RESUMEN
BACKGROUND: In recent years a new concept of health, 'positive health', has been developed, which focusses on a person's resilience instead of merely the absence of disease. A previous survey among a variety of stakeholders in general health care showed that there are differences in how dimensions of positive health are valued. Patients valued the spiritual and societal participation dimension higher than physicians and policymakers. AIM: To investigate how the six dimensions of positive health are valued by patients, health care professionals and policymakers in mental health care in the Netherlands, and to test whether these values differ from such stakeholders in general healthcare. METHOD: In a cross-sectional survey patients (N= 458), healthcare professionals (N=250) and policy makers (N=47) of two mental health care institutions in the Netherlands filled in an online survey. The results were compared to the results of the study by Huber e.a. (2016) by ANCOVA, paired T-tests and cohens' d. RESULTS: Respondents valued all dimensions equally high. No significant differences between groups were found, except for a significant difference on daily functioning. Patients rated this dimension significantly higher than other stakeholders. The equal significance of the six dimensions is in contrast with the findings of a previous survey among stakeholders in general health care. CONCLUSION: In contrast to stakeholders in general health care, those in mental health care valued all dimensions of health of equal and high importance. Only daily functioning was rated lower by professionals and policymakers than by patients.
Asunto(s)
Personal de Salud , Salud Mental , Estudios Transversales , Atención a la Salud , Humanos , Encuestas y CuestionariosRESUMEN
Members of the TNF superfamily, including Fas, Fas ligand, and CD40, have been shown to be expressed on tumor cells. In the studies described in this work, we report that another family member, the ligand for 4-1BB (CD137), is expressed on various human carcinoma cell lines, on cells of solid tumors derived from these cell lines, and cells obtained from human tumors. Expression of 4-1BB ligand (4-1BBL) mRNA was detected by both RT-PCR and Northern blot analysis, and expression of 4-1BBL protein was detected by Western blot analysis of whole cell lysates and by FACS analysis of tumor cells and cell lines. Incubation of tumor cells with a 4-1BB-Ig fusion protein led to the production of IL-8 by the cells, demonstrating that the 4-1BBL is functionally active and signals back into the tumor cells. Furthermore, 4-1BBL expressed on the carcinoma cells functioned as a costimulatory molecule for the production of cytokines (most notably IFN-gamma) in cocultures of T cells and tumor cells. These findings suggest that 4-1BBL expressed on carcinoma cells may significantly influence the outcome of a T cell-tumor cell interaction.
Asunto(s)
Receptores de Factor de Crecimiento Nervioso/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Células Tumorales Cultivadas/inmunología , Células Tumorales Cultivadas/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Ligando 4-1BB , Antígenos CD , Células Cultivadas , Técnicas de Cocultivo , Citocinas/metabolismo , Citometría de Flujo , Células HT29 , Humanos , Interferón gamma/biosíntesis , Ligandos , Activación de Linfocitos , ARN Mensajero/análisis , Transducción de Señal/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Células Tumorales Cultivadas/química , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Integrins play an important role in regulating cell adhesion, motility, and activation. In an effort to identify intracellular proteins expressed by activated T cells that interact with the cytoplasmic domain of beta1-integrin (CD29), we used the beta1-integrin cytoplasmic domain as bait in the yeast two-hybrid system. Here we report that the cytoplasmic domain of beta1-integrin specifically interacts with the cytoskeletal protein filamin. This interaction required all but the most carboxyl-terminal three residues of the cytoplasmic domain of beta1, and the carboxyl-terminal 477 residues of filamin containing the terminal 4. 5 approximately 96-residue tandem repeats of filamin. To verify this interaction in vivo, we showed that filamin specifically coprecipitated with beta1 in mammalian cells. We also showed that recombinant filamin chimeric proteins were able to bind to the beta1 cytoplasmic domain in vitro. We observed that a subset of single point mutations in the cytoplasmic domain of beta1, which had been previously reported to impair its function, disrupt the interaction between beta1 and filamin. Taken together, these findings suggest that the interaction between beta1 and filamin, which in turn can bind actin, provides a mechanism for the interaction of this cell surface receptor with cytoskeletal proteins and that this interaction plays a role in normal receptor function.
Asunto(s)
Proteínas Contráctiles/metabolismo , Integrina beta1/metabolismo , Proteínas de Microfilamentos/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Polaridad Celular , Proteínas Contráctiles/genética , Citoplasma , Filaminas , Humanos , Integrina beta1/genética , Células Jurkat , Activación de Linfocitos , Proteínas de Microfilamentos/genética , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Mutación Puntual , Pruebas de Precipitina , Unión Proteica , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Linfocitos T/metabolismoRESUMEN
Apoptosis, or programmed cell death, is a physiological form of cell death that plays a critical role in the development and maintenance of multicellular organisms. Apoptosis is characterized based on morphological and biochemical criteria. Morphological characteristics include cell shrinkage, cytoplasmic condensation, chromatin segregation and condensation, membrane blebbing, and the formation of membrane-bound apoptotic bodies, whereas the biochemical hallmark of apoptosis is internucleosomal DNA cleavage into oligonucleosome-length fragments. A great deal of research is aimed at defining the molecular mechanisms that play a role in apoptosis. As one of the common end points of experiments related to apoptosis is in fact the death of the cell, it has become important to develop reliable assays to measure cell death that may be compared among the various systems being investigated. This chapter reviews many of the current methods used to measure apoptotic cell death and points out strengths and weaknesses of each approach with respect to the system being examined and the questions being asked. Traditional cell-based methods, including light and electron microscopy, vital dyes, and nuclear stains, are described. Biochemical methods such as DNA laddering, lactate dehydrogenase enzyme release, and MTT/XTT enzyme activity are described as well. Additionally, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling of DNA fragments (TUNEL) and in situ end labeling (ISEL) techniques are reviewed, which when used in conjunction with standard flow cytometric staining methods may yield informative data relating cell death to various cellular parameters, including cell cycle and cell phenotype. The use of one or more of the methods described in this chapter for measuring cell death should enable investigators to accurately assess apoptosis in the context of the various models being examined and help define causal relationships between the mechanisms that regulate apoptosis and the cell death event itself.
Asunto(s)
Apoptosis , Técnicas de Cultivo de Célula/métodos , Animales , HumanosRESUMEN
SFME cells are brain-derived neural precursor cells that are acutely dependent on epidermal growth factor (EGF) for survival, undergoing apoptosis within 24 h after EGF withdrawal. Because the expression of the protooncogene bcl-2 inhibits apoptosis induced by the withdrawal of interleukins or nerve growth factor in some growth factor-dependent haematopoietic or neuronal cell cultures, we examined the effect of Bcl-2 expression on cell death of SFME cells in the absence of EGF. SFME cells expressing human Bcl-2 showed prolonged survival when deprived of EGF compared to control cells not expressing Bcl-2. A significant fraction of Bcl-2-expressing cells remained viable for 4 days in the absence of EGF and resumed proliferation upon readdition of EGF to the cultures. These results suggest that apoptosis induced by EGF withdrawal in SFME cells may share common mechanisms with other growth factor-related apoptotic systems.
Asunto(s)
Apoptosis/genética , Embrión de Mamíferos/citología , Factor de Crecimiento Epidérmico/fisiología , Animales , Medio de Cultivo Libre de Suero , Regulación de la Expresión Génica/fisiología , Genes bcl-2 , Humanos , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/genéticaRESUMEN
The 4-1BB receptor is an inducible type I membrane protein and member of the tumor necrosis factor receptor (TNFR) superfamily that is rapidly expressed on the surface of CD4+ and CD8+ T cells after antigen- or mitogen-induced activation. Cross-linking of 4-1BB and the T cell receptor (TCR) on activated T cells has been shown to deliver a costimulatory signal to T cells. Here, we expand upon previously published studies by demonstrating that CD8+ T cells when compared with CD4+ T cells are preferentially responsive to both early activation events and proliferative signals provided via the TCR and 4-1BB. In comparison, CD28-mediated costimulatory signals appear to function in a reciprocal manner to those induced through 4-1BB costimulation. In vivo examination of the effects of anti-4-1BB monoclonal antibodies (mAbs) on antigen-induced T cell activation have shown that the administration of epitope-specific anti-4-1BB mAbs amplified the generation of H-2d-specific cytotoxic T cells in a murine model of acute graft versus host disease (GVHD) and enhanced the rapidity of cardiac allograft or skin transplant rejection in mice. Cytokine analysis of in vitro activated CD4+ and CD8+ T cells revealed that anti-4-1BB costimulation markedly enhanced interferon-gamma production by CD8+ T cells and that anti-4-1BB mediated proliferation of CD8+ T cells appears to be IL-2 independent. The results of these studies suggest that regulatory signals delivered by the 4-1BB receptor play an important role in the regulation of cytotoxic T cells in cellular immune responses to antigen.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Inmunidad Celular , Activación de Linfocitos/inmunología , Receptores de Factor de Crecimiento Nervioso/inmunología , Receptores del Factor de Necrosis Tumoral/inmunología , Animales , Antígenos CD , Linfocitos T CD8-positivos/citología , División Celular/inmunología , Ratones , Ratones Endogámicos BALB C , Receptores de Factor de Crecimiento Nervioso/biosíntesis , Receptores del Factor de Necrosis Tumoral/biosíntesis , Transducción de Señal/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis TumoralRESUMEN
The T cell activation antigen 4-1BB (CDw137) is a distantly related member of the tumor necrosis factor receptor family of cell surface receptors. We previously reported that murine 4-1BB (m4-1BB) bound to extracellular matrix (ECM) proteins. Recently, a tumor necrosis factor-like ligand of m4-1BB, m4-1BBL, as well as the human counterparts of 4-1BB (ILA) and 4-1BBL (h4-1BB and h4-1BBL, respectively) have been cloned. No information is currently available on how binding of m4-1BB to ECM proteins affects its binding to m4-1BBL and vice versa and if the ability of m4-1BB to bind ECM proteins is conserved across species. We report that binding of m4-1BBL to m4-1BB blocked its ability to bind laminin (LN), while binding of m4-1BB to LN did not block its ability to bind m4-1BBL. Furthermore, binding of m4-1BBL to the m4-1BB.LN complex did not displace LN. These findings suggest the two ligands bind to proximal but distinct sites on m4-1BB. This is supported by the observation that six of eight anti-m4-1BB monoclonal antibodies blocked the interaction between 4-1BB and 4-1BBL, while seven blocked LN binding. Ligand and monoclonal antibody binding studies with a truncated protein lacking the amino-terminal LN-homologous domain of m4-1BB demonstrated that regions downstream of the LN-homologous domain participate in LN binding and that the intact protein is required for m4-1BBL binding. Studies with h4-1BB showed that h4-1BB only bound h4-1BBL, indicating that the ECM binding activity of 4-1BB is not conserved across species. This finding allowed the construction of murine/human 4-1BB chimeras, which permitted further dissection of the regions of 4-1BB involved in LN and 4-1BBL binding and suggests that sequence differences in the LN-homologous domain of h4-1BB in part account for the inability of h4-1BB to bind ECM proteins.
Asunto(s)
Laminina/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Ligando 4-1BB , Secuencia de Aminoácidos , Animales , Antígenos CD , Células COS , Espacio Extracelular , Humanos , Ligandos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes de Fusión , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis TumoralRESUMEN
Serum-free mouse embryo (SFME) cells derived in a defined serum-free medium have been cultured for more than 200 generations and display properties of neural progenitor cells. SFME cells express the neuroepithelial stem cell marker nestin in defined serum-free medium. Exposure of SFME cells to transforming growth factor beta (TGF-beta) or serum decreases nestin expression and induces the astrocyte marker glial fibrillary acidic protein, suggesting that SFME cells differentiate into astrocytes upon exposure to TGF-beta or serum. We examined the expression by SFME cells of the functional central nervous system (CNS) astrocyte marker glutamine synthetase (GS). GS activity is induced in SFME cells upon exposure to TFG-beta or serum. The induction of GS activity was dose- and time-dependent and was reversible. Retinoic acid, hydrocortisone, and dibutyryl cyclic AMP also induced GS expression. The induction of GS activity was accompanied by an increase in the level of GS mRNA and protein. This work provides further evidence that SFME cells represent neural progenitor cells which differentiate into functional astrocytes upon exposure to TGF-beta or serum.
Asunto(s)
Astrocitos/citología , Encéfalo/embriología , Regulación del Desarrollo de la Expresión Génica , Glutamato-Amoníaco Ligasa/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Células Madre/citología , Animales , Astrocitos/enzimología , Biomarcadores , Sangre , Western Blotting , Encéfalo/citología , Bucladesina/farmacología , Bovinos , Diferenciación Celular/efectos de los fármacos , Carbón Orgánico , Medio de Cultivo Libre de Suero/farmacología , Inducción Enzimática/efectos de los fármacos , Glutamato-Amoníaco Ligasa/genética , Hidrocortisona/farmacología , Proteínas de Filamentos Intermediarios/biosíntesis , Proteínas de Filamentos Intermediarios/genética , Ratones , Ratones Endogámicos BALB C , Proteínas del Tejido Nervioso/genética , Nestina , Células Madre/efectos de los fármacos , Células Madre/enzimología , Factor de Crecimiento Transformador beta/farmacología , Tretinoina/farmacología , Ácido Valproico/farmacologíaRESUMEN
A variety of neurodegenerative disease states have been associated with oxidative damage or stress. Such stress is thought to be mediated by excessive exposure of cells to reactive oxygen species such as free radicals, which can be generated following cell lysis, oxidative burst (as part of the immune response) or by the presence of an excess of free transition metals. Since the neuronal death observed in neurodegenerative diseases may be related to free radical damage, we were interested in developing a model system to investigate the mechanisms by which reactive oxygen species may damage or kill neurons. To this end, we have recently reported that brief exposure of cultured cortical neurons to H2O2 can induce neuronal death that proceeds via an apoptotic cell suicide pathway. The studies reported here investigate H2O2-induced cell death in more detail. Our data suggest that exposure of cultured cortical neurons to H2O2 can induce apoptotic cell death within 3 h, as assessed by cell viability, morphological and ultrastructural measures. In addition, experiments presented show that exposure to high concentrations of H2O2 (100 microM) causes increases in intracellular free calcium within 3 h, suggesting that increased intracellular calcium may be associated with some aspects of H2O2-induced cell death. However, at intermediate concentrations of H2O2 (30 microM), intracellular calcium remained stable during a 3 h exposure, during which time membrane blebbing was observed in ultrastructural studies. This suggests that some aspects of apoptotic cell death induced by H2O2 may not be associated with increased intracellular free calcium. Thus, this model appears valuable for studies of the mechanism(s) by which oxidative injury may induce apoptotic cell death and damage to neurons in the CNS.
Asunto(s)
Muerte Celular , Peróxido de Hidrógeno/farmacología , Neuronas/efectos de los fármacos , Animales , Calcio/metabolismo , Células Cultivadas/efectos de los fármacos , Sondas de ADN , Fura-2 , Microscopía Electrónica , Modelos Neurológicos , Ratas , Ratas Endogámicas , Factores de TiempoRESUMEN
The serum-free mouse embryo (SFME) cell line, derived from 16-day-old mouse embryos in medium in which serum was replaced by growth factors and other supplements, has been cultured for more than 200 generations. SFME cells are nontumorigenic, lack gross chromosomal abnormalities, and display characteristics of CNS progenitor cells. SFME cells show reversible induction of the astrocyte-specific marker glial fibrillary acidic protein when cultured in the presence of TGF-beta or serum. In order to determine if SFME cells exhibit further characteristics of CNS progenitor cells we investigated the expression of the gene encoding nestin, an intermediate filament protein expressed by neuroepithelial stem cells of the CNS. SFME cells express nestin in serum-free medium, and nestin expression is reversibly down-regulated by TGF-beta or serum. These results demonstrate that nestin expression is regulated by factors present in serum and support the hypothesis that SFME cells represent a CNS progenitor cell.
Asunto(s)
Astrocitos/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Proteínas de Filamentos Intermediarios/biosíntesis , Proteínas del Tejido Nervioso , Factor de Crecimiento Transformador beta/farmacología , Animales , Astrocitos/ultraestructura , Northern Blotting , Diferenciación Celular , Medio de Cultivo Libre de Suero , Proteína Ácida Fibrilar de la Glía/biosíntesis , Proteínas de Filamentos Intermediarios/genética , Ratones , Ratones Endogámicos BALB C , Nestina , Plasticidad Neuronal/efectos de los fármacos , ARN/biosíntesis , ARN/aislamiento & purificaciónRESUMEN
To investigate whether direct oxidative damage may induce neuronal death via an apoptotic cell suicide pathway, we exposed cultured cortical neurons to the prooxidant hydrogen peroxide. Our experiments demonstrate that brief exposures to low concentrations of hydrogen peroxide can induce neuronal cell death via apoptosis as demonstrated by toxicity assays, specific DNA staining, and the appearance of DNA laddering on agarose gels. These results suggest that direct oxidative injury may serve as a general trigger for apoptosis in the CNS, and that other apoptotic stimuli, such as exposure to amyloid-beta peptides, may also involve oxidative injury. Peroxide-induced apoptosis may serve as a valuable model for further studies of apoptosis in neurons.
Asunto(s)
Apoptosis/efectos de los fármacos , Corteza Cerebral/citología , Peróxido de Hidrógeno/farmacología , Neuronas/citología , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Células Cultivadas , ADN/análisis , ADN/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Agar , Embrión de Mamíferos , Neuronas/efectos de los fármacos , Neuronas/fisiología , RatasRESUMEN
The molecular mechanism responsible for the neurodegeneration in Alzheimer disease is not known; however, accumulating evidence suggests that beta-amyloid peptide (A beta P) contributes to this degeneration. We now report that synthetic A beta Ps trigger the degeneration of cultured neurons through activation of an apoptotic pathway. Neurons treated with A beta Ps exhibit morphological and biochemical characteristics of apoptosis, including membrane blebbing, compaction of nuclear chromatin, and internucleosomal DNA fragmentation. Aurintricarboxylic acid, an inhibitor of nucleases, prevents DNA fragmentation and delays cell death. Our in vitro results suggest that apoptosis may play a role in the neuronal loss associated with Alzheimer disease.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Apoptosis/efectos de los fármacos , Corteza Cerebral/citología , Daño del ADN , Neuronas/citología , Péptidos beta-Amiloides/síntesis química , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , ADN/efectos de los fármacos , ADN/aislamiento & purificación , Embrión de Mamíferos , Ratones , Microscopía Electrónica de Rastreo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Degeneración Nerviosa/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/farmacologíaRESUMEN
Serum-free mouse embryo (SFME) cells, derived in medium in which serum is replaced with growth factors and other supplements, are proastroblasts that are acutely dependent on epidermal growth factor (EGF) for survival. Ultrastructurally, an early change found in SFME cells deprived of EGF was a loss of polysomes which sedimentation analysis confirmed to be a shift from polysomes to monosomes. The ribosomal shift was not accompanied by decreased steady-state level of cytoplasmic actin mRNA examined as an indicator of cellular mRNA level. With time the cells became small and severely degenerate and exhibited nuclear morphology characteristic of apoptosis. Genomic DNA isolated from cultures undergoing EGF deprivation-dependent cell death exhibited a pattern of fragmentation resulting from endonuclease activation characteristic of cells undergoing apoptosis or programmed cell death. Flow cytometric analysis indicated that cultures in the absence of EGF contained almost exclusively G1-phase cells. Some of the phenomena associated with EGF deprivation of SFME cells are similar to those observed upon NGF deprivation of nerve cells in culture, suggesting that these neuroectodermal-derived cell types share common mechanisms of proliferative control involving peptide growth factor-dependent survival.
Asunto(s)
Astrocitos/citología , Factor de Crecimiento Epidérmico/farmacología , Animales , Astrocitos/química , Astrocitos/ultraestructura , Sangre , Núcleo Celular/ultraestructura , Supervivencia Celular , Células Cultivadas , Medios de Cultivo , Citoplasma/ultraestructura , ADN/análisis , Embrión de Mamíferos , Fase G1 , L-Lactato Deshidrogenasa/metabolismo , Ratones , Microscopía Electrónica , Polirribosomas/ultraestructura , ARN Mensajero/análisisRESUMEN
We applied serum-free cell culture methods that allow extended proliferation of mouse astrocyte precursor cells to the multipassage culture of embryonic human brain cells. Cells were cultured in nutrient medium supplemented with insulin, transferrin, epidermal growth factor, fibroblast growth factor, heparin, high-density lipoprotein, and fibronectin. Cultures were maintained for a maximum of 70 population doublings before proliferation ceased. The cells synthesized glial fibrillary acidic protein, an astrocyte marker, and expression of this protein was increased by incubation of the cells with transforming growth factor beta or serum. These results identify extracellular factors important for proliferation and differentiation of embryonic human astrocytes and provide a controlled system for multipassage culture.
Asunto(s)
Astrocitos/citología , Técnicas de Cultivo/métodos , Encéfalo/citología , Diferenciación Celular , División Celular , Células Cultivadas , Medios de Cultivo , Proteína Ácida Fibrilar de la Glía/biosíntesis , Sustancias de Crecimiento/farmacología , HumanosRESUMEN
Mouse embryo cells cultured in vitro in serum-supplemented media undergo growth crisis, resulting in the loss of genomically normal cells prior to the appearance of established, aneuploid cell lines. Mouse embryo cells established and maintained for multiple passages in the absence of serum did not exhibit growth crisis or gross chromosomal aberration. Cells cultured under these conditions were dependent on epidermal growth factor for survival. Proliferation was reversibly inhibited by serum or platelet-free plasma, suggesting that mouse embryo cultures maintained by conventional procedures are under the influence of inhibitory factors.
Asunto(s)
Supervivencia Celular , Células Cultivadas/citología , Animales , Sangre , Ciclo Celular , Medios de Cultivo , Factor de Crecimiento Epidérmico/fisiología , Ratones/embriología , Translocación GenéticaRESUMEN
Human serum spreading factor, an animal cell adhesion-promoting glycoprotein, bound Staphylococcus aureus in an assay in which association of bacteria to protein-precoated microtiter wells was quantitated. Interaction of human serum spreading factor with S. aureus suggests that this protein may play a role in host defenses and in the invasiveness and pathogenicity of microbial infections.