RESUMEN
BACKGROUND: Alterations in the nasal epithelial barrier homeostasis and increased interleukin 33 (IL-33) expression contribute to the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). AIMS: As Notch-1 signaling is crucial in repair processes of mucosa, the current study assessed Notch-1/Jagged-1 signaling and IL-33 in the epithelium of nasal polyps biopsies from allergic (A-CRSwNP; n = 9) and not allergic (NA-CRSwNP; n = 9) subjects by immunohistochemistry. We also assessed, in a model of nasal epithelial cells, the effects of stimulation of Notch-1 with Jagged-1 on the expression of IL-33 (by flow cytometry, immunofluorescence, and immunocytochemistry), Jagged-1 (by flow cytometry), and p-CREB transcription factor (by western blot analysis). RESULTS: Ex vivo (a) in normal epithelium, the expression of Notch-1 and IL-33 were higher in NA-CRSwNP than in A-CRSwNP; (b) in metaplastic epithelium, the expression of Notch-1, Jagged-1, and IL-33 were higher in NA-CRSwNP than in A-CRSwNP; (c) in hyperplastic epithelium, the expression of Notch-1, Jagged-1, and IL-33 were higher in A-CRSwNP than in NA-CRSwNP; and (d) in basal epithelial cells, no differences were observed in the expression of Jagged-1, IL-33, and Notch-1. The expression of Notch-1 significantly correlated with the expression of IL-33. In vitro, stimulation of Notch-1 with Jagged-1 induced the expression of (a) Jagged-1; (b) IL-33; and (c) p-CREB transcription factor. The inhibitor of Notch-1, DAPT, reduced all the effects of Jagged-1 on nasal epithelial cells. CONCLUSIONS: The data herein provided support, for the first time, a putative role of Notch-1/Jagged-1 signaling in the overexpression of IL-33 in the epithelium of nasal polyps from patients with CRSwNP.
Asunto(s)
Células Epiteliales/metabolismo , Interleucina-33/metabolismo , Mucosa Nasal/metabolismo , Pólipos Nasales/metabolismo , Receptor Notch1/metabolismo , Rinitis Alérgica/metabolismo , Sinusitis/metabolismo , Adulto , Línea Celular , Enfermedad Crónica , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Células Epiteliales/inmunología , Células Epiteliales/patología , Femenino , Humanos , Proteína Jagged-1/metabolismo , Masculino , Persona de Mediana Edad , Mucosa Nasal/inmunología , Mucosa Nasal/patología , Pólipos Nasales/inmunología , Pólipos Nasales/patología , Fosforilación , Rinitis Alérgica/inmunología , Rinitis Alérgica/patología , Transducción de Señal , Sinusitis/inmunología , Sinusitis/patología , Regulación hacia Arriba , Adulto JovenRESUMEN
In this chapter, we summarise fundamental findings concerning echinoderms as well as research interests on this phylum for biomedical and evolutionary studies. We discuss how current knowledge of echinoderm biology, in particular of the sea urchin system, can shed light on the understanding of important biological phenomena and in dissecting them at the molecular level. The general principles of sea urchin embryo development are summarised, mainly focusing on cell communication and interactions, with particular attention to the cell-extracellular matrix and cell-cell adhesion molecules and related proteins. Our purpose is not to review all the work done over the years in the field of cellular interaction in echinoderms. On the contrary, we will rather focus on a few arguments in an effort to re-examine some ideas and concepts, with the aim of promoting discussion in this rapidly growing field and opening new routes for research on innovative therapeutic tools.
Asunto(s)
Equinodermos/embriología , Equinodermos/fisiología , Embrión no Mamífero/fisiología , Desarrollo Embrionario , Erizos de Mar/embriología , Erizos de Mar/fisiología , Animales , Tipificación del Cuerpo , Adhesión Celular , Moléculas de Adhesión Celular , Comunicación Celular , Diseño de Fármacos , Modelos AnimalesRESUMEN
Pl-nectin is a glycoprotein first discovered in the extracellular matrix (ECM) of Paracentrotus lividus sea urchin embryo, apically located on ectoderm and endoderm cells. The molecule has been described as functioning as an adhesive substrate for embryonic cells and its contact to ectoderm cells is essential for correct skeletogenesis. The present study was undertaken to elucidate the biochemical characteristics of Pl-nectin and to extend knowledge on its in vivo biological function. Here it is shown that the binding of mesenchyme blastula cells to Pl-nectin-coated substrates was calcium dependent, and reached its optimum at 10 mM Ca2+. Perturbation studies using monoclonal antibody (McAb) to Pl-nectin, which prevent ectoderm cell-Pl-nectin contact, show that dorsoventral axis formation and ectoderm differentiation were retarded. At later stages, embryos recovered and, even if growth and patterning of the skeleton was greatly affected, the establishment of dorsoventral asymmetry was reached. Similarly, the expression of specific ectoderm and endoderm territorial markers was achieved, although occurring with some delay. Endoderm differentiation and patterning was not obviously affected. These results suggest that both endoderm and ectoderm cells have regulative capacities and differentiation of territories is restored after a lag period. On the contrary, failure of inductive differentiation of the skeleton cannot be rescued, even though the ectoderm has recovered.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Huesos/embriología , Moléculas de Adhesión Celular/inmunología , Embrión no Mamífero/efectos de los fármacos , Erizos de Mar/embriología , Animales , Anticuerpos Monoclonales/inmunología , Biomarcadores , Calcio/metabolismo , Desarrollo Embrionario , Técnica del Anticuerpo Fluorescente Indirecta , NectinasRESUMEN
The purification, biochemical characterization and functional features of a novel extracellular matrix protein are described. This protein is a component of the basal lamina found in embryos from the sea urchin species Paracentrotus lividus and Hemicentrotus pulcherrimus. The protein has been named Pl-200K or Hp-200K, respectively, because of the species from which it was isolated and its apparent molecular weight in SDS-PAGE under reducing conditions. It has been purified from unfertilized eggs where it is found packed within cytoplasmic granules, and has different binding affinities to type I collagen and heparin, as assessed by affinity chromatography columns. By indirect immunofluorescence experiments it was shown that, upon fertilization, the protein becomes extracellular, polarized at the basal surface of ectoderm cells, and on the surface of primary mesenchyme cells at the blastula and gastrula stages. The protein serves as an adhesive substrate, as shown by an in vitro binding assay where cells dissociated from blastula embryos were settled on 200K protein-coated substrates. To examine the involvement of the protein in morphogenesis of sea urchin embryo, early blastula embryos were microinjected with anti-200K Fab fragments and further development was followed. When control embryos reached the pluteus stage, microinjected embryos showed severe abnormalities in arms and skeleton elongation and patterning. On the basis of current results, it was proposed that 200K protein is involved in the regulation of sea urchin embryo skeletogenesis.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Erizos de Mar/embriología , Animales , Membrana Basal/metabolismo , Adhesión Celular , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/aislamiento & purificación , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Sea urchin embryos of the species Paracentrotus lividus were treated continuously with different concentrations of all-trans retinoic acid (RA) or dimethylsulfoxide (DMSO) at different developmental stages. A delay in embryonic development was observed when embryos were cultured in the presence of 2 x 10(-5) M RA, between 1 and 12 hours of development. Hence, at 48 hours of development, while control embryos had reached the pluteus stage, RA-treated embryos were at the prism stage. At 72 hours of development RA-treated embryos recovered and continued normal development reaching the pluteus stage. No effect was observed when treatment was performed before 1 hour or after 12 hours of development. DMSO treatment had no effect on normal sea urchin embryo development, although we observed that pigment cells, clearly visible at the pluteus stage, become visible earlier with respect to control embryos. This report confirms the advantages that the sea urchin embryo offers for the study of problems in cellular and developmental biology.
Asunto(s)
Embrión no Mamífero/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Dimetilsulfóxido/farmacología , Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Fertilización , Cinética , Morfogénesis/efectos de los fármacos , Erizos de Mar , Factores de Tiempo , Tretinoina/farmacologíaRESUMEN
1. The mouse neuroblastoma cell line N-115 was used as a model system to study neuronal differentiation induced by treatment of cells with different agents. 2. The extent of morphological differentiation obtained with dibutyryl cyclic AMP (dbc-AMP), dimethyl sulfoxide (DMSO), retinoic acid (RA), and serum-free medium was correlated to the expression of the mRNA for the gamma isoform of the glycolytic enzyme enolase, a recognized neuron-specific marker. 3. A 4-day treatment of the cells with any of the differentiation inducing agents used in this study resulted in the extension of long neurites, though differences in cell body shape were observed depending on the agent used. 4. Northern blot analysis revealed that changes in the level of gamma enolase-specific mRNA correlate with the extent of morphological differentiation, with a 5- to 20-fold increase depending on the differentiation inducing agent used. 5. Finally, we found that a high cell density causes a significative increase in the level of the gamma enolase-specific message in cells maintained in growing conditions.
Asunto(s)
Bucladesina/farmacología , Dimetilsulfóxido/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Isoenzimas/biosíntesis , Neuronas/enzimología , Fosfopiruvato Hidratasa/biosíntesis , Tretinoina/farmacología , Animales , Northern Blotting , Diferenciación Celular/efectos de los fármacos , Inhibición de Contacto , Medio de Cultivo Libre de Suero/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Isoenzimas/genética , Ratones , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Neuroblastoma , Neuronas/efectos de los fármacos , Fosfopiruvato Hidratasa/genética , ARN Mensajero/genética , ARN Neoplásico/genética , Células Tumorales CultivadasRESUMEN
In this study we determined the levels of gamma enolase mRNA in mouse neuroblastoma cell line N-115 at early period of induction of differentiation by serum withdrawal. The expression of gamma enolase was examined by Northern blot analysis of total RNA extracted from cells induced for different lengths of time. We found a 3-fold increase in the level of gamma enolase mRNA after 24 hours of induction of differentiation and higher levels were detected in cells induced for longer time, reaching a 10-fold increase after four days.
RESUMEN
The stroma of ductal infiltrating carcinoma of the human breast shows characteristic and localized areas of collagen rarefaction and fragmentation. This finding has been correlated with a peculiar type of fibrillar damage, observed in a small percentage of collagen fibrils isolated in the native state from the tumour stroma. The same pattern of lesion has been reproduced in vitro by human collagenase digestion on reconstituted fibrils. No effect has been detected by other nonspecific proteases in the same system.
Asunto(s)
Neoplasias de la Mama/metabolismo , Carcinoma Intraductal no Infiltrante/metabolismo , Colágeno/metabolismo , Anciano , Neoplasias de la Mama/ultraestructura , Carcinoma Intraductal no Infiltrante/ultraestructura , Femenino , Humanos , Colagenasa Microbiana/metabolismo , Microscopía Electrónica , Persona de Mediana Edad , Elastasa Pancreática/metabolismo , Tripsina/metabolismoRESUMEN
The tumour stroma in cases of ductal infiltrating carcinoma of the mammary gland contains substantial amounts of collagen type I trimer, besides the regular collagen types. Reconstituted collagen trimer consists of fibrils that are significantly thinner than reconstituted fibrils of type I collagen. The axial periodicity is somewhat longer due to widening of the c-d and d-e regions. Transmission EM of the tumours shows characteristic phenomena at the stromal-tumour cell junctions: frequent absence of a basal lamina and thin disordered collagen fibrils that show frequent direct contacts with tumour cells. On the basis of literature data concerning the interaction between stroma and epithelia under physiological and pathological conditions it is hypothesized that the interaction of collagen type I trimer fibrils with tumour cells is instrumental in augmenting tumour cell progression and that the trimer may provide contact guidance for invasive growth.
Asunto(s)
Adenocarcinoma Escirroso/ultraestructura , Neoplasias de la Mama/ultraestructura , Carcinoma Intraductal no Infiltrante/ultraestructura , Colágeno/análisis , Femenino , Humanos , Sustancias Macromoleculares , Microscopía ElectrónicaRESUMEN
Further analyses about collagen present in ductal infiltrating carcinoma of human mammary gland indicate that a large amount of it is represented by type I omotrimer that has been separated from the two other present species, type I eterotrimer and type III, by means of fractionated saline precipitation. Quantitative determinations of the three types, extracted by mild pepsin digestion, are also reported.
Asunto(s)
Neoplasias de la Mama/análisis , Colágeno/análisis , Humanos , MasculinoRESUMEN
Ductal infiltrating carcinoma of the breast is characterized by a remarkable amount of collagen fibrils surrounding nests and cords of neoplastic cells. Sequential extractions with pepsin release three classes of intact collagen chains, which have been identified as alpha 1 (I), alpha 2 (I) and alpha 1 (III) types. Alpha 1 (I) is prominent among these classes.