RESUMEN
Basic fibroblast growth factor (FGF-2) has been shown to enhance the survival and neurite extension of various types of neurons including spinal ganglion neurons. In addition, endogenous FGF-2 and FGF receptors are upregulated following peripheral nerve lesion in ganglia and at the lesion site. FGF-2 protein is expressed in different isoforms (18 kDa, 21 kDa, 23 kDa) and differentially regulated after nerve injury. In the rat we analyzed the regenerative capacity of the high molecular weight (HMW) FGF-2 isoforms (21/23 kDa) to support the regeneration of the axotomized adult sciatic nerve across long gaps. The nerve stumps were inserted into the opposite ends of a silicone chamber resulting in an interstump gap of 15 mm. Silicone tubes were filled with Matrigel or a mixture of Schwann cells (SC) and Matrigel. SC were prepared from newborn rats and transfected to overexpress HMW FGF-2. Four weeks after the operation procedure, channels were analyzed with regard to tissue cables bridging both nerve stumps and myelinated axons distal to the original proximal nerve stump. Peripheral nerves interposed with HMW Schwann cells displayed significantly enhanced nerve regeneration, with the greatest number of tissue cables containing myelinated axons and the highest number of myelinated axons. These results suggest that a cellular substrate together with a source of a trophic factor could be a promising tool to promote nerve regeneration and, therefore, become useful also for a clinical approach to repair long gaps.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/metabolismo , Regeneración Nerviosa , Neuronas/fisiología , Isoformas de Proteínas/metabolismo , Células de Schwann/fisiología , Siliconas/metabolismo , Animales , Animales Recién Nacidos , Técnicas de Cultivo de Célula/métodos , Colágeno/metabolismo , Combinación de Medicamentos , Factor 2 de Crecimiento de Fibroblastos/química , Factor 2 de Crecimiento de Fibroblastos/genética , Procesamiento de Imagen Asistido por Computador , Laminina/metabolismo , Fibras Nerviosas Mielínicas/metabolismo , Fibras Nerviosas Mielínicas/ultraestructura , Neuronas/ultraestructura , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Proteoglicanos/metabolismo , Ratas , Ratas Sprague-Dawley , Células de Schwann/ultraestructura , Nervio Ciático/citología , Nervio Ciático/metabolismo , Nervio Ciático/cirugíaRESUMEN
Fibroblast growth factor-2 (FGF-2) is an important modulator of cell growth and differentiation and stimulates cell survival of various cells including neurons. Rat FGF-2 occurs in three isoforms, a low molecular weight 18 kD and two high molecular weight forms (21, 23 kD), representing alternative translation products from a single mRNA. The 18 kD isoform shows mainly cytoplasmatic localization, whereas the 21/23 kD FGF-2 are localized in the nucleus. In addition, the FGF-2 isoforms are differentially regulated in the sensory ganglia and peripheral nerve following nerve injury and in the adrenal medulla during post-natal development and after hormonal stimuli. The distinct intracellular distribution and differential regulation of the different FGF-2 isoforms indicate that they have unique biological roles, however, little is known about the biological effects of the high molecular weight FGF-2 isoforms. Immortalized Schwann cells and PC12 cells, which stably overexpress the different FGF-2 isoforms, showed that the different endogenous-overexpressed FGF-2 isoforms lead to dramatic modifications in cell proliferation and survival, when tested in serum-free and serum-containing medium. In contrast, application of recombinant FGF-2 isoforms on normal PC12 and immortalized Schwann cells results in similar biological effects on the proliferation and survival of the cells. Furthermore, we investigated the potential regulatory effects of endogenous-overexpressed and exogenous-applied FGF-2 isoforms on the mRNA level of the FGF-2 receptors and, additionally, on the tyrosin hydroxylase mRNA expression in PC12 cells.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/química , Proteínas Tirosina Quinasas , Animales , División Celular , Supervivencia Celular , Fibroblastos/metabolismo , Células PC12 , Plásmidos/metabolismo , Unión Proteica , Isoformas de Proteínas , ARN Mensajero/metabolismo , Ratas , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/biosíntesis , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células de Schwann/metabolismo , TransfecciónRESUMEN
Basic fibroblast growth factor is expressed in different isoforms which display tissue and species specificity and are differentially regulated during development and after experimental interventions. The differential regulation of the fibroblast growth factor-2 isoforms may indicate specific activities and functions of these molecules. The characterization of fibroblast growth factor-2 effects, however, is almost exclusively based on studies including the 18,000 mol. wt isoform. It is not yet known whether the high molecular weight fibroblast growth factor-2 isoforms (21,000 mol. wt, 23,000 mol. wt) exert similar or distinct activities in the nervous system. In the present study, we investigated the effects of the high molecular weight isoforms on dissociated rat mesencephalic dopaminergic neurons. For this purpose, recombinant fibroblast growth factor-2 isoforms, prepared in a histidine expression system, were administered on dopaminergic neurons in vitro, and Schwann cells over-expressing the high molecular weight isoforms were co-cultured with dopaminergic neurons. This is the first demonstration to show that the high molecular weight isoforms mediate a neurotrophic activity. Exogenous high molecular weight fibroblast growth factor-2 isoforms stimulated the survival of embryonic mesencephalic dopaminergic neurons and protected them from 6-hydroxydopamine neurotoxicity. In addition, co-culture of dopaminergic neurons with high molecular weight fibroblast growth factor-2 over-expressing Schwann cells revealed an increased survival and neurite formation of the mesencephalic dopaminergic neurons. These results suggest that the high molecular weight fibroblast growth factor-2 isoforms may serve as a new tool for the treatment of Parkinson's disease.
Asunto(s)
Dopamina/metabolismo , Factor 2 de Crecimiento de Fibroblastos/química , Factor 2 de Crecimiento de Fibroblastos/farmacología , Mesencéfalo/embriología , Factores de Crecimiento Nervioso/fisiología , Neuronas/fisiología , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo , Embrión de Mamíferos/metabolismo , Mesencéfalo/citología , Mesencéfalo/efectos de los fármacos , Peso Molecular , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neurotoxinas/farmacología , Oxidopamina/farmacología , Isoformas de Proteínas/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Células de Schwann/metabolismo , Células de Schwann/fisiología , Sustancia Negra/citología , Sustancia Negra/efectos de los fármacos , Sustancia Negra/embriologíaRESUMEN
OBJECTIVE: Chronic pancreatitis is a painful chronic inflammatory disease of the exocrine pancreas that is associated with the replacement of functional parenchyma by extended fibrosis and with a massive infiltration of T lymphocytes. However, to date further characterization of infiltrating T cells in chronic pancreatitis has not been undertaken. METHODS: Using the novel method of multiepitope imaging with fluorochrome-tagged specific monoclonal antibodies, which allows the simultaneous localization and characterization of T cells in tissues, we analyzed the distribution and phenotypes of T cells infiltrating the pancreas in chronic pancreatitis. RESULTS: The mean CD4:CD8 ratio in 10 cases of chronic pancreatitis was 2.4:1. In order of decreasing frequency, the following markers were observed: CD45RO, CD18, TCRgammadelta, and CD103. The lymphocytes, especially of the CD4+ subset, were found mainly in the fibrous stroma, but T cells were also observed periductally. A T-cell subset bearing the phenotype CD8+CD103+, analogous to intestinal intraepithelial lymphocytes, was found intracalating between the cells of the ductal epithelium. CONCLUSIONS: Phenotyping of the T lymphocytes in chronic pancreatitis supports the concept of the involvement of cell-mediated cytotoxicity in the pathogenesis of this disease. In addition, intraepithelial lymphocytes were found interspersed between the ductal epithelial cells, pointing to a role of this T-cell subset as a first-line defense against deleterious epithelial events in chronic pancreatitis.
Asunto(s)
Antígenos CD/análisis , Antígenos CD8/análisis , Cadenas alfa de Integrinas , Páncreas/patología , Pancreatitis/patología , Subgrupos de Linfocitos T/patología , Adolescente , Adulto , Antígenos CD18/análisis , Antígenos CD4/análisis , Enfermedad Crónica , Epitelio/patología , Epítopos/inmunología , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Antígenos Comunes de Leucocito/análisis , Masculino , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T gamma-delta/análisisRESUMEN
BACKGROUND: T lymphocytes play a central role in the immune response to cancer, with specific T-cell reactivity provided by the T-cell receptor (TCR) alphabeta-chain heterodimer. Whereas human pancreatic adenocarcinoma is characterized by a massive infiltration of T lymphocytes, to date no analysis of the TCR Valpha-gene expression of the tumor-infiltrating lymphocytes in pancreatic cancer has been performed. METHODS: Using reverse transcriptase polymerase chain reaction (RT-PCR) followed by dot blot hybridization, we determined the TCR alpha-chain repertoire at the mRNA level in pancreatic carcinoma and compared our findings with the TCR Valpha repertoire in the normal pancreas and chronic pancreatitis. RESULTS: A heterogeneous lowly restricted TCR Valpha repertoire was observed in pancreatic carcinomas, different from the TCR Valpha repertoire in chronic pancreatitis. CONCLUSIONS: Pancreas-infiltrating T cells show a distinct TCR Valpha gene expression profile in normal pancreas, chronic pancreatitis, and pancreatic cancer.
Asunto(s)
Adenocarcinoma/metabolismo , Regulación de la Expresión Génica , Neoplasias Pancreáticas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Vitronectina/metabolismo , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Cartilla de ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Immunoblotting , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Páncreas/metabolismo , Pancreatitis/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Vitronectina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Pancreatic cancer is characterized by an increasing incidence and an extremely poor prognosis. It is resistant to most of the conventional treatment modalities. Histomorphologically, it presents with a strong desmoplastic reaction around cancer cells, and lymphocytes are typically localized as aggregates in the fibrotic interstitial tissue. Using the method of multi-epitope imaging with fluorochrome-tagged specific MoAbs which allows the simultaneous localization and characterization of T cells in tissues, we studied phenotypes and distribution of tumour-infiltrating lymphocytes (TIL) in pancreatic cancer. CD3+ T cells comprised up to 90% of the tumour-infiltrating cells which were either CD4+ or CD8+, most of them being memory cells (CD45RO+). In decreasing order of frequency, T lymphocytes carried the markers for CD45RO, CD18, CD103 and TCR gammadelta. Very few natural killer cells (CD56+) were observed. Twenty percent of CD8+ were labelled with CD103. These CD8+CD103+ T cells, analogous to the gut intraepithelial lymphocytes (IEL), were found in the fibrous interstitial tissue. Furthermore, an inverse correlation was found between the expression of CD18, the beta2-integrin, which mediates adhesion of activated lymphocytes, and CD45RO in the CD8+ subset of TIL (P = 0.046). In conclusion, phenotyping of T lymphocytes in pancreatic cancer raises the possibility that pancreatic cancer cells develop several strategies to escape the T cell-induced cytolysis by (i) the aggregation of cytotoxic CD8+CD103+ T cells in the fibrous tissue distant from the tumour cells, and (ii) the presence of CD18-bearing cells which lack the expression of the activation marker CD45RO.