RESUMEN
Some food/food components have been the object of request of authorization to the use of health claims related to cognitive function in adults and compliant with the Regulation (EC) 1924/2006. Most of the requests have received a negative opinion by the European Food Safety Authority (EFSA) also because of the choice of not appropriate outcome variables (OVs) and methods of measurement (MMs) selected in the trials used to substantiate the claim. This manuscript referes to the collection, collation and critical analysis of OVs and MMs related to cognitive function in adults. OVs and MMs were collected from the EFSA Guidance document and the applications for authorization of health claims pursuant to the Articles 13(5). The critical analysis of OVs and MMs, performed by a literature review, was aimed at defining their appropriateness in the context of a specific claimed effect. The results highlight the importance of an adequate choice of OVs and MMs for an effective substantiation of the claims related to cognitive functioning. The information provided in this document may serve to EFSA for updating the guidance on the scientific requirements for health claims related to cognitive functions, but also for a better design of randomized controlled trials aimed at substantiating such health claims.
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Cognición , Dieta , Alimentos , Inocuidad de los Alimentos , Humanos , Legislación de Medicamentos , Pruebas NeuropsicológicasRESUMEN
BACKGROUND AND AIMS: The high number of negative opinions from the European Food Safety Authority (EFSA) to the requests for authorization of health claims is largely due to the design of human intervention studies, including the inappropriate choice of outcome variables (OVs) and of their methods of measurement (MMs). The present manuscript reports the results of an investigation aimed to collect, collate and critically analyse the information in relation to claimed effects, OVs and MMs, in the context of protection against oxidative damage and cardiovascular health compliant with Regulation 1924/2006. METHODS AND RESULTS: Claimed effects, OVs and the related MMs were collected from EFSA Guidance documents and applications for authorization of health claims under Articles 13.5 and 14. The OVs and their MMs were evaluated only if the claimed effect was sufficiently defined and was considered beneficial by EFSA. The collection, collation and critical analysis of the relevant scientific literature consisted in the definition of the keywords, the PubMed search strategies and the creation of databases of references. The critical analysis of the OVs and their MMs was performed on the basis of the literature review and was aimed at defining the appropriateness of OVs and MMs in the context of the specific claimed effects. CONCLUSIONS: The information provided in this document could serve to EFSA for the development of further guidance on the scientific requirements for health claims, as well as to the stakeholders for the proper design of human intervention studies aimed to substantiate such health claims.
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Antioxidantes/administración & dosificación , Enfermedades Cardiovasculares/prevención & control , Inocuidad de los Alimentos , Alimentos Funcionales , Estrés Oxidativo/efectos de los fármacos , Antioxidantes/efectos adversos , Biomarcadores/sangre , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/diagnóstico , Enfermedades Cardiovasculares/epidemiología , Daño del ADN/efectos de los fármacos , Europa (Continente)/epidemiología , Alimentos Funcionales/efectos adversos , Regulación Gubernamental , Análisis de Peligros y Puntos de Control Críticos , Humanos , Legislación Alimentaria , Peroxidación de Lípido/efectos de los fármacos , Factores Protectores , Carbonilación Proteica/efectos de los fármacos , Medición de Riesgo , Factores de RiesgoRESUMEN
OBJECTIVE: Osteoarthritis (OA) is a common and highly debilitating degenerative disease whose complex pathogenesis and the multiplicity of the molecular processes involved, hinder its complete understanding. Protein Kinase C (PKC) novel isozyme PKCε recently proved to be an interesting molecule for further investigations as it can represent an intriguing, new actor in the acquisition of a OA phenotype by the chondrocyte. DESIGN: PKCε was modulated in primary chondrocytes from human OA patient knee cartilage samples by means of short hairpin RNA (ShRNA) and the expression of cartilage specific markers observed at mRNA and protein level. The involvement of Histone deacetylases (HDACs) signaling pathway was also investigated through the use of specific inhibitors MS-275 and Inhibitor VIII. RESULTS: PKCε loss induces up-regulation of Runt-domain transcription factor (RUNX2), Metalloproteinase 13 (MMP13) and Collagen X (COL10) as well as an enhanced calcium deposition in OA chondrocyte cultures. In parallel, PKCε knock-down also leads to SOX9 and Collagen II (COL2) down-modulation and to a lower deposition of glycosaminoglycans (GAGs) in the extracellular matrix (ECM). This novel regulatory role of PKCε over cartilage hypertrophic phenotype is exerted via an HDAC-mediated pathway, as HDAC2 and HDAC4 expression is modulated by PKCε. HDAC2 and HDAC4, in turn, are at least in part responsible for the modulation of the master transcription factors RUNX2 and SOX9, key regulators of chondrocyte phenotype. CONCLUSIONS: PKCε prevents the phenotypic progression of the OA chondrocyte, acting on cartilage specific markers through the modulation of the transcription factors SOX9 and RUNX2. The loss of PKCε enhances, in fact, the OA hypertrophic phenotype, with clear implications in the pathophysiology of the disease.
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Osteoartritis , Benzamidas , Cartílago Articular , Condrocitos , Humanos , Proteína Quinasa C-epsilon , PiridinasRESUMEN
INTRODUCTION: Satellite cells are muscle resident stem cells and are responsible for muscle regeneration. In this study we investigate the involvement of PKCε during muscle stem cell differentiation in vitro and in vivo. Here, we describe the identification of a previously unrecognized role for the PKCε-HMGA1 signaling axis in myoblast differentiation and regeneration processes. METHODS: PKCε expression was modulated in the C2C12 cell line and primary murine satellite cells in vitro, as well as in an in vivo model of muscle regeneration. Immunohistochemistry and immunofluorescence, RT-PCR and shRNA silencing techniques were used to determine the role of PKCε and HMGA1 in myogenic differentiation. RESULTS: PKCε expression increases and subsequently re-localizes to the nucleus during skeletal muscle cell differentiation. In the nucleus, PKCε blocks Hmga1 expression to promote Myogenin and Mrf4 accumulation and myoblast formation. Following in vivo muscle injury, PKCε accumulates in regenerating, centrally-nucleated myofibers. Pharmacological inhibition of PKCε impairs the expression of two crucial markers of muscle differentiation, namely MyoD and Myogenin, during injury induced muscle regeneration. CONCLUSION: This work identifies the PKCε-HMGA1 signaling axis as a positive regulator of skeletal muscle differentiation.
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Diferenciación Celular , Desarrollo de Músculos/fisiología , Músculo Esquelético/citología , Mioblastos/citología , Proteína Quinasa C-epsilon/metabolismo , Regeneración/fisiología , Células Satélite del Músculo Esquelético/citología , Animales , Western Blotting , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Proteína HMGA1a/genética , Proteína HMGA1a/metabolismo , Técnicas para Inmunoenzimas , Ratones , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Proteína Quinasa C-epsilon/antagonistas & inhibidores , Proteína Quinasa C-epsilon/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Satélite del Músculo Esquelético/metabolismo , Transducción de SeñalRESUMEN
Among the three classic Philadelphia chromosome-negative myeloproliferative neoplasms, primary myelofibrosis (PMF) is the most severe in terms of disease biology, survival and quality of life. Abnormalities in the process of differentiation of PMF megakaryocytes (MKs) are a hallmark of the disease. Nevertheless, the molecular events that lead to aberrant megakaryocytopoiesis have yet to be clarified. Protein kinase CÉ (PKCÉ) is a novel serine/threonine kinase that is overexpressed in a variety of cancers, promoting aggressive phenotype, invasiveness and drug resistance. Our previous findings on the role of PKCÉ in normal (erythroid and megakaryocytic commitment) and malignant (acute myeloid leukemia) hematopoiesis prompted us to investigate whether it could be involved in the pathogenesis of PMF MK-impaired differentiation. We demonstrate that PMF megakaryocytic cultures express higher levels of PKCÉ than healthy donors, which correlate with higher disease burden but not with JAK2V617F mutation. Inhibition of PKCÉ function (by a negative regulator of PKCÉ translocation) or translation (by target small hairpin RNA) leads to reduction in PMF cell growth, restoration of PMF MK differentiation and inhibition of PKCÉ-related anti-apoptotic signaling (Bcl-xL). Our data suggest that targeting PKCÉ directly affects the PMF neoplastic clone and represent a proof-of-concept for PKCÉ inhibition as a novel therapeutic strategy in PMF.
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Megacariocitos/citología , Mielofibrosis Primaria/tratamiento farmacológico , Proteína Quinasa C-epsilon/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mielofibrosis Primaria/etiología , Mielofibrosis Primaria/patologíaRESUMEN
RATIONALE: Vessel formation is a crucial event in tissue repair after injury. Thus, one assumption of innovative therapeutic approaches is the understanding of its molecular mechanisms. Notwithstanding our knowledge of the role of Protein Kinase C epsilon (PKCε) in cardio-protection and vascular restenosis, its role in vessel progenitor differentiation remains elusive. OBJECTIVE: Given the availability of PKCε pharmacological modulators already tested in clinical trials, the specific aim of this study is to unravel the role of PKCε in vessel progenitor differentiation, with implications in vascular pathology and vasculogenesis. METHODS AND RESULTS: Mouse Peri-Vascular Adipose Tissue (PVAT) was used as source of mesenchymal vessel progenitors. VEGF-induced differentiation of PVAT cells down-regulates both PKCε and p-PAK1 protein expression levels. PKCε overexpression and activation: i) reduced the expression levels of SMA and PECAM in endothelial differentiation of PVAT cells; ii) completely abrogated tubules formation in collagen gel assays; iii) increased the expression of p-PAK1. CONCLUSION: PKCε negatively interferes with vessel progenitor differentiation via interaction with PAK-1.
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Tejido Adiposo/citología , Células Endoteliales/citología , Neovascularización Fisiológica/fisiología , Proteína Quinasa C-epsilon/metabolismo , Quinasas p21 Activadas/biosíntesis , Actinas/biosíntesis , Adventicia/citología , Animales , Proteínas de Unión al Calcio/biosíntesis , Diferenciación Celular , Células Cultivadas , Reestenosis Coronaria/enzimología , Regulación hacia Abajo , Activación Enzimática , Ratones , Proteínas de Microfilamentos/biosíntesis , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesis , Proteína Quinasa C-epsilon/biosíntesis , Proteína Quinasa C-epsilon/farmacología , Proteínas Smad/biosíntesis , Factor A de Crecimiento Endotelial Vascular/metabolismo , CalponinasRESUMEN
Protein kinase Cepsilon (PKCε) exerts a well-known cardio-protective activity in ischemia-reperfusion injury and plays a pivotal role in stem cell proliferation and differentiation. Although many studies have been performed on physiological and morphological effects of PKCε mis-expression in cardiomyocytes, molecular information on the role of PKCε on early cardiac gene expression are still lacking. We addressed the molecular role of PKCε in cardiac cells using mouse cardiomyocytes and rat bone marrow mesenchymal stem cells. We show that PKCε is modulated in cardiac differentiation producing an opposite regulation of the cardiac genes NK2 transcription factor related, locus 5 (nkx2.5) and GATA binding protein 4 (gata4) both in vivo and in vitro. Phospho-extracellular regulated mitogen-activated protein kinase 1/2 (p-ERK1/2) levels increase in PKCε over-expressing cells, while pkcε siRNAs produce a decrease in p-ERK1/2. Indeed, pharmacological inhibition of ERK1/2 rescues the expression levels of both nkx2.5 and gata4, suggesting that a reinforced (mitogen-activated protein kinase) MAPK signaling is at the basis of the observed inhibition of cardiac gene expression in the PKCε over-expressing hearts. We demonstrate that PKCε is critical for cardiac cell early gene expression evidencing that this protein is a regulator that has to be fine tuned in precursor cardiac cells.
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Diferenciación Celular , Células Madre Mesenquimatosas/enzimología , Miocitos Cardíacos/enzimología , Proteína Quinasa C-epsilon/metabolismo , Transducción de Señal , Animales , Células Cultivadas , Factor de Transcripción GATA4/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Proteína Quinasa C-epsilon/genética , Interferencia de ARN , Ratas , Ratas Wistar , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
Physical activity in schools is declining in many countries and inactivity in childhood has become a recognized risk factor. Data from a program of professionally guided physical exercise in primary school children were collected before and after the academic year of training. Four thousand five hundred children (6-10 years) were enrolled, and conditional and coordinative motor abilities (speed, trunk flexibility, long jumping, somersault, Harre circuit test) were measured. Anthropometric measurements were focused on body mass index (BMI), weight and height. Females never showed a significant variation of BMI, whereas males in the first and fourth grades showed significant differences. On the contrary, when considering the motor abilities studied, all the comparisons were highly significant. At the end of training, both males and females did better than at the beginning, and males were constantly faster than females. Our data, generated on a large number of children, show that professionally guided programs of physical education in the primary school lead to significant progresses in the development of conditional and coordinative abilities, without altering BMI values, thus not interfering with the balanced progression of body weight and height.
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Ejercicio Físico/fisiología , Actividad Motora/fisiología , Educación y Entrenamiento Físico/métodos , Antropometría , Rendimiento Atlético , Índice de Masa Corporal , Niño , Femenino , Humanos , Italia , MasculinoRESUMEN
The morphogenetic events leading to the transendothelial passage of lymphoid and tumoral cells are analyzed in light of a very recent and global theory of intercellular communication designated as the Triune Information Network (TIN). The TIN system is based on the assumption that cell-cell interactions primarily occur through cell surface informations or topobiological procesess, whose mechanisms rely upon expression of adhesion molecules, and are regulated by an array of locally-borne (autocrine/paracrine signals and autonomic inputs) and distantly-borne (endocrine secretions) messages. The final aim of the TIN is to control homeostatic functions crucial for the organism survival, like morphogenesis. Knowledge of the TIN signals involved in lymphoid and tumoral cell intravasation might offer a new perspetive to study the mechanisms of tumor immunity. Recognition of tumor target cells by immune cytotoxic effectors, in fact, can be considered a notable case of TIN-mediated cell to cell interaction. In particular, Natural Killer (NK) cells play a role in the cell-mediated control of tumor growth and metastatic spreading. Cell targeting and killing are dependent on the different NK cell receptors and on the efficacy of NK cells after cytokine and monoclonal antibody administration in cancer therapy. Since efficacy of NK cell-based immunotheraphy has been proven in KIR-mismatch regimens or in TRAIL-dependent apoptosis, the ability to manipulate the balance of activating and inhibitory receptors on NK cells and of their cognate ligands as well as the sensitivity of tumor cells to apoptosis, opens new perspectives for NK cell based immunotherapy.
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Tolerancia Inmunológica , Células Asesinas Naturales/inmunología , Neoplasias/inmunología , Sistemas Neurosecretores/fisiología , Animales , Citotoxicidad Inmunológica , Humanos , Inmunidad Celular , Células Asesinas Naturales/patología , Neoplasias/patología , Receptores Inmunológicos/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/patología , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismoRESUMEN
The regulation of the hematopoietic stem cell pool size and the processes of cell differentiation along the hematopoietic lineages involve apoptosis. Among the different factors with a recognized activity on blood progenitor cells, TRAIL - a member of the TNF family of cytokines - has an emerging role in the modulation of normal hematopoiesis.PKC(epsilon) levels are regulated by EPO in differentiating erythroid progenitors and control the protection against the apoptogenic effect of TRAIL. EPO-induced erythroid CD34 cells are insensitive to the apoptogenic effect of TRAIL between day 0 and day 3, due to the lack of specific surface receptors expression. Death receptors appear after day 3 of differentiation and consequently erythroid cells become sensitive to TRAIL up to day 9/10, when the EPO-driven up-regulation of PKC epsilon intracellular levels inhibits the TRAIL-mediated apoptosis, via Bcl-2. In the time interval between day 3 and 9, therefore, the number of erythroid progenitors can be limited by the presence of soluble or membrane-bound TRAIL present in the bone marrow microenvironment.
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Proteínas Reguladoras de la Apoptosis/fisiología , Eritropoyesis/fisiología , Glicoproteínas de Membrana/fisiología , Proteína Quinasa C-epsilon/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Apoptosis/fisiología , Diferenciación Celular/fisiología , Linaje de la Célula , Eritropoyetina/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF , Factores de TiempoRESUMEN
Natural killer cells play a key role in the defence of organisms against virus infections and in the control of tumor onset. Interleukin-2 is a multifunctional inflammatory cytokine able to activate natural killer cells, essentially inducing cell proliferation, lymphokine-activated-killer cell generation and cytokine production. Here we discuss some signaling events generated by interleukin-2 in the cell nucleus of primary human natural killer cells, specifically focusing on the lipid signal transduction and the induction of the cyclic adenosine-5'-monophosphate response element binding protein transcription factor. The implications of these nuclear events in the response of natural killer cells to interleukin-2 are also discussed.
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Interleucina-2/fisiología , Células Asesinas Naturales/fisiología , Animales , Núcleo Celular/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Humanos , Isoenzimas/fisiología , Células Asesinas Naturales/ultraestructura , Proteínas Quinasas Activadas por Mitógenos/fisiología , Fosfolipasa C beta , Fosforilación , Serina/metabolismo , Serina/fisiología , Transducción de Señal/fisiología , Translocación Genética/genética , Translocación Genética/fisiología , Fosfolipasas de Tipo C/fisiologíaRESUMEN
Human herpesvirus (HHV)-7 encodes a unique 65-kDa heparin-binding glycoprotein, designated gp65. This molecule is thought to play a role in virus attachment and entry. To obtain reagents to map the structure and function of HHV-7 gp65, we produced monoclonal antibodies to this molecule. Ten monoclonal antibodies reacting with gp65 on ELISA were subdivided in four groups on the basis of their isotype and differential reactivity with (i) native versus denatured forms of gp65, and (ii) mature (virion-associated) versus immature (cell-associated) forms of the molecule. We were able to map the binding epitopes for eight of these ten antibodies, and these were found to cluster to one site on gp65 (amino acids 239-278); within this region, the antibodies reacted with at least three distinct domains (244-251, 255-262, 263-278). The reasons for the apparent immunodominance of this region are uncertain. Taken together, this panel of antibodies constitutes an extensive and well-characterized set of HHV-7 specific antibodies that may have utility for future analyses of the structure/function of gp65, and for studies on the virus life cycle.
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Anticuerpos Monoclonales/inmunología , Mapeo Epitopo , Glicoproteínas/inmunología , Herpesvirus Humano 7/inmunología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Glicoproteínas/química , Humanos , Isotipos de Inmunoglobulinas , Ratones , Datos de Secuencia Molecular , Proteínas del Envoltorio Viral/químicaRESUMEN
Human herpesvirus 7 (HHV-7) is endemic in the adult human population. Although HHV-7 preferentially infects activated CD4(+) T lymphocytes, the consequence of T-cell infection for viral pathogenesis and immunity are still largely unknown. HHV-7 infection induces apoptosis mostly in uninfected bystander cells but not in productively infected CD4(+) T cells. To dissect the underlying molecular events, the role of death-inducing ligands belonging to the tumor necrosis factor (TNF) cytokine superfamily was investigated. HHV-7 selectively up-regulated the expression of TNF-related apoptosis-inducing ligand (TRAIL), but not that of CD95 ligand or TNF-alpha in lymphoblastoid (SupT1) or primary activated CD4(+) T cells. Moreover, in a cell-to-cell-contact assay, HHV-7-infected CD4(+) T lymphocytes were cytotoxic for bystander uninfected CD4(+) T cells through the TRAIL pathway. By contrast, HHV-7 infection caused a marked decrease of surface TRAIL-R1, but not of TRAIL-R2, CD95, TNF-R1, or TNF-R2. Of note, the down-regulation of TRAIL-R1 selectively occurred in cells coexpressing HHV-7 antigens that became resistant to TRAIL-mediated cytotoxicity. These findings suggest that the TRAIL-mediated induction of T-cell death may represent an important immune evasion mechanism of HHV-7, helping the virus to persist in the host organism throughout its lifetime.
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Apoptosis , Linfocitos T CD4-Positivos/inmunología , Regulación de la Expresión Génica , Herpesvirus Humano 7/inmunología , Glicoproteínas de Membrana/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Proteínas Reguladoras de la Apoptosis , Western Blotting , Antígenos CD4/genética , Linfocitos T CD4-Positivos/virología , Línea Celular , Citotoxicidad Inmunológica , Humanos , Glicoproteínas de Membrana/genética , Proteínas Recombinantes/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/genética , Receptor fas/inmunologíaRESUMEN
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induced both cytotoxic (apoptosis) and cytostatic (cell cycle perturbation) effects on the human myeloid K562 cell line. TRAIL stimulated caspase 3 and nitric oxide synthase (NOS) activities, and both pathways cooperate in mediating inhibition of K562 survival/growth. This was demonstrated by the ability of z-VAD-fmk, a broad inhibitor of effector caspases, and N-nitro-L-arginine methyl ester (L-NAME), an NOS pharmacologic inhibitor, to completely (z-VAD-fmk) or partially (L-NAME) suppress the TRAIL-mediated inhibitory activity. Moreover, z-VAD-fmk was able to block TRAIL-mediated apoptosis and cell cycle abnormalities and increase of NOS activity. The addition of the NO donor sodium nitroprusside (SNP) to K562 cells reproduced the cytostatic effect of TRAIL without inducing apoptosis. When TRAIL was associated to SNP, a synergistic increase of apoptosis and inhibition of clonogenic activity was observed in K562 cells as well as in other myeloblastic (HEL, HL-60), lymphoblastic (Jurkat, SupT1), and multiple myeloma (RPMI 8226) cell lines. Although SNP greatly augmented TRAIL-mediated antileukemic activity also on primary leukemic blasts, normal erythroid and granulocytic cells were less sensitive to the cytotoxicity mediated by TRAIL with or without SNP. These data indicate that TRAIL promotes cytotoxicity in leukemic cells by activating effector caspases, which directly lead to apoptosis and stimulate NO production, which mediates cell cycle abnormalities. Both mechanisms seem to be essential for TRAIL-mediated cytotoxicity.
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Apoptosis/efectos de los fármacos , Neoplasias Hematológicas/enzimología , Glicoproteínas de Membrana/farmacología , Óxido Nítrico Sintasa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Reguladoras de la Apoptosis , Inhibidores de Caspasas , Caspasas/metabolismo , Ciclo Celular/efectos de los fármacos , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patología , Humanos , Células K562/efectos de los fármacos , Glicoproteínas de Membrana/antagonistas & inhibidores , Células Madre Neoplásicas/efectos de los fármacos , Óxido Nítrico Sintasa/efectos de los fármacos , Óxido Nítrico Sintasa/farmacología , Nitroprusiato/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF , Células Tumorales Cultivadas/efectos de los fármacos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the catabolism of tryptophan. By creating a local microenvironment in which levels of tryptophan are low, IDO-expressing antigen-presenting cells (APC) could regulate T cell activation. This may be relevant to control both viral and bacterial replication as well as neoplastic cell growth. Interferon-alpha (IFN-alpha) is an antiviral cytokine affecting cellular differentiation. In addition, it reduces proliferation of CD4(+) T cells by several molecular mechanisms. To dissect the molecular steps responsible for the INF-mediated antiproliferative activity, we sought to determine whether activated primary CD4(+) T cells in the presence of IFN-alpha would produce IDO. We demonstrate here that IDO mRNA is not present in resting CD4(+) T cells. Stimulation with anti-CD3 plus interleukin-2 (IL-2) induces expression of IDO mRNA (about 2000 copies/150,000 cells), as determined by semiquantitative RT-PCR. When cells were stimulated in the presence of IFN-alpha, expression of IDO mRNA was significantly increased (more than 12,000 copies/150,000 cells). Functional analysis of IDO activity paralleled the results obtained with RT-PCR, demonstrating increased production of active enzyme in CD4(+) T cells stimulated in the presence of IFN-alpha. Our results indicate that IFN-alpha modulates levels of IDO produced by activated CD4(+) T cells. This would likely affect bystander cells by modifying levels of tryptophan in the local microenvironment.
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Linfocitos T CD4-Positivos/enzimología , Linfocitos T CD4-Positivos/inmunología , Interferón-alfa/farmacología , Triptófano Oxigenasa/genética , Triptófano Oxigenasa/metabolismo , Secuencia de Bases , Complejo CD3/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Cartilla de ADN/genética , Expresión Génica , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa , Interferón alfa-2 , Quinurenina/metabolismo , Activación de Linfocitos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Triptófano/metabolismoRESUMEN
The addition of low concentrations (0.1-1 nM) of extracellular HIV-1 Tat protein to PC12 neuronal cells stimulated a rapid (peak at 5 min) elevation of the cAMP intracellular levels, which in turn induced the phosphorylation of CREB transcription factor (peak at 15 min) on serine-133 (Ser-133). On the contrary, at later time points (60-120 min) Tat induced a significant decline of intracellular cAMP with respect to the basal levels observed in control cells treated with bovine serum albumin. In blocking experiments performed with pharmacological inhibitors, Tat decreased the intracellular levels of cAMP and CREB Ser-133 phosphorylation through a signal transduction pathway involving the sequential activation of phosphatidylinositol 3-kinase, AKT, and cyclic nucleoside phosphodiesterases. Moreover, in transient transfection experiments, Tat inhibited transcription of CREB promoter in a manner strictly dependent on the presence of the cAMP-responsive elements (CRE) in the CREB promoter. Consistently, the expression of endogenous CREB protein was significantly reduced in PC12 cells by prolonged (24-48 h) treatment with Tat. This decline in the expression of CREB, which plays an essential role in the survival and function of neuronal cells, anticipated a progressive increase of apoptosis in Tat-treated cells. Although obtained in a neuronal cell line, our findings might help to explain some aspects of the pathogenesis of HIV-1-associated dementia.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Regulación de la Expresión Génica , Productos del Gen tat/farmacología , VIH-1/fisiología , Neuronas/fisiología , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Androstadienos/farmacología , Animales , Bovinos , Cromonas/farmacología , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Morfolinas/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Células PC12 , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Fosfoserina/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-akt , Ratas , Albúmina Sérica Bovina/farmacología , Transcripción Genética , Transfección , Wortmanina , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
We show here that HIV type 1 (HIV-1) Tat protein, in combination with anti-CD3/CD28 mAbs, promotes IL-2 production and proliferation of primary CD4(+) T lymphocytes, obtained from HIV-1-seronegative donors. This effect was observed when Tat was immobilized on a solid support, but it was not observed with soluble Tat. Such hyperactivation was accomplished by recruiting the rolipram-sensitive cyclic nucleoside phosphodiesterase 4 and resulted in increased susceptibility to HIV-1 infection. Accordingly, rolipram potently inhibited HIV-1 replication in cultures stimulated by anti-CD3/CD28 +/- Tat. These results add to the concept that decreasing Tat activity is an important addition to anti-HIV-1 therapy, and they suggest a target for anti-HIV-1 chemotherapy, phosphodiesterase 4.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/fisiología , Linfocitos T CD4-Positivos/inmunología , Productos del Gen tat/metabolismo , VIH-1/inmunología , Activación de Linfocitos/inmunología , Replicación Viral/inmunología , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , División Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Interleucina-2/biosíntesis , Líquido Intracelular/metabolismo , Rolipram/farmacología , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
To investigate the tropism of the T-lymphotropic human herpesvirus 7 (HHV-7) for hematopoietic progenitors, cord blood CD34(+) cells were inoculated in vitro with HHV-7 and then induced to differentiate along the granulocytic and erythroid lineages by the addition of appropriate cytokine cocktails. In semisolid assays, HHV-7 modestly affected the growth of committed (granulocytic/macrophagic and erythroid) progenitors, whereas it significantly decreased the number of pluripotent (granulocytic/erythroid/ monocytic/megakaryocytic) progenitors. Such inhibitory effect was completely abrogated by incubating HHV-7 inoculum with anti-HHV-7 neutralizing serum. In liquid cultures, HHV-7 hastened maturation along the myeloid but not the erythroid lineage, as demonstrated by the up-regulation of CD33 early myeloid antigen at day 7 of culture, and of CD15 and CD14 antigens at day 15. Moreover, HHV-7 messenger RNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in cells maturating along both the myeloid and the erythroid lineages. To evaluate the relevance of these in vitro findings, the presence of HHV-7 was investigated in bone marrow (BM) unfractionated mononuclear cells (MCs) as well as in purified CD34(+) and CD34(-) cell subsets, obtained from 14 normal adult donors. HHV-7 DNA was detected by DNA-PCR in 4 of 7 BMMC samples, and it was found to be associated with both the CD34(-) (2 of 7) and the CD34(+ )(1 of 7) fractions. These data indicate that HHV-7 infects BM cells in vivo and shows the ability to affect the survival/differentiation of CD34(+) hematopoietic progenitors in vitro by inhibiting more ancestral progenitors and perturbing the maturation of myeloid cells.
Asunto(s)
Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/virología , Herpesvirus Humano 7/fisiología , Adulto , Antígenos CD/análisis , Antígenos CD34/análisis , Células de la Médula Ósea/citología , Diferenciación Celular , Línea Celular , Replicación del ADN , ADN Viral/análisis , Eritrocitos/citología , Eritrocitos/virología , Sangre Fetal/citología , Granulocitos/citología , Granulocitos/virología , Hematopoyesis , Herpesvirus Humano 7/genética , Humanos , Recién Nacido , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T , Replicación ViralRESUMEN
Treatment of dopaminergic rat PC12 cells with human immunodeficiency virus, type 1 (HIV-1) Tat protein or tat cDNA inhibited the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme for the dopamine biosynthetic pathway, as well as the production and release of dopamine into the culture medium. Moreover, the Tat addition to PC12 cells up-regulated the expression of the inducible cAMP early repressor (ICER), a specific member of the cAMP-responsive element modulator transcription factor family, in a cAMP-dependent manner. In turn, ICER overexpression abrogated the transcription activity of the TH promoter in PC12 cells, strongly suggesting ICER involvement in Tat-mediated inhibition of TH gene expression. In vivo injection of synthetic HIV-1 Tat protein into the striatum of healthy rats induced a subclinical Parkinson's-like disease that became manifested only when the animals were treated with amphetamine. As early as one week postinjection, the histochemical examination of the rat substantia nigra showed a reduced staining of neurons expressing TH followed by a loss of TH(+) neurons at later time points. As Tat protein can be locally released into the central nervous system by HIV-1-infected microglial cells, our findings may contribute to the explanation of the pathogenesis of the motorial abnormalities often reported in HIV-1 seropositive individuals.