RESUMO
Entamoeba histolytica, the protozoan parasite causing human amoebisis, has recently been found to comprise two genetically distinct forms, potentially pathogenic and constitutively nonpathogenic ones. Host tissue destruction by pathogenic forms is belived to result from cell functions mediaed by a lectin-type adherence receptor, a pore-forming peptide involved in host cell lysis, and abundant expression of cysteine proteinase(s). Isolation and molecular cloning of these amoeba products have provided the tools for structural analyses and manipulations of cell functions including comparisons between pathogenic and nonpathogenic forms
Assuntos
Entamebíase/classificação , Entamoeba histolytica/análise , Entamoeba/classificação , Entamoeba histolytica/patogenicidadeAssuntos
Animais , Amebíase/diagnóstico , Amebíase/fisiopatologia , Amebíase/terapia , Entamoeba histolytica/análise , Entamoeba histolytica/isolamento & purificação , Entamoeba histolytica/patogenicidade , Iodoquinol/administração & dosagem , Iodoquinol/farmacocinética , Iodoquinol/uso terapêutico , Metronidazol/administração & dosagem , Metronidazol/farmacocinética , Primatas/parasitologiaAssuntos
Amebíase/diagnóstico , Amebíase/imunologia , Células Clonais/análise , Células Clonais/imunologia , Células Clonais/ultraestrutura , Entamoeba histolytica/análise , Entamoeba histolytica/imunologia , Entamoeba histolytica/ultraestrutura , Ensaio de Imunoadsorção Enzimática/instrumentação , Técnicas In VitroAssuntos
Camundongos , Coelhos , Animais , Aglutininas/análise , Aglutininas/imunologia , Aglutininas/isolamento & purificação , Entamoeba histolytica/análise , Entamoeba histolytica/enzimologia , Entamoeba histolytica/ultraestrutura , Soros Imunes/análise , Soros Imunes/imunologia , Técnicas In VitroAssuntos
Humanos , Ensaios Enzimáticos Clínicos , Ensaios Enzimáticos Clínicos/instrumentação , Estudos de Coortes/instrumentação , Eletroforese , Eletroforese/instrumentação , Entamoeba histolytica/análise , Entamoeba histolytica/enzimologia , Entamoeba histolytica/ultraestrutura , Inquéritos EpidemiológicosAssuntos
Humanos , Amebíase/diagnóstico , Amebíase/fisiopatologia , Entamoeba histolytica/análise , Entamoeba histolytica/patogenicidade , Entamoeba histolytica/ultraestrutura , Inquéritos Epidemiológicos , Imunoeletroforese , Imunoeletroforese/instrumentação , Testes Sorológicos , Testes de Hemaglutinação/instrumentação , Testes de Hemaglutinação/métodos , VirulênciaAssuntos
Amebíase/fisiopatologia , Células Clonais/fisiopatologia , Células Clonais/parasitologia , Células Epiteliais/fisiopatologia , Células Epiteliais/parasitologia , Eletroforese , Eletroforese/instrumentação , Entamoeba histolytica/análise , Entamoeba histolytica/fisiologia , Entamoeba histolytica/ultraestrutura , Soros Imunes/farmacocinética , Técnicas In VitroRESUMO
Se realizó una experiencia para evaluar la efectividad de las preparaciones permanentes de muestras obtenidas previa purga en fijador de Brooke-Goldman (PVA), con muestras espontáneas de los mismos pacientes, en fijador de Junod (SAF). También fueron comparados los porcentajes obtenidos para E. histolytica y D. fragilis, con los modelos de recolección y procesamiento de heces más usados en nuestro país. Fueron estudiados 248 pacientes. Se tomaron tres muestras de cada uno de ellos. La primera (M1) y segunda muestra (M2) fueron recolectados con heces espontáneas, de días no consecutivos, en fijador de Junod (SAF) y, una tercera (M3), previa purga, en fijador de Brooke-Goldman (PVA). Las muestras recolectadas en SAF (M1 y M2) fueron analizadas por microscopia de preparaciones húmedas directas y previo enriquecimiento por los métodos Ritchie y Faust. Las muestras fijadas en PVA se colorearon con la técnica tricrómica modificada. El análisis de los datos obtenidos mostró diferencias significativas entre las muestras espontáneas de días no consecutivos (M1 y M2) para: Entamoeba histolytica, Giordia lamblia y Endolimax nana. También el examen estadístico reveló diferencias significativas entre las muestras uno y dos, tomadas juntamente, con respecto a los resultados obtenidos en PVA (M3), para: Entamoeba histolytica, Giardia lambia, Dientamoeba fragilis y Endolimax nana. Se compararon los porcentajes obtenidos por coloraciones permanentes para E. hitolytica y D. fragilis, con los correspondientes a otras formas de diagnóstico: 9.088 muestras procesadas por los métodos directo y Ritchie, y 4.228 estudiados por los métodos directo, Ritchie, Faust y coloraciones sólo en casos sospechosos. Estos datos indicaron una mayor eficiencia de la recolección en PVA, previa purga y posterior coloración para el diagnóstico de los dos protozoarios. (AU)
Assuntos
Humanos , Estudo Comparativo , Fezes/microbiologia , Infecções por Protozoários/diagnóstico , Enteropatias Parasitárias/diagnóstico , Fezes/análise , Fezes/parasitologia , Entamoeba histolytica/análise , Dientamoeba/análise , Técnicas de Laboratório Clínico/métodos , Coloração e Rotulagem , Fixadores/uso terapêuticoRESUMO
Se realizó una experiencia para evaluar la efectividad de las preparaciones permanentes de muestras obtenidas previa purga en fijador de Brooke-Goldman (PVA), con muestras espontáneas de los mismos pacientes, en fijador de Junod (SAF). También fueron comparados los porcentajes obtenidos para E. histolytica y D. fragilis, con los modelos de recolección y procesamiento de heces más usados en nuestro país. Fueron estudiados 248 pacientes. Se tomaron tres muestras de cada uno de ellos. La primera (M1) y segunda muestra (M2) fueron recolectados con heces espontáneas, de días no consecutivos, en fijador de Junod (SAF) y, una tercera (M3), previa purga, en fijador de Brooke-Goldman (PVA). Las muestras recolectadas en SAF (M1 y M2) fueron analizadas por microscopia de preparaciones húmedas directas y previo enriquecimiento por los métodos Ritchie y Faust. Las muestras fijadas en PVA se colorearon con la técnica tricrómica modificada. El análisis de los datos obtenidos mostró diferencias significativas entre las muestras espontáneas de días no consecutivos (M1 y M2) para: Entamoeba histolytica, Giordia lamblia y Endolimax nana. También el examen estadístico reveló diferencias significativas entre las muestras uno y dos, tomadas juntamente, con respecto a los resultados obtenidos en PVA (M3), para: Entamoeba histolytica, Giardia lambia, Dientamoeba fragilis y Endolimax nana. Se compararon los porcentajes obtenidos por coloraciones permanentes para E. hitolytica y D. fragilis, con los correspondientes a otras formas de diagnóstico: 9.088 muestras procesadas por los métodos directo y Ritchie, y 4.228 estudiados por los métodos directo, Ritchie, Faust y coloraciones sólo en casos sospechosos. Estos datos indicaron una mayor eficiencia de la recolección en PVA, previa purga y posterior coloración para el diagnóstico de los dos protozoarios.
Assuntos
Humanos , Fezes/microbiologia , Enteropatias Parasitárias/diagnóstico , Infecções por Protozoários/diagnóstico , Técnicas de Laboratório Clínico , Dientamoeba/análise , Entamoeba histolytica/análise , Fezes/análise , Fezes/parasitologia , Fixadores/uso terapêutico , Coloração e RotulagemRESUMO
To detect molecules of Entamoeba histolytica involved in the trophozoite-target cell interaction, three different antisera were generated: (a) two rabbit antisera, one against total amebic proteins and another directed specifically to the 112-kDa adhesin; and (b) a mouse antiserum against amebic molecules adhering to the red blood cell (RBC) surface after incubation of RBCs with total soluble protein from trophozoites (anti-adhesion serum). All three antisera recognized the 112-kDa adhesion. Adhesion of this molecule to the RBC surface was temperature-dependent. More of the 112-kDa adhesion was found on the surface of RBCs incubated with trophozoites at 37 degrees C than on RBCs incubated at room temperature or at 0 degree C. Experiments using both anti-adhesin and anti-total ambebic protein sera revealed the presence of 210, 160, 112, 90, 70, 50, and 24-kDa proteins on RBC incubated with trophozoites. Surface proteins obtained from iodinated MDCK cells recognized amebic proteins of 112, 90, and 48-50 kDa. Virulence-deficient mutants presented a similar amount of the 112-kDa adhesin to the wild-type strain. However, in mutants, the adhesion was not functional, since they did not adhere to RBCs. 90- and 24-kDa proteins were also found to be altered in mutants.
Assuntos
Entamoeba histolytica/análise , Interações Hospedeiro-Parasita , Proteínas de Membrana/análise , Proteínas de Protozoários/análise , Animais , Aderência Bacteriana , Adesão Celular , Entamoeba histolytica/imunologia , Entamoeba histolytica/patogenicidade , Células Epiteliais , Eritrócitos/análise , Imunofluorescência , Proteínas de Membrana/imunologia , Fagocitose , Proteínas de Protozoários/imunologia , Coelhos , VirulênciaRESUMO
Entamoeba histolytica kills cells by contact dependent cytolysis. The mechanism underlying this process must be of rapid onset because target cells round up and show marked zeiosis within 15 min of contact. In earlier work, we identified a remarkable ion-channel forming protein which we named amoebapore, that may contribute to the amoeba-induced target cell killing. Within the amoeba it exists as part of a supramolecular aggregate together with other proteins of unknown function. In this work we report the purification of a solubilized form of the amoebapore. Amoebapore was found to exist as an apparent dimer of the previously reported protein whose molecular weight had been determined under denaturing conditions. Two isoforms of this dimer, with pI values of 6.8 and 5.3 present at a ratio of 7 to 1, were identified and purified. Both isoforms demonstrate ion-channel forming activity in planar lipid membranes. These channels show a unit conductance of 5-20 pS and remain open for less than 1 s. Upon lateral aggregation, opening becomes concerted to a greater degree with channel conductance are observed. The isolated particulate form of amoebapore depolarizes cells.
Assuntos
Entamoeba histolytica/análise , Canais Iônicos/análise , Proteínas de Membrana/isolamento & purificação , Proteínas de Protozoários , Animais , Linhagem Celular , Cromatografia em Gel , Cromatografia por Troca Iônica , Entamoeba histolytica/ultraestrutura , Fluorometria , Bicamadas Lipídicas , Proteínas de Membrana/análiseRESUMO
The galactose and N-acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica is a cell surface protein which mediates parasite adherence to human colonic mucus, colonic epithelial cells, and other target cells. The amebic lectin was purified in 100-micrograms quantities from detergent-solubilized trophozoites by monoclonal antibody affinity chromatography. The adherence lectin was purified 500-fold as judged by radioimmunoassay. The nonreduced lectin had a molecular mass of 260 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of pH 6.2. The amebic lectin reduced with beta-mercaptoethanol consisted of 170- and 35-kDa subunits. Both subunits could be labeled on the cell surface with 125I, and both were metabolically labeled with [3H]glucosamine. The amino termini of the subunits had unique amino acid sequences, and polyclonal antisera to the heavy subunit did not cross-react with the light subunit. The yield of phenylthiohydantoin derivatives from the second and third positions in the sequence of the heavy and light subunits gave a molar ratio of one 170- to one 35-kDa subunit. Antibodies directed to the heavy subunit inhibited amebic adherence to Chinese hamster ovary cells by 100%, suggesting that the heavy subunit is predominantly responsible for mediating amebic adherence.
Assuntos
Acetilgalactosamina , Entamoeba histolytica/análise , Galactosamina , Galactose , Lectinas , Sequência de Aminoácidos , Animais , Anticorpos , Anticorpos Monoclonais , Complexo Antígeno-Anticorpo , Linhagem Celular , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Galactosamina/análogos & derivados , Lectinas/isolamento & purificação , Substâncias Macromoleculares , Dados de Sequência Molecular , Peso MolecularRESUMO
Histamine receptors on the surface of E. histolytica could be demonstrated by histochemical method using three isolates of the protozoa grown and maintained in modified Boeck and Drbohlav's medium. Prior treatment of E. histolytica with cimetidine a H2 blocker, blocked the histamine uptake. Similar treatment with mepyramine maleate, a H1 blocker, did not prevent histamine uptake by the protozoa. It is postulated that E. histolytica has H2 receptors on its surface.
Assuntos
Entamoeba histolytica/análise , Histamina/metabolismo , Receptores Histamínicos/análise , Animais , Cimetidina/farmacologia , Pirilamina/farmacologiaRESUMO
Amoeba-bacterium cultures of Entamoeba histolytica transferred to a hypoosmotic medium depleted of nutrients changed morphologically and biochemically. The cells ejected grains of rice starch, rounded up, and formed a distinct cell wall that was resistant to detergent, bound the sialic acid-specific lectin from Limulus polyphemus, and became fluorescent with Calcofluor M2R. A subpopulation of these cells displayed more than one nucleus. All these signs are characteristic of encysting cells and were also observed in cysts obtained from a human patient. The morphological changes were accompanied by the appearance of two new glycoproteins with apparent molecular sizes of 100 and 150 kilodaltons which contained sialic acid. Sialic acid has been reported to be absent from trophozoites of Entamoeba species. The presence of this sugar residue on cyst-specific proteins parallels recently reported findings during the encystation of the related reptilian parasite Entamoeba invadens. This may indicate a basic role for sialic acid in the encystation of Entamoeba parasites.