RESUMEN
Tenascin-C (TNC) and tenascin-W (TNW), large hexameric glycoproteins overexpressed in the tumor microenvironment, are useful tumor biomarkers for theranostic applications. For now, polyclonal and monoclonal antibodies, as well as aptamers targeting TNC and TNW have been developed. However, the immunostaining sensitivity of antibodies is very heterogenous. The main aim of this study was to generate antibodies in dromedary that detect TNC and TNW, respectively. We show that immune sera from immunized dromedaries are able to specifically bind native TNC and TNW by ELISA and also to detect TNC and TNW in matrix tracks of mammary tumors by immunostaining. Furthermore, we demonstrate that purified IgG subtypes are able to interact specifically with TNC or TNW by ELISA and immunostaining. These camelid antibodies are a good basis to develop tools for the detection of TNC and TNW in the tumor microenvironment and could potentially have a broader application for early diagnosis of solid cancers.
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Anticuerpos/inmunología , Camelus/inmunología , Tenascina/inmunología , Animales , Anticuerpos/análisis , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/inmunología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Ensayo de Inmunoadsorción Enzimática , Femenino , Células HEK293 , Humanos , Inmunización , Ratones , Microscopía Fluorescente , Tenascina/análisis , Microambiente TumoralRESUMEN
Background Brevican, neurocan, tenascin-C and tenascin-R are extracellular matrix proteins present in brain that show increased expression in experimental animal models of brain injury. However, little is known about the dynamics of these proteins in human body fluids, such as cerebrospinal fluid (CSF) and serum, after traumatic brain injury (TBI). The aims of this study were to investigate if matrix proteins in CSF and serum are associated with functional outcome following traumatic brain injury, if their concentrations change over time and to compare their levels between brain injured patients to controls. Methods In total, 42 traumatic brain injury patients, nine healthy controls and a contrast group consisting of 38 idiopathic normal pressure hydrocephalus patients were included. Enzyme-linked immunosorbent assays (ELISAs) were used to measure the concentrations of proteins. Results Increased concentrations of brevican, tenascin-C and tenascin-R in CSF correlated with unfavourable outcome, with stronger outcome prediction ability compared to other biomarkers of brain tissue injury. CSF brevican, tenascin-R and serum neurocan gradually decreased with time (p = 0.04, p = 0.008, p = 0.005, respectively), while serum tenascin-C (p = 0.01) increased. CSF concentrations of brevican, neurocan and tenascin-R (only in time point 3) after TBI were lower than in the idiopathic normal pressure hydrocephalus group (p < 0.0001, p < 0.0001, and p = 0.0008, respectively). In serum, tenascin-C concentration was higher and neurocan lower compared to healthy controls (p = 0.02 and p = 0.0009). Conclusions These findings indicate that levels of extracellular matrix proteins are associated with clinical outcome following TBI and may act as markers for different pathophysiology than currently used protein biomarkers.
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Lesiones Traumáticas del Encéfalo/metabolismo , Proteínas de la Matriz Extracelular/análisis , Adulto , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Encéfalo/metabolismo , Lesiones Traumáticas del Encéfalo/sangre , Lesiones Traumáticas del Encéfalo/líquido cefalorraquídeo , Proteínas de la Matriz Extracelular/sangre , Proteínas de la Matriz Extracelular/líquido cefalorraquídeo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neurocano/análisis , Neurocano/sangre , Neurocano/líquido cefalorraquídeo , Tenascina/análisis , Tenascina/sangre , Tenascina/líquido cefalorraquídeo , Resultado del TratamientoRESUMEN
BACKGROUND: Both the extracellular matrix molecule tenascin-C (Tn-C) and tissue inhibitors of metalloproteinases (TIMPs) have a role in tissue injury, inflammation, and remodeling. In this pilot study, we tried to evaluate the role of these markers in acute kidney injury (AKI). METHODS: A total of 52 subjects were enrolled in this study. Group 1 consisted of 27 patients with AKI (stage 1, 2, and 3), and Group 2 consisted of 25 age- and gender-matched healthy subjects. Serum and urine samples (to determine Tn-C and TIMP-1) were obtained from the participants at the beginning of the study. Second samples were obtained from Group 1 patients when renal function improved (at discharge). RESULTS: Serum TIMP-1 concentrations (admission and discharge) were higher in Group 1 than Group 2 (p = 0.0001 for both comparisons). Tn-C excretion in spot urine was significantly higher in healthy controls than at the admission levels of the patient group (p = 0.036). However, TIMP-1 excretion in spot urine was lower in healthy controls than in admission and discharge levels of the patient group (p = 0.0001 for both comparisons). CONCLUSIONS: Our results show that these biomarkers (especially TIMP-1) may have a role in the pathophysiology of AKI. Further studies are needed in this field.
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Lesión Renal Aguda/patología , Biomarcadores/análisis , Tenascina/análisis , Inhibidor Tisular de Metaloproteinasa-1/análisis , Lesión Renal Aguda/sangre , Lesión Renal Aguda/orina , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Biomarcadores/orina , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Índice de Severidad de la Enfermedad , Tenascina/sangre , Tenascina/orina , Inhibidor Tisular de Metaloproteinasa-1/sangre , Inhibidor Tisular de Metaloproteinasa-1/orina , Adulto JovenRESUMEN
Although Tenascin-C (TNC) as an extracellular matrix protein involved in various cancers, the mechanisms by which TNC leads to decreased survival time remain to be clarified in CRC. We assessed the expression of TNC and its relationship with cancer associated fibroblasts (CAFs) markers, epithelial-to-mesenchymal transition (EMT) and cell cycle markers in 100 paraffin-embedded CRC tissue samples using immunohistochemistry. TNC expression was higher in CRC tissue samples than in adjacent non-tumor-tissues (Pâ¯<â¯.001). In addition, TNC was involved in clinical stage (Pâ¯=â¯.030), pT stage (Pâ¯=â¯.049), distant metastasis (Pâ¯=â¯.004), tumor recurrence (Pâ¯=â¯.007), and tumor budding (Pâ¯<â¯.001). TNC play crucial roles in regulating the poor 5-year CRC survival rate by Kaplan-Meier analysis, and was an independent predictor of poor overall survival (Pâ¯=â¯.007) and disease-free survival (Pâ¯=â¯.004) in CRC. Moreover, it was postively correlated with CAF (SMA (Pâ¯<â¯.001) and FSP1 (Pâ¯=â¯.005)) and cell cycle marker p27 (Pâ¯=â¯.013) along with EMT (E-cadherin, Pâ¯=â¯.599; Snail, Pâ¯<â¯.001; vimentin, Pâ¯=â¯.012). TNC may promote EMT-like change and proliferation, which lead to poor prognosis for patients with CRC.
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Neoplasias Colorrectales/genética , Tenascina/metabolismo , Adulto , Anciano , Biomarcadores de Tumor , Cadherinas/metabolismo , Proliferación Celular/genética , China , Neoplasias del Colon/metabolismo , Neoplasias Colorrectales/metabolismo , Supervivencia sin Enfermedad , Transición Epitelial-Mesenquimal/genética , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Fibroblastos , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Metástasis Linfática , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/metabolismo , Pronóstico , Tenascina/análisis , Tenascina/genéticaRESUMEN
Kidney fibrosis initiates at certain focal sites in which the fibrogenic niche provides a specialized microenvironment that facilitates fibroblast activation and proliferation. However, the molecular identity of these fibrogenic niches is poorly characterized. Here, we determined whether tenascin-C (TNC), an extracellular matrix glycoprotein, is a component of the fibrogenic niche in kidney fibrosis. In vivo, TNC expression increased rapidly in kidneys subjected to unilateral ureteral obstruction or ischemia/reperfusion injury and predominantly localized at the foci rich in fibroblasts in renal interstitium. In vitro, TNC selectively promoted renal interstitial fibroblast proliferation, bromodeoxyuridine incorporation, and the expression of proliferation-related genes. The mitogenic activity of TNC required the integrin/focal adhesion kinase/mitogen-activated protein kinase signaling cascade. Using decellularized extracellular matrix scaffolds, we found that TNC-enriched scaffolds facilitated fibroblast proliferation, whereas TNC-deprived scaffolds inhibited proliferation. Matrix scaffold prepared from fibrotic kidney also promoted greater ex vivo fibroblast proliferation than did scaffolds prepared from healthy kidney. Conversely, small interfering RNA-mediated knockdown of TNC in vivo repressed injury-induced fibroblast expansion and renal fibrosis. These studies identify TNC as a major constituent of the fibrogenic niche that promotes fibroblast proliferation, and illustrate a pivotal role for the TNC-enriched microenvironment in kidney fibrogenesis.
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Enfermedades Renales/etiología , Enfermedades Renales/patología , Riñón/patología , Tenascina/análisis , Animales , Proliferación Celular , Fibroblastos/patología , Fibrosis/etiología , Riñón/química , Masculino , Ratones , Ratones Endogámicos C57BL , Tenascina/fisiologíaRESUMEN
Tenascin C (TNC) is a key of extracellular matrix glycoprotein and highly express in numerous human malignancies. Herein, we attempted to clarify the clinicopathological significance of TNC as a prognostic determinant of breast ductal carcinoma. Then, we investigated TNC immunohistochemical expression in 150 breast ductal carcinomas and 27 normal breast tissue samples. Clinical relevance of TNC expression and the association TNC expression with other factors related to cancer-associated fibroblasts were also examined. In results, TNC expression was significantly higher in breast ductal carcinoma (56.0%) than normal breast tissues (25.9%). The upregulation TNC in cancer stromal were associated with pT stage (P=0.003), lymph node metastasis (P=0.002) and tumor node metastasis stage (P=0.001), also was correlated with an increase in tumor-associated macrophage population (P<0.001). The microvessel density (MVD) was significantly higher in TNC positive group than in negative group (P<0.001). In both univariate and multivariate Cox regression analyses, TNC was an independent poor prognostic factor for overall survival (OS) in breast ductal carcinoma patients. Importantly, over-expression TNC (P<0.001), FSP1 (P<0.001), SMA (P=0.002) and Vimentin (P=0.049) were significantly correlation with the lower OS (P<0.005). In addition, TNC expression in breast ductal carcinoma stromal was positively correlated with FSP1 (P<0.001), SMA (P=0.001) and Vimentin (P<0.001). In conclusion, the high expression of TNC could be a useful cancer-associated fibroblasts marker for the prediction of prognosis of breast ductal carcinoma patients.
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Neoplasias de la Mama/diagnóstico , Fibroblastos Asociados al Cáncer/metabolismo , Carcinoma Ductal de Mama/diagnóstico , Metástasis Linfática/diagnóstico , Tenascina/análisis , Biomarcadores de Tumor/análisis , Mama/citología , Mama/metabolismo , Neoplasias de la Mama/patología , Fibroblastos Asociados al Cáncer/citología , Carcinoma Ductal de Mama/patología , Estudios de Casos y Controles , Femenino , Humanos , Metástasis Linfática/patología , Persona de Mediana Edad , Pronóstico , Modelos de Riesgos Proporcionales , Manejo de EspecímenesRESUMEN
Glioblastoma multiforme (GBM) is a highly aggressive brain tumor, with dismal survival outcomes. Recently, cancer stem cells (CSCs) have been demonstrated to play a role in therapeutic resistance and are considered to be the most likely cause of cancer relapse. The identification of CSCs is an important step toward finding new and effective ways to treat GBM. Tenascin-C (TNC) protein has been identified as a potential marker for CSCs in gliomas based on previous work. Here, we have investigated the expression of TNC in tissue microarrays including 17 GBMs, 18 WHO grade III astrocytomas, 15 WHO grade II astrocytomas, 4 WHO grade I astrocytomas, and 7 normal brain tissue samples by immunohistochemical staining. TNC expression was found to be highly associated with the grade of astrocytoma. It has a high expression level in most of the grade III astrocytomas and GBMs analyzed and a very low expression in most grade II astrocytomas, whereas it is undetectable in grade I astrocytomas and normal brain tissues. Double-immunofluorescence staining for TNC and CD133 in GBM tissues revealed that there was a high overlap between theses two positive populations. The results were further confirmed by flow cytometry analysis of TNC and CD133 in GBM-derived stem-like neurospheres in vitro. A limiting dilution assay demonstrated that the sphere formation ability of CD133(+)/TNC(+) and CD133(-)/TNC(+) cell populations is much higher than that of the CD133(+)/TNC(-) and CD133(-)/TNC(-) populations. These results suggest that TNC is not only a potential prognostic marker for GBM but also a potential marker for glioma CSCs, where the TNC(+) population is identified as a CSC population overlapping with part of the CD133(-) cell population.
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Biomarcadores de Tumor/análisis , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Tenascina/análisis , Análisis de Matrices Tisulares/métodos , Adolescente , Adulto , Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/química , Femenino , Glioblastoma/química , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Células Madre Neoplásicas , Tenascina/metabolismo , Células Tumorales Cultivadas , Adulto JovenRESUMEN
PURPOSE: Fibronectin (Fn) and tenascin-C (TnC) are two extracellular matrix proteins associated with remodeling changes. Fn and TnC gene and protein expression in lung tissue, including their predominant location in bronchial and pulmonary artery structures, have not yet been fully evaluated. The aim of the present study was to assess: (1) gene expression of Fn and TnC in lung samples from chronic obstructive pulmonary disease (COPD) and non-COPD subjects; and (2) protein content and location of Fn and TnC in both groups. METHODS: Consecutive subjects requiring lung resection due to lung cancer surgery were included. Lung specimens were examined for gene expression by quantitative real-time PCR (values expressed as fold change ratio). The analysis of their protein content and location was performed by western blot and immunohistochemical studies, respectively. Patients were divided into two cohorts according to COPD status. RESULTS: A total of 41 patients (20 with COPD and 21 without COPD) were included. An enhanced Fn gene expression was observed in the COPD group compared to the non-COPD group (4.73 ± 0.54 vs. 2.65 ± 0.57; P = 0.012), whereas no differences in gene TnC expression were observed (2.91 ± 0.44 vs. 2.60 ± 0.48; P = 0.633). No differences in lung protein content and location were found between groups. Immunohistochemical evaluation showed a predominantly vascular and bronchial location of Fn and TnC in both groups. CONCLUSIONS: An enhanced lung gene expression of Fn was observed in COPD subjects compared to non-COPD subjects. No differences were found in Fn protein expression or in TnC gene or protein expression among groups.
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Fibronectinas/análisis , Fibronectinas/genética , Pulmón/química , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Tenascina/análisis , Tenascina/genética , Anciano , Remodelación de las Vías Aéreas (Respiratorias) , Western Blotting , Estudios de Casos y Controles , Femenino , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Pulmón/fisiopatología , Pulmón/cirugía , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Enfermedad Pulmonar Obstructiva Crónica/cirugía , Reacción en Cadena en Tiempo Real de la Polimerasa , Índice de Severidad de la EnfermedadRESUMEN
Studying gene expression profile in a single cancer cell is important because multiple genes are associated with cancer development. Quantum dots (QDs) have been utilized as biological probes for imaging and detection. QDs display specific optical and electrical properties that depend on their size that can be applied for imaging and sensing applications. In this study, simultaneous imaging of the cancer biomarkers, tenascin-C and nucleolin, was performed using two types of aptamer-conjugated QDs. The simultaneous imaging of these two different cancer markers in three cancer cell lines was reliable and cell line-specific. Current requirements for cancer imaging technologies include the need for simple preparation methods and the ability to detect multiple cancer biomarkers and evaluate their intracellular localizations. The method employed in this study is a feasible solution to these requirements.
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Biomarcadores de Tumor/análisis , Diagnóstico por Imagen/métodos , Puntos Cuánticos/metabolismo , Línea Celular Tumoral , Humanos , Fosfoproteínas/análisis , Proteínas de Unión al ARN/análisis , Tenascina/análisis , NucleolinaRESUMEN
The BM microenvironment is required for the maintenance, proliferation, and mobilization of hematopoietic stem and progenitor cells (HSPCs), both during steady-state conditions and hematopoietic recovery after myeloablation. The ECM meshwork has long been recognized as a major anatomical component of the BM microenvironment; however, the molecular signatures and functions of the ECM to support HSPCs are poorly understood. Of the many ECM proteins, the expression of tenascin-C (TN-C) was found to be dramatically up-regulated during hematopoietic recovery after myeloablation. The TN-C gene was predominantly expressed in stromal cells and endothelial cells, known as BM niche cells, supporting the function of HSPCs. Mice lacking TN-C (TN-C(-/-)) mice showed normal steady-state hematopoiesis; however, they failed to reconstitute hematopoiesis after BM ablation and showed high lethality. The capacity to support transplanted wild-type hematopoietic cells to regenerate hematopoiesis was reduced in TN-C(-/-) recipient mice. In vitro culture on a TN-C substratum promoted the proliferation of HSPCs in an integrin α9-dependent manner and up-regulated the expression of the cyclins (cyclinD1 and cyclinE1) and down-regulated the expression of the cyclin-dependent kinase inhibitors (p57(Kip2), p21(Cip1), p16(Ink4a)). These results identify TN-C as a critical component of the BM microenvironment that is required for hematopoietic regeneration.
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Células de la Médula Ósea/citología , Médula Ósea/metabolismo , Hematopoyesis , Células Madre Hematopoyéticas/citología , Tenascina/metabolismo , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/efectos de la radiación , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Proliferación Celular , Células Endoteliales/citología , Células Endoteliales/metabolismo , Fluorouracilo/administración & dosificación , Fluorouracilo/efectos adversos , Eliminación de Gen , Células Madre Hematopoyéticas/metabolismo , Cadenas alfa de Integrinas/genética , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , Células del Estroma/citología , Células del Estroma/metabolismo , Tenascina/análisis , Tenascina/genética , Regulación hacia Arriba , Irradiación Corporal TotalRESUMEN
BACKGROUND: Bronchial asthma often starts in early childhood. Clinical manifestation of the disease is likely due to inflammatory processes in the airways initiated by various stimuli. Developed remodelling is regularly observed in the bronchial mucosa of adult asthmatics but we still lack information about its onset and latter development with the natural course of the disease. In this study, we analysed histological findings in bronchial biopsies obtained from very young children (under 4 yr of age). We hypothesized that initial undetectable changes in the airway epithelium of children predisposed to asthma may be one of the first mechanisms leading to morphological changes in the bronchial mucosa. METHODS: We measured the thickness of the basement membrane using a light microscope and analysed the presence of its three basic structural glycoproteins: laminin, tenascin and collagen IV, using immunohistochemical techniques. We compared these findings in children predisposed to asthma according to the selected clinical criteria of the Asthma Predictive Index and in a control group of children. RESULTS: We found a significant difference in the thickness of the basement membrane between the two groups. We also found a difference in the subepithelial deposition of laminin and collagen IV in the basement membrane but no difference in the deposition of tenascin. CONCLUSIONS: We conclude that initial changes leading to further remodelling may start at a very early age even before clinical manifestation of the disease.
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Remodelación de las Vías Aéreas (Respiratorias) , Asma/etiología , Bronquios/patología , Mucosa Respiratoria/patología , Factores de Edad , Asma/metabolismo , Asma/patología , Membrana Basal/química , Membrana Basal/patología , Biopsia , Bronquios/química , Broncoscopía , Estudios de Casos y Controles , Preescolar , Colágeno Tipo IV/análisis , Diagnóstico Precoz , Femenino , Humanos , Inmunohistoquímica , Lactante , Laminina/análisis , Masculino , Valor Predictivo de las Pruebas , Mucosa Respiratoria/química , Factores de Riesgo , Tenascina/análisisRESUMEN
A series of bis-, tris- and tetra-phosphonated pyridine ligands is presented. In view of their potential use as chelates for radiopharmaceutical applications, the physico-chemical properties of the ligands and of their Co(II), Ni(II), Cu(II), and Zn(II) complexes were studied by means of potentiometry and UV-Vis absorption spectroscopy. The pKa values of the ligands and of the complexes, as well as the stability constants for the formation of the complexes, are presented. The kinetic aspects of the formation of Cu(II) complexes and of their dissociation in acidic media were studied by means of stopped flow experiments, and the stability of the Cu(II) complex toward reduction to Cu(I) was investigated by cyclic voltammetry and by titration with different reducing agents. The different thermodynamic and kinetic aspects of the polyphosphonated ligands were compared with regard to the impact of the number of phosphonic acid functions. Considering the very promising properties for complexation, preliminary SPECT/CT imaging experiments were carried out on mice with (99m)Tc using the bis- and tetra-phosphonated ligands L(2) and L(1). Finally, a bifunctional version of chelate L(1), L*, was used to label MTn12, a rat monoclonal antibody with both specificity and relatively high affinity for murine tenascin-C. The labeling was monitored by MALDI/MS spectrometry and the affinity of the labeled antibody was checked by immunostaining experiments. After chelation with (99m)Tc, the (99m)Tc-L*-MTn12 antibody was injected into a transgenic mouse with breast cancer and the biodistribution of the labeled antibody was followed by SPECT/CT imaging.
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Quelantes/química , Complejos de Coordinación/química , Organofosfonatos/química , Piridinas/química , Radiofármacos/química , Animales , Anticuerpos Monoclonales/química , Neoplasias de la Mama/diagnóstico , Femenino , Ligandos , Ratones , Ratones Transgénicos , Ratas , Tenascina/análisis , Termodinámica , Distribución Tisular , Tomografía Computarizada de Emisión de Fotón ÚnicoRESUMEN
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive parenchymal lung disease of unknown aetiology and poor prognosis, characterized by altered tissue repair and fibrosis. The extracellular matrix (ECM) is a critical component in regulating cellular homeostasis and appropriate wound healing. The aim of our study was to determine the expression profile of highlighted ECM proteins in IPF lungs. METHODS: ECM gene and protein expression was analyzed by cDNA microarrays, rt-PCR, immunohistochemistry and western-blot in lungs from idiopathic pulmonary fibrosis (IPF), hypersensitivity pneumonitis (HP), categorized as chronic (cHP) and subacute (saHP), and healthy lung tissue. Primary fibroblast cultures from normal subjects and fibrotic patients were studied to evaluate tenascin-C (TNC) synthesis. RESULTS: A total of 20 ECM proteins were upregulated and 6 proteins downregulated in IPF. TNC was almost undetected in normal lungs and significantly upregulated in fibrotic lungs (IPF and cHP) compared to saHP. Furthermore, it was located specifically in the fibroblastic foci areas of the fibrotic lung with a subepithelial gradient pattern. TNC levels were correlated with fibroblastic foci content in cHP lungs. Versican and fibronectin glycoproteins were associated with TNC, mainly in fibroblastic foci of fibrotic lungs. Fibroblasts from IPF patients constitutively synthesized higher levels of TNC than normal fibroblasts. TNC and α-sma was induced by TGF-ß1 in both fibrotic and normal fibroblasts. TNC treatment of normal and fibrotic fibroblasts induced a non-significant increased α-sma mRNA. CONCLUSIONS: The difference in ECM glycoprotein content in interstitial lung diseases could contribute to the development of lung fibrosis. The increase of TNC in interstitial areas of fibrotic activity could play a key role in the altered wound healing.
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Fibroblastos/metabolismo , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/metabolismo , Tenascina/biosíntesis , Tenascina/genética , Actinas/genética , Actinas/metabolismo , Adulto , Anciano , Alveolitis Alérgica Extrínseca/genética , Alveolitis Alérgica Extrínseca/metabolismo , Células Cultivadas , Enfermedad Crónica , Regulación hacia Abajo , Femenino , Fibronectinas/genética , Fibronectinas/metabolismo , Expresión Génica , Humanos , Fibrosis Pulmonar Idiopática/patología , Pulmón/química , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Tenascina/análisis , Factor de Crecimiento Transformador beta1/farmacología , Regulación hacia Arriba , Versicanos/genética , Versicanos/metabolismoRESUMEN
Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is characterized by progressive fatty and fibrous replacement. A female in her 70s, suddenly found in cardiopulmonary arrest. The heart weighed 452 g and yellow discoloration was observed. Histological examination revealed the replacement of the right ventricular myocardium by adipose tissue and fibrosis. The cause of death was fatal arrhythmia caused by ARVC/D. Tenascin C staining was useful in evaluating myocardial remodeling.
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Displasia Ventricular Derecha Arritmogénica/patología , Tenascina/análisis , Anciano , Displasia Ventricular Derecha Arritmogénica/metabolismo , Autopsia , Resultado Fatal , Femenino , Humanos , Inmunohistoquímica , Miocardio/química , Miocardio/patología , Tenascina/metabolismo , Remodelación VentricularRESUMEN
The extracellular matrix (ECM) protein tenascin-C (TN-C) is upregulated within glioma tissues and cultured glioma cell lines. TN-C possesses a multi-modular structure and a variety of functional properties have been reported for its domains. We describe five novel monoclonal antibodies identifying different domains of TN-C. The epitopes for these antibodies were investigated by using recombinantly expressed fibronectin type III domains of TN-C. The biological effects of TN-C fragments on glioma cell proliferation and adhesion were analyzed. The expression pattern of TN-C in human glioma tissue sections and in glioma cell lines was studied with the novel library of monoclonal antibodies. The immunocytochemical analyses of the established human glioma cell lines U-251-MG, U-373-MG and U-87-MG revealed distinct staining patterns for each antibody. Robust expression of TN-C was found within the tumor mass of surgery specimens from glioblastoma. In many cases, the expression of this ECM molecule was clearly associated with blood vessels, particularly with microvessels. Three of the new antibodies highlighted individual TN-C-expressing single cells in glioma tissues. The effect of TN-C domains on glioma cells was examined by a BrdU-proliferation assay and an adhesion assay. Short fragments of constitutively expressed TN-C-domains did not exert significant effects on the proliferation of glioma cells, whereas the intact molecule increased cell division rates. In contrast, the long fragment TNfnALL containing all of the FNIII domains of TN-C decreased proliferation. Additionally, we found strong differences between the adhesion-influencing properties of the recombinant fragments on glioma cells.
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Neoplasias Encefálicas/irrigación sanguínea , Neoplasias del Sistema Nervioso Central/irrigación sanguínea , Glioma/irrigación sanguínea , Tenascina/análisis , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Neoplasias del Sistema Nervioso Central/genética , Neoplasias del Sistema Nervioso Central/patología , Regulación Neoplásica de la Expresión Génica , Glioblastoma/irrigación sanguínea , Glioblastoma/genética , Glioblastoma/patología , Glioma/genética , Glioma/patología , Humanos , Inmunohistoquímica , Tenascina/genéticaRESUMEN
OBJECTIVE: To determine the role of insulin-like growth factor binding protein 3 (IGFBP-3) in mediating the effects of transforming growth factor ß (TGFß) on tenascin-C (TN-C) production and to assess the levels of TN-C in vivo in patients with systemic sclerosis (SSc)-associated pulmonary fibrosis. METHODS: Human primary lung fibroblasts were stimulated with TGFß or IGFBP-3 in the presence or absence of specific small interfering RNAs and chemical inhibitors of the signaling cascade. TN-C levels in lung tissue specimens obtained from patients with SSc-associated pulmonary fibrosis were assessed using immunohistochemical analysis and were compared with the levels in specimens obtained from normal donors. TN-C levels were quantified in sera from normal donors and patients with SSc with or without pulmonary fibrosis, using an enzyme-linked immunosorbent assay. RESULTS: IGFBP-3 mediated the induction of TN-C by TGFß. Direct induction of TN-C by IGFBP-3 occurred in a p38 MAP kinase-dependent manner. TN-C levels were abundant in lung tissues from patients with SSc and were localized to subepithelial layers of the distal airways. No TN-C was detectable around the proximal airways. Patients with SSc-associated pulmonary fibrosis had significantly higher levels of circulating TN-C compared with SSc patients without pulmonary fibrosis. Longitudinal samples obtained from patients with SSc before and after the onset of pulmonary fibrosis showed increased levels of TN-C after the onset of pulmonary fibrosis. CONCLUSION: IGFBP-3, which is overexpressed in fibrotic lungs, induces production of TN-C by subepithelial fibroblasts. The increased lung tissue levels of TN-C parallel the levels detected in the sera of SSc patients with pulmonary fibrosis, suggesting that TN-C may be a useful biomarker for SSc-related pulmonary fibrosis.
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Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/farmacología , Fibrosis Pulmonar/metabolismo , Esclerodermia Sistémica/metabolismo , Tenascina/metabolismo , Células Cultivadas , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Fibrosis Pulmonar/complicaciones , Fibrosis Pulmonar/patología , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Proteínas Recombinantes/farmacología , Esclerodermia Sistémica/complicaciones , Esclerodermia Sistémica/patología , Tenascina/análisis , Factor de Crecimiento Transformador beta/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Focal cortical dysplasias (FCDs) of the brain are recognized as a frequent cause of intractable epilepsy. To contribute to the current understanding of the mechanisms of epileptogenesis in FCD, our study provides evidence that not only cellular alterations and synaptic transmission, but also changed diffusion properties of the extracellular space (ECS), induced by modified extracellular matrix (ECM) composition and astrogliosis, might be involved in the generation or spread of seizures in FCD. The composition of the ECM in FCD and non-malformed cortex (in 163 samples from 62 patients) was analyzed immunohistochemically and correlated with the corresponding ECS diffusion parameter values determined with the real-time iontophoretic method in freshly resected cortex (i.e. the ECS volume fraction and the geometrical factor tortuosity, describing the hindrances to diffusion in the ECS). The ECS in FCD was shown to differ from that in non-malformed cortex, mainly by the increased accumulation of certain ECM molecules (tenascin R, tenascin C, and versican) or by their reduced expression (brevican), and by the presence of an increased number of astrocytic processes. The consequent increase of ECS diffusion barriers observed in both FCD type I and II (and, at the same time, the enlargement of the ECS volume in FCD type II) may alter the diffusion of neuroactive substances through the ECS, which mediates one of the important modes of intercellular communication in the brain - extrasynaptic volume transmission. Thus, the changed ECM composition and altered ECS diffusion properties might represent additional factors contributing to epileptogenicity in FCD.
Asunto(s)
Encefalopatías/patología , Matriz Extracelular/química , Espacio Extracelular/química , Malformaciones del Desarrollo Cortical/patología , Adolescente , Adulto , Astrocitos/metabolismo , Encefalopatías/metabolismo , Brevicano/análisis , Niño , Preescolar , Difusión , Epilepsia , Matriz Extracelular/metabolismo , Espacio Extracelular/metabolismo , Femenino , Humanos , Iontoforesis/métodos , Masculino , Malformaciones del Desarrollo Cortical/metabolismo , Malformaciones del Desarrollo Cortical de Grupo I , Persona de Mediana Edad , Neocórtex/patología , Tenascina/análisis , Versicanos/análisis , Adulto JovenRESUMEN
The synthesis of a phosphonated acyclic bifunctional chelate L* for the labeling of biomaterial is described. L* is based on a pyridine backbone, functionalized in ortho positions by aminomethyl-bis-methylphosphonic acids, and, in the para position, by a side chain containing a reactive NHS carbamate function. The stability of L* in aqueous solutions at different pH values was studied by mass spectrometry, showing the activated function to be sensitive to hydrolysis above neutral pH. The reactivity of L* towards amine functions was tested using ethylamine under different conditions of pH and concentrations, and by the labeling of two reference peptides containing both an N-terminal amino function and a ε-amino group of a lysine residue in the backbone, and a supplementary thiol group of a cysteine residue for one of these two peptides. The results showed the coupling to be efficient at pH 8.0, with a total selectivity for the terminal amine function with respect to lysine and cysteine. The labeling was further performed on B28-13, a mouse monoclonal antibody specifically recognizing tenascin-C protein in human cancer. The labeled antibody was characterized by means of mass spectrometry and spectrofluorimetry, unraveling a labeling ratio of one chelate per antibody. Finally, the affinity of the labeled antibody towards its target was controlled by immunofluorescence staining experiments on human colon cancer biopsies, confirming the affinity of the labeled peptide for tenascin-C.
Asunto(s)
Quelantes/síntesis química , Neoplasias del Colon/patología , Proteínas de Neoplasias/análisis , Organofosfonatos/química , Péptidos/química , Piridinas/química , Tenascina/análisis , Anticuerpos Monoclonales/química , Biopsia , Neoplasias del Colon/diagnóstico por imagen , Cisteína/química , Etilaminas/química , Técnica del Anticuerpo Fluorescente , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Lisina/química , Imagen Multimodal , Tomografía de Emisión de Positrones , Radiofármacos/síntesis química , Espectrometría de Fluorescencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Coloración y Etiquetado/métodos , Tomografía Computarizada por Rayos XRESUMEN
BACKGROUND: Sarcoidosis is a disease characterized by granulomatous lesions involving multiple organ systems. The etiology of sarcoidosis remains unknown, and reliable biomarkers have not been identified. Tenascin-C is an extracellular matrix molecule expressed during wound healing in various tissues. The present study aimed to investigate the role of tenascin-C in sarcoidosis. METHODS: Enzyme-linked immunosorbent assays were used to measure tenascin-C levels in serum and bronchoalveolar lavage fluid (BALF) from 31 patients with sarcoidosis and 15 healthy individuals. Relationships between tenascin-C concentrations in BALF and serum samples and clinical parameters in patients with sarcoidosis were evaluated. RESULTS: BALF tenascin-C levels were significantly higher in patients with sarcoidosis than in healthy individuals, but serum levels were no different. BALF tenascin-C levels showed positive correlations with serum lactic dehydrogenase levels and the ratio of lymphocytes in BALF. BALF tenascin-C levels were also higher in patients with parenchymal infiltration on chest radiographs than in those without. CONCLUSIONS: The present results demonstrated that the BALF tenascin-C level was correlated with pulmonary infiltrates on chest radiographs in patients with sarcoidosis. Although measurement of serum tenascin-C levels has a limited role and measurement of BALF tenascin-C levels might be impractical, tenascin-C in the lung might play a role in the pathogenesis of sarcoidosis. Further studies are necessary to determine the role of BALF tenascin-C in sarcoidosis.