RESUMEN
SiO2/PEG organic-inorganic hybrid materials, which differ in polyethylene glycol (PEG) content, were synthesized by sol-gel technique and the characterization of their structure and biological properties was carried out in order to evaluate the possible use in biomedical field. FT-IR spectroscopy detected that the two components of the hybrids (SiO2 and PEG) are linked by hydrogen bonds between the Si-OH groups of the inorganic phase and the terminal alcoholic groups and/or the ethereal oxygen atoms in the repeating units of polymer. X-ray diffraction analysis ascertained the amorphous nature of the gels and the observation of their morphology by SEM microscopy confirmed that the interpenetration of the two phases (organic and inorganic) occurs on nanometric scale. The biological characterization was carried out as a function of the polymer amount to study its influence on material behavior. The results showed that the synthesized materials were bioactive and biocompatible. The formation of a hydroxyapatite layer, indeed, was observed on their surface by SEM/EDX analysis after soaking in simulated body fluid. Moreover, the biocompatibility of SiO2/PEG hybrids was assessed performing MTT and SRB cytotoxicity tests on fibroblast cell NIH 3T3 after 24 and 48h of exposure, as well as Trypan Blue dye exclusion test. The response to the presence of the investigated materials was positive. The cell growth and proliferation showed dependence on polymer amount and time of exposure to the material extracts. Therefore, the obtained results are encouraging for the use of the obtained hybrids in dental or orthopedic applications.
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Proliferación Celular/efectos de los fármacos , Fibroblastos/metabolismo , Hidrogeles , Ensayo de Materiales , Polietilenglicoles , Dióxido de Silicio , Animales , Supervivencia Celular/efectos de los fármacos , Fibroblastos/citología , Hidrogeles/síntesis química , Hidrogeles/química , Hidrogeles/farmacología , Ratones , Células 3T3 NIH , Polietilenglicoles/química , Polietilenglicoles/farmacología , Dióxido de Silicio/química , Dióxido de Silicio/farmacologíaRESUMEN
The objective of the following study has been the synthesis via sol-gel and the characterization of novel organic-inorganic hybrid materials to be used in biomedical field. The prepared materials consist of an inorganic zirconia matrix containing as organic component the polyethylene glycol (PEG), a water-soluble polymer used in medical and pharmaceutical fields. Various hybrids have been synthesized changing the molar ratio between the organic and inorganic parts. Fourier transform spectroscopy suggests that the structure of the interpenetrating network is realized by hydrogen bonds between the Zr-OH group in the sol-gel intermediate species and both the terminal alcoholic group and ethereal oxygen atoms in the repeating units of polymer The amorphous nature of the gels has been ascertained by X-ray diffraction analysis. The morphology observation has been carried out by using the Scanning Electron Microscope and has confirmed that the obtained materials are nanostructurated hybrids. The bioactivity of the synthesized system has been shown by the formation of a hydroxyapatite layer on the surface of samples soaked in a fluid simulating the human blood plasma. The potential biocompatibility of hybrids has been assessed as performing indirect MTT cytotoxicity assay towards 3T3 cell line at 24, 48, and 72 h exposure times.
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Materiales Biocompatibles/síntesis química , Geles/química , Polietilenglicoles/química , Circonio/química , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/toxicidad , Supervivencia Celular/efectos de los fármacos , Durapatita/sangre , Durapatita/química , Humanos , Ratones , Células 3T3 NIH , Nanoestructuras/química , Propiedades de SuperficieRESUMEN
BACKGROUND AND PURPOSE: We investigated the ability of natural and synthetic selective NK receptors agonists and antagonists to modulate cyclooxygenase-2 (COX-2) expression in human polymorphonuclear leucocytes (PMNs). EXPERIMENTAL APPROACH: The presence of all three tachykinin in PMNs was assessed by Western blot and PCR techniques. Natural and synthetic ligands selective for the tachykinin receptors were used to modulate COX-2 protein (measured with Western blotting) and activity [as prostaglandin E(2) (PGE(2)) output]. Effects of substance P (SP) on phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-kappaB) activation were studied to analyse the signalling pathway involved in COX-2 up-regulation mediated by SP. KEY RESULTS: Stimulation of NK receptors with the natural ligands SP, neurokinin A (NKA) and neurokinin B, in the pmol.L(-1)-micromol.L(-1) concentration range, modulated COX-2 expression and PGE(2) release in a concentration- and time-dependent manner. Experiments with synthetic selective agonists [Sar(9), Met(O(2))(11)]SP, [beta-Ala(8)] NKA(4-10), senktide or selective antagonists L703,606, SR48,968 or SR142801, confirmed that COX-2 up-regulation was mediated by NK receptors. We found that mainly p38, p42 and p46 MAPKs were phosphorylated by SP and SB202190, PD98059 and SP600125, which are selective inhibitors of these kinases, blocked SP-induced COX-2 expression. SP also induced nuclear translocation of NF-kappaB concentration-dependently, with a maximum effect at 1 nmol.L(-1). CONCLUSIONS AND IMPLICATIONS: Human PMNs possess functional NK(1), NK(2) and NK(3) receptors, which mediate the induction of COX-2 expression and NF-kappaB activation by SP.
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Ciclooxigenasa 2/biosíntesis , Neutrófilos/enzimología , Neutrófilos/metabolismo , Receptores de Taquicininas/fisiología , Western Blotting , Células Cultivadas , Dinoprostona/metabolismo , Relación Dosis-Respuesta a Droga , Inducción Enzimática , Humanos , Ligandos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Neutrófilos/efectos de los fármacos , Fosforilación , Reacción en Cadena de la Polimerasa , Receptores de Taquicininas/agonistas , Receptores de Taquicininas/antagonistas & inhibidores , Sustancia P/farmacología , Sustancia P/fisiologíaAsunto(s)
Ciclooxigenasa 2/metabolismo , Inducción Enzimática , Neutrófilos/efectos de los fármacos , Sustancia P/farmacología , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Humanos , Neutrófilos/metabolismo , Receptores de Neuroquinina-1/metabolismo , Sustancia P/agonistasRESUMEN
BACKGROUND AND PURPOSE: Cyclooxygenase-2 (COX-2) is highly expressed during inflammation and can promote the progression of colorectal cancer. Interactions between cancer cells and vascular endothelial cells are key events in this process. Recently, the selective COX-2 inhibitor, celecoxib, was shown to inhibit expression of the adhesion molecules, ICAM-1 and VCAM-1, in the human colon cancer cell line HT29 and to inhibit adhesion of HT29 cells to FCS-coated plastic wells. Here, we evaluated the effects of celecoxib on adhesion of HT29 cells to human umbilical vein endothelial cells (HUVEC), mediated by ICAM-1 and VCAM-1, to assess further the potential protective effects of celecoxib on cancer development. EXPERIMENTAL APPROACH: Celecoxib was incubated for 4 h with HT29 cells and HUVEC and adhesion was quantified by a computerized micro-imaging system. Expression analysis of ICAM-1 and VCAM-1 cell adhesion molecules was performed by western blot. KEY RESULTS: Celecoxib (1 nM-10 microM) inhibited, with the same potency, adhesion of HT29 cells to resting HUVEC or to HUVEC stimulated by tumour necrosis factor-alpha (TNF-alpha), mimicking inflammatory conditions. Analysis of ICAM-1 and VCAM-1 expression showed that celecoxib inhibited expression of both molecules in TNF-alpha-stimulated HUVEC, but not in resting HUVEC; inhibition was concentration-dependent and maximal (about 50%) at 10 microM celecoxib. CONCLUSIONS AND IMPLICATIONS: In conclusion, our data show that celecoxib inhibits HT29 cell adhesion to HUVEC and expression of ICAM-1 and VCAM-1, in stimulated endothelial cells. These effects may contribute to the chemopreventive activity of celecoxib in the development of colorectal cancer.
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Inhibidores de la Ciclooxigenasa/farmacología , Molécula 1 de Adhesión Intercelular/efectos de los fármacos , Pirazoles/farmacología , Sulfonamidas/farmacología , Molécula 1 de Adhesión Celular Vascular/efectos de los fármacos , Western Blotting , Celecoxib , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Neoplasias del Colon/fisiopatología , Neoplasias del Colon/prevención & control , Inhibidores de la Ciclooxigenasa/administración & dosificación , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células HT29 , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Pirazoles/administración & dosificación , Sulfonamidas/administración & dosificación , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Venas Umbilicales , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
BACKGROUND AND PURPOSE: We investigated the ability of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, to modulate expression of ICAM-1 and VCAM-1 in the colon cancer cell line HT29. EXPERIMENTAL APPROACH: We analysed the effect of celecoxib on ICAM-1 and VCAM-1 protein and mRNA expression in HT29 cells. Experiments were performed in the presence of mitogen-activated protein kinases (MAPK) inhibitors to evaluate the involvement of these kinases in this phenomenon. We evaluated adhesion of HT29 cells to FCS-coated plastic wells in the presence of celecoxib or MAPK inhibitors. Furthermore, we studied the effect of celecoxib on apoptosis. KEY RESULTS: Celecoxib down-regulated ICAM-1 and VCAM-1 expression in HT29 cells in a time- and dose-dependent way. Celecoxib reduced activation of p38 and p55 c-Jun terminal NH(2) kinase (JNK) MAPKs, but did not affect p46 JNK or p42/44 MAPK phosphorylation. Pretreatment with SB202190 or SP600125, specific inhibitors of p38 and JNK MAPKs, respectively, reduced ICAM-1 and VCAM-1 expression in HT29 cells dose-dependently. Adhesion of HT29 cells to FCS-coated plastic wells was inhibited dose-dependently by celecoxib, and also by SB202190 and SP600125. Celecoxib showed a pro-apoptotic effect, inducing Bax and BID but down-regulating Bcl-2. CONCLUSIONS AND IMPLICATIONS: Our findings show that celecoxib caused down-regulation of ICAM-1 and VCAM-1, affecting the adhesive properties of HT29 cells in a COX-2 independent way, inhibiting p38 and p55 MAPKs and activating a pro-apoptotic pathway.
Asunto(s)
Inhibidores de la Ciclooxigenasa/farmacología , Molécula 1 de Adhesión Intercelular/efectos de los fármacos , Pirazoles/farmacología , Sulfonamidas/farmacología , Molécula 1 de Adhesión Celular Vascular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Celecoxib , Adhesión Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/fisiopatología , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa/administración & dosificación , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Células HT29 , Humanos , Molécula 1 de Adhesión Intercelular/genética , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Pirazoles/administración & dosificación , Sulfonamidas/administración & dosificación , Factores de Tiempo , Molécula 1 de Adhesión Celular Vascular/genética , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Rhabdomyosarcomas secrete high levels of insulin-like growth factor-II, suggesting this autocrine growth factor plays a major role in the unregulated growth of this childhood cancer. Treatment of Rh30 rhabdomyosarcoma cells with insulin-like growth factor binding protein-6 (IGFBP-6; 1000 ng/ml), which binds insulin-like growth factor-II with high affinity, inhibited growth in vitro (p < 0.001). Co-incubation of cells with rapamycin (1.56 ng/ml), an inhibitor of p70 S6 kinase, and IGFBP-6 (200 ng/ml) resulted in a significant reduction in Rh30 cell number compared to rapamycin or IGFBP-6 alone (p < 0.05 for both). Co-treatment of Rh30 cells with CCI-779 (5 and 50 ng/ml), an ester analogue of rapamycin, and IGFBP-6 (200 or 1000 ng/ml) also inhibited growth in vitro relative to CCI-779 alone (p < 0.01 and p < 0.001, respectively). In a nude mouse model, xenografts of Rh30 cells transfected with a recombinant vector encoding IGFBP-6 (phBP6-E3) showed delayed growth relative to vector control xenografts (27 days vs. 19 days to reach an average tumour volume of 0.5 cm (3); p < 0.001). Treatment with CCI-779 (10 mg/kg) of mice inoculated with vector control xenografts, also delayed growth (to 31 days; p = 0.0055) relative to untreated mice with vector control xenografts. Co-treatment with CCI-779 (10 mg/kg) reduced phBP6-E3 transfected xenograft growth even further (to 45 days) compared to vector control xenografts (p < 0.001, day 33). CCI-779 thus acts additively with IGFBP-6 to reduce rhabdomyosarcoma growth both in vitro and in vivo.
Asunto(s)
División Celular/efectos de los fármacos , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/farmacología , Rabdomiosarcoma/patología , Sirolimus/análogos & derivados , Sirolimus/farmacología , Animales , Línea Celular , Línea Celular Tumoral , Humanos , Riñón , Cinética , Ratones , Ratones Desnudos , Proteínas Recombinantes/farmacología , Trasplante HeterólogoRESUMEN
Rhabdomyosarcoma is the most common soft-tissue sarcoma of childhood. Rhabdomyosarcoma cell lines overexpress insulin-like growth factor-II (IGF-II), an autocrine growth factor that is inhibited by insulin-like growth factor binding protein-6 (IGFBP-6). IGFBP-6 is associated with myoblast quiescence, and expression in rhabdomyosarcoma cells is low. The effect of IGFBP-6 on 2 rhabdomyosarcoma cell lines, RD and Rh30, was studied. IGFBP-6 inhibited anchorage-dependent growth of RD and Rh30 cells in a dose-dependent manner (p < 0.0001). IGFBP-6 also inhibited anchorage-independent growth of RD cells in soft agar in a dose-dependent manner (p < 0.01). Anchorage-independent growth of RD cells on polyhydroxyethylmethacrylate-coated plates was decreased to a minimum of 48% of control after treatment with IGFBP-6 (p < 0.001). In this system, IGFBP-6 increased apoptosis 4-fold (p < 0.001). IGF-II partially reversed the IGFBP-6-induced decrease in growth and increase in apoptosis. Rh30 cells were stably transfected with an IGFBP-6 cDNA and subcutaneous xenografts established in BALB/c nude mice. After 18 days, sizes of 2 independent clones of IGFBP-6-overexpressing Rh30 cells were reduced to 12% and 26% of vector control-transfected tumors (p = 0.0006 and 0.002, respectively). IGFBP-6 therefore inhibits proliferation and promotes apoptosis of rhabdomyosarcoma in vitro and dramatically inhibits xenograft growth in vivo, at least in part by inhibiting IGF-II. Low expression of IGFBP-6 may therefore contribute to rhabdomyosarcoma growth and metastasis.
Asunto(s)
Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/fisiología , Rabdomiosarcoma/prevención & control , Animales , Apoptosis , División Celular , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Rabdomiosarcoma/patología , Células Tumorales CultivadasRESUMEN
The wall of a human artery consists of three distinct tunics. The tunica intima is lined by a layer of endothelial cells facing the lumen. Smooth muscle cells (SMCs) are the predominant cell type in the tunica media of arteries. They are surrounded by a basal lamina containing collagen IV, proteoglycans, glycosaminoglycans, glycoproteins, and extracellular matrix (ECM) molecules: collagens type 1, III, V, and VI, and elastin. The external tunica adventitia consists primarily of collagen fibers, elastic tissue, and fibroblasts. Because smooth muscle cells play a dominant role in the development of intimal hyperplasia during atherosclerosis, these cells have been studied extensively in vitro.
RESUMEN
Endothelial cells line the luminal surface of all blood vessels in the body. The endothelial surface in adult humans is composed of approximately l-6×l0(13) cells and covers an area of 1-7 m(2). Endothelium serves many functions, including fluid and solute exchange through cell contraction, provision of an antithrombogenic surface through tissue plasminogen activator (tPA) and prostacyclin release, synthesis of angiogenic factors such as adenosine, allowance of leukocyte trafficking through adhesion molecule synthesis, presentation of antigens to the immune system, maintenance of vascular tone through nitric oxide and endothelin synthesis, and metabolism of circulating molecules through the release of enzymes such as lipoprotein lipase.
RESUMEN
BACKGROUND: Primary effusion lymphoma (PEL) associates with HHV-8 infection, preferentially develops in immunodeficient patients and grows in the serous body cavities. PEL derives from post-germinal center, pre-terminally differentiated B-cells. The pathogenesis of PEL is unclear and the sole identified genetic lesions are human herpesvirus type-8 (HHV-8) infection in all cases and EBV infection in 70% of cases. Epstein-Barr virus (EBV) infection in PEL displays a latency I phenotype. OBJECTIVES: To clarify the pathogenesis and histogenesis of PEL by investigating (1) the lymphoma karyotype; (2) the expression status of the Met tyrosine kinase receptor and of its ligand hepatocyte growth factor (HGF); (3) the molecular profile of EBV, with particular focus on mutations of EBNA-1 genes, which are thought to affect viral tumorigenicity in EBV-infected neoplasms displaying the latency I phenotype. STUDY DESIGN: Twenty-four PEL (nine cell lines and 15 primary specimens) formed the basis of the study. Karyotypes were investigated by conventional cytogenetics and fluorescent in situ hybridization (FISH) in selected cases. The expression status of Met and HGF was defined by multiple techniques, including RT-PCR, FACS analysis, immunocytochemistry, Western blot studies and ELISA. The molecular profile of EBNA-1 genes of EBV were investigated by DNA direct sequencing. RESULTS: Trisomy 7, trisomy 12 and breaks at 1q21-q25 are recurrently associated with PEL. PEL consistently co-express Met and HGF both at the mRNA and protein level. Among aggressive B-cell lymphomas, Met/HGF co-expression appears to be relatively specific for PEL. The EBNA-1 gene of EBV displays a high degree of genetic heterogeneity in PEL, with no preferential association with one specific variant. CONCLUSIONS: PEL associates with recurrent chromosomal alterations, suggesting that viral infection is not sufficient for tumor development and that lesions of cellular genes may be required. The expression of Met/HGF by PEL cells may bear implications for the lymphoma proliferation and growth pattern, since Met/HGF interactions influence cell mitogenesis and motogenesis. EBV infection in PEL displays a latency I phenotype and fails to associate with specific EBNA-1 variants, suggesting that the role of EBV in PEL is not mediated by the major transforming pathways currently known in EBV positive lymphomas.
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Herpesvirus Humano 8/genética , Linfoma de Células B/patología , Linfoma de Células B/virología , Antígenos Nucleares del Virus de Epstein-Barr/genética , Femenino , Variación Genética , Factor de Crecimiento de Hepatocito/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 8/aislamiento & purificación , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Linfoma de Células B/metabolismo , Masculino , Proteínas Proto-Oncogénicas c-met/metabolismo , Células Tumorales CultivadasRESUMEN
Primary effusion lymphoma (PEL) harbors consistent infection by human herpesvirus-8, preferentially develops in immunodeficient patients and selectively localizes to the serous body cavities. Histogenetic analysis has suggested that PEL originates from post-germinal center, pre-terminally differentiated B cells sharing phenotypic features with plasma cells. Here we have investigated the expression status and functional integrity of the Met tyrosine kinase receptor and of its ligand hepatocyte growth factor (HGF). Thirteen PEL (nine cell lines and four primary specimens) were analyzed for Met and HGF expression and function by multiple assays. For comparison, a panel of 34 high grade B cell non-Hodgkin lymphomas (NHL) other than PEL was also investigated. Co-expression of Met and HGF was found in all PEL analyzed, whereas it was restricted to 1/34 B cell NHL other than PEL (P < 0.001; chi2 test). The Met protein expressed by PEL displays biochemical characteristics typical of Met expressed by other cell types and is capable of tyrosine autophosphorylation. By using a combination of immunological and biological assays, production and secretion of a functional HGF species was identified in all PEL cell lines analyzed. HGF stimulation of PEL cells rapidly induces Met tyrosine phosphorylation, demonstrating the functional integrity of the Met/HGF loop. Because of the well known mitogenic and motogenic properties of Met/HGF interactions, these data may bear implications for PEL growth and dissemination. Among B cell neoplasms, Met/HGF co-expression selectively clusters with PEL and, as demonstrated by previous studies, with multiple myeloma plasma cells, thus reinforcing the notion that PEL displays biologic similarities with tumors derived from late stages of B cell differentiation.
Asunto(s)
Factor de Crecimiento de Hepatocito/análisis , Infecciones por Herpesviridae/virología , Herpesvirus Humano 8 , Linfoma de Células B/química , Linfoma de Células B/virología , Proteínas Proto-Oncogénicas c-met/análisis , Regulación Neoplásica de la Expresión Génica , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Infecciones por Herpesviridae/complicaciones , Herpesvirus Humano 8/aislamiento & purificación , Humanos , Inmunohistoquímica , Linfoma de Células B/metabolismo , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin's lymphomas (AIDS-NHLs) consistently derive from B cells, are histologically heterogeneous, and are associated with distinct molecular pathways depending upon histology. Recently, it has been proposed that inactivating mutations of the bax death agonist may contribute to the pathogenesis of human tumors. In particular, among B-cell malignancies, BAX mutations have been detected at a certain frequency in Burkitt lymphomas. This study is aimed at defining the status of the BAX gene throughout the clinicopathologic spectrum of AIDS-NHL (n = 54), including AIDS-related Burkitt lymphoma (n = 14), AIDS-related Burkitt-like lymphoma (n = 8), AIDS-related diffuse large cell lymphoma (n = 15), AIDS-related primary central nervous system lymphoma (n = 6), and AIDS-related primary effusion lymphoma (n = 11). All 6 BAX exons and flanking sequences were subjected to mutational analysis by polymerase chain reaction-single strand conformation polymorphism followed by DNA direct sequencing of positive cases. Mutations of BAX among AIDS-NHL were restricted to a cell line of AIDS-related primary effusion lymphoma, which harbored a frameshift mutation causing the introduction of a proximal stop codon. All other AIDS-NHL displayed wild-type BAX alleles. In order to investigate whether BAX inactivation in AIDS-NHL may occur through mechanisms other than gene mutation, bax protein expression was investigated by Western blot analysis or immunohistochemistry in selected cases. All AIDS-NHL analyzed expressed normal bax proteins. Overall, this study indicates that deregulation of apoptotic control in AIDS-NHL is not caused by BAX alterations. Genes Chromosomes Cancer 27:177-182, 2000.
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Linfoma Relacionado con SIDA/genética , Linfoma no Hodgkin/genética , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/genética , Western Blotting , Análisis Mutacional de ADN , ADN de Neoplasias/química , ADN de Neoplasias/genética , Humanos , Mutación , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Proteínas Proto-Oncogénicas/metabolismo , Eliminación de Secuencia , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteína X Asociada a bcl-2RESUMEN
Hematopoietic growth factors (HGFs) sustain the survival, proliferation and differentiation of hematopoietic stem cells and some functions of mature blood cells. In man several HGFs have been characterised and cloned so far, and this has allowed investigators to confer the rationale for the clinical application of these molecules in hematology and oncology. In particular G-CSF and GM-CSF are currently utilised to abrogate the hematological toxicity of chemotherapy for standard and dose-intensified therapy, neutropenia following bone marrow and peripheral blood stem cell transplantation. Moreover there has recently been great interest in the ex vivo expansion of hematopoietic stem and progenitor cells for a variety of applications, such as in vitro tumor cell purging or for reducing the volume of blood processed by the leukapheresis. Several combinations of HGFs have been described to sustain the ex vivo survival and proliferation of these cells disclosing new opportunities in the field of stem cells transplants.
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Sustancias de Crecimiento/farmacología , Sistema Hematopoyético/fisiología , Trasplante Autólogo/fisiología , Animales , Factor Estimulante de Colonias de Granulocitos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Proteínas Recombinantes/farmacologíaRESUMEN
GAS6 is a ligand for the tyrosine kinase receptors Rse, Axl, and Mer, but its function is poorly understood. Previous studies reported that both GAS6 and Axl are expressed by vascular endothelial cells (EC), which play a key role in leukocyte extravasation into tissues during inflammation through adhesive interactions with these cells. The aim of this work was to evaluate the GAS6 effect on the adhesive function of EC. Treatment of EC with GAS6 significantly inhibited adhesion of polymorphonuclear cells (PMN) induced by phorbol 12-myristate 13-acetate (PMA), platelet-activating factor (PAF), thrombin, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), but not that induced by FMLP and IL-8. GAS6 did not affect adhesion to resting EC. Titration experiments showed that high concentrations of GAS6 were needed to inhibit PMN adhesion and that inhibition was dose-dependent at the concentration range of 0.1 to 1 microg/mL. One possibility was that high concentrations were needed to overwhelm the effect of endogenous GAS6 produced by EC. In line with this possibility, treatment of resting EC with soluble Axl significantly potentiated PMN adhesion. Analysis of localization of GAS6 by confocal microscopy and cytofluorimetric analysis showed that it is concentrated along the plasma membrane in resting EC and treatment with PAF induces depletion and/or redistribution of the molecule. These data suggest that GAS6 functions as a physiologic antiinflammatory agent produced by resting EC and depleted when proinflammatory stimuli turn on the proadhesive machinery of EC.
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Endotelio Vascular/citología , Granulocitos/citología , Péptidos y Proteínas de Señalización Intercelular , Proteínas/farmacología , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Citocinas/farmacología , Endotelio Vascular/metabolismo , Granulocitos/metabolismo , Humanos , Proteínas de la Membrana/farmacología , Factor 2 de la Biogénesis del Peroxisoma , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
GAS6, a gene previously identified as growth arrest specific, has been demonstrated to be the ligand of Axl, a novel tyrosine kinase receptor widely expressed in both normal and neoplastic hematopoietic tissue. We have observed previously that GAS6 mRNA was present in whole bone marrow. This preliminary finding prompted us to investigate the presence of GAS6 in hematopoietic tissue and the possible role of this molecule in controlling the proliferation of hematopoietic precursors. We report here that the protein GAS6 is diffusely present in hematopoietic tissue, both in stromal and in hematopoietic cells, and that, among these cells, positivity is observed in megakaryocytes and myelomonocytic precursors. Furthermore, our data suggest that GAS6 is not a growth factor for hematopoietic progenitors or stromal fibroblasts. Despite the fact that both the Axl receptor and its ligand, GAS6, are expressed in hematopoietic tissue, the biological role of their interactions remains to be determined.
Asunto(s)
Células de la Médula Ósea/metabolismo , Hematopoyesis , Péptidos y Proteínas de Señalización Intercelular , Proteínas/metabolismo , Biopsia , División Celular/efectos de los fármacos , Células Cultivadas , Fibroblastos/citología , Expresión Génica , Factores de Crecimiento de Célula Hematopoyética/farmacología , Humanos , Mitógenos , Proteínas Oncogénicas/fisiología , Proteínas/genética , Proteínas Proto-Oncogénicas , ARN Mensajero/genética , Proteínas Tirosina Quinasas Receptoras/fisiología , Proteínas Recombinantes/farmacología , Tirosina Quinasa del Receptor AxlRESUMEN
Many chemotherapy regimens are associated with variable periods of myelosuppression. In cancer patients, neutropenia (less than 500 neutrophils/microL) is the most important risk factor for infections. The incidence and severity of infectious complications are related to depth and duration of neutropenia, with the highest risk if neutrophils are less than 100/microL for more than a week. The period required for neutrophil recovery is usually short with standard regimens, but prolonged after high dose chemotherapy followed by autologous bone marrow transplant (-ABMT) or peripheral blood stem cell (PBSC) infusion. Under these conditions, the administration of granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) accelerates neutrophil recovery and shortens the duration of hospitalization. In standard chemotherapy settings, prophylactic use of CSF's is a matter of debate. Several studies have reached contrasting conclusion, but, combining effectiveness and costs, it results that this use of CSF'S is not to be recommended unless the risk of infections (elderly patients, reduced marrow reserve) is high. The administration of G-CSF or GM-CSF to a febrile neutropenic patient (cfr CSF's therapy) shortens the duration of neutropenia, although no great clinical benefits are evident. Nevertheless the identification of subsets of patients with additional risk factors (i.e. absolute neutrophil count < 100/microL at the onset of fever or delayed neutrophil recovery) should be helpful in establishing the role of CSF's therapy. When prolonged periods of severe neutropenia (less than 500 neutrophils/microL) are expected, antibiotics should be prophylactically administered. Fluoroquinolones seem to be the optimal choice in heavily myelosuppressed patients (ie. bone marrow transplant recipients). Fluoroquinolones are effective in reducing the frequency of gram-negative bacteremia, but, because of incomplete coverage, gram-positive infections are becoming increasingly problematic. The association with an agent that can be absorbed orally, active against gram-positive cocci, seems to be an effective strategy. Fungal infections are an important cause of morbility and mortality in severely neutropenic patients. Safety and efficacy of antifungal triazoles and the lipid formulations of amphotericin B used prophylactically still require investigation. In patients at high risk for fungal infections, monitoring cultures are predictive for systemic mycoses and should guide prophylactic and therapeutic choices. The standard treatment of oncologic patients with potential infectious neutropenia complications is admission to the hospital and treatment with broad-spectrum intravenous antibiotics. Until third generation cephalosporin and carbapenems became available, most neutropenic febrile patients were treated with associations of an aminoglycoside plus a beta-lactam. Monotherapy with the new antibiotics has proven to be effective as an association therapy and offers advantages in terms of cost and tolerability. Whether or not vancomycin is included in the initial antibiotic regimen should be decided on the basis of epidemiological consideration (i.e. prevalence of meticillin-resistant Staphylococcus aureus or Staphylococcus mitis in certain centers). Antifungal therapy is indicated in neutropenic patients who remain febrile after one week of broad-spectrum antibiotics or have recurrent fever. Amphotericin B should be promptly administered in patients suspected of invasive mycoses. Selected patients with fever and neutropenia, that can be identified on the basis of reduced risk of severe complications, do not need hospitalization. In the first reports, outpatient treatment has proven to be effective, cost saving and well received by patients, but further studies are needed to accurately define low risk status and the optimal home antibiotic regimens.
Asunto(s)
Antineoplásicos/efectos adversos , Fiebre/etiología , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Neutropenia/tratamiento farmacológico , Atención Ambulatoria , Antiinfecciosos/uso terapéutico , Antineoplásicos/administración & dosificación , Esquema de Medicación , Humanos , Infecciones/complicaciones , Infecciones/tratamiento farmacológico , Infecciones/etiología , Neutropenia/inducido químicamente , Neutropenia/prevención & controlRESUMEN
The plasmin/plasminogen system of enzymes may be involved in leukocyte migration through the endothelial cell layer of the vascular wall during inflammatory processes associated with vascular injury, atherosclerosis, and sepsis. Synthesis of plasminogen activator inhibitor type 1 (PAI-1) by the endothelium may protect these cells and the subendothelial cell matrix from excessive degradation and retard leukocyte migration. We report in this work for the first time the down-regulation of both basal and thrombin- or endotoxin-induced PAI-1 in cultured human endothelial cells by the activated T cell product, IFN-gamma. Down-regulation of basal and thrombin- or endotoxin-induced endothelial PAI-1 protein by IFN-gamma was found to be both time and dose dependent. Decreases of up to 71% relative to thrombin- or endotoxin-treated controls, using an optimal IFN-gamma concentration of between 20 and 200 U/ml, were found for human macrovascular and microvascular endothelial cells. However, IFN-gamma did not appear to affect IL-1 alpha- and TNF-alpha-induced levels of PAI-1 protein or mRNA in these cells. Northern blot analysis paralleled protein results, showing decreases in specific endothelial cell thrombin- or LPS-induced PAI-1 mRNA expression, respectively, after incubation with IFN-gamma for 24 h. These results suggest a means by which the migration of circulating leukocytes through endothelial cell layers during inflammation may be facilitated.
Asunto(s)
Regulación hacia Abajo/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotoxinas/farmacología , Interferón gamma/farmacología , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Inhibidor 1 de Activador Plasminogénico/metabolismo , Trombina/farmacología , Células Cultivadas , Endotelio Vascular/citología , Humanos , Inhibidores de Serina Proteinasa/biosíntesis , Inhibidores de Serina Proteinasa/metabolismoRESUMEN
Regulation of endothelial cell (EC) plasminogen activator inhibitor type-1 (PAI-1), the primary physiological inhibitor of tissue-type plasminogen activator (TPA) and urokinase-type plasminogen activator (UPA), by various stimuli has been well characterized. We report the upregulation of secreted and intracellular PAI-1 in human umbilical ECs when cocultured with human smooth muscle cells (SMCs) on amniotic membranes or incubated with SMC conditioned medium (CM) under serum-free conditions as determined by enzyme-linked immunosorbent assay. Cocultured human umbilical vein ECs and SMCs, or human umbilical artery ECs and SMCs, displayed a 73% and 68% increase, respectively, in released PAI-1. SMC-derived stimulatory factor release showed tissue specificity, since only human aortic, umbilical vein, and umbilical artery SMCs upregulated PAI-1 synthesis, whereas SMCs from human mammary artery, pulmonary artery, and saphenous vein did not. Stimulation of EC PAI-1 by SMC CM was both time and concentration dependent, with as much as five- and fourfold increases in supernatants and lysates, respectively. PAI-1 synthesis and activity in ECs from other vascular beds were also upregulated by SMC CM. Northern blot analysis paralleled the protein results, showing as much as a 2.7-fold increase in specific EC PAI-1 mRNA expression after incubation with SMC CM for 8 hours. PAI-1 stimulatory activity in SMC CM was completely abolished by boiling or incubation with protamine sulfate and was reduced by transient acidification or heparin-Sepharose pretreatment by 33% or 48%, respectively. The stimulatory factor(s) appeared to have a molecular mass of 23 kD as determined by gel filtration.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Comunicación Celular , Endotelio Vascular/metabolismo , Músculo Liso Vascular/fisiología , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Células Cultivadas , Cromatografía en Gel , Medios de Cultivo Condicionados , Endotelio Vascular/citología , Factor 2 de Crecimiento de Fibroblastos/análisis , Humanos , Interleucina-1/análisis , Músculo Liso Vascular/citología , Inhibidor 1 de Activador Plasminogénico/genética , ARN Mensajero/análisis , Factor de Necrosis Tumoral alfa/análisisRESUMEN
We investigated the effect of interferon-alpha 2 (IFN-alpha 2) on interleukin-1 alpha (IL-1 alpha)-induced up-regulation of urokinase type plasminogen activator (u-PA) expression in human foreskin microvascular endothelial cells (HFMEC) and human umbilical vein endothelial cells (HUVEC) in vitro. When IFN-alpha 2 and IL-1 alpha were added to the cells simultaneously, IFN-alpha 2 inhibited IL-1 alpha-induced up-regulation of u-PA antigen in a dose- and time-dependent fashion in HFMEC, whereas in HUVEC no effect of IFN-alpha 2 on IL-1 alpha-induced u-PA was seen. IL-1 alpha-induced up-regulation of PAI-1 antigen in HFMEC was not counteracted by IFN-alpha 2. When IFN-alpha 2 was added to HFMEC 1 or 2 h after IL-1 alpha a significant inhibition in u-PA synthesis was seen, whereas when IFN-alpha 2 was added to the cells 8 h after IL-1 alpha no effect on the induction of u-PA synthesis by IL-1 alpha was seen. IFN-alpha 2 also inhibited significantly the IL-1 alpha stimulated up-regulation of specific u-PA mRNA expression. In conclusion, our data show that IFN-alpha 2 can counteract the IL-1 alpha-induced up-regulation of u-PA in a similar way as IFN-gamma. This effect, which seems to be specific for microvascular endothelial cells, could contribute to the modulation of endothelial cell-mediated extravascular proteolysis in processes such as wound healing, neovascularisation, and endothelial cell migration.