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OBJECTIVE: The study aimed determining whether assessment of cartilage oligomeric matrix protein (COMP) degradation products could serve as a serological disease course and therapeutic response predictor in arthritis. METHODS: We generated a panel of monoclonal antibodies against COMP fragments and developed a novel capture enzyme-linked immunosorbent assay (ELISA) for detecting COMP fragments in patients with osteoarthritis (OA) and rheumatoid arthritis (RA). This test was also used to monitor COMP fragments in surgically-induced OA, collagen-induced arthritis (CIA), and tumor necrosis factor (TNF) transgenic animal models. RESULTS: Compared with a commercial COMP ELISA kit that detected no significant difference in COMP levels between OA and control groups, a significant increase of the COMP fragments were noted in the serum of OA patients assayed by this newly established ELISA. In addition, serum COMP fragment levels were well correlated with severity in OA patients and the progression of surgically-induced OA in murine models. Furthermore, the serum levels of COMP fragments in RA patients, mice with CIA, and TNF transgenic mice were significantly higher when compared with their controls. Interestingly, treatment with TNFα inhibitors and methotrexate led to a significant decrease of serum COMP fragments in RA patients. Additionally, administration of Atsttrin [Tang, et al., Science 2011;332(6028):478] also resulted in a significant reduction in COMP fragments in arthritis mice models. CONCLUSION: A novel sandwich ELISA is capable of reproducibly measuring serum COMP fragments in both arthritic patients and rodent arthritis models. This test also provides a valuable means to utilize serum COMP fragments for monitoring the effects of interventions in arthritis.
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Artritis Experimental/metabolismo , Artritis Reumatoide/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Osteoartritis/metabolismo , Adolescente , Adulto , Animales , Antirreumáticos/farmacología , Proteína de la Matriz Oligomérica del Cartílago , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas de la Matriz Extracelular/efectos de los fármacos , Femenino , Glicoproteínas/efectos de los fármacos , Humanos , Masculino , Proteínas Matrilinas , Metotrexato/farmacología , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Proteínas Recombinantes de Fusión/farmacología , Líquido Sinovial/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Adulto JovenRESUMEN
We report the experimental realization and the characterization of polarization and momentum hyperentangled two-photon states, generated by a new parametric source of correlated photon pairs. By adoption of these states an "all-versus-nothing" test of quantum mechanics was performed. The two-photon hyperentangled states are expected to find at an increasing rate a widespread application in state engineering and quantum information.
RESUMEN
We describe herein a novel in vitro approach that can be used effectively to obtain valuable insights into the role of platelets, various coagulation proteins as well as proteins of the subendothelial extracellular matrix involved in the hemostatic and thrombotic processes occurring under microgravity. At difference with other experimental approaches proposed in the past our device operates in a closed system and under different shear forces, which better mimics flow conditions occurring in vessels. Furthermore our device by allowing real time monitoring of the thrombotic process and its underlying mechanisms can be regarded as a reliable system for the precise assessment of platelet function.
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Medicina Aeroespacial/instrumentación , Coagulación Sanguínea/fisiología , Ingravidez , Plaquetas/fisiología , Pruebas Hematológicas/instrumentación , Hemostasis/fisiología , Humanos , Microfluídica/instrumentación , Microfluídica/métodos , Microscopía Fluorescente , Trombosis/fisiopatologíaRESUMEN
The human mesonephros is currently regarded as a simplified version of the foetal metanephros, primarily due to the close morphological resemblance between these two structures. The aim of the present study was to define whether human mesonephric and foetal metanephric nephrons share immunophenotypical traits in their corresponding structures (glomeruli, proximal and distal tubules). For this purpose we first investigated immunohistochemically the overall expression and topographical distribution of cytokeratins 7, 8, 18, 19, and 20, vimentin and alpha-smooth muscle actin in mature mesonephric nephrons and compared the results with those obtained in maturing-stage foetal metanephric nephrons. No expression of cytokeratins 7 and 20 was found. Cytokeratins 8, 18, and 19 and vimentin showed a restricted and basically coincident expression along the different components of both mesonephric and metanephric nephrons. These findings indicate that the intermediate filament protein profile of human mature mesonephric nephrons closely recapitulates that observed in developing metanephros and thereby strengthens the concept that human mesonephros, a transient ontogenic structure, is largely similar to the foetal metanephros. The sole difference between human mesonephros and foetal metanephros was the divergent expression of alpha-smooth muscle actin. This protein exhibited an increasingly accentuated mesangial expression paralleling the morphological maturation of metanephric glomerulus, whereas it was absent from the mesonephric one. This would suggest that the mesangial cells in these two renal structures have a different function during the foetal life.
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Actinas/biosíntesis , Queratinas/biosíntesis , Mesonefro/metabolismo , Músculo Liso/metabolismo , Nefronas/metabolismo , Vimentina/biosíntesis , Adulto , Femenino , Humanos , Inmunohistoquímica , Glomérulos Renales/embriología , Glomérulos Renales/metabolismo , Túbulos Renales Distales/enzimología , Túbulos Renales Distales/metabolismo , Túbulos Renales Proximales/enzimología , Túbulos Renales Proximales/metabolismo , Mesonefro/embriología , Músculo Liso/embriología , Nefronas/embriología , Fenotipo , EmbarazoRESUMEN
To determine whether subendothelial laminins (LNs) could be implicated in the extravasation of neoplastic lymphocytes, we have examined the distribution of a number of LN isoforms in human vascular structures of adult individuals and have assayed the ability of the isolated LN molecules to promote adhesion of lymphoma and leukemic cells in vitro using a novel cell adhesion assay, CAFCA, Centrifugal Assay for Fluorescence-based Cell Adhesion (E. Giacomello et al., Biotechniques, 26: 758-762, 1999; P. Spessotto et al., Methods Mol. Biol., 139: 321-343, 2000). The use of previously characterized LN chain-specific antibodies showed that the vast majority of the smaller vascular compartments, known to correspond to sites of lymphocyte transmigration, expressed the subunits involved in the structuring of 9 of the 12 LN isoforms known to date. Eight LN isoforms (i.e., LN-1, -2, -4, -5, -8, -9, -10, and -11) and four naturally occurring LN complexes were isolated from various tissues and cultured cells by combined gel filtration, ion exchange, and immunoaffinity chromatographies, and the identity/composition of the isolated LNs/LN complexes was asserted by immunochemical means and amino-acid sequencing. Notwithstanding the widespread colocalization of LN isoforms, a panel of neoplastic B- and T-cell lines and lymphocytes isolated from patients affected by chronic lymphocytic B-cell leukemia attached preferentially and with high avidity to purified LN-8, purified LN-10, and LN-10-containing protein complexes, whereas lymphocytes derived from patients diagnosed with acute lymphocytic leukemia failed to bind to these LNs. All of the tested neoplastic lymphocytes failed to adhere to the isolated LN-1, LN-4, LN-9, and LN-11 and attached moderately well to purified LN-2 and LN-5. The interaction of transformed lymphocytes with LNs was cation-dependent and interchangeably mediated by the alpha3beta1 and alpha6beta1 integrins. The degree of engagement of the two LN receptors was dependent upon their relative levels of cell surface expression, whereas, irrespective of the phenotype, lymphocytes deprived of either of these receptors were incapable of LN binding. The findings suggest that LN-8 and LN-10 may act in an independent or complementary fashion as primary components of the endothelial basement membrane favoring the interaction of extravasating neoplastic lymphocytes. Thus, our results would demonstrate that different LN isoforms may evoke diverse cellular responses in different cell types and that this divergence may be the basis for the redundancy of LN distribution in a number of vascular structures.
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Linfocitos B/patología , Endotelio Vascular/metabolismo , Laminina/metabolismo , Linfocitos T/patología , Animales , Anticuerpos/farmacología , Anticuerpos Monoclonales , Linfocitos B/metabolismo , Membrana Basal/metabolismo , Bovinos , Adhesión Celular/fisiología , Endotelio Vascular/citología , Humanos , Integrinas/antagonistas & inhibidores , Integrinas/inmunología , Integrinas/metabolismo , Laminina/inmunología , Leucemia Linfocítica Crónica de Células B/patología , Leucemia de Células T/patología , Linfoma de Células T/patología , Linfocitos T/metabolismoRESUMEN
Cartilage oligomeric matrix protein has been implicated as an important component of endochondral ossification because of its direct effects on chondrocytes. The importance of this protein for skeletal development and growth has been recently illustrated by the identification of mutations in cartilage oligomeric protein genes in two types of inherited chondrodysplasias and osteoarthritic phenotypes: multiple epiphyseal dysplasia and pseudoachondroplasia. In the present study, we report the presence of cartilage oligomeric protein in embryonic and adult osteoblasts. A foot from a 21-week-old human fetus, subchondral bone obtained from knee replacement surgery in an adult patient, and a limb from a 19-day-postcoital mouse embryo were analyzed with immunostaining and in situ hybridization. In the human fetal foot, cartilage oligomeric protein was localized to osteoblasts of the bone collar and at the newly formed bone at the growth plate and bone diaphyses. Immunostaining was performed on the adult subchondral bone and showed positive intracellular staining for cartilage oligomeric protein of the osteoblasts lining the trabecular bone. There was no staining of the osteocytes. Immunostaining of the mouse limb showed the most intense staining for cartilage oligomeric protein in the hypertrophic chondrocytes and in the surrounding osteoblast cells of the developing bone. Cartilage oligomeric protein mRNA and protein were detected in an osteoblast cell line (MG-63), and cartilage oligomeric protein mRNA was detected from human cancellous bone RNA. These results suggest that the altered structure of cartilage oligomeric protein by the mutations seen in pseudoachondroplasia and multiple epiphyseal dysplasia may have direct effects on osteoblasts, contributing to the pathogenesis of these genetic disorders.
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Cartílago/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Osteoblastos/metabolismo , Animales , Cartílago/citología , Proteína de la Matriz Oligomérica del Cartílago , Células Cultivadas , Condrocitos/metabolismo , Cartilla de ADN/química , Proteínas de la Matriz Extracelular/genética , Femenino , Feto , Huesos del Pie/embriología , Huesos del Pie/metabolismo , Miembro Anterior/embriología , Miembro Anterior/metabolismo , Edad Gestacional , Glicoproteínas/genética , Humanos , Hibridación in Situ , Proteínas Matrilinas , Ratones , ARN Mensajero/metabolismoRESUMEN
Mouse cartilage oligomeric matrix protein cDNA was cloned and sequenced by a reverse transcription-polymerase chain reaction. The open reading frame encoded a product of 755 amino acids that shares a high degree of identity to and possesses all the characteristic molecular features of both rat and human cartilage oligomeric matrix protein. This suggests that cartilage oligomeric matrix protein is highly conserved during evolution. The clone was 83, 84, and 95% identical to human, bovine, and rat cartilage oligomeric matrix protein cDNA, respectively. In tissues from the adult mouse, cartilage oligomeric matrix protein was expressed not only in cartilage and tendon but in trachea, bone, skeletal muscle, eye, heart, and placenta as well, and no expression was found in other tissues. Immunohistology revealed that cartilage oligomeric matrix was deposited as early as 10 days post coitus in predifferentiated mouse embryo mesenchyme. It was detected in all cartilaginous tissues and in the skeletal muscles of the embryo at day 13. As development progressed, accumulation of cartilage oligomeric matrix protein was marked in the growth plate. At 19 days post coitus, it was prominently deposited in the hypertrophic zone of the growth plate, perichondrium, and periosteum and in the superficial layer of the articular cartilage surface but was absent in the more central areas of the epiphyseal cartilage. The restricted tissue distribution and expression of cartilage oligomeric matrix protein in developing as well as adult mouse tissues suggest the regulation of this protein at the transcriptional level. The findings reported herein are the first detailed characterization of the distribution of cartilage oligomeric matrix protein during early skeletal development of the mouse.
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Cartílago/embriología , Cartílago/fisiología , Proteínas de la Matriz Extracelular/genética , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas/genética , Animales , Secuencia de Bases , Northern Blotting , Cartílago/química , Proteína de la Matriz Oligomérica del Cartílago , Clonación Molecular , ADN Complementario , Proteínas Matrilinas , Ratones , Datos de Secuencia Molecular , ARN Mensajero/análisis , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Matrix metalloproteinase (MMP)-19 and MMP-20 (enamelysin) are two recently discovered members of the MMP family. These enzymes are involved in the degradation of the various components of the extracellular matrix (ECM) during development, haemostasis and pathological conditions. Whereas MMP-19 mRNA is found widely expressed in body tissues, including the synovium of normal and rheumatoid arthritic patients, MMP-20 expression is restricted to the enamel organ. In this study we investigated the ability of MMP-19 and MMP-20 to cleave two of the macromolecules characterising the cartilage ECM, namely aggrecan and the cartilage oligomeric matrix protein (COMP). Both MMPs hydrolysed aggrecan efficiently at the well-described MMP cleavage site between residues Asn(341) and Phe(342), as shown by Western blotting using neo-epitope antibodies. Furthermore, the two enzymes cleaved COMP in a distinctive manner, generating a major proteolytic product of 60 kDa. Our results suggest that MMP-19 may participate in the degradation of aggrecan and COMP in arthritic disease, whereas MMP-20, due to its unique expression pattern, may primarily be involved in the turnover of these molecules during tooth development.
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Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Metaloendopeptidasas/metabolismo , Proteoglicanos/metabolismo , Agrecanos , Secuencia de Aminoácidos , Animales , Artritis Reumatoide/enzimología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Western Blotting , Cartílago/citología , Cartílago/enzimología , Cartílago/metabolismo , Proteína de la Matriz Oligomérica del Cartílago , Dominio Catalítico , Bovinos , Electroforesis en Gel de Poliacrilamida , Matriz Extracelular/química , Matriz Extracelular/enzimología , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/química , Glicoproteínas/química , Humanos , Lectinas Tipo C , Proteínas Matrilinas , Metaloproteinasa 20 de la Matriz , Metaloproteinasas de la Matriz/química , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz Secretadas , Metaloendopeptidasas/química , Metaloendopeptidasas/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteoglicanos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia/genética , Especificidad por Sustrato , Porcinos , Diente/citología , Diente/enzimología , Diente/metabolismoRESUMEN
Once specified to become neural crest (NC), cells occupying the dorsal portion of the neural tube disrupt their cadherin-mediated cell-cell contacts, acquire motile properties, and embark upon an extensive migration through the embryo to reach their ultimate phenotype-specific sites. The understanding of how this movement is regulated is still rather fragmentary due to the complexity of the cellular and molecular interactions involved. An additional intricate aspect of the regulation of NC cell movement is that the timings, modes and patterns of NC cell migration are intimately associated with the concomitant phenotypic diversification that cells undergo during their migratory phase and the fact that these changes modulate the way that moving cells interact with their microenvironment. To date, two interplaying mechanisms appear central for the guidance of the migrating NC cells through the embryo: one involves secreted signalling molecules acting through their cognate protein kinase/phosphatase-type receptors and the other is contributed by the multivalent interactions of the cells with their surrounding extracellular matrix (ECM). The latter ones seem fundamental in light of the central morphogenetic role played by the intracellular signals transduced through the cytoskeleton upon integrin ligation, and the convergence of these signalling cascades with those triggered by cadherins, survival/growth factor receptors, gap junctional communications, and stretch-activated calcium channels. The elucidation of the importance of the ECM during NC cell movement is presently favoured by the augmenting knowledge about the macromolecular structure of the specific ECM assembled during NC development and the functional assaying of its individual constituents via molecular and genetic manipulations. Collectively, these data propose that NC cell migration may be governed by time- and space-dependent alterations in the expression of inhibitory ECM components; the relative ratio of permissive versus non-permissive ECM components; and the supramolecular assembly of permissive ECM components. Six multidomain ECM constituents encoded by a corresponding number of genes appear to date the master ECM molecules in the control of NC cell movement. These are fibronectin, laminin isoforms 1 and 8, aggrecan, and PG-M/version isoforms V0 and V1. This review revisits a number of original observations in amphibian and avian embryos and discusses them in light of more recent experimental data to explain how the interaction of moving NC cells with these ECM components may be coordinated to guide cells toward their final sites during the process of organogenesis.
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Movimiento Celular , Matriz Extracelular , Cresta Neural , Animales , Embrión de Pollo , Proteínas de la Matriz Extracelular , Cresta Neural/citología , Cresta Neural/embriologíaAsunto(s)
Movimiento Celular/fisiología , Animales , Adhesión Celular/fisiología , Fluorescencia , HumanosRESUMEN
It has been proposed that hyaluronan-binding proteoglycans play an important role as guiding cues during neural crest (NC) cell migration, but their precise function has not been elucidated. In this study, we examine the distribution, structure and putative role of the two major hyaluronan-binding proteoglycans, PG-M/versicans and aggrecan, during the course of avian NC development. PG-M/versicans V0 and V1 are shown to be the prevalent isoforms at initial and advanced phases of NC cell movement, whereas the V2 and V3 transcripts are first detected following gangliogenesis. During NC cell dispersion, mRNAs for PG-M/versicans V0/V1 are transcribed by tissues lining the NC migratory pathways, as well as by tissues delimiting nonpermissive areas. Immunohistochemistry confirm the deposition of the macromolecules in these regions and highlight regional differences in the density of these proteoglycans. PG-M/versicans assembled within the sclerotome rearrange from an initially uniform distribution to a preferentially caudal localization, both at the mRNA and protein level. This reorganization is a direct consequence of the metameric NC cell migration through the rostral portion of the somites. As suggested by previous in situ hybridizations, aggrecan shows a virtually opposite distribution to PG-M/versicans being confined to the perinotochordal ECM and extending dorsolaterally in a segmentally organized manner eventually to the entire spinal cord at axial levels interspacing the ganglia. PG-M/versicans purified from the NC migratory routes are highly polydispersed, have an apparent M(r) of 1,200-2,000 kDa, are primarily substituted with chondroitin-6-sulfates and, upon chondroitinase ABC digestion, are found to be composed of core proteins with apparent M(r )of 360-530, 000. TEM/rotary shadowing analysis of the isolated PG-M/versicans confirmed that they exhibit the characteristic bi-globular shape, have core proteins with sizes predicted for the V0/V1 isoforms and carry relatively few extended glycosaminoglycan chains. Orthotopical implantation of PG-M/versicans immobilized onto transplantable micromembranes tend to 'attract' moving cells toward them, whereas similar implantations of a notochordal type-aggrecan retain both single and cohorts of moving NC cells in close proximity of the implant and thereby perturb their spatiotemporal migratory pattern. NC cells fail to migrate through three-dimensional collagen type I-aggrecan substrata in vitro, but locomote in a haptotactic manner through collagen type I-PG-M/versican V0 substrata via engagement of HNK-1 antigen-bearing cell surface components. The present data suggest that PG-M/versicans and notochordal aggrecan exert divergent guiding functions during NC cell dispersion, which are mediated by both their core proteins and glycosaminoglycan side chains and may involve 'haptotactic-like' motility phenomena. Whereas aggrecan defines strictly impenetrable embryonic areas, PG-M/versicans are central components of the NC migratory pathways favoring the directed movement of the cells.
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Proteoglicanos Tipo Condroitín Sulfato/fisiología , Proteínas de la Matriz Extracelular , Ácido Hialurónico/metabolismo , Cresta Neural/citología , Proteoglicanos/fisiología , Agrecanos , Animales , Anticuerpos/química , Western Blotting , Bovinos , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Embrión de Pollo , Proteoglicanos Tipo Condroitín Sulfato/química , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Epítopos , Fibronectinas/metabolismo , Inmunohistoquímica , Hibridación in Situ , Membranas Intracelulares , Lectinas Tipo C , Microscopía Electrónica , Cresta Neural/embriología , Isoformas de Proteínas , Proteoglicanos/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Distribución Tisular , Células Tumorales Cultivadas , VersicanosRESUMEN
Engagement of cell surface adhesion receptors with extracellular constituents and with cellular counter-receptors is crucial for the extravasation of blood-borne neoplastic cells and their seeding at distant sites; however, the early events of tumor dissemination-ie, the intravasation step(s)-have been largely neglected. A role for the alpha4beta7 integrin was hypothesized to explain the high leukemogenicity exhibited by one (NQ22) among several T-cell lymphomas studied. To clarify the mechanisms of early aggressivity, the behavior of highly and poorly leukemogenic cell lines were compared in vitro. Cocultivation of physically separated leukemic cells with resting endothelial cells resulted in the up-regulation of VCAM-1 expression. NQ22 cells expressed mRNA of different cytokines that up-regulate VCAM-1 and at higher levels than cells of a nonaggressive lymphoma, and they migrated more efficiently through an activated endothelial cell layer. With the use of neutralizing antibodies against interferon-gamma, granulocyte macrophage colony-stimulating factor, and tumor necrosis factor (TNF)-alpha, it was determined that TNF-alpha is one of the soluble factors released by NQ22 cells involved in the up-regulation of VCAM-1. The finding that vascular cells within the early local growth were strongly positive for VCAM-1 indicated that NQ22 cells could activate endothelial cells also in vivo. Finally, cocultivation of preleukemic alpha4(-)NQ22 cells with TNF-alpha-activated endothelial cells induced the expression of alpha4 integrins on the former cells. Reciprocal up-regulation and engagement of alpha4/VCAM-1 pairs determined the sequential transmigration and intravasation steps, and similar mechanisms might affect the aggressivity of human T lymphoblastic lymphomas.
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Integrinas/genética , Linfoma de Células T/patología , Invasividad Neoplásica , Molécula 1 de Adhesión Celular Vascular/genética , Animales , Anticuerpos , Adhesión Celular , Movimiento Celular , Técnicas de Cocultivo , Endotelio Vascular/fisiología , Femenino , Regulación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Integrinas/fisiología , Interferón gamma/inmunología , Interferón gamma/fisiología , Masculino , Ratones , Ratones Endogámicos AKR , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/farmacología , Factor de Necrosis Tumoral alfa/fisiología , Molécula 1 de Adhesión Celular Vascular/fisiologíaRESUMEN
Synovium and cartilage from patients with osteoarthritis or rheumatoid arthritis were analyzed for expression of cartilage oligomeric matrix protein. Immunostaining of synovium with antiserum to cartilage oligomeric matrix protein demonstrated positive staining in both diseases. In osteoarthritis, there was positive staining within the synovial cells and immediately subjacent connective tissue, with less intense staining in the deeper connective tissue. In rheumatoid arthritis, there was less intense staining within the synovial cells and marked intense staining in the deeper connective tissue. In situ hybridization performed with an antisense digoxigenin-labeled riboprobe to human cartilage oligomeric matrix protein confirmed the presence of cartilage oligomeric matrix protein mRNA in the cells of the synovial lining in both types of synovium. Quantitative polymerase chain reaction with a cartilage oligomeric matrix protein MIMIC demonstrated increased cartilage oligomeric matrix protein mRNA in rheumatoid cartilage and synovium as compared with osteoarthritic cartilage and synovium, respectively; mRNA levels in rheumatoid synovium were similar to those from osteoarthritic chondrocytes. As a result of the high expression of cartilage oligomeric matrix protein from rheumatoid synovium, inflammatory synovium should be considered as a potential tissue source of cartilage oligomeric matrix protein in any investigation of biological markers of cartilage metabolism. The upregulated expression of cartilage oligomeric matrix protein in inflammatory tissues suggests its in vivo regulation by cytokines.
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Artritis Reumatoide/metabolismo , Cartílago Articular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Osteoartritis/metabolismo , Membrana Sinovial/metabolismo , Proteína de la Matriz Oligomérica del Cartílago , Proteínas de la Matriz Extracelular/genética , Glicoproteínas/genética , Humanos , Técnicas Inmunológicas , Hibridación in Situ , Proteínas Matrilinas , ARN Mensajero/metabolismo , Distribución Tisular/fisiologíaRESUMEN
Cell adhesion assays are widely used to identify novel cellular ligands, novel cell surface receptors for these ligands and to elucidate the mechanisms responsible for the underlying cellular and molecular interactions. We report here the development of a novel centrifugal assay for fluorescence-based cell adhesion (CAFCA) that offers a number of advantages over the currently available assays. CAFCA is based on two centrifugation steps: one to allow for the synchronization of the initial cell-substratum contact and one to enable both a defined removal force to be exerted onto the cells for displacement of unbound cells and determination of the relative binding strengths of adhering cells. The fluorescently tagged cells are monitored in specifically devised, disposable microplate assemblies by a two-sided fluorescence detection through the computer-interfaced SPECTRAFLUOR microplate fluorometer. The assay is rapid, accurate, reproducible and adaptable to small numbers of delicate primary cells that can ideally be labeled with the fluorochrome calcein AM (or analogous vital fluorescent dyes). Most uniquely, CAFCA provides (i) means of assessing the precise number of cells bound to a given substratum out of the total amount of cells contained within the population to be analyzed and (ii) a means of establishing the attachment strengths (i.e., dynes/cell) in a high number of samples/conditions simultaneously. CAFCA is therefore expected to make a substantial methodological and conceptual contribution to the range of available assays aimed at examining cellular interactions in vitro and promises the potential of being able to transpose automated versions of these tests for routine use in laboratories.
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Adhesión Celular , Centrifugación/métodos , Animales , Biotecnología , Línea Celular , Células Cultivadas , Centrifugación/instrumentación , Medios de Cultivo , Fluorescencia , Humanos , Leucemia Linfocítica Crónica de Células B/patología , RatonesRESUMEN
We have quantitated the relative contributions of the constitutively active alpha4beta1 and alpha4beta7 integrins and the domains embodying their cognate binding sites in mediating human B-cell lymphoma adhesion and chemotaxis on fibronectin. By cooperating, the central cell-binding and IIICS carboxy-terminal domains were entirely responsible for the adhesion activity displayed by fibronectin, and their relative contribution to this process was estimated to be 30% versus 70%. Assessment of the leukocyte-substrate binding strength (ie, dynes/cell) indicated a 10-fold higher avidity of the cell-IIICS domain interaction. The two integrins interchangeably recognized both domains, but differed quantitatively in their participation in the adhesive event, as well as in domain preference. The use of 3Fn (according to the nomenclature proposed by Bork and Koonin [Curr Opin Struct Biol 6:366, 1996] for the type III fibronectin modules) module-specific antibodies and recombinant polypeptides showed that alpha4 integrins recognized both the RGD sequence (3Fn10) and an apparently novel synergistic site located within the 3Fn8 module; even in this case, the integrins displayed a distinct binding site preference. Interleukin-1beta (IL-1beta)/IL-2-induced chemotaxis also involved cooperative function of the central cell-binding and IIICS domains, but the mechanisms regulating this phenomenon differed markedly from those controlling cell adhesion. First, the relative contribution of the individual domains was comparable, but neither of the individual domains promoted migration to the extent observed on intact fibronectin. Secondly, alpha4beta1 and alpha4beta7 integrins were both involved in the domain-binding necessary for initiation of migration, but the relative contribution of each receptor in the chemotactic process was less disparate than for initial cell adhesion. Thirdly, the mode by which chemotactic B-lymphoma movement was supported by the central cell-binding domain differed from that sustaining cell adhesion in that it involved independent recognition of either the 3Fn8 or the 3Fn9 module, which acted in synergy with the 3Fn10 module. Our data provide novel evidence concerning the relative importance of the constitutively active alpha4beta1 and alpha4beta7 integrins for the interaction of B-cell lymphoma cells with fibronectin, and they emphasize a multiple and diverse recognition of sites responsible for either anchorage or locomotion of tumor leukocytes on this matrix molecule.
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Quimiotaxis , Fibronectinas , Integrinas , Linfoma de Células B/patología , Receptores Mensajeros de Linfocitos , Sitios de Unión , Adhesión Celular , Humanos , Integrina alfa4beta1 , Células Tumorales CultivadasRESUMEN
We have identified type VI collagen (Col VI) as a primary subendothelial extracellular matrix component responsible for von Willebrand factor (vWF)-dependent platelet adhesion and aggregation under high tensile strength. Intact tetrameric Col VI was the form of the collagen found to be capable of promoting vWF-mediated platelet adhesion/aggregation under this shear condition, whereas removal of the predominant portion of the terminal globules by pepsin treatment abrogated its activity. The inability of the pepsin-digested Col VI to support any platelet interaction at high flow was because of the failure of the A3(vWF) domain to bind to this form of collagen, suggesting a stringent requirement of a tridimensional conformation or of intactness of its macromolecular structure. In contrast, the A1(vWF) domain bound to both intact and pepsin-digested Col VI tetramers but, in accordance with the cooperating function of the two vWF domains, failed to support platelet adhesion/aggregation under high shear onto Col VI by itself. The putative A1(vWF) binding site resided within the A7(VI) module (residues 413-613) of the globular amino-terminal portion of the alpha3(VI) chain. Soluble recombinant A7(VI) polypeptide strongly perturbed the vWF-mediated platelet adhesion to Col VI under high shear rates, without affecting the binding of the vWF platelet receptor glycoprotein Ibalpha to its cognate ligand A1(vWF). The findings provide evidence for a concerted action of the A1(vWF) and A3(vWF) domains in inducing platelet arrest on Col VI. This is accomplished via an interaction of the A1(vWF) domain with a site contained in the alpha3 chain A7(VI) domain and via a conformation-dependent interaction of the A3(vWF) domain with the intact tetrameric collagen. The data further emphasize that Col VI microfilaments linking the subendothelial basement membrane to the interstitial collagenous network may play a pivotal role in the hemostatic process triggered upon damage of the blood vessel wall.
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Colágeno/metabolismo , Adhesividad Plaquetaria , Factor de von Willebrand/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Membrana Basal/metabolismo , Sitios de Unión , Plaquetas/metabolismo , Colágeno/inmunología , Humanos , Integrinas/metabolismo , Ratones , Pepsina A/metabolismo , Conformación Proteica , Receptores de Colágeno , Proteínas Recombinantes/metabolismo , Ristocetina/metabolismoRESUMEN
Chondroblastoma is defined as a 'benign tumour, characterized by highly cellular and relatively undifferentiated tissue composed of rounded or polygonal chondroblast-like cells' and the 'presence of cartilaginous intercellular matrix' (WHO). An extensive analysis of the extracellular matrix composition and gene expression pattern of a large series of chondroblastoma cases shows, however, that type II collagen, which is the main component of any cartilage matrix, is not expressed by the neoplastic cells of this tumour entity and is not deposited into the extracellular tumour matrix. Instead, osteoid and fibrous matrix is formed, with its typical biochemical composition. The multifocal expression of aggrecan proteoglycan in most chondroblastomas explains the bluish, pseudo-chondroid appearance of some of the matrix-rich areas of chondroblastomas. This study did not show chondroid matrix formation or chondroblastic cell differentiation in chondroblastomas, suggesting that chondroblastoma should be classified as a specific bone-forming, rather than cartilage-forming neoplasm.
Asunto(s)
Neoplasias Óseas/patología , Condroblastoma/patología , Proteínas de la Matriz Extracelular , Agrecanos , Neoplasias Óseas/genética , Condroblastoma/genética , Colágeno/análisis , Colágeno/genética , Humanos , Inmunohistoquímica , Hibridación in Situ , Lectinas Tipo C , Proteínas de Neoplasias/análisis , Proteoglicanos/análisis , Proteoglicanos/genética , Proteínas S100/análisis , Vimentina/análisisRESUMEN
We report the molecular cloning of the human laminin alpha3b chain variant and its mRNA expression pattern in adult human tissues when compared to the alpha3a variant. The mRNA encoding for the alpha3b variant is about 11 kb and the predicted translation product carries the complete set of domains typical for a 'full-sized' laminin alpha chain. Apart from the similar domain structure of alpha3b also the sequence of alpha3 resulted more closely related to the alpha5 than to the alpha4 chain. Quantitative analysis of the RNA expression in a broad panel of adult human tissues indicated that the alpha3b variant is more widely distributed than the alpha3a shorter variant.
Asunto(s)
Variación Genética , Laminina/química , Laminina/genética , Adulto , Secuencia de Aminoácidos , Animales , Línea Celular Transformada , Clonación Molecular , Humanos , Laminina/biosíntesis , Ratones , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Distribución TisularRESUMEN
Human synovium was analyzed for the possible expression of cartilage oligomeric matrix protein (COMP). Immunostaining with polyclonal antiserum to COMP demonstrated positive staining within the synovial cells and immediately subjacent connective tissue, with less intense staining in the deeper connective tissue. Western blot analysis using either polyclonal or monoclonal antibodies to human COMP confirmed the presence of COMP by immunoreactive bands with the same molecular mass (approximately 110 kDa) as purified articular cartilage COMP. PCR using oligonucleotides that span human COMP exons 7-13 revealed identical amplification products from cDNA prepared from either human chondrocytes or synovium. Northern blot analysis using a biotinylated-probe to human COMP, spanning exons 12-13, also reveal an identical hybridization product to either human chondrocyte or synovium total RNA. Human synovium should be considered as a potential tissue source of COMP in any investigation of biological markers of cartilage metabolism.