RESUMEN
Intermediate filaments are frequently used in studies of developmental biology as markers of cell differentiation. To assess whether they can be useful to identify differentiating pancreatic endocrine cells, we examined the pattern of expression of nestin, cytokeratin 20, and vimentin on acetone-fixed cryosections of rat adult and developing pancreas. We also studied vimentin expression in mouse embryonic pancreas at E19. Cytokeratin 20 was found in all pancreatic epithelial cell lineages during the entire development of the rat gland and in the adult animals. Under our experimental conditions, therefore, cytokeratin 20 is not an exclusive marker of rat duct cells. Nestin was detected exclusively in stromal cells either in the adult or developing rat pancreas. Vimentin was observed within cells located in the primitive ducts of rat pancreas starting from E12.5. Their number rapidly increased, reaching its highest level in newborn animals. Vimentin was also spotted in alpha cells starting from E12.5 but disappeared soon after birth, likely identifying immature or recently differentiated alpha cells. In addition, vimentin was observed in duct and alpha cells of mouse developing pancreas showing that its expression in such cells is not an event restricted to the rat. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
Asunto(s)
Proteínas de Filamentos Intermediarios/biosíntesis , Páncreas/metabolismo , Animales , Animales Recién Nacidos , Diferenciación Celular , Linaje de la Célula , Femenino , Edad Gestacional , Células Secretoras de Glucagón/metabolismo , Queratina-20/biosíntesis , Masculino , Ratones , Ratones Endogámicos C3H , Proteínas del Tejido Nervioso/biosíntesis , Nestina , Páncreas/embriología , Páncreas/crecimiento & desarrollo , Ratas , Ratas Sprague-Dawley , Especificidad de la Especie , Vimentina/biosíntesisRESUMEN
Nestin is considered a marker of neurogenic and myogenic precursor cells. Its arrangement is regulated by cyclin-dependent kinase 5 (CDK5), which is expressed in murine podocytes. We investigated nestin expression in human adult and fetal kidney as well as CDK5 presence in adult human podocytes. Confocal microscopy demonstrated that adult glomeruli display nestin immunoreactivity in vimentin-expressing cells with the podocyte morphology and not in cells bearing the endothelial marker CD31. Glomerular nestin-positive cells were CDK5 immunoreactive as well. Western blotting of the intermediate filament-enriched cytoskeletal fraction and coimmunoprecipitation of nestin with anti-CDK5 antibodies confirmed these results. Nestin was also detected in developing glomeruli within immature podocytes and a few other cells. Confocal microscopy of experiments conducted with antibodies against nestin and endothelial markers demonstrated that endothelial cells belonging to capillaries invading the lower cleft of S-shaped bodies and the immature glomeruli were nestin immunoreactive. Similar experiments carried out with antibodies raised against nestin and alpha-smooth muscle actin showed that the first mesangial cells that populate the developing glomeruli expressed nestin. In conclusion, nestin is expressed in the human kidney from the first steps of glomerulogenesis within podocytes, mesangial, and endothelial cells. This expression, restricted to podocytes in mature glomeruli, appears associated with CDK5.
Asunto(s)
Feto Abortado/química , Proteínas de Filamentos Intermediarios/análisis , Riñón/química , Proteínas del Tejido Nervioso/análisis , Adulto , Anciano , Autopsia , Western Blotting , Quinasa 5 Dependiente de la Ciclina/análisis , Humanos , Inmunohistoquímica , Riñón/citología , Microscopía Confocal , Persona de Mediana Edad , Nestina , Podocitos/metabolismoRESUMEN
In the past three decades, many studies have analyzed ultrastructural and molecular markers of differentiation in squamous stratified epithelial tissues. In these tissues, epithelial cells migrating from the basal layer to the upper layers undergo drastic changes, which involve membrane-associated proteins, DNA synthesis, phenotypic aspects, lipid composition, and cytoskeletal components. Cytoskeletal components include a large and heterogeneous group, including intermediate filaments, components of the cornified envelope, and of the stratum corneum. When grown in mono- and multilayer cell cultures, epithelial cells isolated from the oral mucosa may reproduce many of the biochemical and morphological aspects of epithelial tissue in vivo. In the present paper, we examine phenotypic changes, development of suprabasal layer, and Involucrin expression occurring in differentiating oral epithelial cells, based on literature review and original data.
Asunto(s)
Diferenciación Celular/fisiología , Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Queratinas/metabolismo , Mucosa Bucal/metabolismo , Animales , Agregación Celular/fisiología , Células Cultivadas/metabolismo , Células Cultivadas/ultraestructura , Citoesqueleto/ultraestructura , Células Epiteliales/citología , Humanos , Mucosa Bucal/citología , Organogénesis/fisiología , Precursores de Proteínas/metabolismoRESUMEN
The activation of the molecular cascade leading to Ca++ -induced differentiation in cultured epithelial cells might be provided by the establishment of intercellular junctions between cells. In the present paper, we tested the hypothesis that Ca++ concentration would determine morphological and biochemical changes in intercellular junctions of cultured human gingival cells. Triplicate samples of monolayer cultures of human oral gingival cells were grown with two different Ca++ concentrations (0.3 and 1.8 mM), and examined by transmission (TEM) and scanning (SEM) electron microscopy at different time periods. To determine the role of the E-cadherin/beta-catenin complex in intercellular junction formation, oral epithelial cell cultures were grown in 0.3 mM Ca++ in presence of a blocking antibody anti human E-cadherin, stained with antibodies anti human beta-catenin, and examined by confocal laser scanning microscopy (CLSM). By TEM and SEM, cells grown at physiologic Ca++ concentrations (i.e., 1.8 mM) showed a subjective increase of the size of microvilli and of the number of intercellular junctions, which was more evident after 3 days in culture. Desmosome-like junctions were observed in cells grown in 1.8 mM Ca++, not in cells grown in 0.3 mM. By CLSM, development of intercellular adhesion was marked by membranous localization of E-cadherin and beta-catenin within the first hours in both culture types. When cell-cell adhesion was prevented, cells showed round shape and no membranous localization of beta-catenin. Restoring cell adhesion brought about polygonal cell shape and membranous localization of beta-catenin. We can conclude that increased Ca++ concentration may determine biochemical and morphological changes at membranous level in human oral epithelial cells. These changes may facilitate the development of intercellular junctions.
Asunto(s)
Comunicación Celular/fisiología , Diferenciación Celular/fisiología , Células Epiteliales/ultraestructura , Uniones Intercelulares/ultraestructura , Mucosa Bucal/ultraestructura , Cadherinas/metabolismo , Calcio/metabolismo , Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Comunicación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Desmosomas/efectos de los fármacos , Desmosomas/metabolismo , Desmosomas/ultraestructura , Relación Dosis-Respuesta a Droga , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Inmunohistoquímica , Uniones Intercelulares/efectos de los fármacos , Uniones Intercelulares/metabolismo , Microscopía Electrónica , Microvellosidades/efectos de los fármacos , Microvellosidades/metabolismo , Microvellosidades/ultraestructura , Mucosa Bucal/efectos de los fármacos , Mucosa Bucal/metabolismo , beta Catenina/metabolismoRESUMEN
Despite many studies on the topic, plasma cells found in human periapical chronic inflammatory lesions (granulomas) continue to present unresolved issues. In this study, we tried to assess quantitatively and qualitatively the nature of plasma cells of 4 human periapical granulomas. Samples were analyzed for relative amounts of IgG-, IgM-, IgA-, and IgE-positive plasma cells by immunohistochemistry, and for morphological changes by transmission electron microscopy (TEM). By immunohistochemistry, many plasma cells stained positively with anti-IgG and anti-IgM antibodies; fewer cells reacted with anti-IgE and anti-IgA. Russell Bodies, controversial aspects of plasma cell maturation, showed positive reactivity of the superficial layer only to antibodies against IgG and IgM. By TEM analysis, phenotypes of normal and dysfunctional plasma cells (Mott cells) were evident. Russell Bodies appeared as intra- or extracellular round vesicles, with an homogeneous internal core, and an external membrane, resembling rough endoplasmatic reticulum (RER). We can conclude that mucosal immune response is not the predominant type in the periapical lesions examined. Positive immunoreaction for IgG and IgM of Russell Bodies may be due to the residual RER membrane, whereas components of yet unidentified nature may occupy the internal core.
Asunto(s)
Anticuerpos/inmunología , Granuloma Periapical/inmunología , Células Plasmáticas/inmunología , Pulpitis/inmunología , Enfermedad Crónica , Vesículas Citoplasmáticas/inmunología , Vesículas Citoplasmáticas/ultraestructura , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Inmunohistoquímica , Cuerpos de Inclusión/inmunología , Cuerpos de Inclusión/ultraestructura , Membranas Intracelulares/inmunología , Membranas Intracelulares/ultraestructura , Microscopía Electrónica de Transmisión , Granuloma Periapical/patología , Células Plasmáticas/ultraestructura , Pulpitis/patologíaRESUMEN
M-cells are believed to play a pivotal role in initiation of the immune response. These cells, located in the epithelia that overlie mucosal lymphoid follicles, are responsible for the active uptake of particulate antigens and for their translocation to the underlying lymphoid tissue. The identification of reliable markers for M-cells is therefore extremely important for the study of the initial steps that lead to the immune response. For this purpose, we studied cytokeratin 20 (CK20) expression in the epithelium of rabbit palatine tonsils by immunofluorescence, confocal microscopy, and Western blotting. CK20+ cells were observed in all rabbit palatine tonsils examined. By Western blotting, one CK20-immunoreactive band was identified at 46 kD on samples of proteins from the intermediate filament-enriched cytoskeletal fraction of tonsil epithelium. Double labeling of CK20+ cells with cell-specific markers confirmed that such cells were actually M-cells. Moreover, CK20+ M-cells displayed a mature phenotype (they formed pockets harboring lymphoid cells) and were functionally competent because they could take up particulate antigens from the pharyngeal lumen. We conclude that CK20 is an M-cell marker for rabbit palatine tonsils. Moreover, we can hypothesize the use of M-cells as a possible site for antigen delivery of particle-based vaccines.
Asunto(s)
Proteínas de Filamentos Intermediarios/biosíntesis , Tonsila Palatina/metabolismo , Animales , Western Blotting , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes/química , Queratina-20 , Masculino , Microscopía Confocal , Microesferas , Tonsila Palatina/citología , Conejos , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismoRESUMEN
In order to evaluate the influence of specific factors on mercury (P-Hg) levels and antioxidant power (P-FRAP) in human plasma, 26 healthy donors were examined by a dentist, their plasma analyzed for Hg by atomic absorption spectrometry and for total antioxidant activity by FRAP method. Hg plasma concentration was found to be correlated with the number of amalgam fillings, suggesting that Hg released from fillings is a source of Hg in non-occupational exposed subjects. P-FRAP correlated negatively with P-Hg suggesting a pro-oxidant role of the Hg released from amalgam fillings. Though age by itself was not significantly correlated with P-FRAP, when considered together with P-Hg in multivariate analysis, it was found to be a major related cofactor. Multivariate analysis showed no influence of fish consumption or cigarette smoking on P-FRAP.
Asunto(s)
Antioxidantes/farmacología , Amalgama Dental/efectos adversos , Mercurio/sangre , Adulto , Amalgama Dental/química , Femenino , Recuperación de Fluorescencia tras Fotoblanqueo , Humanos , Masculino , Persona de Mediana Edad , Espectrofotometría AtómicaRESUMEN
The constituents of the intermediate filament network of adrenal gland cells have not been deeply investigated in vivo. Adrenocortical cells have been reported to express cytokeratins and vimentin, but the intermediate filament components of the adrenomedullary cells are still unknown. Nestin is an intermediate filament protein that is mainly expressed in the developing nervous and muscle systems. It has been reported to be unable to form filaments by itself and it co-assembles with vimentin. Using immunocytochemical and biochemical approaches, the present study demonstrates that nestin is expressed in situ either in the cortex or in the medulla of adult rat adrenal glands. Nestin-negative cells prevalently form the zona glomerulosa whereas the zona fasciculata and the zona reticularis are mainly nestin-immunoreactive. Nestin-positive cells always express vimentin-like immunoreactivity but several cells apparently expressing only vimentin are detectable too. Nestin is also expressed by adrenomedullary cells that also display a faint vimentin-like immunoreactivity. We hypothesise that the inconstant detection of nestin in adrenocortical cells depends on their different functional moments. Moreover, even though our data do not allow to confirm vimentin in adrenomedullary cells, in situ detection of nestin in the adrenal medulla indirectly supports in vivo expression of vimentin in chromaffin cells.