RESUMEN
UNLABELLED: The phenotypic and functional characteristics of immune cells of osteoporotic women compared to healthy controls similar for age and estrogen level showed for the first time significant changes in several B lymphocytes populations in postmenopausal osteoporosis, related to bone mineral density (BMD) and fractures, and a significant lower basal secretion of interferon-gamma (IFN-gamma) by CD4(+). INTRODUCTION: To investigate the interactions between bone and immune system, we studied the phenotypic and functional characteristics of immune cells of 26 postmenopausal women with osteoporotic (OP) fractures compared to 24 healthy controls. METHODS: We analyzed surface markers of peripheral B, CD4(+) and CD8(+) lymphocytes and cytokine secretion in supernatants of these cells cultured with or without stimulation. Body composition was assessed by dual energy X-ray absorptiometry. RESULTS: The two groups were similar for age and estrogen level. OP women had a significantly lower body mass index, fat mass, and lean mass. The number of CD19(+), CD19(+)/CD27(+), CD19(+)/CD27(+)/CD5(-)/CD38(+) and CD19(+)/CD27(+)/RANK(+), CD4(+)/CD27(+)/CD45RA(-)/RANK(+), and CD4(+)/CD27(+)/CD45RA(-)/CD28(+) was lower in OP women and positively correlated to BMD. In OP women, under basal conditions, CD4(+) secreted less IFN-gamma and B lymphocytes more granulocyte macrophage colony-stimulating factor (GM-CSF). GM-CSF was positively correlated to fracture rate and negatively to BMD. CONCLUSIONS: Our results suggest that, regardless of age and estrogen status, postmenopausal OP is associated with immune changes, highlighting a possible role of IFN-gamma in the pathophysiology of OP and reporting, for the first time, changes in several B lymphocyte populations. These alterations may reflect the frailty observed after fracture, providing new insight into the mechanisms of morbidity and mortality associated with OP fractures.
Asunto(s)
Osteoporosis Posmenopáusica/inmunología , Anciano , Anciano de 80 o más Años , Subgrupos de Linfocitos B/inmunología , Composición Corporal/fisiología , Estudios de Casos y Controles , Células Dendríticas/inmunología , Estrógenos/sangre , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Humanos , Inmunidad Celular , Inmunofenotipificación , Interferón gamma/biosíntesis , Persona de Mediana Edad , Osteoporosis Posmenopáusica/sangre , Osteoporosis Posmenopáusica/fisiopatología , Fracturas Osteoporóticas/inmunología , Fracturas Osteoporóticas/fisiopatología , Proyectos Piloto , Subgrupos de Linfocitos T/inmunologíaRESUMEN
T-cell apoptosis during septic shock (SS) has been associated with deleterious outcome, but the mechanisms of apoptosis are not well understood. As T-cells are not infected in bacterial infection, our hypothesis was that deleterious interactions between lymphocytes and monocytes could be involved. This is a cross-sectional study of 27 patients presenting with community-acquired SS, 23 infected patients without SS and 18 controls. Cytofluorometric techniques were used to study apoptosis, the costimulatory pathway and cytokine synthesis. Apoptosis was increased in SS compared to infected patients without SS and controls: the median values were 18, 2 and 3%, respectively, for CD4(+) T-cells (P < 0.001), and 12, 5 and 2%, respectively, for CD8(+) T-cells (P < 0.001). Patients with SS exhibited significant CD152 over-expression on T-cells, while CD86 expression was decreased on monocytes (P = 0.004). The synthesis of interleukin-2 was decreased in patients with SS compared to the other groups, while secretions of interferon-gamma and TNF-alpha were not altered. Ten surviving patients with SS showed a trend towards the normalisation of these parameters on day 7. In SS, T-cell apoptosis is related, at least in part, to the alteration of the costimulatory pathway, which, in turn, leads to significant modification of the cytokine network.
Asunto(s)
Apoptosis , Choque Séptico/inmunología , Linfocitos T/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/biosíntesis , Antígeno B7-2/biosíntesis , Antígeno CTLA-4 , Femenino , Citometría de Flujo , Humanos , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Imatinib targets the Bcr-Abl oncogene that causes chronic myelogenous leukemia (CML) in humans. Recently, we demonstrated that besides triggering apoptosis in K562 cells, imatinib also mediated their erythroid differentiation. Although both events appear to proceed concomitantly, it is not known at present whether or not imatinib-induced apoptosis and differentiation are interdependent processes. Hence, we investigated the requirements for Bcr-Abl inhibitor-mediated apoptosis and erythroid differentiation in several established and engineered CML cell lines. Imatinib triggered apoptosis and erythroid differentiation of different CML cell lines, but only apoptosis exhibited sensitivity to ZVAD-fmk inhibition. Conversely, the p38 mitogen-activated protein (MAP) kinase inhibitor, SB202190, significantly slowed down erythroid differentiation without affecting caspase activation. Furthermore, imatinib and PD166326, another Bcr-Abl inhibitory molecule, triggered erythroid differentiation of K562 cell clones, nevertheless resistant to Bcr-Abl inhibitor-induced apoptosis. Finally, short hairpin RNA inhibitor (shRNAi) silencing of caspase 3 efficiently inhibited caspase activity but had no effect on erythroid differentiation, whereas silencing of Bcr-Abl mimicked imatinib or PD166326 treatment, leading to increased apoptosis and erythroid differentiation of K562 cells. Taken together, our findings not only demonstrate that Bcr-Abl inhibitor-mediated apoptosis and differentiation are fully distinguishable events, but also that caspases are dispensable for erythroid differentiation of established CML cell lines.
Asunto(s)
Apoptosis/efectos de los fármacos , Inhibidores de Caspasas , Diferenciación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Benzamidas , Caspasas/metabolismo , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Células Eritroides/citología , Células Eritroides/enzimología , Células Eritroides/patología , Proteínas de Fusión bcr-abl , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/sangre , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Piperazinas/farmacología , Proteínas Tirosina Quinasas/sangre , Piridinas/farmacología , Pirimidinas/farmacologíaRESUMEN
Hepatitis C virus (HCV) infection leads to liver injury, which is thought to be immune-mediated. Apoptosis of hepatic T cells could influence histological damage. We quantified peripheral and intrahepatic T-cell apoptosis in 28 patients with chronic hepatitis C by using cytofluorometric techniques. METAVIR score and HCV plasma viral load were determined. Six liver biopsies, obtained from controls without chronic hepatitis during hepatobiliary surgery, served as controls. In patients, liver T-cell apoptosis was upregulated compared to peripheral T cells: 35 versus 7% for CD4+ and 56 versus 13% for CD8+ T cells (P < 0.001). Liver T-cell apoptosis levels from patients were increased compared to controls for both CD4+ (P = 0.041) and CD8+ T cells (P = 0.007). Nine patients exhibiting METAVIR scores A and F < or = 1 showed higher intrahepatic CD4+ T-cell apoptosis compared to the 19 patients with a higher METAVIR score (P = 0.001) and both histological activity and fibrosis were related to apoptosis level. There was also an inverse relationship between the level of intrahepatic CD8+ T-cell apoptosis and serum transaminase activity (P = 0.023). Our study shows immune compartmentalization, suggesting that the study of peripheral blood lymphocytes may not be fully relevant to the pathophysiology of HCV hepatitis, and that the severity of liver injury is inversely correlated with intrahepatic CD4+ T-cell apoptosis.
Asunto(s)
Apoptosis/inmunología , Linfocitos T CD4-Positivos/inmunología , Hepacivirus/inmunología , Hepatitis C/inmunología , Hígado/inmunología , Adulto , Anciano , Bencimidazoles/química , Biopsia con Aguja Fina , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Femenino , Citometría de Flujo , Colorantes Fluorescentes/química , Hepatitis C/patología , Hepatitis C/virología , Hepatocitos/inmunología , Hepatocitos/virología , Humanos , Hígado/patología , Cirrosis Hepática/inmunología , Cirrosis Hepática/virología , Masculino , Persona de Mediana Edad , Transaminasas/sangre , Carga ViralRESUMEN
Incubation of Jurkat cells in the presence of H2O2 either directly added to the culture medium or generated with glucose oxidase, menadione or the couple xanthine/xanthine oxidase induced a marked decrease of phosphatidylserine synthesis in the absence of changes in the synthesis of phosphatidylcholine and phosphatidylethanolamine. Concentration dependent response curves indicated that H2O2 induced inhibition of phosphatidylserine synthesis with an IC(50)=5 microM while both induction of tyrosine phosphorylation of proteins and Ca(2+) signals were obtained with an EC(50)=300 microM. The tyrosine kinase and Ca(2+) independent mechanism was confirmed by comparing the H2O2-induced and the CD3-induced inhibition of phosphatidylserine synthesis using several Jurkat clones differing in the expression of cell surface receptors such as CD3/TCR and CD45 and protein tyrosine kinase such as p72syk, ZAP-70 and p56lck. While CD3-induced inhibition of phosphatidylserine synthesis necessitates protein tyrosine phosphorylation and Ca(2+) signals, H2O2 provoked its effect in all the clones studied independently of the presence or absence of the proteins previously shown to be key elements in T cell signal transduction. Conversely, the antioxidant molecule, butylated hydroxanisole, generates an increased PtdSer synthesis, suggesting that the synthesis of this phospholipid is regulated by the redox status of the cells.
Asunto(s)
Peróxido de Hidrógeno/farmacología , Fosfatidilserinas/biosíntesis , Linfocitos T/efectos de los fármacos , Antioxidantes/farmacología , Transporte Biológico , Hidroxianisol Butilado/farmacología , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Interacciones Farmacológicas , Humanos , Células Jurkat , Proteínas Tirosina Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Linfocitos T/metabolismoRESUMEN
Apoptosis is one of the mechanisms involved in the persistent CD4(+) T cell lymphopenia occurring in human immunodeficiency virus (HIV)-infected patients treated with highly active antiretroviral therapy (HAART). The aim of this study was to look at the relationship between the level of T cell apoptosis in patients on HAART and their HIV viral load measurement. Forty-five patients receiving HAART for a median time of 5 months were included: 13 had an undetectable viral load and 32 had a viral load > or = 200 copies/mL. No relationship was observed between the level of spontaneous T cell apoptosis and the viral load measurement. Patients with a viral load > or = 200 copies/mL exhibited a significantly higher level of CD4-induced apoptosis of CD4(+) T cells, compared with patients with undetectable viraemia (38 versus 15%, respectively, P = 0.03). Activation-induced apoptosis may be involved in the paucity of the immune reconstitution observed in HIV-infected patients treated with HAART.
Asunto(s)
Apoptosis , Linfocitos T CD4-Positivos/patología , Infecciones por VIH/patología , VIH/fisiología , Carga Viral , Adulto , Terapia Antirretroviral Altamente Activa/efectos adversos , Femenino , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Derivatives of salicylic acid (SA) and benzoic acid prevent endothelin-1 (ET-1) binding to ETA receptors. This study analyzed actions of 30 derivatives of benzoic acid and salicylic acid on (125)I-ET-1 binding to recombinant rat ETA receptors. The most active compounds were 3,5-dibromosalicylic acid (Br2SA, K(i) = 0.5 mM) and 3,5-diiodosalicylic acid (K(i) = 0.3 mM). They were about 50 times more potent than SA and aspirin. Br2SA inhibited equilibrium (125)I-ET-1 binding in an apparently competitive manner. It accelerated 8-fold the dissociation of (125)I-ET-1 receptor complexes and did not modify the second order rate constant of association of (125)I-ET-1 to its receptors. Br2SA also decreased the affinity of ETA receptors for receptor antagonists BQ-123 and bosentan. Br2SA accelerated dissociation of (125)I-ET-1-solubilized ETA receptor complexes and decreased the apparent molecular size of solubilized receptors. Br2SA and 3,5-diiodosalicylic acid inhibited two cellular actions of ET-1: the mobilization of intracellular Ca(2+) stores in isolated cells and contractions of rat aortic rings. They accelerated the relaxing action of BQ-123 and bosentan in ET-1-treated aortic rings. The results suggest the existence of an allosteric modifier site on ETA receptors that recognizes selected derivatives of SA. SA derivatives might be of therapeutic interest to relieve tight ET-1 binding and to favor actions of receptor antagonists.
Asunto(s)
Antagonistas de los Receptores de Endotelina , Endotelina-1/metabolismo , Salicilatos/farmacología , Regulación Alostérica , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Ácido Benzoico/química , Ácido Benzoico/farmacología , Transporte Biológico , Bosentán , Bromobenzoatos , Calcio/metabolismo , Células Cultivadas , Cromatografía en Gel , Interacciones Farmacológicas , Radioisótopos de Yodo , Péptidos Cíclicos/farmacología , Ratas , Receptor de Endotelina A , Receptores de Endotelina/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Ácido Salicílico/química , Ácido Salicílico/farmacología , Solubilidad , Sulfonamidas/farmacología , Vasodilatación/efectos de los fármacosRESUMEN
Infection of BALB/c mice with Leishmania major results in the rapid accumulation of IL-4 transcripts within CD4(+) T cells that react to the parasite Leishmania homologue of mammalian RACK1 (LACK) Ag. Because memory/effector cells secrete IL-4 more rapidly than naive cells, we sought to analyze the phenotype of these lymphocytes before infection. Indeed, a fraction of LACK-specific CD4(+) T cells expressed a typical CD62 ligand(low)CD44(high)CD45RB(low) phenotype in uninfected mice. LACK-specific T cells were primed in gut-associated lymphoid tissues by cross-reactive microbial Ags as demonstrated by their reactivity with bacterial extracts and by the ability of APCs from the mesenteric LN of BALB/c mice to induce their proliferation. Also, mice in which the digestive tract has been decontaminated exhibited a reduced proportion of LACK-specific T cells expressing a memory/effector phenotype and did not exhibit the early accumulation of IL-4 transcripts induced by L. major. Thus, LACK-specific T cells represent a subset of CD4(+) T cells which have acquired the ability to rapidly secrete IL-4 as the result of their priming by cross-reactive microbial Ags. Tracking the fate of these cells may provide information about the regulation of cell-mediated immune responses to gut Ags in physiological and pathological situations.
Asunto(s)
Antígenos Bacterianos/inmunología , Linfocitos T CD4-Positivos/inmunología , Interleucina-4/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Activación de Linfocitos/inmunología , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/inmunología , Secuencia de Aminoácidos , Animales , Antibacterianos/farmacología , Anticuerpos Monoclonales/administración & dosificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/inmunología , Susceptibilidad a Enfermedades , Enterococcus faecalis/crecimiento & desarrollo , Enterococcus faecalis/inmunología , Epítopos de Linfocito T/inmunología , Escherichia coli/crecimiento & desarrollo , Escherichia coli/inmunología , Femenino , Vida Libre de Gérmenes/inmunología , Hibridomas , Inyecciones Intraperitoneales , Interleucina-4/biosíntesis , Interleucina-4/genética , Interleucina-4/inmunología , Mucosa Intestinal/efectos de los fármacos , Leishmaniasis Cutánea/microbiología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones Transgénicos , Datos de Secuencia Molecular , ARN Mensajero/biosíntesisRESUMEN
1. Diadenosine polyphosphates (Ap(n)As, n=2 - 7) are considered as stress mediators in the cardiovascular system. They act both via identified P2 purinoceptors and via yet to be characterized receptors. This study analyses the actions of Ap(n)As in clones of rat brain capillary endothelial cells that express P2Y(1) receptors (B10 cells) or both P2Y(1) and P2Y(2) receptors (B7 cells). 2. B10 cells responded to Ap(3)A with rises in intracellular Ca(2+) concentration ([Ca(2+)](i)). This response was prevented by adenosine-3'-phosphate-5'-phosphate, an antagonist of P2Y(1) receptors. It was largely suppressed by a treatment with apyrase VII or with creatine phosphokinase/creatine phosphate to degrade contaminating ADP. 3. Ap(n)As inhibited ADP induced increases in [Ca(2+)](i) mediated by P2Y(1) receptors by shifting ADP concentration-response curves to larger concentrations. Apparent K(i) values were estimated to be 6 microM for Ap(4)A, 10 microM for Ap(5)A and 47 microM for Ap(6)A. Ap(2)A and Ap(3)A were much less active. 4. Ap(n)As were neither agonists nor antagonists of the endogenous P2Y(2) receptor in B7 cells. 5. Ap(n)As are neither agonists nor antagonists of the G(i)-coupled, ADP receptor in B10 cells. 6. The results suggest that most actions of Ap(n)As in B7 and B10 cells can be accounted for by endogenous P2Y(1) receptors. Ap(4)A, Ap(5)A and Ap(6)A are specific antagonists of endogenous Ca(2+)-coupled P2Y(1) receptors.
Asunto(s)
Fosfatos de Dinucleósidos/farmacología , Endotelio Vascular/efectos de los fármacos , Antagonistas del Receptor Purinérgico P2 , Adenosina Difosfato/farmacología , Adenilil Ciclasas/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Animales , Encéfalo/irrigación sanguínea , Encéfalo/citología , Calcio/metabolismo , Células Cultivadas , Células Clonales , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Ratas , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y1RESUMEN
Aspirin is a commonly used drug with a wide pharmacological spectrum including antiplatelet, anti-inflammatory, and neuroprotective actions. This study shows that aspirin and sodium salicylate, its major blood metabolite, reverse contractile actions of endothelin-1 (ET-1) in isolated rat aorta and human mammary arteries. They also prevent the intracellular Ca(2+) mobilizing action of ET-1 in cultured endothelial cells but not those of neuromedin B or UTP. Inhibition of the actions of ET-1 by salicylates is apparently competitive. Salicylates inhibit (125)I-ET-1 binding to recombinant rat ETA receptors. Salicylic acid promotes dissociation of (125)I-ET-1 ETA receptor complexes both in the absence and the presence of unlabeled ET-1. It has no influence on the rate of association of (125)I-ET-1 to ETA receptors. Salicylates do not promote dissociation of (125)I-ET-1 ETB receptor complexes. Salicylates potentiate relaxing actions of receptor antagonists such as bosentan. It is concluded that salicylates are allosteric inhibitors of ETA receptors. The results also suggest that: 1) irreversible ET-1 binding probably limits actions of receptor antagonists in vivo, and 2) an association of salicylates and ETA receptor antagonists should be used to evaluate the physiopathological role of ET-1 and may be of therapeutic interest in the treatment of ischemic heart disease.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Aspirina/farmacología , Antagonistas de los Receptores de Endotelina , Salicilato de Sodio/farmacología , Regulación Alostérica/efectos de los fármacos , Animales , Aorta , Transporte Biológico/efectos de los fármacos , Bosentán , Calcio/metabolismo , Células Cultivadas , Sinergismo Farmacológico , Endotelina-1/metabolismo , Humanos , Arterias Mamarias/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Ratas , Receptores de Endotelina/metabolismo , Sulfonamidas/farmacologíaRESUMEN
Early in the apoptotic process, CD95 induces a translocation of phosphatidylserine (PtdSer) from the inner to the outer leaflet of the cellular plasma membrane. In mammalian cells, PtdSer is only synthesized through a calcium-dependent exchange of the polar head group of pre-existing phospholipids, either phosphatidylcholine or phosphatidylethanolamine, by a serine. Using a pharmacological approach, we examined the influence of PtdSer synthesis on CD95-induced PtdSer exposure at the surface of Jurkat cells. We found that CD3/TCR triggering or thapsigargin treatment of Jurkat cells was accompanied both by a decreased PtdSer synthesis and by a strong reduction of CD95-induced PtdSer at the cell surface, as monitored by fluorescence-activated cell sorting (FACS) analysis of annexin V-fluorescein isothiocyanate (FITC)-labeled cells. PtdSer synthesis through the serine-base exchange enzyme system thus appeared as one of the mechanisms implicated in the recently discovered CD3/TCR-induced down-regulation of CD95-induced apoptosis. Conversely, increasing the activity of the serine-base exchange enzyme system with different drugs, either the K+ channel blocker quinine, the cationic amphiphil stearylamine, or three different calmodulin antagonists, chlorpromazine, trifluoperazine, and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7), resulted in an increased appearance of PtdSer at the surface of CD95-treated cells. Both PtdSer synthesis and CD95-induced annexin V-FITC reactivity were abrogated in ATP-depleted cells. Also, modifying the membrane potential with valinomycin (hyperpolarization) or either gramicidin or KCl (depolarization) demonstrated a strong relationship between PtdSer synthesis and annexin V-FITC reactivity in CD95-treated cells. Together, our results indicate that CD95-induced exposure of PtdSer at the cell surface could be regulated by the activity of the serine-base exchange enzyme system.
Asunto(s)
Apoptosis , Transferasas de Grupos Nitrogenados/metabolismo , Fosfatidilserinas/fisiología , Receptor fas/metabolismo , Adenosina Trifosfato/metabolismo , Humanos , Células Jurkat , Cinética , Activación de Linfocitos , Potenciales de la Membrana , Fosfatidilserinas/biosíntesis , Serina , Linfocitos T/efectos de los fármacos , Linfocitos T/fisiologíaRESUMEN
Anchorage removal like growth factor removal induces apoptosis. In the present study we have characterized signaling pathways that can prevent this cell death using a highly growth factor- and anchorage-dependent line of lung fibroblasts (CCL39). After anchorage removal from exponentially growing cells, annexin V-FITC labeling can be detected after 8 h. Apoptosis was confirmed by analysis of sub-G1 DNA content and Western blotting of the caspase substrate poly (ADP-ribose) polymerase. Growth factor withdrawal accelerates and potentiates suspension-induced cell death. Activation of Raf-1 kinase in suspension cultures of CCL39 or Madin-Darby canine kidney cells stably expressing an estrogen-inducible activated-Raf-1 construct (DeltaRaf-1:ER) suppresses apoptosis induced by growth factor and/or anchorage removal. This protective effect appears to be mediated by the Raf, mitogen- or extracellular signal-regulated kinase kinase (MEK), and mitogen-activated protein kinase module because it is sensitive to pharmacological inhibition of MEK-1 and it can be mimicked by expression of constitutively active MEK-1 in CCL39 cells. Finally, apoptosis induced by disruption of the actin cytoskeleton with the Rho-directed toxin B (Clostridium difficile) is prevented by activation of the DeltaRaf-1:ER chimeric construct. These findings highlight the ability of p42/p44 mitogen-activated protein kinase to generate survival signals that counteract cell death induced by loss of matrix contact, cytoskeletal integrity, and extracellular mitogenic factors.
Asunto(s)
Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Apoptosis , Sangre , Adhesión Celular , Células Cultivadas , Cricetinae , Perros , Activación Enzimática , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Sodium ortho-vanadate (Na3VO4), an inhibitor of protein tyrosine phosphatase, induces a rapid (15 min) and strong inhibition of phosphatidylserine synthesis with an IC50 = 100 microM. The mode of action of Na3VO4 was compared to that of CD3 mAbs. It was found that Na3VO4 bypasses the major CD3-induced T cell activation signals including protein tyrosine phosphorylation, p56lck activation and the generation of second messengers including inositol phosphates and its subsequent Ca2+ mobilization as well as diacylglycerol production. These facts were confirmed by using a panel of Jurkat clones that differs by the expression of either tyrosine kinases involved in the CD3-induced T cell activation pathway such as p56lck, p72syk and ZAP-70 or some cell surface receptors such as the CD3/TCR complex or the CD45 phosphatase.
Asunto(s)
Complejo CD3/metabolismo , Señalización del Calcio/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Fosfatidilserinas/biosíntesis , Linfocitos T/inmunología , Vanadatos/farmacología , Anticuerpos Monoclonales , Calcio/metabolismo , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Diglicéridos/análisis , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/metabolismo , Humanos , Inositol 1,4,5-Trifosfato/análisis , Células Jurkat , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación/efectos de los fármacos , Fosfotirosina/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Sistemas de Mensajero Secundario , Especificidad por Sustrato , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
Institutions in France are not yet well prepared to respond to allegations of scientific misconduct. Following a serious allegation in late 1997, INSERM, the primary organization for medical and health-related research in France, began to reflect on this subject, aided by scientists and jurists. The conclusions have resulted in establishing a procedure to be followed in cases of alleged misconduct, and also in reinforcing the application of good laboratory practices within each laboratory. Guidelines for authorship practices and scientific assessment must also be considered. Even though each institution must remain responsible for responding to allegations of scientific misconduct within its doors, INSERM would like to see national, European, and international co-ordination about the methods of such response.
Asunto(s)
Investigación Biomédica , Regulación Gubernamental , Mala Conducta Científica , Academias e Institutos , Comités Consultivos , Ética , Ética en Investigación , Europa (Continente) , Francia , Guías como Asunto , Cooperación InternacionalRESUMEN
HAART may increase CD4+ T cell counts despite a persistently detectable HIV load. The impact of HAART on apoptosis, which may play a role in the disease process in HIV-infected patients, has not been extensively studied. We performed a study to compare the level of spontaneous T cell apoptosis and anti-retroviral treatments in a cohort of HIV-1-infected patients. Data were obtained from a computerized medical record. Quantification of apoptotic cells was by cytofluorometric technique. From November 1995 to December 1997 we studied T cell apoptosis in 112 HIV-infected patients. Forty patients were classified A, 36 B and 36 C. Thirty patients were naive and 82 received an anti-retroviral treatment, 49 including a protease inhibitor (PI). The median plasma viraemia determined in 63 patients was 3.6 (range 1.3-5.6) log10. The median apoptotic cell count was 22% (range 2-73%) and 12% (range 2-60%) for CD4+ and CD8+ T cells, respectively. We did not observe any correlation between the HIV viraemia and the level of apoptosis of T cell subsets. Patients with HAART showed a lower percentage of apoptotic CD4+ T cells only: 16% (range 2-61%) versus 25% (range 5-73%) for patients receiving two nucleoside analogues (P = 0.02). This effect was significant in stage A patients and remained observable during the whole course of HIV disease. In conclusion, HAART, without any relation to plasma viraemia, is able to reduce apoptosis of CD4+ T cells.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Apoptosis , Linfocitos T CD4-Positivos/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Adulto , Anciano , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Didanosina/uso terapéutico , Quimioterapia Combinada , Femenino , Infecciones por VIH/inmunología , Infecciones por VIH/patología , Inhibidores de la Proteasa del VIH/uso terapéutico , Humanos , Lamivudine/uso terapéutico , Masculino , Persona de Mediana Edad , Análisis Multivariante , Estudios Retrospectivos , Estavudina/uso terapéutico , Carga Viral , Zalcitabina/uso terapéutico , Zidovudina/uso terapéuticoRESUMEN
Phosphatidylserine synthesis as measured by the incorporation of [(3)H]serine into phosphatidylserine (PtdSer) through the serine-base exchange enzyme system (serine-BEES) is markedly inhibited in Jurkat cells treated with caffeine. The caffeine-induced inhibition was compared to that observed in cells treated with either CD3 mAb or thapsigargin. While CD3- and thapsigargin-induced inhibition was related to the release of Ca(2+) from the endoplasmic reticulum (ER), a process that deprives the serine-BEES of its major cofactor, caffeine modified PtdSer synthesis in the absence of decreased Ca(2+) content of ER. Using Jurkat clones differing by the expression of cell surface markers or protein tyrosine kinases implicated in the CD3/TCR signal transmission pathway, we have shown that CD3 mAb-induced inhibition of PtdSer synthesis necessitates the expression of both the CD3/TCR and the protein tyrosine phosphatase CD45 at the cell surface as well as the presence of p56(lck) and ZAP-70 protein tyrosine kinases. By contrast, thapsigargin, a blocker of the Ca(2+)-ATPase of the ER, known for its Ca(2+) releasing properties, inhibited PtdSer synthesis in all the Jurkat clones tested, indicating that this compound bypasses the CD3/TCR-induced signals. Despite its lack of effect on Ca(2+) release from ER and on protein tyrosine phosphorylations, caffeine inhibited PtdSer synthesis in all the Jurkat clones. The use of several cAMP-inducing drugs and of others xanthine derivatives indicated that caffeine modify PtdSer synthesis either by a direct action on the serine-BEES or by a modification of the structure of the phospholipids used as substrate by the enzyme.
Asunto(s)
Complejo CD3/inmunología , Cafeína/farmacología , Señalización del Calcio , Fosfatidilserinas/biosíntesis , Proteínas Tirosina Quinasas/metabolismo , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Calcio/metabolismo , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Humanos , Ionomicina/farmacología , Células Jurkat , Fosforilación/efectos de los fármacos , Transducción de Señal , Tapsigargina/farmacologíaRESUMEN
The mechanisms of apoptosis have become better understood, in part with the discovery of Fas/CD95. We report the case of a patient characterized by a decreased CD4+ T cell count and an overexpression of Fas/CD95 resulting in apoptosis. A 54-year-old man presented with disseminated Mycobacterium xenopi infection. Analysis showed CD4+ T lymphopenia. Tests for human immunodeficiency virus (HIV) types 1 and 2 were negative. We compared the patient with eight healthy controls and five HIV-infected patients in terms of the expression of Fas/CD95 and Fas-mediated apoptosis of peripheral T lymphocytes. The percent of CD95+ cells in lymphocytes was 98% for the patient, and the mean percent of CD95+ cells in lymphocytes +/- SD for HIV-infected patients and healthy controls was 75% +/- 16% and 36% +/- 26%, respectively. The patient had a high level of spontaneous apoptosis, and apoptotic cells were all identified as being CD4+ T cells. Monoclonal antibodies to CD95 dramatically increased apoptosis of CD4+ T cells exclusively. CD4+ T lymphopenia observed in our patient correlated with an overexpression of Fas together with spontaneous and Fas-induced apoptosis.
Asunto(s)
Apoptosis/inmunología , Linfocitos/inmunología , Linfocitopenia-T Idiopática CD4-Positiva/inmunología , Receptor fas , Recuento de Linfocito CD4 , Infecciones por VIH/inmunología , Humanos , Masculino , Persona de Mediana Edad , Infecciones por Mycobacterium no Tuberculosas/complicaciones , Infecciones por Mycobacterium no Tuberculosas/inmunología , Mycobacterium xenopi , Receptores de Interleucina-2 , Valores de Referencia , Linfocitopenia-T Idiopática CD4-Positiva/etiología , Receptor fas/análisis , Receptor fas/inmunología , Receptor fas/fisiologíaRESUMEN
Ligation of Fas with its natural ligand or with anti-Fas antibodies induces an apoptotic program in Fas sensitive cells. We report here the identification of the tyrosine kinase p59Fyn as a substrate for CPP32-like proteinases and more particularly caspase 3 during Fas-mediated apoptosis in Jurkat T cells. Inhibition of CPP32-like proteinases by Ac-Asp-Glu-Val-Asp-aldehyde but not by Ac-Tyr-Val-Ala-Asp-aldehyde prevents CPP32, PARP and p59Fyn cleavage indicating that CPP32 or CPP32-like proteinases are responsible for the cleavage of p59Fyn. Cleavage occurs in the N-terminal domain of p59Fyn between Asp19 and Gly20 and is accompanied by relocation of an active p57Fyn kinase to cytoplasm of Fas-stimulated Jurkat cells as judged by both biochemical and confocal microscopy experiments. Thus, p59Fyn relocation and activity may play an important role during Fas-mediated cell death in human T lymphocytes.
Asunto(s)
Apoptosis , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Linfocitos T/enzimología , Receptor fas/fisiología , Caspasa 3 , Caspasa 8 , Caspasa 9 , Caspasas/metabolismo , Células Clonales , Humanos , Hidrólisis , Células Jurkat , Ácido Mirístico/metabolismo , Ácido Palmítico/metabolismo , Fragmentos de Péptidos/metabolismo , Pruebas de Precipitina , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-fyn , Transducción de Señal , Especificidad por Sustrato , Linfocitos T/patología , Familia-src Quinasas/metabolismoRESUMEN
The effects of 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) on intracellular Ca2+ mobilization and cyclic AMP accumulation were investigated using rat brain capillary endothelial cells which express an endogenous P2Y1 receptor, human platelets which are known to express a P2Y1 receptor, and Jurkat cells stably transfected with the human P2Y1 receptor. In endothelial cells, BzATP was a competitive inhibitor of 2-methylthio ADP (2-MeSADP) and ADP induced [Ca2+]i responses (Ki = 4.7 microM) and reversed the inhibition by ADP of adenylyl cyclase (Ki = 13 microM). In human platelets, BzATP inhibited ADP-induced aggregation (Ki = 5 microM), mobilization of intracellular Ca2+ stores (Ki = 6.3 microM), and inhibition of adenylyl cyclase. In P2Y1-Jurkat cells, BzATP inhibited ADP and 2-MeSADP-induced [Ca2+]i responses (Ki = 2.5 microM). It was concluded that BzATP is an antagonist of rat and human P2Y1 receptors and of platelet aggregation. In contrast to other P2Y1 receptor antagonists (A2P5P and A3P5P) which inhibit only ADP-induced Ca2+ mobilization, BzATP inhibits both the Ca2+- and the cAMP-dependent intracellular signaling pathways of ADP. These results provide further evidence that P2Y1 receptors contribute to platelet ADP responses.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Antagonistas del Receptor Purinérgico P2 , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/antagonistas & inhibidores , Adenosina Difosfato/farmacología , Adenosina Trifosfato/farmacología , Inhibidores de Adenilato Ciclasa , Adenilil Ciclasas/metabolismo , Alprostadil/farmacología , Animales , Plaquetas/efectos de los fármacos , Plaquetas/fisiología , Encéfalo/irrigación sanguínea , Calcio/metabolismo , Toxina del Cólera/farmacología , AMP Cíclico/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Células Jurkat , Ratas , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2/fisiología , Receptores Purinérgicos P2Y1 , Transducción de Señal/efectos de los fármacos , Tionucleótidos/antagonistas & inhibidores , Tionucleótidos/farmacología , TransfecciónRESUMEN
1. ADP is a potent agonist of rat and human P2Y1 purinoceptors. ATP is a weak competitive antagonist. This study analyses the situation in which P2Y1 receptors are exposed to ATP in the presence of exogenous ecto-nucleotidases (apyrases) that have high or low ATPase/ADPase activity ratio. 2. Rat brain capillary endothelial cells of the B10 clone express P2Y1 receptors that couple to intracellular Ca2+ mobilization. They have low endogenous ecto-ATPase and ecto-ADPase activities. 3. ATP did not raise intracellular Ca2+ in B10 cells. Addition of apyrases III or VII (1 u ml(-1)) to ATP treated cells induced large intracellular Ca2+ transients. Apyrases had no action in the absence of ATP. 4. A 1 u ml(-1) apyrase III solution generated 20 microM ADP from 0.1 mM ATP within 15 s. This concentration of ADP was sufficient to produce maximal activation of P2Y1 receptors. 5. ATP was a full agonist of P2Y1 receptors in the presence of 1 u ml(-1) apyrase III. Dose response curves for the apparent actions of ATP were bell shaped in the presence of 0.1 u ml(-1) apyrase III. Apyrase III did not alter ADP dose response curves when coincubated with ADP for 15 s. 6. Apyrase VII (1 u ml(-1)) shifted dose response curves for the actions of ADP to larger concentrations. It induced a bell shaped ATP dose response curve. 7. Results suggest that ATPDases prevent P2Y1 receptor activation by degrading ADP but may contribute to P2Y1 receptor activation by generating ADP from ATP.