RESUMEN
This study used a mixed-method research design to examine public attitudes toward sexual advance directives (SADs), a theoretical legal document intended to protect the sexual rights of individuals across the lifespan. Respondents (N = 537) largely indicated that SADs should exist (65.4%) to preserve autonomy and protect resident rights, but they would not sign one right now (60.4%) because they were too young. Linear regression analyses revealed statistical associations between education level, relationship length, and sexual instrumentality for the existence of SADs, while gender, age, perceived importance of sexuality, and sexual self-disclosure were associated with whether a respondent would sign the document now. Overall analyses indicate that valuing sexual expression as a right, autonomy, sexuality in older age, and sexual communication led to a greater amount of general support for SADs. Respondents were sensitive to the concerns of SADs in the context of fluid consent between partners and their safety.
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Demencia , Opinión Pública , Directivas Anticipadas , Anciano , Humanos , Conducta Sexual , SexualidadRESUMEN
BACKGROUND: Men are less likely than women to seek out mental health treatment, despite men also having mental health struggles. AIM: We examined typologies of men's reasons for not seeking treatment even though they perceived a need for treatment. METHOD: We conducted a latent class analysis using a U.S. national sample of 836 men who specifically indicated a need for mental health services, but did not seek treatment from the National Study on Drug Use and Health. RESULTS: We identified three distinct types of men and their unique rationales for not seeking treatment: positive attitudes class, perceived high behavioral control class, and negative attitudes class. CONCLUSIONS: Unique strategies to market clinical services to each of the various needs and concerns of these types of men may prove more beneficial than a generic outreach approach. We encourage mental health professionals to proactively educate men about their services and aim to reduce barriers for men seeking treatment.
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Servicios de Salud Mental , Salud Mental , Femenino , Humanos , Masculino , Aceptación de la Atención de Salud/psicología , PsicoterapiaRESUMEN
Intense few-cycle laser pulses have a breadth of applications in high energy density science, including particle acceleration and x-ray generation. Multi-amplifier laser system pulses have durations of tens of femtoseconds or longer. To achieve high intensities at the single-cycle limit, a robust and efficient post-compression scheme is required. We demonstrate a staged compression technique using self-phase modulation in thin dielectric media, in which few-cycle pulses can be produced. The few-cycle pulse is then used to generate extreme ultravoilet light via high harmonic generation at strong field intensities and to generate MeV electron beams via laser solid interactions at relativistic intensities.
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Demencia , Personal de Salud/psicología , Opinión Pública , Conducta Sexual , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Cuidados a Largo Plazo , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
BACKGROUND: The purpose of this study was to examine whether SB 203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor, is effective in reversing the pathogenic effects of antiphospholipid antibodies. METHODS: The adhesion of THP-1 monocytes to cultured endothelial cells (EC) treated with immunoglobulin G (IgG) from a patient with antiphospholipid syndrome (IgG-APS) or control IgG (IgG-NHS) in the presence and absence of SB 203580 was examined. The size of an induced thrombus in the femoral vein, the adhesion of leukocytes to EC of cremaster muscle, tissue factor (TF) activity in carotid artery and in peritoneal macrophages, the ex vivo expression of vascular cell adhesion molecule-1 (VCAM-1) in aorta preparations and platelet aggregation were studied in mice injected with IgG-APS or control IgG-NHS and with or without SB 203580. RESULTS: SB 203580 significantly reduced the increased adhesion of THP-1 to EC in vitro, the number of leukocytes adhering to EC, the thrombus size, the TF activity in carotid arteries and in peritoneal mononuclear cells, and the expression of VCAM-1 in aorta of mice, and completely abrogated platelet aggregation induced by IgG-APS. CONCLUSION: These data suggest that targeting the p38 MAPK pathway may be valuable in designing new therapy modalities for treating thrombosis in patients with APS.
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Anticuerpos Antifosfolípidos/inmunología , Endotelio Vascular/metabolismo , Trombosis/enzimología , Trombosis/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Adhesión Celular , Células Cultivadas , Endotelio Vascular/citología , Humanos , Imidazoles/farmacología , Leucocitos/citología , Ratones , Activación Plaquetaria , Piridinas/farmacologíaRESUMEN
Antiphospholipid (aPL) antibodies, detected in patients with antiphospholipid syndrome (APS) are associated with thrombosis, pregnancy loss and thrombocytopenia. Studies have shown that aPL are thrombogenic in vivo, but the mechanism(s) involved are not completely understood. Several studies have demonstrated that aPL antibodies activate endothelial cells (ECs) in vitro, as determined by up-regulation of adhesion molecules: E-selectin (E-sel); intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and in vivo. The objectives of these study were to determine the effects of aPL antibodies on the expression of E-selectin on ECs, on the adhesion of monocytes to ECs and to study the role of E-selectin on aPL antibodies enhanced thrombus formation and activation of ECs in vivo. We demonstrated that the surface expression of E-selectin on HUVEC by ELISA was increased 400-fold when treated with tumor necrosis factor-alpha (TNF-alpha) and 421-fold when treated with aPL antibodies during 4 h. APL antibodies also induced activation of the nuclear factor-kappa B (NF-kappaB). APL antibodies increased significantly the number of adhering leukocytes to ECs in vivo in C57BL/6 J mice when compared to IgG-NHS treated mice. This effect was abrogated in E-selectin-deficient mice. The thrombus size was significantly increased in C57BL/6 J mice treated with aPL antibodies when compared to mice treated with IgG-NHS. This enhancement in thrombus size by aPL antibodies was abrogated in E-selectin-deficient mice treated with aPL antibodies.
Asunto(s)
Anticuerpos Antifosfolípidos/farmacología , Selectina E/fisiología , Trombosis/etiología , Animales , Anticuerpos Antifosfolípidos/aislamiento & purificación , Adhesión Celular/efectos de los fármacos , Selectina E/efectos de los fármacos , Selectina E/genética , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/patología , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/metabolismoRESUMEN
OBJECTIVE: To investigate changes in the oral healthcare of adults with learning disability after transference from long stay hospital care to community-based care. SUBJECTS: Adults with learning disability who were former residents of a single long stay hospital and who had been resettled into the community during the period April 1995 to April 1998. DESIGN: Structured questionnaire with a covering letter sent to community-based carers. Hospital notes were reviewed to assess oral healthcare received as in-patients. RESULTS: There was a 68% response rate to the questionnaire from community-based carers with details obtained from 106 out of a possible 157 subjects. As residents in the hospital, all subjects were examined regularly by a dentist--yearly for edentulous and six-monthly for dentate individuals. However, attendance patterns were less regular as residents in the community. In the community, individuals were also less likely to receive operative dental treatment. Although oral hygiene regimes were generally on a daily basis only 37% of the subjects and/or their carers had received oral health education from dental professionals in the community. CONCLUSION: Changes from institutional living to community-based housing for adults with learning disability may be associated with changes in dental attendance and treatment patterns.
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Desinstitucionalización , Atención Dental para la Persona con Discapacidad , Discapacidades para el Aprendizaje , Adulto , Anciano , Anciano de 80 o más Años , Citas y Horarios , Atención Dental para la Persona con Discapacidad/clasificación , Atención Dental para la Persona con Discapacidad/estadística & datos numéricos , Profilaxis Dental , Operatoria Dental , Inglaterra , Femenino , Conductas Relacionadas con la Salud , Educación en Salud Dental , Hospitales Especializados , Humanos , Londres , Masculino , Persona de Mediana Edad , Higiene Bucal , Instituciones Residenciales , Factores de TiempoRESUMEN
Hyperpolarization in human leukemia THP-1 monocytes adherent to vascular cell adhesion molecule (VCAM)-1 is due to an induction of inwardly rectifying K(+) currents (I(ir)) (Colden-Stanfield M and Gallin EK, Am J Physiol Cell Physiol 275: C267-C277, 1998). We determined whether the VCAM-1-induced hyperpolarization is sufficient to augment the increase in intracellular free calcium ([Ca(2+)](i)) produced by Ca(2+) store depletion with thapsigargin (TG) and readdition of external CaCl(2) in fura 2-loaded THP-1 monocytes. Whereas there was a 2.1-fold increase in [Ca(2+)](i) in monocytes bound to glass for 5 h in response to TG and CaCl(2) addition, adherence to VCAM-1 produced a 5-fold increase in [Ca(2+)](i). Depolarization of monocytes adherent to VCAM-1 by I(ir) blockade or exposure to high [K(+)] abolished the enhancement of the peak [Ca(2+)](i) response. In monocytes bound to glass, hyperpolarization of the membrane potential with valinomycin, a K(+) ionophore, to the level of hyperpolarization seen in cells adherent to VCAM-1 produced similar changes in peak [Ca(2+)](i). Adherence of monocytes to E-selectin produced a similar peak [Ca(2+)](i) to cells bound to glass. Thus monocyte adherence to the physiological substrate VCAM-1 produces a hyperpolarization that is sufficient to enhance Ca(2+) entry and may impact Ca(2+)-dependent monocyte function.
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Calcio/metabolismo , Integrinas/metabolismo , Monocitos/metabolismo , Canales de Potasio/metabolismo , Receptores Mensajeros de Linfocitos/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Integrina alfa4beta1 , Integrinas/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Monocitos/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Receptores Mensajeros de Linfocitos/efectos de los fármacos , Tapsigargina/farmacología , Molécula 1 de Adhesión Celular Vascular/efectos de los fármacosRESUMEN
Antiphospholipid (aPL) Abs are associated with thrombosis, pregnancy loss, and thrombocytopenia in patients with systemic lupus erythematosus or primary antiphospholipid syndrome (APS). beta2-Glycoprotein I (beta2GPI), a phospholipid-binding serum protein, is involved in aPL binding to phospholipids. aPL can be generated in mice by immunization with beta2GPI, and these Abs are thrombogenic and cause pregnancy loss in mice. The objective of this study is to determine whether aPL induced by immunization with the phospholipid-binding site of beta2GPI are thrombogenic and whether they activate endothelial cells (EC) in vivo and in vitro. Murine monoclonal aPL were generated from spleen cells of a mouse immunized with GDKV, a synthetic 15-aa peptide spanning Gly274-Cys288 in the fifth domain of human beta2GPI, which represents the phospholipid-binding site of beta2GPI. The Abs generated had aPL and anti-beta2GPI activities. The effect of these Abs on thrombus formation and on EC activation in vivo was determined using a mouse model of thrombosis and microcirculation that enables examination of the adhesion of leukocyte to EC as an indication of EC activation as well as adhesion molecule expression using in vitro ELISA analysis. Mice injected with this monoclonal aPL showed a significant increase in leukocyte sticking and also produced larger thrombi that persisted longer. Exposure to GDKV-induced aPL for 4 h significantly increased surface Ag expression of E-selectin, ICAM-1, and VCAM-1. These data indicate that aPL induced by immunization with the phospholipid binding site of beta2GPI are thrombogenic and activate endothelial cells.
Asunto(s)
Adyuvantes Inmunológicos/biosíntesis , Adyuvantes Inmunológicos/fisiología , Anticuerpos Antifosfolípidos/biosíntesis , Anticuerpos Antifosfolípidos/fisiología , Endotelio Vascular/metabolismo , Oligopéptidos/inmunología , Trombosis/etiología , Adyuvantes Inmunológicos/administración & dosificación , Secuencia de Aminoácidos , Animales , Anticuerpos Antifosfolípidos/administración & dosificación , Sitios de Unión/inmunología , Moléculas de Adhesión Celular/biosíntesis , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/inmunología , Femenino , Glicoproteínas/metabolismo , Humanos , Inyecciones Intraperitoneales , Masculino , Ratones , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Fosfolípidos/inmunología , Fosfolípidos/metabolismo , Trombosis/inmunología , beta 2 Glicoproteína IRESUMEN
BACKGROUND: Antiphospholipid (aPL) antibodies are associated with thrombosis in patients diagnosed with antiphospholipid syndrome (APS) and enhance thrombus formation in vivo in mice, but the mechanism of thrombosis by aPL is not completely understood. Although aPL antibodies have been shown to inhibit protein C activation and activate endothelial cells (ECs) in vitro, no study has examined whether these antibodies activate ECs in vivo. Therefore, human affinity-purified aPL (ap aPL) antibodies from APS patients were tested in a mouse model of microcirculation using the cremaster muscle that allows direct microscopic examination of thrombus formation and adhesion of white blood cells (WBCs) to ECs as an indication of EC activation in vivo. Adhesion molecule expression on human umbilical vein endothelial cells (HUVECs) after aPL exposure was performed to confirm EC activation in vitro. METHODS AND RESULTS: All 6 ap aPL antibodies significantly increased the expression of VCAM-1 (2.3- to 4.4-fold), with one of the antibodies also increasing the expression of E-selectin (1.6-fold) on HUVECs in vitro. In the in vivo experiments, each ap aPL antibody except for 1 preparation increased WBC sticking (mean number of WBCs ranged from 22.7 to 50.6) compared with control (14.4), which correlated with enhanced thrombus formation (mean thrombus size ranged from 1098 to 6476 versus 594 microm2 for control). CONCLUSIONS: Activation of ECs by aPL antibodies in vivo may create a prothrombotic state on ECs, which may be the first pathophysiological event of thrombosis in APS.
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Anticuerpos Antifosfolípidos/farmacología , Síndrome Antifosfolípido/inmunología , Enfermedades Autoinmunes/inmunología , Endotelio Vascular/efectos de los fármacos , Trombofilia/etiología , Adulto , Animales , Anticuerpos Antifosfolípidos/inmunología , Especificidad de Anticuerpos , Síndrome Antifosfolípido/fisiopatología , Enfermedades Autoinmunes/fisiopatología , Adhesión Celular , Células Cultivadas , Relación Dosis-Respuesta Inmunológica , Selectina E/análisis , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Femenino , Glicoproteínas/inmunología , Humanos , Inmunización Pasiva , Inmunoglobulina G/farmacología , Molécula 1 de Adhesión Intercelular/análisis , Inhibidor de Coagulación del Lupus/inmunología , Inhibidor de Coagulación del Lupus/farmacología , Masculino , Ratones , Microcirculación/efectos de los fármacos , Trombosis/etiología , Molécula 1 de Adhesión Celular Vascular/análisis , beta 2 Glicoproteína IRESUMEN
We conducted a large population-based survey of fragile X (FRAXA) syndrome in ethnically diverse metropolitan Atlanta. The eligible study population consisted of public school children, aged 7-10 years, in special education-needs (SEN) classes. The purpose of the study was to estimate the prevalence among whites and, for the first time, African Americans, among a non-clinically referred population. At present, 5 males with FRAXA syndrome (4 whites and 1 African American), among 1,979 tested males, and no females, among 872 tested females, were identified. All males with FRAXA syndrome were mentally retarded and had been diagnosed previously. The prevalence for FRAXA syndrome was estimated to be 1/3,460 (confidence interval [CI] 1/7,143-1/1,742) for the general white male population and 1/4, 048 (CI 1/16,260-1/1,244) for the general African American male population. We also compared the frequency of intermediate and premutation FRAXA alleles (41-199 repeats) and fragile XE syndrome alleles (31-199 repeats) in the SEN population with that in a control population, to determine if there was a possible phenotype consequence of such high-repeat alleles, as has been reported previously. No difference was observed between our case and control populations, and no difference was observed between populations when the probands were grouped by a rough estimate of IQ based on class placement. These results suggest that there is no phenotype consequence of larger alleles that would cause carriers to be placed in an SEN class.
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Alelos , Etnicidad , Síndrome del Cromosoma X Frágil/genética , Vigilancia de la Población , Niño , Femenino , Síndrome del Cromosoma X Frágil/epidemiología , Tamización de Portadores Genéticos , Humanos , Masculino , Fenotipo , PrevalenciaRESUMEN
Resting membrane potential (RMP) and whole cell currents were recorded in human THP-1 monocytes adherent to polystyrene, unstimulated human umbilical vein endothelial cells (HUVECs), lipopolysaccharide (LPS)-treated HUVECs, immobilized E-selectin, or vascular cell adhesion molecule 1 (VCAM-1) using the patch-clamp technique. RMP after 5 h on polystyrene was -24.3 +/- 1.7 mV (n = 42) with delayed rectifier K+ (Idr) and Cl- currents (ICl) present in >75% of the cells. Inwardly rectifying K+ currents (Iir) were present in only 14% of THP-1 cells. Adherence to unstimulated HUVECs or E-selectin for 5 h had no effect on Iir or ICl but decreased Idr. Five hours after adherence to LPS-treated HUVECs, outward currents were unchanged, but Iir was present in 81% of THP-1 cells. A twofold increase in Iir and a hyperpolarization (-41.3 +/- 3.7 mV, n = 16) were abolished by pretreatment of THP-1 cells with cycloheximide, a protein synthesis inhibitor, or herbimycin A, a tyrosine kinase inhibitor, or by pretreatment of the LPS-treated HUVECs with anti-VCAM-1. Only a brief (15-min) interaction between THP-1 cells and LPS-treated HUVECs was required to induce Iir expression 5 h later. THP-1 cells adherent to VCAM-1 exhibited similar conductances to cells adherent to LPS-treated HUVECs. Thus engagement of specific integrins results in selective modulation of different K+ conductances.
Asunto(s)
Selectina E/farmacología , Endotelio Vascular/fisiología , Monocitos/fisiología , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/fisiología , Molécula 1 de Adhesión Celular Vascular/farmacología , Adhesión Celular , Línea Celular , Células Cultivadas , Canales de Cloruro/efectos de los fármacos , Canales de Cloruro/fisiología , Canales de Potasio de Tipo Rectificador Tardío , Endotelio Vascular/citología , Humanos , Cinética , Lipopolisacáridos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Monocitos/efectos de los fármacos , Técnicas de Placa-Clamp , Poliestirenos , Canales de Potasio/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factores de Tiempo , Venas UmbilicalesRESUMEN
Three studies were conducted to determine the effects of electrical stimulation, hot processing and carrageenan usage on sensory, shear force and cooking properties of low-fat (5, 10%) beef patties. The right or left sides of beef carcasses assigned to electrical stimulation (ES) received 600 V pulsating current for 120 s. Non-stimulated (NS) sides were also included for comparisons. Sides subjected to hot processing (HP) were boned 90 min post-exsanguination. Cold processing (CP) was initiated 48 h post mortem. Formulations for the manufacture of patties included the use of carrageenan [none, 0.5% iota (ι), 0.5% kappa (κ)]. Patties from NSHP beef had higher pH and cooking yields, less shrink in patty dimensions during cooking and lower shear force values than patties from NSCP controls (P < 0.05). ES improved tenderness and juiciness of CP patties, while ESHP patties exhibited higher cooking yields than NSCP patties. Tenderness was improved in two of the three studies as a result of increasing the fat content from 5 to 10%. Use of ι-carrageenan provided improvements in tenderness and juiciness over patties receiving no carrageenan or κ-carrageenan. Processors should consider HP with 1.0% added salt (improved cooking yields, patty configuration, tenderness) and the use of ι-carrageenan (improved tenderness and juiciness) for low-fat beef patties.
RESUMEN
The effects of bone morphogenetic protein-2 (BMP-2), -4, and -6 were tested on the differentiation of rat osteoprogenitor cells using a bone nodule-forming assay system, and the kinetics of their actions were investigated by double labeling for alkaline phosphatase (ALP) and bromodeoxyuridine (BrdU) uptake in log phase cultures. All BMPs stimulated bone nodule formation, with an optimal concentration of 25 ng/ml resulting in nodule numbers of approximately 250% of controls using BMP-4 and -6. BMP-2 showed reduced potency compared to either BMP-4 or -6. No evidence of chondrocytic differentiation was found in any of the cultures. The effect of BMPs on nodule formation was seen after only 24 h of exposure to BMPs, but only affected nodule numbers when added to early cultures. Nodule size and number of cells per nodule were increased with BMP6 only. Continuous or 24-h exposure to BMP-2 or -4 increased the number of postmitotic ALP-positive cells in log phase cultures, whereas BMP-6 increased the number of postmitotic ALP-negative cells. The results demonstrate that BMP-6, like other BMPs, can stimulate osteoblast differentiation independent of any chondrogenic effects and suggest that an early osteoprogenitor cell is an important target cell for the action of BMPs during bone induction. Overall, BMP-2 and -4 showed differences in potency in the assay systems used, but had qualitatively similar effects. In contrast, the qualitative differences found with BMP-6 suggest that BMP-6 may be acting principally on an early stage osteoprogenitor cell.
Asunto(s)
Osteoblastos/efectos de los fármacos , Proteínas/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Proteínas Morfogenéticas Óseas , Diferenciación Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Técnicas In Vitro , Osteoblastos/citología , Osteoblastos/enzimología , Ratas , Ratas Wistar , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/enzimologíaRESUMEN
To characterize the effect of 60Co gamma radiation on cell-cell and pathogen-cell interactions, the adherence of undifferentiated HL-60 cells to HUVEC monolayers was tested in the absence and presence of LPS or influenza virus type A. Basal HL-60 cell adherence to uninfected HUVEC monolayers (3.0 +/- 1.6%, n = 30) was not altered when HUVECs were exposed to 1- to 10-Gy gamma irradiation 4 to 72 h before the adhesion assay. LPS treatment of HUVEC monolayers (0.5 microgram/ml, 4 h) produced a 6.9-fold increase in adherence that was not altered by previous irradiation. However, when HUVEC monolayers were subjected to 1-10 Gy 41 h before influenza virus infection (10(6) pfu/ml) for 7 h, virus-induced adherence was enhanced in a dose-dependent manner. Increased virus hemagglutinin (HA) protein expression mediated the radiation-induced adherence for the following reasons: 1) HA Ag increases paralleled increases in leukocyte adherence. 2) Northern blot analysis demonstrated a time-dependent increase in mRNA HA levels. 3) Anti-HA blocked HL-60 cell adherence to irradiated and virus-infected HUVEC monolayers. These changes were associated with an increased virus titer yield and virus-induced HUVEC killing. In contrast, cytotoxicity produced by vesicular stomatitis virus, which unlike influenza virus replicates cytoplasmically, was not altered by radiation in HUVECs. In related studies, the canine kidney epithelial (MDCK) cell line showed a similar increased influenza virus production after gamma radiation, indicating that the radiation-induced increase in production of influenza virus is not cell-specific and probably involves a nuclear mechanism.
Asunto(s)
Adhesión Celular/efectos de la radiación , Endotelio Vascular/efectos de la radiación , Endotelio Vascular/virología , Virus de la Influenza A/fisiología , Leucocitos/efectos de la radiación , Animales , Antígenos de Superficie/biosíntesis , Northern Blotting , Adhesión Celular/inmunología , Células Cultivadas , Perros , Ensayo de Inmunoadsorción Enzimática , Epitelio/efectos de la radiación , Rayos gamma , Humanos , Virus de la Estomatitis Vesicular Indiana/fisiologíaRESUMEN
The adherence of undifferentiated 51Cr-labeled HL-60 (0.5 x 10(6) HL-60 cells/well) cells was monitored on influenza virus-infected HUVEC monolayers. Whereas only 3.0 +/- 1.6% (n = 36) of HL-60 cells adhered to uninfected HUVEC, adherence was increased to 41.7 +/- 2.2% (n = 6), 79.7 +/- 1.2% (n = 6), 83.9 +/- 0.7% (n = 6), and 84.4 +/- 0.5% (n = 6) on HUVEC infected for 7 h at a MOI of 1, 3, 6, and 9, respectively. In comparison, HL-60 cell adherence increased to 35% when HUVEC monolayers were stimulated with LPS (0.2-20 micrograms) for 4 h. Increased adherence to infected HUVEC occurred at 5 h postinfection, peaked at 7 h, and was maintained at 24 h postinfection. Active virus and metabolically active endothelial cells were required to mediate the virus-induced adherence. E-selectin and ICAM-1 Ag were upregulated 78.3- and 4.1-fold, respectively, by LPS (0.02-20 micrograms, 4 h) whereas virus infection (7 h) only increased these proteins 2.6- and 1.4-fold with a MOI > or = 16. Although the time courses of expression for both adhesion molecules after LPS treatment of virus infection were similar, the difference in the magnitude of upregulation suggests that virus-induced adherence is not a result of upregulation of E-selectin and ICAM-1. In contrast, surface expression of HA is involved in HL-60 cell adherence to virus-infected HUVEC because (1) the time course and magnitude of HA AG expression paralleled the time course and magnitude of HL-60 cell adherence after virus infection of HUVEC; (2) HL-60 cell aggregates were absent on infected HUVEC monolayers in the presence of anti-HA; (3) HL-60 cells competed with RBC for infected endothelial cells stained for cellular HA Ag and (4) anti-HA abolished the virus-induced adherence. Furthermore, it appears that HL-60 cells are binding directly to HA because HL-60 cell adherence to a cell-free surface was increased if virus was prebound and neuraminidase treatment of HL-60 cells prevented the HL-60 cell adherence to influenza virus-infected endothelial monolayers.
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Adhesión Celular , Endotelio Vascular/citología , Hemaglutininas Virales/metabolismo , Gripe Humana/inmunología , Leucocitos/citología , Moléculas de Adhesión Celular/metabolismo , Selectina E , Humanos , Técnicas In Vitro , Virus de la Influenza A , Gripe Humana/patología , Molécula 1 de Adhesión Intercelular , Neuraminidasa/farmacología , Células Tumorales Cultivadas , Venas Umbilicales/citologíaRESUMEN
The distribution of inwardly rectifying (Ki) and calcium-activated (KCa) potassium channels on the apical and basal surfaces of bovine aortic endothelial cells (BAECs) was examined by inverting BAEC monolayers onto polylysine-coated cover slips. To monitor cellular polarity, we examined human red blood cell adherence (hemadsorption) to the influenza virus protein, hemagglutinin (HA), and virus budding on the surface of infected BAECs. Hemadsorption and virus budding occurred on the apical surface but were not apparent on the basal surface of monolayers 1 and 5 h after inversion, although cellular HA antigen localization confirmed that all monolayers were infected. In contrast, by 9.5 and 24 h after inversion, hemadsorption was evident on the "new" apical surface. Single-channel patch-clamp analysis revealed the presence of both Ki and KCa channels on the apical surface and basal surface of BAEC monolayers 2-5 h after inversion. K channel conductance and kinetics were similar regardless of the surface monitored. This nonenzymatic mechanical technique of exposing the basal surface of endothelium provides a useful tool to study the distribution of ion channels in endothelium and in other polarized cell types grown in tissue culture.
Asunto(s)
Endotelio Vascular/fisiología , Orthomyxoviridae/fisiología , Animales , Membrana Celular/microbiología , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Polaridad Celular , Supervivencia Celular , Técnicas Citológicas , Electrofisiología , Endotelio Vascular/citología , Endotelio Vascular/microbiología , Hemabsorción , Hemaglutininas/metabolismo , Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/microbiología , Infecciones por Orthomyxoviridae/patología , Canales de Potasio/metabolismoRESUMEN
Bovine aortic endothelial cells (BAECs) respond to bradykinin with an increase in cytosolic-free Ca2+ concentration, [Ca2+]i, accompanied by an increase in surface membrane K+ permeability. In this study, electrophysiological measurement of K+ current was combined with 86Rb+ efflux measurements to characterize the K+ flux pathway in BAECs. Bradykinin- and Ca2(+)-activated K+ currents were identified and shown to be blocked by the alkylammonium compound, tetrabutylammonium chloride and by the scorpion toxin, noxiustoxin, but not by apamin or tetraethylammonium chloride. Whole-cell and single-channel current analysis suggest that the threshold for Ca2+ activation is in the range of 10 to 100 nM [Ca2+]i. The whole-cell current measurements show voltage sensitivity only at the membrane potentials more positive than 0 mV where significant current decay occurs during a sustained depolarizing pulse. Another K+ current present in control conditions, an inwardly rectifying K+ current, was blocked by Ba2+ and was not affected by noxiustoxin or tetrabutylammonium chloride. Efflux of 86Rb+ from BAEC monolayers was stimulated by both bradykinin and ionomycin. Stimulated efflux was blocked by tetrabutyl- and tetrapentyl-ammonium chloride and by noxiustoxin, but not by apamin or furosemide. Thus, 86Rb+ efflux stimulated by bradykinin and ionomycin has the same pharmacological sensitivity as the bradykinin- and Ca2(+)-activated membrane currents. The results confirm that bradykinin-stimulated 86Rb+ efflux occurs via Ca2(+)-activated K+ channels. The blocking agents identified may provide a means for interpreting the role of the Ca2(+)-activated K+ current in the response of BAECs to bradykinin.
Asunto(s)
Bradiquinina/farmacología , Endotelio Vascular/metabolismo , Canales de Potasio/metabolismo , Potasio/metabolismo , Animales , Aorta/citología , Calcio/farmacología , Bovinos , Células Cultivadas , Conductividad Eléctrica , Endotelio Vascular/citología , Potenciales de la Membrana , Compuestos de Amonio Cuaternario/farmacología , Rubidio/metabolismo , Venenos de Escorpión/farmacologíaRESUMEN
The resting intracellular pH (pHi) of BAECs, by using BCECF fluorescence, at 37 degrees C in Na+ Hanks' was 7.22 +/- 0.03. Cells which had been acid-loaded recovered from the intracellular acidification in Na+ Hanks' in a [Na+]o-dependent and amiloride-sensitive manner. Recovery from acidification had an apparent Km for Na+ of 40 +/- 10 mM and Ki for amiloride of 26 +/- 4 microM. Morphine (50 microM, 20 min, 37 degrees C) increased the pHi to 7.55 +/- 0.05. Naloxone (50 microM) given 5 min before morphine (50 microM) blocked this effect, indicating that this was an opiate receptor-mediated phenomenon. To determine if morphine activated the Na+/H+ exchanger, pHi was monitored in Na+-free Hanks', acidic Na+ Hanks' or amiloride-containing Na+ Hanks'. The alkalinization produced by morphine was not observed under all these circumstances. These data suggest morphine activates the Na+/H+ exchanger via opiate receptors.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Morfina/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Proteínas Portadoras/metabolismo , Bovinos , Células Cultivadas , Endotelio Vascular/metabolismo , Concentración de Iones de Hidrógeno , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Cinética , Receptores Opioides/efectos de los fármacos , Receptores Opioides/metabolismo , Intercambiadores de Sodio-HidrógenoRESUMEN
A four year regional screening programme to detect cystic fibrosis using measurement of immunoreactive trypsinogen is described. During this period 60 infants were diagnosed; 34 by screening, 12 born with meconium ileus, and 14 not identified by the screening assay but who presented with clinical symptoms at a later age, giving an incidence of cyst fibrosis in the region during this time of 1/1807. Screening has resulted in earlier detection of cystic fibrosis in many infants, thus allowing treatment to be instituted at an early age, and genetic counseling offered to the parents. There were a number of false positives and false negatives with the immunoreactive trypsinogen screening assay. In addition, eight infants who were sweat tested at an early age had a sweat sodium concentration of less than 70 mmol/l, although they were subsequently shown to have cystic fibrosis. These results confirm other published data showing that sweat sodium results may be low in very young infants with cystic fibrosis. At the time of diagnosis seven (20%) of the infants identified by screening were totally asymptomatic and several additional children had symptoms of such a type that the diagnosis of cystic fibrosis had not been considered at the time of screening. Despite the problems experienced it has been decided to continue screening.