RESUMEN
Fogo Selvagem (FS), the endemic form of pemphigus foliaceus, is mediated by pathogenic IgG4 autoantibodies against the amino-terminal extracellular cadherin domain of the desmosomal cadherin desmoglein 1 (Dsg1). Here we define the detailed epitopes of these pathogenic antibodies. Proteolytic footprinting showed that IgG4 from 95% of FS donor sera (19/20) recognized a 16-residue peptide (A129LNSMGQDLERPLELR144) from the EC1 domain of Dsg1 that overlaps the binding site for an adhesive-partner desmosomal cadherin molecule. Mutation of Dsg1 residues M133 and Q135 reduced the binding of FS IgG4 autoantibodies to Dsg1 by â¼50%. Molecular modeling identified two nearby EC1 domain residues (Q82 and V83) likely to contribute to the epitope. Mutation of these residues completely abolished the binding of FS IgG4 to Dsg1. Bead aggregation assays showed that native binding interactions between Dsg1 and desmocollin 1 (Dsc1), which underlie desmosome structure, were abolished by Fab fragments of FS IgG4. These results further define the molecular mechanism by which FS IgG4 autoantibodies interfere with desmosome structure and lead to cell-cell detachment, the hallmark of this disease.
Asunto(s)
Autoanticuerpos/metabolismo , Desmogleína 1/inmunología , Desmosomas/metabolismo , Epítopos de Linfocito B/inmunología , Inmunoglobulina G/metabolismo , Pénfigo/inmunología , Péptidos/inmunología , Animales , Autoanticuerpos/inmunología , Brasil/epidemiología , Células Cultivadas , Enfermedades Endémicas , Mapeo Epitopo , Humanos , Inmunización Pasiva , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , Mutagénesis Sitio-Dirigida , Pénfigo/epidemiología , Unión Proteica , Conformación ProteicaRESUMEN
Entamoeba histolytica trophozoites adhere to epithelium at the cell-cell contact and perturb tight junctions disturbing the transepithelial electrical resistance. Behind tight junctions are the adherens junctions (AJs) that reinforce them and the desmosomes (DSMs) that maintain the epithelium integrity. The damage produced to AJs and DMSs by this parasite is unknown. Here, we studied the effect of the trophozoites, the EhCPADH complex, and the EhCP112 recombinant enzyme (rEhCP112) on AJ and DSM proteins. We found that trophozoites degraded ß-cat, E-cad, Dsp l/ll, and Dsg-2 with the participation of EhCPADH and EhCP112. After contact of epithelial cells with trophozoites, immunofluorescence and transmission electron microscopy assays revealed EhCPADH and rEhCP112 at the intercellular space where they colocalised with ß-cat, E-cad, Dsp l/ll, and Dsg-2. Moreover, our results suggested that rEhCP112 could be internalised by caveolae and clathrin-coated vesicles. Immunoprecipitation assays showed the interaction of EhCPADH with ß-cat and Dsp l/ll. Besides, in vivo assays demonstrated that rEhCP112 concentrates at the cellular borders of the mouse intestine degrading E-cad and Dsp I/II. Our research gives the first clues on the trophozoite attack to AJs and DSMs and point out the role of the EhCPADH and EhCP112 in the multifactorial event of trophozoites virulence.
Asunto(s)
Uniones Adherentes/metabolismo , Cisteína Endopeptidasas/metabolismo , Entamoeba histolytica/enzimología , Entamoeba histolytica/metabolismo , Entamebiasis/patología , Uniones Estrechas/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antiprotozoarios/inmunología , Células CACO-2 , Cadherinas/metabolismo , Línea Celular , Desmosomas/metabolismo , Perros , Entamoeba histolytica/inmunología , Entamebiasis/parasitología , Células Epiteliales/metabolismo , Humanos , Mucosa Intestinal/parasitología , Células de Riñón Canino Madin Darby , Masculino , Ratones , Ratones Endogámicos C57BL , beta Catenina/metabolismoRESUMEN
Even though the liver synthesizes most of circulating IGF-1, it lacks its receptor under physiological conditions. However, according to previous studies, a damaged liver expresses the receptor. For this reason, herein, we examine hepatic histology and expression of genes encoding proteins of the cytoskeleton, extracellular matrix, and cell-cell molecules and inflammation-related proteins. A partial IGF-1 deficiency murine model was used to investigate IGF-1's effects on liver by comparing wild-type controls, heterozygous igf1+/-, and heterozygous mice treated with IGF-1 for 10 days. Histology, microarray for mRNA gene expression, RT-qPCR, and lipid peroxidation were assessed. Microarray analyses revealed significant underexpression of igf1 in heterozygous mice compared to control mice, restoring normal liver expression after treatment, which then normalized its circulating levels. IGF-1 receptor mRNA was overexpressed in Hz mice liver, while treated mice displayed a similar expression to that of the controls. Heterozygous mice showed overexpression of several genes encoding proteins related to inflammatory and acute-phase proteins and underexpression or overexpression of genes which coded for extracellular matrix, cytoskeleton, and cell junction components. Histology revealed an altered hepatic architecture. In addition, liver oxidative damage was found increased in the heterozygous group. The mere IGF-1 partial deficiency is associated with relevant alterations of the hepatic architecture and expression of genes involved in cytoskeleton, hepatocyte polarity, cell junctions, and extracellular matrix proteins. Moreover, it induces hepatic expression of the IGF-1 receptor and elevated acute-phase and inflammation mediators, which all resulted in liver oxidative damage.
Asunto(s)
Proteínas de Fase Aguda/metabolismo , Regulación de la Expresión Génica , Hepatitis/metabolismo , Mediadores de Inflamación/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Hígado/metabolismo , Receptores de Somatomedina/metabolismo , Proteínas de Fase Aguda/genética , Animales , Cadherinas/genética , Cadherinas/metabolismo , Cruzamientos Genéticos , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Desmosomas/inmunología , Desmosomas/metabolismo , Desmosomas/patología , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Perfilación de la Expresión Génica , Hepatitis/inmunología , Hepatitis/patología , Hepatitis/prevención & control , Inyecciones Subcutáneas , Factor I del Crecimiento Similar a la Insulina/administración & dosificación , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/uso terapéutico , Peroxidación de Lípido , Hígado/inmunología , Hígado/patología , Masculino , Ratones , Ratones Transgénicos , Estrés Oxidativo , Receptores de Somatomedina/genética , Proteínas de Uniones Estrechas/genética , Proteínas de Uniones Estrechas/metabolismoRESUMEN
Giardia duodenalis is a parasitic protozoan that causes diarrhea and other symptoms which together constitute a disease known as giardiasis. Although the disease has been well defined, the mechanisms involving the establishment of the infection have not yet been fully elucidated. In this study, we show that after 24h of interaction between parasites and intestinal Caco-2 cells, there was an alteration of the paracellular permeability, as observed by an approximate 42% of reduction in the transepithelial electrical resistance and permeation to ruthenium red, which was concomitant with ultrastructural changes. Nevertheless, epithelium viability was not affected. We also demonstrate that there was no change in expression of junctional proteins (tight and adherens) but that the distribution of these proteins in Caco-2 cells after parasite adhesion was significantly altered, as observed via laser scanning confocal microscopy 3D reconstruction. The present work shows that adhesion of Giardia duodenalis trophozoites to intestinal cells in vitro induces disturbances of the tight, adherens and desmosomal junctions.
Asunto(s)
Uniones Adherentes/metabolismo , Desmosomas/metabolismo , Giardia/fisiología , Giardiasis/parasitología , Uniones Estrechas/metabolismo , Citoesqueleto de Actina/metabolismo , Uniones Adherentes/parasitología , Uniones Adherentes/ultraestructura , Animales , Células CACO-2 , Permeabilidad de la Membrana Celular , Supervivencia Celular , Desmosomas/parasitología , Desmosomas/ultraestructura , Células Epiteliales/metabolismo , Células Epiteliales/parasitología , Interacciones Huésped-Parásitos , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitología , Uniones Estrechas/parasitología , Uniones Estrechas/ultraestructura , TrofozoítosRESUMEN
Class I-restricted T cell associated molecule (CRTAM) is a member of the immunoglobulin superfamily that complies with the structural characteristics of the JAM family of proteins and is phylogenetically more closely related to nectin-like proteins. Here we demonstrate for the first time, that CRTAM is expressed in epithelial cells along the lateral membrane and is important for early cell-cell contacts and cell-substrate interactions. CRTAM is sensitive to intermediate filament disruption and treatment of monolayers with soluble CRTAM enhances cell-cell dissociation and lowers transepithelial electrical resistance. Incubation of newly plated cells with anti-CRTAM antibody decreases the formation of cell aggregates and promotes cell detachment. Co-cultures of epithelial cells and fibroblasts that lack CRTAM expression and in vitro binding assays, demonstrate the participation of CRTAM in homotypic and heterotypic trans-interactions. Hence we conclude that CRTAM is a molecule involved in epithelial cell adhesion.
Asunto(s)
Adhesión Celular/fisiología , Células Epiteliales/fisiología , Inmunoglobulinas/metabolismo , Animales , Calcio/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Desmosomas/metabolismo , Células Epiteliales/citología , Fibroblastos/citología , Fibroblastos/fisiología , Humanos , Inmunoglobulinas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Uniones Estrechas/metabolismoRESUMEN
Intercellular junctions are studied in the epithelium lining the testis of the freshwater snail Pomacea canaliculata by conventional staining and lanthanum tracer techniques. The junctional complex consists of belt desmosomes and septate junctions. Septate junctions are of the pleated-sheet type and they are constantly associated with mitochondria. Gap and tight junctions appear to be absent. These septate junctions seem to be the structural correlate of an epithelial permeability barrier that separate the testis from the extrapallial space where the shell elements are deposited. These junctions may contribute to a functional barrier in the male gonad of Pomacea canaliculata. The results indicate that freshwater prosobranchs have junctional structures very close to those found in other molluscs.
Asunto(s)
Desmosomas/metabolismo , Células Epiteliales/metabolismo , Testículo/metabolismo , Animales , Permeabilidad de la Membrana Celular/fisiología , Desmosomas/ultraestructura , Células Epiteliales/ultraestructura , Masculino , Microscopía Electrónica , Microvellosidades/metabolismo , Microvellosidades/ultraestructura , Mitocondrias/ultraestructura , Caracoles , Testículo/citologíaRESUMEN
Ultrastructural features and cytokeratin expression of inverted ductal papillomas of minor salivary gland origin were studied. Under the electron microscope, an increased number of desmosomes and mucus-like granules in some cells were the most striking features. Immunohistochemical study revealed that tumor cells displayed strongly positive reactions with cytokeratins 13 and 14, and less strong reactions with cytokeratins 7, 8, 18 and 5D3. These results support the hypothesis that an inverted ductal papilloma can be derived from the proximal portion of a salivary gland excretory duct.