardiocytes in a syngeneic system with spleen lymphocytes as effector cells. Myocarditis with electrocardiographic alterations was induced in naive recipients by transfer of lymphocytes from chagasic syngeneic mice. Immunization with subcellular fractions of T. cruzi led to a chronic myocarditis similar to that observed in infected animals; mice with autoimmune myocarditis induced by immunization with heart antigens developed antibodies which bind to T. cruzi epitopes. Glucocorticoids administered with trypanocidal drugs dramatically reduce the electrocardiographic alterations and to a lesser extent the myocarditis. Parasitological [quot ]cure[quot ] by treatment with trypanocidal drugs led to loss of the premunition state associated with the disappearance of antibodies able to induce complement-mediated lysis or ADCC against circulating forms of T. cruzi. However, both resistance against a challenge with a small number of parasites and cell-mediated anti T. cruzi immunity persisted. Our studies suggest that more than a single immunopathological mechanism is involved since anti-heart T cell cytotoxicity delayed type hypersensitivity and antiheart ADCC have been observed in chronically infected mice; this could be due to the existence of epitopes shared by the heart and the parasite.
RESUMO
Reinfection of chronic chagasic mice after treatment with nifurtimox resulted in different outcomes according to the number of parasites used for inoculation. Nifurtimox-treated chagasic animals injected with 2,500 trypomastigotes developed higher parasitemia and increased mortality compared with nontreated chagasic mice. When reinfection was done with 25 trypomastigotes, treated and nontreated animals showed similar parasitemias and mortalities, which were significantly higher in nonchagasic controls infected for the first time. Immunological studies showed that treatment with nifurtimox led to a decrease in anti-Trypanosoma cruzi antibodies engaged in parasite destruction, inducing either complement-dependent lysis or antibody-dependent cytotoxicity, but no difference in anti-T. cruzi cell-mediated immunity was found between treated and nontreated chagasic animals. It is concluded that treatment with nifurtimox leads to a loss of resistance to reinfection with a large number of trypanosomes, which is maintained with challenge with a few parasites, and that these two thresholds of premunition are probably associated with humoral and cell-mediated anti-T. cruzi immune responses, respectively.