RESUMEN
Gonadotrophin-releasing hormone (GnRH) agonists are used to treat gonadal steroid-dependent disorders in humans and to contracept animals. These agonists are considered to work by desensitising gonadotrophs to GnRH, thereby suppressing follicle-stimulating hormone (FSH) and luteinising hormone (LH) secretion. It is not known whether changes occur in the cellular composition of the pituitary gland after chronic GnRH agonist exposure. Adult male Sprague-Dawley rats were treated with a sham, deslorelin, or deslorelin plus testosterone implant for 41.0 ± 0.6 days. In a second experiment, rats were castrated and treated with deslorelin and/or testosterone. Pituitary sections were labelled immunocytochemically for FSHß and LHß, or gonadotrophin α subunit (αGSU). Deslorelin suppressed testis weight by two-thirds and reduced plasma FSH and LH in intact rats. Deslorelin decreased the percentage of gonadotrophs, although the effect was specific to the FSHß-immunoreactive (-ir) cells. Testosterone did not reverse the deslorelin-induced reduction in the overall gonadotroph population. However, in the presence of testosterone, the proportion of gonadotrophs that was FSHß-ir increased in the remaining gonadotrophs. There was no effect of treatment on the total LHß-ir cell population, although the loss of FSHß in bi-hormonal cells increased the proportion of mono-hormonal LHß-ir gonadotrophs. The castration-induced plasma LH and FSH increases were suppressed by deslorelin, testosterone or both. Castration increased both LH-ir and FSH-ir without increasing the overall gonadotroph population, thus increasing the proportion of bi-hormonal cells. Deslorelin suppressed these increases. Testosterone increased FSH-ir in deslorelin-treated castrate rats. Deslorelin did not affect αGSU immunoreactivity, suggesting that the gonadotroph population per se is not eliminated by deslorelin, although the ability of gonadotrophs to synthesise FSHß is compromised. We hypothesise that the FSH dominant suppression may be central to the long-term contraceptive efficacy of deslorelin in the male.
Asunto(s)
Hormona Folículo Estimulante/metabolismo , Pamoato de Triptorelina/análogos & derivados , Animales , Regulación hacia Abajo/efectos de los fármacos , Hormona Folículo Estimulante/sangre , Gonadotrofos/efectos de los fármacos , Gonadotrofos/metabolismo , Hormona Liberadora de Gonadotropina/agonistas , Antagonistas de Hormonas/farmacología , Inmunohistoquímica , Masculino , Orquiectomía , Tamaño de los Órganos/efectos de los fármacos , Hipófisis/anatomía & histología , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Ratas , Ratas Sprague-Dawley , Testículo/anatomía & histología , Testículo/efectos de los fármacos , Testículo/metabolismo , Factores de Tiempo , Pamoato de Triptorelina/farmacologíaRESUMEN
BACKGROUND: We analyzed the results of combined heart-kidney transplantation (CHKTx) over a 10-year period. METHODS: Between September 1996 and May 2007 at Mayo Clinic, 12 patients (age 52 ± 12.2 years) underwent CHKTx as a simultaneous procedure in 10 recipients and as a staged procedure in two recipients with unstable hemodynamics after heart transplantation. RESULTS: There was no operative mortality. Patient survival rates for the CHKTx recipients at 1 and 3 months and 6 years were 91%, 83%, and 83% and did not differ from isolated heart transplantation (IHTx) recipients (97%, 95%, and 79%, P = 0.61). The freedom from cardiac allograft rejection (≥ grade 2) at 3 months was 73% for CHKTx and had not changed during further follow-up; for IHTx, freedom from rejection at 3 months and 1 and 6 years was 61%, 56%, and 42% (P = .08). Heart and renal allograft survival was 100% with and left ventricular ejection fraction 66% ± 8.4% and glomerular filtration rate 61 ± 25 at last follow-up. There were no signs of cardiac allograft vasculopathy in the CHKTx recipients. CONCLUSION: CHKTx yields favorable long-term outcome, with a low incidence of cardiac rejection and vasculopathy. Simultaneous CHKTx appears feasible, if hemodynamics is satisfactory. This approach expands the selection criteria for transplantation in patients with coexisting end-stage cardiac and renal disease.
Asunto(s)
Vasos Coronarios/trasplante , Rechazo de Injerto , Trasplante de Corazón , Trasplante de Riñón , Adulto , Vasos Coronarios/patología , Femenino , Humanos , Pruebas de Función Renal , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Riesgo , Tasa de SupervivenciaRESUMEN
The aim of this study was to assess the patterns, predictors and outcomes of left ventricular remodeling after heart transplantation (HTX). Routine echocardiographic studies were performed and analyzed at 1 week, 1 year and 3-5 years after HTX in 134 recipients. At each study point the total cohort was divided into three subgroups based on determination of left ventricle mass and relative wall thickness: (1) NG-normal geometry (2) CR-concentric remodeling and (3) CH-concentric hypertrophy. Abnormal left ventricular geometry was found as early as 1 week after HTX in 85% of patients. Explosive mode of donor brain death was the most significant determinant of CH (OR 2.9, p = 0.01) at 1 week. CH at 1 week (OR 2.72, p = 0.01), increased body mass index (OR 1.1, p = 0.01) and cytomegalovirus viremia (OR - 4.06, p = 0.02) were predictors of CH at 1 year. CH of the cardiac allograft at 1 year was associated with increased mortality as compared to NG (RR 1.87, p = 0.03). CR (RR 1.73, p = 0.027) and CH (RR 2.04, p = 0.008) of the cardiac allograft at 1 year is associated with increased subsequent graft arteriosclerosis as compared to NG.
Asunto(s)
Vasos Coronarios/fisiopatología , Trasplante de Corazón , Tasa de Supervivencia , Remodelación Ventricular , Adulto , Estudios de Cohortes , Electrocardiografía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Trasplante HomólogoRESUMEN
BACKGROUND: Although evidence supports the efficacy of reducing immunosuppression for transplant-associated skin cancer, clinical thresholds for and risks associated with reduction are not well defined. OBJECTIVES: In this study, experienced transplant physicians were surveyed regarding appropriate thresholds for consideration of reduction of immunosuppression and the likelihood of rejection and allograft compromise associated with various levels of reduction. PATIENTS AND METHODS: Fifty-two transplant physicians reviewed 13 hypothetical patient scenarios with graduated morbidity and mortality risk and provided opinions on the degree of reduction of immunosuppression that was warranted and the risks associated with various degrees of reduction. RESULTS: Renal, liver and cardiac transplant physicians generally concurred on the level of reduction of immunosuppression warranted by various degrees of skin cancer. As morbidity and mortality from skin cancer increased, physicians were more likely to accept risk to allograft function from more aggressive reduction. CONCLUSIONS: Reduction of immunosuppression is considered a reasonable adjuvant strategy in recipients of solid organ transplants who have substantial morbidity and mortality risk from skin cancer. Physicians are willing to accept an increased risk of allograft compromise when confronted by severe or extensive skin cancer. Further research is needed to define the precise correlation among levels of reduction of immunosuppression, therapeutic efficacy, and concomitant risks.
Asunto(s)
Terapia de Inmunosupresión/efectos adversos , Neoplasias Cutáneas/prevención & control , Esquema de Medicación , Femenino , Humanos , Huésped Inmunocomprometido , Inmunosupresores/administración & dosificación , Masculino , Trasplante de Órganos , Factores de Riesgo , Neoplasias Cutáneas/inmunología , Tolerancia al Trasplante/inmunologíaRESUMEN
BACKGROUND: Eosinophils contribute to the pathogenesis of asthma and localize to the lung after allergen exposure by uncertain mechanisms. METHODS: We used intrabronchial instillation of allergen to model the interaction between inhaled allergen and the lung. We measured the number of peripheral blood leukocytes and the expression of VLA-4 (CD49d), Mac-1 (CD11b) and PSGL-1 (CD162) up to 4 h after instillation of allergen into a bronchus of eight atopic asthmatics. For controls, we instilled normal saline into a subset of the asthmatic subjects, and allergen into nonatopic, nonasthmatic subjects. RESULTS: There were changes of total leukocyte number, number of polymorphonuclear leukocytes, lymphocytes, monocytes and eosinophils in all three groups (atopic asthmatics instilled with allergen, atopic asthmatics instilled with saline, nonatopic nonasthmatic subjects instilled with allergen), which were likely related to bronchoscopy. However, the decrease of eosinophils was significant only in the atopic asthmatics instilled with allergen. The remaining eosinophils in the allergen challenged asthmatics were not activated as defined by cell density or change of expression of VLA-4, Mac-1 and PSGL-1. CONCLUSIONS: While eosinophils rapidly and specifically leave the circulation after allergen challenge of atopic asthmatics, the remaining circulating eosinophils are not activated.
Asunto(s)
Alérgenos/administración & dosificación , Asma/inmunología , Movimiento Celular/inmunología , Eosinófilos/inmunología , Administración por Inhalación , Adulto , Alérgenos/inmunología , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Antígeno CD11b/análisis , Antígeno CD11b/inmunología , Femenino , Humanos , Integrina alfa4/análisis , Integrina alfa4/inmunología , Interleucina-5/análisis , Interleucina-5/inmunología , Masculino , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/inmunología , Mucosa Respiratoria/inmunologíaRESUMEN
The molecular assemblies of signal transduction components, for example kinases and their target proteins or receptor-ligand complexes and intracellular signaling molecules, are critical for biological functions in cells. To better understand the interactions of these molecular assemblies and to screen for new pharmaceutics that could control and modulate these types of interactions, we have focused on developing high throughput approaches for the analysis of G-protein coupled receptors via flow cytometry. Flow cytometry offers a number of advantages including real-time collection of multicomponent data, and together with improvements in sample handling, the high throughput sampling rate is up to 100 samples per minute. For our targets, assemblies of solubilized GPCRs, a screening platform of a dextran bead has proven to be flexible, allowing different surface chemistries on the beads. The bead can be either ligand-labeled or have epitope-linked proteins attached to the bead surface, enabling several molecular assemblies to be constructed and analyzed. A major improvement with this system is that for screening ligands for GPCRs the underlying mechanism of action for these compounds can be investigated and incorporated into the definition of a 'hit'. Our current screening system is capable of simultaneously distinguishing GPCR agonists and antagonists.
Asunto(s)
Citometría de Flujo/métodos , Proteínas de Unión al GTP/análisis , Receptores de Superficie Celular/análisis , Animales , Técnicas Químicas Combinatorias , Citometría de Flujo/instrumentación , Proteínas de Unión al GTP/agonistas , Proteínas de Unión al GTP/antagonistas & inhibidores , Humanos , Receptores de Superficie Celular/agonistas , Receptores de Superficie Celular/antagonistas & inhibidoresRESUMEN
We describe a micromixing approach that is compatible with commercial autosamplers, flow cytometry, and other detection schemes that require the mixing of components that have been introduced into laminarflow. The scheme is based on high-throughput flow cytometry (HyperCyt) where samples from multi-well plates that have been picked up by an autosampler can be separated during delivery by the small air bubbles introduced during the transit of the autosampler probe from well to well. Here, either cell or particle samplesflowing continuously and driven by a syringe are brought together in a Y with reagent samples from wells driven by a peristaltic pump. The mixing is driven by a magnetic microstirrer contained within the sample line. The mixing is assessed using fluorescence of both cell calcium responses and bead-based fluorescence unquenching. In the analysis stream, the particles and reagents are mixed with eithera "wire" or "bar". The bar is more efficient than the wire, and the efficiency of either depends on the spinning action. The high-throughput approach and mixing in HyperCyt integrate autosamplers with submicroliter detection volumes for analysis in flow cytometry or in microfluidic channels.
Asunto(s)
Citometría de Flujo/instrumentación , Citometría de Flujo/métodos , Microesferas , Diseño de Equipo , Análisis de Falla de Equipo , Poliestirenos , Reología/instrumentación , Reología/métodosRESUMEN
BACKGROUND: Online mixing for continuous high-throughput flow cytometry has not been previously described. A simple, general high-throughput method for mixing and delivery of submicroliter volumes in laminar flow at low Reynolds numbers would be widely useful. MATERIALS AND METHODS: We describe a micromixing approach that is compatible with commercial autosamplers, flow cytometry, and other detection schemes that require mixing of components that have been introduced into laminar flow. The scheme is based on a previous approach to high-throughput flow cytometry (HyperCyt, Kuckuck et al.: Cytometry 44:83-90, 2001). We showed that samples from multiwell plates that have been picked up by an autosampler can be separated during delivery by the small air bubbles introduced during the transit of the autosampler probe from well to well. Here, a particle sample flowing continuously is brought together in a Y with reagent samples from wells, which have been separated by bubbles. RESULTS: In the effluent stream, the particles and reagents are mixed, most likely as a result of peristaltic action, and reagents from individual wells can be resolved. The sample volumes that can be mixed with this technology are submicroliter in volume, and samples can be mixed at rates up to at least 100/samples per minute. With the current device, carryover between samples can be eliminated if the mixing system is flushed with several volumes of buffer. The anticipated throughput for screening is expected to be at least 20 samples per minute. CONCLUSIONS: The high-throughput approach and peristaltic mixing in HyperCytTM serve to integrate autosamplers with submicroliter detection volumes for analysis in flow cytometry or in microfluidic channels.
Asunto(s)
Citometría de Flujo/instrumentación , Citometría de Flujo/métodos , Procesamiento de Señales Asistido por Computador/instrumentación , Avidina , Biotina , Difusión , Inmunoquímica/métodos , Bombas de Infusión/normas , MicroesferasRESUMEN
The flow cytometer is unique among biomedical analysis instruments because it makes simultaneous and multiple optical measurements on individual cells or particles at high rates. High throughput flow cytometry represents a potentially important multifactorial approach for screening large combinatorial libraries of compounds. Limiting this approach has been the availability of instrumentation and methods in flow cytometry for automated sample handling on the scale required for drug discovery applications. Here, we describe an automated system in which a novel patented fluidics-based pharmacology platform, the HTPS (High Throughput Pharmacological System), is coupled to a flow cytometer using a recently described plug flow-coupling valve technology. Individual samples are aspirated sequentially from microplate wells and delivered to a flow cytometer for rapid multiparametric analysis. For primary screening to detect and quantify cell fluorescence in endpoint assays, a high-speed no-wash protocol enabled processing of 9-10 cell samples/min from 96-well microplates. In an alternate primary screening format, soluble receptor ligands were sampled from microplate wells at rates of 3-4 samples/minute and successfully assessed for the ability to elicit intracellular calcium responses. Experiments with fluorescent beads validated the accurate automated production by the HTPS of exponential and linear gradients of soluble compounds. This feature enabled rapid (2- to 3-min) characterization of the intracellular calcium dose response of myeloid cells to formyl peptide as well as the quantitative relationship between formyl peptide receptor occupancy and cell response. HTPS flow cytometry thus represents a powerful high throughput multifactorial approach to increase the efficiency with which novel bioresponse-modifying drugs may be identified and characterized.
Asunto(s)
Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Citometría de Flujo/métodos , Automatización , Humanos , Péptidos/química , Factores de Tiempo , Células U937RESUMEN
This work examines the affinity of alpha(4)beta(1)-integrin and whether affinity regulation by G protein-coupled receptor (GPCR) and chemokines receptors is compatible with cell adhesion mediated between alpha(4)-integrin and vascular cell adhesion molecule-1. We used flow cytometry to examine the binding of a fluorescent derivative of an LDV peptide (Chen, L. L., Whitty, A., Lobb, R. R., Adams, S. P., and Pepinsky, R. B. (1999) J. Biol. Chem. 274, 13167-13175) to several cell lines and leukocytes with alpha(4)-integrin ranging from about 2,000 to 100,000 sites/cell. The results support the idea that alpha(4)-integrins exhibit multiple affinities and that affinity changes are regulated by the dissociation rate and conformation. The affinity varies by 3 orders of magnitude with the affinity induced by binding mAb TS2/16 plus Mn(2+) > Mn(2+) ' TS2/16 > activation because of occupancy of GPCR or chemokines receptor > resting receptors. A significant fraction of the receptors respond to the activating process. The change in alpha(4)-integrin affinity and the corresponding change in off rates mediated by GPCR receptor activation are rapid and transient, and their duration depends on GPCR desensitization. The affinity changes mediated by IgE receptor or interleukin-5 receptor persist longer. It appears that the physiologically active state of the alpha(4)-integrin, determined by inside-out signaling, has similar affinity in several cell types.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Integrinas/metabolismo , Péptidos/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Mensajeros de Linfocitos/metabolismo , Anticuerpos Monoclonales/metabolismo , Línea Celular , Citometría de Flujo , Colorantes Fluorescentes/metabolismo , Humanos , Integrina alfa4beta1 , Cinética , Leucocitos/metabolismo , Manganeso/metabolismo , Estructura Molecular , Péptidos/genética , Unión Proteica , Receptores de Superficie Celular/genética , Factores de Tiempo , TransfecciónRESUMEN
BACKGROUND: Conventional flow cytometry does not allow the rapid analysis of multiple samples. This has limited its uses in drug discovery, for which the standard for throughput is 100,000 samples per day. METHODS: We describe a simple method in which commercial peristaltic tubing is connected from a commercial autosampler to a flow cytometer. The samples are delivered via a peristaltic pump from source wells in a multiwell plate. The samples are separated by air bubbles. RESULTS: Throughput rates approach the limit of the autosampler (up to 100 wells per minute). Using optimal tubing and flow rates, particles remain within appropriate light scatter and fluorescence gates. The carryover between wells is typically less than 5% without and 1% with a wash step. The volumes of sample delivered are in the microliter scale. The approach has been validated with instruments from three manufacturers. CONCLUSIONS: Flow cytometry has potential throughput of 100,000 samples or more per day starting with the method described. The method is currently best suited to end-point assays. However, combined with high-speed sorting and single- cell assays, the number of assays could approach 1 billion per day.
Asunto(s)
Citometría de Flujo/métodos , Citometría de Flujo/instrumentación , Humanos , Sensibilidad y Especificidad , Factores de Tiempo , Células U937RESUMEN
BACKGROUND: Plug flow cytometry is a recently developed system for the automated delivery of multiple small boluses or "plugs" of cells or particles to the flow cytometer for analysis. Important system features are that sample plugs are of precisely defined volume and that the sample vessel need not be pressurized. We describe how these features enable direct cell concentration determinations and novel ways to integrate flow cytometers with other analytical instruments. METHODS: Adhesion assays employed human polymorphonuclear neutrophils (PMNs) loaded with Fura Red and Chinese hamster ovary (CHO) cells cotransfected with genes for green fluorescent protein (GFP) and human P-selectin. U937 cells expressing the human 7-transmembrane formyl peptide receptor were loaded with the fluorescent probe indo-1 for intracellular ionized calcium determinations. A computer-controlled syringe or peristaltic pump loaded the sample into a sample loop of the plug flow coupler, a reciprocating eight-port valve. When the valve position was switched, the plug of sample in the sample loop was transported to the flow cytometer by a pressure-driven fluid line. RESULTS: In stirred mixtures of PMNs and CHO cells, we used plug flow cytometry to directly quantify changes in concentrations of nonadherent singlet PMNs. This approach enabled accurate quantification of adherent PMNs in multicell aggregates. We constructed a novel plug flow interface between the flow cytometer and a cone-plate viscometer to enable real-time flow cytometric analysis of cell-cell adhesion under conditions of uniform shear. The High Throughput Pharmacology System (HTPS) is an instrument used for automated programming of complex pharmacological cell treatment protocols. It was interfaced via the plug flow coupling device to enable rapid (< 5 min) flow cytometric characterization of the intracellular calcium dose-response profile of U937 cells to formyl peptide. CONCLUSIONS: By facilitating the coupling of flow cytometers to other fluidics-based analytical instruments, plug flow cytometry has extended analytical capabilities in cell adhesion and pharmacological characterization of receptor-ligand interactions.
Asunto(s)
Adhesión Celular/fisiología , Citometría de Flujo/métodos , Neutrófilos/fisiología , Animales , Benzofuranos , Células CHO , Cricetinae , Diseño de Equipo , Citometría de Flujo/instrumentación , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes , Humanos , Imidazoles , Técnicas In Vitro , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Selectina-P/análisis , Selectina-P/genética , Reproducibilidad de los Resultados , Estrés Mecánico , Transfección , Células U937 , ViscosidadRESUMEN
Infectious complications are a major cause of morbidity and mortality in transplant recipients. We describe a case of fatal disseminated aspergillosis immediately following autologous peripheral stem cell reconstitution in a patient who had undergone orthotopic heart transplantation for systemic amyloidosis. The case described suggests that the infectious risks in patients undergoing these sequential procedures may be distinct from those occurring in patients undergoing either procedure independently. Potential prophylactic and therapeutic interventions are discussed. Since this experimental and evolving approach for the management of systemic amyloidosis is potentially applicable to a limited number of patients, multicenter collaboration may be needed to further define the infectious risks in this unique subset of transplant recipients.
Asunto(s)
Amiloidosis/terapia , Aspergilosis/patología , Trasplante de Corazón/patología , Trasplante de Células Madre Hematopoyéticas , Adulto , Anfotericina B/uso terapéutico , Amiloidosis/cirugía , Antifúngicos/uso terapéutico , Resultado Fatal , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Humanos , Complicaciones Posoperatorias/patología , Proteínas RecombinantesRESUMEN
Although flow cytometry has the powerful ability to rapidly screen large collections of cells, the technology has yet to be efficiently applied to large-scale screening operations involving multiple discrete suspensions. High-throughput flow cytometry would be beneficial to many areas of biological investigation, such as modern drug discovery, which involves testing of cellular targets against millions of potentially valuable compounds. The authors have developed a flow injection analysis approach to automated sample handling in which individual sample suspensions are sequentially inserted as plugs of precisely defined volumes into a flowing fluid which delivers them to the laser beam. The unit describes the basic elements and concepts of this plug flow system and discusses representative applications.
Asunto(s)
Citometría de Flujo/instrumentación , Animales , Biotecnología/métodos , Separación Celular/métodos , Diseño de Fármacos , Diseño de Equipo , Humanos , Rayos Láser , Programas Informáticos , Factores de TiempoRESUMEN
Intracardiac ectopic thyroid is a rare lesion. We present a case of successful excision of thyroid tissue obstructing the right ventricular outflow tract and provide a literature review. In all cases reported to date, the ectopic tissue arose from the ventricular septum and extended to the outflow tract.
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Coristoma/cirugía , Cardiopatías/cirugía , Glándula Tiroides/cirugía , Anciano , Coristoma/complicaciones , Femenino , Cardiopatías/complicaciones , Humanos , Obstrucción del Flujo Ventricular Externo/etiologíaRESUMEN
Radiation-induced heart disease is an increasingly recognized late sequela of mediastinal radiation therapy for malignant neoplasms. We report four cases of heart transplantation for end-stage heart failure induced by mediastinal radiation therapy. Short-term and intermediate-term results are excellent with all four patients currently surviving a mean of 48 months after transplantation. Neither a second malignancy nor recurrence of the primary malignancy has been observed to date. The early results of heart transplantation for end-stage, radiation-induced heart disease are encouraging.
Asunto(s)
Gasto Cardíaco Bajo/etiología , Gasto Cardíaco Bajo/cirugía , Trasplante de Corazón , Neoplasias Hematológicas/radioterapia , Radioterapia/efectos adversos , Adulto , Niño , Neoplasias Hematológicas/complicaciones , Humanos , Masculino , Persona de Mediana Edad , Trasplante HomólogoRESUMEN
Scientists seeking hard evidence of prayer's curative powers misunderstand the nature of prayer in the Western theistic traditions. Yet theistically consonant ways in which religious belief may influence health do not figure as they should in current professional practice.
Asunto(s)
Curación Mental , Religión y Medicina , Espiritualidad , Actitud del Personal de Salud , Beneficencia , Terapias Complementarias , Diversidad Cultural , Investigación Empírica , Humanos , Curación Mental/psicología , Autonomía Personal , Ensayos Clínicos Controlados Aleatorios como Asunto/métodos , TeologíaRESUMEN
Flow cytometry offers numerous advantages over traditional techniques for measuring intracellular Ca(2+) in lymphoid and nonlymphoid cells. In particular, the heterogeneity of cell responses can be defined by flow cytometry, and multiparameter analyses permit the determination of intracellular Ca(2+) in surface-marker-defined target cells as well as correlation of changes in Ca(2+) with other biochemical markers, including ligand binding. This article presents several established methods for measuring intracellular Ca(2+) by flow cytometry in lymphoid and nonlymphoid cells. Examples are provided for determination of Ca(2+) in human peripheral blood leukocytes and two human epithelial cell lines grown in monolayer. In addition, applications are reviewed or presented for correlating changes in intracellular Ca(2+) with other cell parameters, including cell cycle analysis, changes in cell membrane integrity, and the induction of apoptosis markers. Finally, a number of novel sample handling capabilities useful for performing kinetic analyses of Ca(2+) changes by flow cytometry are now available and one application is presented which is finding utility in pharmacologic studies.
Asunto(s)
Calcio/análisis , Calcio/metabolismo , Citometría de Flujo/métodos , Líquido Intracelular/química , Líquido Intracelular/metabolismo , Compuestos de Anilina/metabolismo , Benzofuranos/metabolismo , Calibración , Adhesión Celular , Línea Celular , Membrana Celular/metabolismo , Células Cultivadas , Quelantes/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Citometría de Flujo/instrumentación , Colorantes Fluorescentes/metabolismo , Humanos , Imidazoles/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Propidio/metabolismo , Receptores de Formil Péptido , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Receptores de Péptidos/genética , Receptores de Péptidos/metabolismo , Espectrometría de Fluorescencia , Transfección , Xantenos/metabolismoRESUMEN
The selective interaction of neutrophils with E-selectin and eosinophils with P-selectin has been previously reported, but the relevance of selectin site density and fluid shear has not been studied in detail. We have developed a new approach to examine these interactions in cell suspensions that integrates an on-line cone-plate viscometer with a flow cytometer. We find that eosinophils and neutrophils both use P-selectin glycoprotein ligand-1 to form stable conjugates with P-selectin Chinese hamster ovary cell transfectants, with a preferential adhesion of eosinophils. Further, the difference in cell adhesion between neutrophils and eosinophils is magnified at P-selectin expression levels below approximately 20 sites/microm2, a range likely to be relevant to endothelial cell expression levels in conditions associated with eosinophilia. The unique behavior is retained over shear rates ranging from 100 to 1500/s but is magnified at low shear. Results from parallel-plate flow chamber assays suggest that preferential eosinophil adhesion reflects an enhanced efficiency of initial PSGL-1 bond formation with P-selectin rather than a unique ability of eosinophils to mediate rolling interactions of longer duration on low-density P-selectin substrates. These differences may account in part for the increase in eosinophil accumulation in allergic diseases.
Asunto(s)
Movimiento Celular/inmunología , Eosinófilos/metabolismo , Neutrófilos/metabolismo , Selectina-P/biosíntesis , Selectina-P/inmunología , Animales , Sitios de Unión de Anticuerpos/genética , Velocidad del Flujo Sanguíneo/inmunología , Células CHO , Adhesión Celular/genética , Adhesión Celular/inmunología , Comunicación Celular/genética , Comunicación Celular/inmunología , Movimiento Celular/genética , Cricetinae , Eosinófilos/inmunología , Citometría de Flujo , Humanos , Neutrófilos/inmunología , Selectina-P/genética , Selectina-P/metabolismo , Análisis de Regresión , TransfecciónRESUMEN
The L-selectin adhesion molecule mediates leukocyte recruitment to inflammatory sites and lymphocyte trafficking through the peripheral lymph nodes. In response to leukocyte activation, L-selectin is proteolytically released from the cell surface, disabling leukocytes from the subsequent L-selectin-dependent interactions. We have found that L-selectin shedding is sensitive to sulfhydryl chemistry; it is promoted by thiol-oxidizing or -blocking reagents and inhibited by reducing reagents. Phenylarsine oxide (PAO), a trivalent arsenical that interacts with vicinal dithiols, is most potent in inducing rapid shedding of L-selectin from isolated neutrophils, eosinophils, and lymphocytes as well as from neutrophils in whole blood. PAO does not cause cell activation, nor does it interfere with integrin function or alter the expression of several other cell surface molecules at the low concentrations that induce L-selectin shedding. PAO is not required to enter the cell to induce L-selectin shedding. TAPI-2 ((N-(D,L-[2-(hydroxyaminocarbonyl)-methyl]-4-methylpentanoyl)-L-3-(tert-butyl)-alanyl-l -alanine, 2-aminoethyl amide), which has previously been shown to inhibit the activation-dependent L-selectin shedding, is also capable of inhibiting PAO-induced L-selectin shedding. We hypothesize that PAO-induced L-selectin shedding involves a regulatory molecule, such as protein disulfide isomerase (PDI), an enzyme that plays a role in the formation and rearrangement of disulfide bonds, contains PAO-binding, vicinal dithiol-active sites, and is expressed on the neutrophil surface. Cell surface expression of PDI, L-selectin shedding induced by PDI-blocking Abs and by bacitracin, a known inhibitor of PDI activity, and direct binding of PDI to PAO, provide supporting evidence for this hypothesis.