RESUMO
The study was focused on the relationship of Fasciola hepatica-secreted proteinases and human IgG subclasses. Each IgG was incubated at different pH values and lengths of time with either the adult parasite excretion-secretion products or the purified cysteinyl proteinases cathepsin L1 and cathepsin L2. The Ig fragments produced were isolated and characterized by Western blot analysis, and the specific cleavage sites were determined by amino acid sequence analysis. Parasite excretion-secretion products and both cathepsins L produced similar degradation patterns and cleaved all human IgG subclasses at the hinge region, yielding at pH 7.3 and 37 degrees C Fab and Fc fragments in the case of IgG1 and IgG3 or Fab(2) and Fc in IgG2 and IgG4. While IgG1 and IgG3 were readily degraded by E/S products either in the presence or in the absence of reducing agents, IgG2 and IgG4 were resistant to proteolysis and were only digested in the presence of 0.1 M dithiothreitol. The cathepsins L needed the presence of dithiothreitol to digest IgG1, IgG2, and IgG4 whereas IgG3 was identically cleaved under both reducing and nonreducing conditions. The main cleavage sites produced by E/S products, CL1, or CL2 were located at the positions peptide bonds: His237-Thr238, Glu237-Cys239, Gly233-Asp234, and Ser241-Cys242 for gamma1, gamma2, gamma3, or gamma4, respectively. The enzymes gave additional splitting sites on the middle hinge of IgG3 to produce shorter Fc fragments and also produce Fd degradation of the IgG4. No cleavage specificity differences were found between CL1 and CL2, but they differed in the kinetics of IgG3 degradation. By lowering the pH, only the E/S products produced concomitant destruction of the Fc while preserving the Fab portion. Under all the conditions assayed the enzymes produced an Fc'-like fragment of 14-15 kDa corresponding to the whole CH3 domain of the immunoglobulin. Contrary to the extensive degradation produced by cathepsins on digested proteins, its actions on IgG subclasses were specific and restricted; thus, all the fragments produced could be potentially involved in the mechanisms used by the parasite to evade the host immune response.
Assuntos
Endopeptidases/metabolismo , Fasciola hepatica/enzimologia , Fragmentos de Imunoglobulinas/análise , Imunoglobulina G/metabolismo , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Fasciola hepatica/imunologia , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/química , Imunoglobulina G/classificaçãoRESUMO
The invasive stages of the parasitic trematode Fasciola hepatica release proteinases into the medium in which they are maintained. In this study, we investigated the interaction of F. hepatica excretory/secretory (E/S) products and 2 cysteine proteinases (CL1 and CL2) purified from these products with extracellular matrix and basement membrane macromolecules. Fasciola hepatica E/S products contained collagenolytic activity on fibrillar types I and III collagen as well as basement membrane type IV collagen. CL1 and CL2 were capable of degrading acid-soluble type III and type IV collagen but not insoluble type I collagen. In contrast, neither the E/S products nor the purified CL1 and CL2 showed elastinolytic activity. Fibronectin and laminin were degraded by E/S products and by CL1 and CL2. Sequence analysis of fibronectin degradation products showed that the fragments obtained corresponded to complete biologically active domains. These results indicate that the cysteine proteinases secreted by F. hepatica may be involved in the process of tissue invasion by the parasite.
Assuntos
Colágeno/metabolismo , Endopeptidases/metabolismo , Fasciola hepatica/enzimologia , Fibronectinas/metabolismo , Laminina/metabolismo , Sequência de Aminoácidos , Animais , Membrana Basal/química , Membrana Basal/metabolismo , Bovinos , Elastina/metabolismo , Fibronectinas/químicaRESUMO
We report the clinical, SPET, immunohistochemical and DNA features of an early-onset familial Alzheimer's disease (FAD) in an Argentine pedigree of South American indian ethnic background. Pedigree spans 5 generations comprising more than 110 biological relatives. Clinical data supported the diagnosis of early onset FAD (mean age at onset 38.9 years) in 10 family members, including 3 with pathological confirmation (mean age at death 48.5). The pattern of transmission suggested autosomal dominant inheritance. Prominent features were mood changes, early language impairment, myoclonus, seizures and cerebellar signs. SPET displayed bilateral frontal, temporo-parietal and cerebellar hypoperfusion in early stages and in an asymptomatic member at risk, suggesting that SPET may have predictive value in this family. Immunohistochemistry showed beta amyloid deposits within neuritic plaques and vessel walls and no anti-PrP immunoreactivity. DNA analysis showed no abnormalities in the beta amyloid precursor protein gene. The identification of additional genetic defects in well characterized independent FAD pedigrees will contribute to the understanding of the pathogenesis of Alzheimer's disease.
Assuntos
Idade de Início , Doença de Alzheimer/genética , Doença de Alzheimer/imunologia , Encéfalo/fisiopatologia , Linhagem , Adulto , Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/imunologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Anticorpos Monoclonais , Argentina , Western Blotting , Encéfalo/diagnóstico por imagem , Transtornos Cognitivos , DNA/análise , Eletroencefalografia , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Estudos Retrospectivos , Tomografia Computadorizada de Emissão de Fóton Único , Tomografia Computadorizada por Raios XRESUMO
Amiloidosis abarca a un gran espectro de enfermedades que se caracterizan por el depósito de proteínas fibrilares en uno o mas sitios del organismo. A pesar de los avances producidos en los últimos 20 anos en el conocimiento de las proteínas depositadas así como los genes que las codifican, el mecanismo etiopatogénico que conduce a la formación de fibras insolubles se desconoce. Cardiomegalia, polineuropatía periférica, hepato-esplenomagalia, proteinuria, púrpura notrombocitopénica, sindrome de mala absorción, y sidrome del tunel carpiano son algunas de las carcterísticas clínicas que deben alertar al médico en el diagnóstico de amiloidosis. Frecuentemente, no se diagnostica clínicamente hasta que la enfermedad esta en un grado avanzado. El examen microscópico de secciones de tejido apropiadamente teñidas generalmente confirma el diagnóstico. Recientemente ha surgido el interés en el estudio de la amiloidosis, fundamentalmente debido a su asociación con el envejecimiento y con la enfermedad de Aizheimer (A.D). El hecho de que solo ciertas áreas del Sistema Nervioso Central se encuentren afectadas sugiere que factores propios de cada tejido estan involucrados en el mecanismo de formación y depósito de fibras. Los tipos de amiloidosis hereditaria pueden contribuir a el estudio de los mecanismos patogénicos que desencadenan la enfermedad, ya que estan relacionados a un defecto genético y pueden ser usados para desarrollar modelos animales y líneas terapéuticas.
Assuntos
Humanos , Amiloidose/diagnósticoRESUMO
Amiloidosis abarca a un gran espectro de enfermedades que se caracterizan por el depósito de proteínas fibrilares en uno o mas sitios del organismo. A pesar de los avances producidos en los últimos 20 anos en el conocimiento de las proteínas depositadas así como los genes que las codifican, el mecanismo etiopatogénico que conduce a la formación de fibras insolubles se desconoce. Cardiomegalia, polineuropatía periférica, hepato-esplenomagalia, proteinuria, púrpura notrombocitopénica, sindrome de mala absorción, y sidrome del tunel carpiano son algunas de las carcterísticas clínicas que deben alertar al médico en el diagnóstico de amiloidosis. Frecuentemente, no se diagnostica clínicamente hasta que la enfermedad esta en un grado avanzado. El examen microscópico de secciones de tejido apropiadamente teñidas generalmente confirma el diagnóstico. Recientemente ha surgido el interés en el estudio de la amiloidosis, fundamentalmente debido a su asociación con el envejecimiento y con la enfermedad de Aizheimer (A.D). El hecho de que solo ciertas áreas del Sistema Nervioso Central se encuentren afectadas sugiere que factores propios de cada tejido estan involucrados en el mecanismo de formación y depósito de fibras. Los tipos de amiloidosis hereditaria pueden contribuir a el estudio de los mecanismos patogénicos que desencadenan la enfermedad, ya que estan relacionados a un defecto genético y pueden ser usados para desarrollar modelos animales y líneas terapéuticas.(AU)