RESUMEN
BACKGROUND: Systemic inflammatory response syndrome (SIRS) can occur in association with cardiopulmonary bypass (CPB) surgery, resulting in multiple organ dysfunction (MOD). Activated neutrophils have been implicated as major inciting factors in this process. Neutrophil-depleting filters incorporated within the extracorporeal blood circuit during CPB have been developed and evaluated, with inconsistent clinical results. METHODS: A novel, biomimetic, selective cytopheretic device (SCD) was tested in vitro within a blood circuit to assess safety and interactions with blood components and further evaluated ex vivo in a bovine model of CPB surgery during ventricular assist device implantation. RESULTS: In vitro blood circuit studies demonstrated that the SCD reduces circulating neutrophils while maintaining low rates of hemolysis compared to current leukocyte-reduction filters. In the bovine CPB model, animals without SCD treatment (No SCD) demonstrated an increase in circulating white blood cell (WBC) and neutrophil counts, steadily increasing throughout CPB. SCD with only systemic heparin anticoagulation (SCD-H) acutely reduced neutrophils for the first 2 hrs of CPB, but followed with a greater than 6-fold increase in neutrophil counts. SCD treatment with regional citrate anticoagulation along the SCD circuit (SCD-C) reduced systemic neutrophil counts throughout 4 hrs of CPB despite lower amounts of eluted cells from the SCD. When analyzed for immature neutrophils, the control and SCD-H showed increasing counts at later time-points, not seen in the SCD-C group, suggesting a more complex mechanism of action than simple leukoreduction. CONCLUSIONS: These results suggest that SCD-C therapy may disrupt the systemic leukocyte response during CPB, leading to improved outcomes for CPB-mediated MOD.
Asunto(s)
Puente Cardiopulmonar , Leucaféresis/instrumentación , Leucaféresis/métodos , Animales , Bovinos , Humanos , Recuento de Leucocitos , Insuficiencia Multiorgánica/prevención & control , Neutrófilos/citología , Recuperación de Sangre Operatoria/instrumentación , Recuperación de Sangre Operatoria/métodosRESUMEN
PURPOSE: Chemotherapy for advanced pancreatic cancer has limited efficacy due to the difficultly of treating established tumours and the evolution of tumour resistance. Chemotherapies for pancreatic cancer are typically studied for their cytotoxic properties rather than for their ability to increase the immunogenicity of pancreatic tumour cells. In this study Gemcitabine in combination with immune modulatory chemotherapies Oxaliplatin, zoledronic acid and pomalidomide was studied to determine how combination therapy alters the immunogenicity of pancreatic tumour cell lines and subsequent T-cell responses. METHODS: Pancreatic tumour cell lines were stimulated with the chemotherapeutic agents and markers of immune recognition were assessed. The effect of chemotherapeutic agents on DC function was measured using uptake of CFSE-stained PANC-1 cells, changes in markers of maturation and their ability to activate CD8+ T-cells. The effect of chemotherapeutic agents on T-cell priming prior to activation using anti-CD3 and anti-CD28 antibodies was determined by measuring IFN-γ expression and Annexin V staining using flow cytometry. RESULTS: These agents demonstrate both additive and inhibitory properties on a range of markers of immunogenicity. Gemcitabine was notable for its ability to induce the upregulation of human leukocyte antigen and checkpoints on pancreatic tumour cell lines whilst inhibiting T-cell activation. Pomalidomide demonstrated immune modulatory properties on dendritic cells and T-cells, even in the presence of gemcitabine. DISCUSSION: These data highlight the complex interactions of different agents in the modulation of tumour immunogenicity and immune cell activation and emphasise the complexity in rationally designing chemo immunogenic combinations for use with immunotherapy.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Desoxicitidina/análogos & derivados , Inmunomodulación/efectos de los fármacos , Neoplasias Pancreáticas/inmunología , Anexina A5/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Desoxicitidina/farmacología , Interacciones Farmacológicas/inmunología , Antígenos de Histocompatibilidad Clase I/efectos de los fármacos , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunomodulación/inmunología , Interferón gamma/metabolismo , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Oxaliplatino/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Talidomida/análogos & derivados , Talidomida/farmacología , Ácido Zoledrónico/farmacología , GemcitabinaRESUMEN
The secondary B cell (SB) antigens are polymorphic HLA-linked antigens on human B cells and macrophages that are identified by primed T cell responses but are genetically distinct from the HLA-DR, MB, and MT antigens. Serologic identification of the SB molecule, using the monoclonal antibody ILR1, now makes it possible to correlate the function of these determinants in human T cell recognition with an Ia-like molecular structure and a genetic locus that marks a new HLA subregion. Three lines of evidence indicate that the ILR1 molecule identifies an epitope on some alleles of the SB gene: (a) the polymorphism of ILR1 -reactivity in the population correlates with SB2 SB3; (b) T cell proliferative response to SB2 and SB3 are specifically inhibited by ILR1; and (c) ILR1 reactivity is exactly concordant with the expression of SB2 in a panel of HLA-deletion mutant lymphoblastoid cell line. Together with previous studies, these results indicate that the SB antigens are on Ia-like molecules. Furthermore, the serologic studies of HLA-deletion mutant cell lines demonstrate that there are two HLA regions centromeric to HLA-B controlling expression of Ia-like molecules: a region toward HLA-B that controls expression of HLA-DR, and a region toward GLO that controls expression of SB.
Asunto(s)
Linfocitos B/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Complejo Mayor de Histocompatibilidad , Anticuerpos Monoclonales , Línea Celular , Mapeo Cromosómico , Genes MHC Clase II , Antígenos HLA-D , Antígenos HLA-DR , Antígeno HLA-DR5 , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Mutación , Linfocitos T/inmunologíaRESUMEN
To determine how the alpha(1,3)fucosyltransferases Fuc-TIV and Fuc-TVII, and the selectin ligands they control may contribute to the adaptive immune response, contact hypersensitivity (CHS) was characterized in mice deficient in either or both enzymes. We find a substantial CHS deficiency in Fuc-TVII(-/-) mice, and a complete deficiency in Fuc-TIV(-/-)/Fuc-TVII(-/-) mice. These defects are not accounted for by alterations in the number or function of epidermal Langerhans cells required for cutaneous antigen processing and presentation. By contrast, defective CHS in Fuc-TVII(-/-) mice or Fuc-TIV(-/-)/Fuc-TVII(-/-) mice is attributed in part to prominent, or nearly complete deficiencies, respectively, in the complement of naive T lymphocytes available in lymph nodes for antigen-dependent activation, expansion, differentiation, and dissemination. Fuc-TVII deficiency also deletes expression of E- and P-selectin ligands by Th1 and T cytotoxic 1 (Tc1) lymphocytes, annuls T cell trafficking to inflamed cutaneous sites in vivo, and thereby controls an essential component of the efferent phase of the cutaneous immune response. These observations indicate that collaborative contributions of Fuc-TIV and Fuc-TVII to L-selectin ligand synthesis, and to lymphocyte recruitment, are requisite components of the primary cellular immune response, and assign an essential role to Fuc-TVII in control of E- and P-selectin ligand expression by Th1 and Tc1 lymphocytes.
Asunto(s)
Fucosiltransferasas/metabolismo , Ganglios Linfáticos/inmunología , Selectinas/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T/inmunología , Células TH1/inmunología , Animales , Diferenciación Celular , Polaridad Celular , Selectina E/metabolismo , Citometría de Flujo , Fucosiltransferasas/deficiencia , Fucosiltransferasas/genética , Inflamación/genética , Inflamación/fisiopatología , Células de Langerhans/citología , Células de Langerhans/fisiología , Ligandos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Selectina-P/metabolismo , Linfocitos T/citología , Linfocitos T Citotóxicos/citología , Células TH1/citologíaRESUMEN
Costimulation mediated by the CD28 receptor has been shown to play an important role in the development of a vigorous T cell immune response. Nevertheless, CD28-deficient mice can mount effective T cell-dependent immune responses. These data suggest that other costimulatory molecules may play a role in T cell activation. In a search for other costimulatory receptors on T cells, we have characterized a monoclonal antibody (mAb) that can costimulate T cells in the absence of accessory cells. Similar to CD28 antibodies, this mAb, R2/60, was found to synergize with T cell receptor engagement in inducing proliferation. Independent ligation of CD3 and the ligand recognized by R2/60 results in T cell proliferation, suggesting that the two molecules do not have to colocalize to activate the R2/60 costimulatory pathway. R2/60 does not react with CD28, and furthermore, R2/60 costimulates in a CD28-independent fashion since the mAb costimulates T cells from the CD28-deficient mice as well as wild-type mice. Expression cloning of the R2/60 antigen identified the ligand as murine CD43. Together, these data demonstrate that CD43 can serve as a receptor on T cells that can provide CD28-independent costimulation.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD , Antígenos CD28/fisiología , Activación de Linfocitos , Sialoglicoproteínas/fisiología , Subgrupos de Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD28/genética , Complejo CD3/inmunología , Leucosialina , Ligandos , Ganglios Linfáticos/citología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Mutantes , Sialoglicoproteínas/inmunología , Transducción de Señal , Organismos Libres de Patógenos Específicos , Timo/citologíaRESUMEN
Upper airway obstruction (UAO) can elicit neuromuscular responses that mitigate and/or compensate for the obstruction. It was hypothesised that flow-limited breathing elicits specific timing responses that can preserve ventilation due to increases in inspiratory duty cycle rather than respiratory rate. By altering nasal pressure during non-rapid eye movement (non-REM) sleep, similar degrees of UAO were induced in healthy males and females (n = 10 each). Inspiratory duty cycle, respiratory rate and minute ventilation were determined for each degree of UAO during non-REM sleep and compared with the baseline nonflow-limited condition. A dose-dependent increase in the inspiratory duty cycle and respiratory rate was observed in response to increasing severity of UAO. Increases in the inspiratory duty cycle, but not respiratory rate, helped to acutely maintain ventilation. Heterogeneity in these responses was associated with variable degrees of ventilatory compensation, allowing for the segregation of individuals at risk for hypoventilation during periods of inspiratory airflow limitation. Upper airway obstruction constitutes a unique load on the respiratory system. The inspiratory duty cycle, but not the respiratory rate, determine the individual's ability to compensate for inspiratory airflow limitation during sleep, and may represent a quantitative phenotype for obstructive sleep apnoea susceptibility.
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Hipoventilación/fisiopatología , Capacidad Inspiratoria/fisiología , Mecánica Respiratoria/fisiología , Sueño/fisiología , Adulto , Obstrucción de las Vías Aéreas/fisiopatología , Resistencia de las Vías Respiratorias/fisiología , Antropometría , Ritmo Circadiano , Femenino , Humanos , Masculino , Valor Predictivo de las Pruebas , Pruebas de Función Respiratoria , Apnea Obstructiva del Sueño/fisiopatología , VigiliaRESUMEN
The glycoprotein hormones lutropin (LH) and follitropin (FSH), which have common alpha-subunits but hormone-specific beta-subunits, are both synthesized in the gonadotroph. However, they bear Asn-linked oligosaccharides that differ in structure. Those on LH terminate with the sequence SO4-4GalNAc beta 1----4GlcNAc beta 1----2Man alpha, whereas those on FSH terminate with the sequence sialic acid alpha-Gal beta 1----4GlcNAc beta 1----2Man alpha. A GalNAc-transferase was identified in bovine pituitary membranes that recognizes features of the alpha-subunit peptide and adds GalNAc to its oligosaccharides with an apparent Michaelis constant of 25 micromolar. The different patterns of glycosylation for LH and FSH indicate that access to the protein recognition marker on the alpha-subunit is modulated by the associated beta-subunit. The tightly regulated synthesis of sulfated and sialylated oligosaccharides on the pituitary glycoprotein hormones suggests these oligosaccharides have an important biological role.
Asunto(s)
Hormona Folículo Estimulante/biosíntesis , Galactosiltransferasas/metabolismo , Hormona Luteinizante/biosíntesis , N-Acetilgalactosaminiltransferasas , Hipófisis/enzimología , Acetilgalactosamina/metabolismo , Animales , Secuencia de Carbohidratos , Bovinos , Membrana Celular/enzimología , Hormona Folículo Estimulante/metabolismo , Hormona Folículo Estimulante de Subunidad beta , Hormonas Glicoproteicas de Subunidad alfa/metabolismo , Glicosilación , Humanos , Hormona Luteinizante/metabolismo , Datos de Secuencia Molecular , Oligosacáridos/metabolismo , Placenta/enzimología , Sulfatos/metabolismoRESUMEN
Interleukin-2 (IL-2) induces cytolytic activity and proliferation of human blood lymphocytes. Yet, prior to activation, these cells do not express IL-2 receptors recognized by monoclonal antibodies to the Tac antigen. A novel glycoprotein (IL-2R beta), identified on several lymphocytoid cell lines, has the ability to bind IL-2 alone and to associate with Tac antigen (IL-2R alpha) to form high-affinity IL-2 receptors. It is now reported that IL-2R beta is expressed on both circulating T lymphocytes and large granular lymphocytes in quantities approximately proportional to their responsiveness to IL-2. Studies of the responses of these cells to IL-2 suggest that IL-2R beta mediates the initial phase of induction of lymphokine activated killer (LAK), natural killer (NK), and proliferative activities. Subsequently, IL-2R alpha is induced and functional high-affinity IL-2 receptors are expressed.
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Células Asesinas Naturales/inmunología , Activación de Linfocitos , Linfocinas/fisiología , Receptores Inmunológicos/fisiología , Anticuerpos Monoclonales , Citotoxicidad Inmunológica , Humanos , Inmunidad Celular , Inmunidad Innata , Linfocitos/clasificación , Sustancias Macromoleculares , Receptores de Interleucina-2 , Linfocitos T/inmunologíaRESUMEN
The site of adenosine 3',5'-monophosphate-mediated fluid and electrolyte secretion across mammalian large intestine was found to be the crypts of Lieberkühn by means of two techniques. First, the formation of fluid droplets was visualized on the oil-covered mucosal surface directly over crypt duct openings when secretion was stimulated. Second, microelectrode impalement of individual surface and crypt cells revealed that only crypts cells produced a pattern of secretagogue induced alterations in membrane potential and resistance that was characteristic of secretory epithelia.
Asunto(s)
Intestino Grueso/fisiología , Equilibrio Hidroelectrolítico , Amilorida/farmacología , Animales , Cloruros/metabolismo , AMP Cíclico/fisiología , Técnicas In Vitro , Intestino Grueso/anatomía & histología , Prostaglandinas E/farmacología , Conejos , Tasa de Secreción/efectos de los fármacos , Sodio/fisiologíaRESUMEN
In the course of a controlled study to evaluate different forms of immunotherapy for subjects with insect-sting hypersensitivity, we observed 11 subjects who had systemic cutaneous urticarial reactions and 3 subjects who experienced systemic anaphylaxis. With the exception of tachycardia, there were no cardiopulmonary changes in the subjects with urticaria, whereas the major manifestation of anaphylactic shock in the other three subjects was severe hypotension that was probably secondary to peripheral vasodilation. Significant abnormalities in gas exchange developed in two subjects. In one, bronchospasm precipitated a respiratory arrest followed by endotracheal intubation with mechanical ventilation. Although plasma histamine levels were not related to the development of cutaneous reactions, the plasma histamine levels correlated with the severity and duration of the cardiopulmonary changes observed during anaphylactic shock. The two subjects with the most severe shock showed evidence of intravascular coagulation characterized by a diminution of Factor V, Factor VIII, fibrinogen, and high molecular weight kininogen, as well as changes in components of the complement system. Standard therapy with epinephrine and fluids, usually recommended for the treatment of systemic anaphylaxis, did not immediately reverse either the hemodynamic or the respiratory abnormalities in the two subjects with the most severe anaphylactic shock. Hemodynamic recovery was gradual and did not seem directly related to any specific therapeutic intervention.
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Anafilaxia/etiología , Histamina/sangre , Hipotensión/etiología , Mordeduras y Picaduras de Insectos/complicaciones , Anafilaxia/tratamiento farmacológico , Proteínas del Sistema Complemento/análisis , Epinefrina/uso terapéutico , Factor V/análisis , Factor VIII/análisis , Fibrinógeno/análisis , Volumen Espiratorio Forzado , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Hipotensión/tratamiento farmacológico , Inmunoterapia , Quininógenos/análisis , Urticaria/etiologíaRESUMEN
BACKGROUND/OBJECTIVE: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma carboxypeptidase that renders a fibrin-containing thrombus less sensitive to lysis. In the present study, we describe the development of a murine model of vena cava thrombosis and its use to characterize the antithrombotic activity of potato carboxypeptidase inhibitor (PCI) of TAFIa (activated TAFI) in mice. METHODS/RESULTS: Vena cava thrombosis was induced by various concentrations of FeCl(3) in C57BL/6 mice. A relatively mild stimulus (3.5% FeCl(3)) induced thrombosis that was consistent and sensitive to reference antithrombotic agents such as clopidogrel and heparin. Dose-response studies identified a PCI dose (5 mg kg(-1) bolus plus 5 mg kg(-1) h(-1), i.v.) that produced a maximum 45% decrease in vena cava thrombus mass as assessed by protein content (n = 8, P < 0.01 compared to vehicle) in the 3.5% FeCl(3)-induced model without exogenous tissue plasminogen activator administration. In contrast, PCI had no effect on 3.5% FeCl(3)-induced carotid artery thrombosis in mice. In a tail transection bleeding model, the 5 mg kg(-1) bolus plus 5 mg kg(-1) h(-1) dose of PCI increased tail-bleeding time up to 3.5 times control (n = 8, P < 0.05). The ex vivo activity of antithrombotic doses of PCI was also demonstrated by the enhanced lysis of whole blood clots formed in a thrombelastograph with the addition of a sub-threshold concentration of tPA. CONCLUSION: These studies provide evidence for a role of TAFIa in venous thrombosis in mice, and describe an optimized vena cava injury model appropriate for the evaluation of antithrombotic drugs and the characterization of novel therapeutic targets.
Asunto(s)
Trombosis de la Vena/tratamiento farmacológico , Animales , Carboxipeptidasa B2/sangre , Carboxipeptidasas/antagonistas & inhibidores , Trombosis de las Arterias Carótidas/sangre , Trombosis de las Arterias Carótidas/inducido químicamente , Trombosis de las Arterias Carótidas/tratamiento farmacológico , Cloruros , Modelos Animales de Enfermedad , Compuestos Férricos/toxicidad , Fibrinolíticos/administración & dosificación , Fibrinolíticos/farmacología , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Plantas/administración & dosificación , Proteínas de Plantas/farmacología , Inhibidores de Proteasas/administración & dosificación , Inhibidores de Proteasas/farmacología , Solanum tuberosum , Terapia Trombolítica , Venas Cavas , Trombosis de la Vena/sangre , Trombosis de la Vena/inducido químicamenteRESUMEN
BACKGROUND: Increased plasma levels of coagulation factor (F) XI are a risk factor for venous thrombosis. OBJECTIVE: To further explore the relationship between FXI and venous thrombosis, we evaluated FXI-deficient and wild-type mice in a ferric chloride (FeCl(3))-induced vena cava thrombosis model. METHODS AND RESULTS: Thrombosis was induced by 3-min topical application of filter papers containing increasing concentrations of FeCl(3) and the thrombus was measured at 30 min. In contrast to wild-type mice, FXI-deficient mice failed to form a thrombus with 5% FeCl(3,) and were partially protected against 7.5% and 10% FeCl(3,) respectively. The protective effect was substantially stronger than a high dose of heparin (1,000 units kg(-1), i.v.), clopidogrel (30 mg kg(-1), p.o.) or argatroban (30 mg kg(-1), i.p.). These antithrombotic agents resulted in off-scale bleeding in a tail bleeding time assay, whereas the bleeding time of FXI-deficient mice was unchanged compared to wild-type mice. In addition to its known effect on the coagulation cascade, enhanced clot lysis was demonstrated in FXI-deficient mouse and human plasma compared to those supplemented with FXIa. CONCLUSION: Given the strong antithrombotic efficacy (possibly contributed by strong anticoagulant activity associated with increased fibrinolytic activity) and mild bleeding diathesis associated with FXI deficiency, therapeutic inhibition of FXI may be a reasonable therapeutic strategy to treat or prevent venous thrombosis.
Asunto(s)
Deficiencia del Factor XI/complicaciones , Compuestos Férricos/farmacología , Venas Cavas/patología , Trombosis de la Vena/prevención & control , Animales , Cloruros , Modelos Animales de Enfermedad , Fibrinólisis , Fibrinolíticos/farmacología , Ratones , Trombosis de la Vena/inducido químicamenteRESUMEN
Purpose Chemotherapy for advanced pancreatic cancer has limited efficacy due to the difficultly of treating established tumours and the evolution of tumour resistance. Chemotherapies for pancreatic cancer are typically studied for their cytotoxic properties rather than for their ability to increase the immunogenicity of pancreatic tumour cells. In this study Gemcitabine in combination with immune modulatory chemotherapies Oxaliplatin, zoledronic acid and pomalidomide was studied to determine how combination therapy alters the immunogenicity of pancreatic tumour cell lines and subsequent T-cell responses. Methods Pancreatic tumour cell lines were stimulated with the chemotherapeutic agents and markers of immune recognition were assessed. The effect of chemotherapeutic agents on DC function was measured using uptake of CFSE-stained PANC-1 cells, changes in markers of maturation and their ability to activate CD8+ T-cells. The effect of chemotherapeutic agents on T-cell priming prior to activation using anti-CD3 and anti-CD28 antibodies was determined by measuring IFN-γ expression and Annexin V staining using flow cytometry. Results These agents demonstrate both additive and inhibitory properties on a range of markers of immunogenicity. Gemcitabine was notable for its ability to induce the upregulation of human leukocyte antigen and checkpoints on pancreatic tumour cell lines whilst inhibiting T-cell activation. Pomalidomide demonstrated immune modulatory properties on dendritic cells and T-cells, even in the presence of gemcitabine. Discussion These data highlight the complex interactions of different agents in the modulation of tumour immunogenicity and immune cell activation and emphasise the complexity in rationally designing chemo immunogenic combinations for use with immunotherapy (AU)
Asunto(s)
Humanos , Neoplasias Pancreáticas/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica , Antineoplásicos Inmunológicos , Citometría de Flujo , Línea Celular TumoralRESUMEN
Obese leptin-deficient (ob/ob) mice demonstrate defects in upper airway structural and neuromuscular control. We hypothesized that these defects predispose to upper airway obstruction during sleep, and improve with leptin administration. High-fidelity polysomnographic recordings were conducted to characterize sleep and breathing patterns in conscious, unrestrained ob/ob mice (23 wk, 67.2 ± 4.1 g, n = 13). In a parallel-arm crossover study, we compared responses to subcutaneous leptin (1 µg/h) vs. vehicle on respiratory parameters during NREM and REM sleep. Upper airway obstruction was defined by the presence of inspiratory airflow limitation (IFL), as characterized by an early inspiratory plateau in airflow at a maximum level (VÌImax) with increasing effort. The severity of upper airway obstruction (VÌImax) was assessed along with minute ventilation (VÌE), tidal volume (VT), respiratory rate (RR), inspiratory duty cycle, and mean inspiratory flow at each time point. IFL occurred more frequently in REM sleep (37.6 ± 0.2% vs. 1.1 ± 0.0% in NREM sleep, P < 0.001), and leptin did not alter its frequency. VÌImax (3.7 ± 1.1 vs. 2.7 ± 0.8 ml/s, P < 0.001) and VÌE increased (55.4 ± 22.0 vs. 39.8 ± 16.4 ml/min, P < 0.001) with leptin vs. vehicle administration. The increase in VÌE was due to a significant increase in VT (0.20 ± 0.06 vs. 0.16 ± 0.05 ml, P < 0.01) rather than RR. Increases in VÌE were attributable to increases in mean inspiratory flow (2.5 ± 0.8 vs. 1.8 ± 0.6 ml/s, P < 0.001) rather than inspiratory duty cycle. Similar increases in VÌE and its components were observed in non-flow-limited breaths during NREM and REM sleep. These responses suggest that leptin stabilized pharyngeal patency and increased drive to both the upper airway and diaphragm during sleep.
Asunto(s)
Leptina/deficiencia , Leptina/uso terapéutico , Obesidad/genética , Síndromes de la Apnea del Sueño/tratamiento farmacológico , Síndromes de la Apnea del Sueño/genética , Animales , Estudios Cruzados , Diafragma/fisiopatología , Leptina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Faringe/fisiopatología , Polisomnografía , Pruebas de Función Respiratoria , Mecánica Respiratoria , Sueño , Sueño REMRESUMEN
Some enteric strains of Escherichia coli release a heat-stable enterotoxin which, in contrast to cholera and heat-labile E. coli enterotoxins, stimulates guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2). We have examined the tissue spcificity of its action and the relation of its action to those of the 8-bromo analogues of cyclic GMP and cyclic AMP. Heat-stable enterotoxin stimulated guanylate cyclase activity and increased cyclic GMP concentration throughout the small and large intestine. It increased transepithelial electric potential difference and short-circuit current in the jejunum, ileum and caecum but not in the duodenum or distal colon. This pattern of electrical responses was mimicked by 8-bromo-cyclic GMP. However, 8-bromo-cyclic AMP produced an electrical response in all intestinal segments. The enterotoxin failed to stimulate guanylate cyclase inliver, lung, pancreas or gastric antral mucosa. In the intestines, it stimulated only the particulate and not the soluble form of the enzyme. Preincubation of the toxin with intestinal membranes did not render it capable of stimulating pancreatic guanylate cyclase. Cytosol factors did not enhance the toxin's stimulation of intestinal guanylate cyclase. This study supports the role of cyclic GMP as intracellular mediator for heat-stable enterotoxin and suggests that the toxin affects a membrane-mediated mechanism for guanylate cyclase activation that is unique to the intestines.
Asunto(s)
GMP Cíclico/metabolismo , Enterotoxinas/farmacología , Guanilato Ciclasa/metabolismo , Intestinos/efectos de los fármacos , Animales , Ciego/efectos de los fármacos , Colon/efectos de los fármacos , Citosol/fisiología , Duodeno/efectos de los fármacos , Escherichia coli/metabolismo , Potenciales Evocados , Íleon/efectos de los fármacos , Técnicas In Vitro , Yeyuno/efectos de los fármacos , Masculino , Microvellosidades/efectos de los fármacos , ConejosRESUMEN
NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) has been reported to block Cl- channels in isolated rabbit nephrons with high potency (IC50 = 80 nM). The effects of this compound on Cl(-)-mediated transport processes in intestinal tissues have been studied using agonist-stimulated short-circuit current (T84) in Ussing chamber experiments and 36Cl- fluxes in monolayers of a colonic cell line (T84). NPPB inhibited PGE1-stimulated Isc in rabbit distal colon and ileum at concentrations in the range 20 to 100 microM. However, NPPB at the same concentrations also inhibited glucose-stimulated Isc in rabbit ileum, suggesting that its effects were not restricted to those on Cl- transport. Consistent with this, exposure of rabbit distal colon to 100 microM NPPB was found to reduce endogenous ATP levels by 69%, implying that, at these concentrations, NPPB could impair active transport processes by an effect on cellular energy metabolism. Clear evidence for a direct effect of NPPB on epithelial chloride channels was found in studies on Cl- fluxes in T84 cell monolayers. NPPB inhibited VIP-stimulated Cl- uptake into T84 cells with an IC50 of 414 microM. NPPB (1 mM) also inhibited Cl- efflux from pre-loaded cells confirming its effect as a weak Cl- channel blocker in this system.
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Cloruros/metabolismo , Colon/metabolismo , Neoplasias del Colon/metabolismo , Íleon/metabolismo , Absorción Intestinal/efectos de los fármacos , Nitrobenzoatos/farmacología , Adenocarcinoma/metabolismo , Animales , Transporte Biológico Activo , Línea Celular , Canales de Cloruro , Humanos , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Masculino , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , ConejosRESUMEN
Adriamycin increases (a) the rate of agglutination of Sarcoma 180 cells by concanavalin A after brief exposure of 2-3 h and (b) membrane fluidity as measured by ESR within 30 min of exposure at concentrations of the anthracycline of 10(-7)-10(-5) M. The effect of adriamycin on agglutination is not due to an increase in the number of surface receptors for concanavalin A, since the extent of binding of the lectin is not altered by adriamycin and no change occurs in the rate of occupancy of the concanavalin A binding sites by the lectin in cells treated with the antibiotic. The order parameter, a measurement of membrane fluidity, decreases in cells exposed to adriamycin and is dose-related. The results indicate that adriamycin can induce changes in the surface membrane of Sarcoma 180 cells within a brief period of exposure to a low but cytotoxic level of this agent.
Asunto(s)
Doxorrubicina/farmacología , Fluidez de la Membrana/efectos de los fármacos , Sarcoma 180/metabolismo , Aglutinación , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Concanavalina A/metabolismo , Cinética , Ratones , Receptores de Concanavalina A/metabolismoRESUMEN
Small organic molecules, like quaternary ammonium compounds, have long been used to probe both the permeation and gating of voltage-dependent K+ channels. For most K+ channels, intracellularly applied quaternary ammonium (QA) compounds such as tetraethylammonium (TEA) and decyltriethylammonium (C10) behave primarily as open channel blockers: they can enter the channel only when it is open, and they must dissociate before the channel can close. In some cases, it is possible to force the channel to close with a QA blocker still bound, with the result that the blocker is "trapped." Armstrong (J. Gen. Physiol. 58:413-437) found that at very negative voltages, squid axon K+ channels exhibited a slow phase of recovery from QA blockade consistent with such trapping. In our studies on the cloned Shaker channel, we find that wild-type channels can trap neither TEA nor C10, but channels with a point mutation in S6 can trap either compound very efficiently. The trapping occurs with very little change in the energetics of channel gating, suggesting that in these channels the gate may function as a trap door or hinged lid that occludes access from the intracellular solution to the blocker site and to the narrow ion-selective pore.
Asunto(s)
Activación del Canal Iónico/fisiología , Canales de Potasio/metabolismo , Animales , Línea Celular , Electrofisiología , Metabolismo Energético , Humanos , Activación del Canal Iónico/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Canales de Potasio/efectos de los fármacos , Canales de Potasio/genética , Proteínas Recombinantes/metabolismo , Compuestos de Tetraetilamonio/metabolismoRESUMEN
Factor XI (FXI) and factor IX (FIX) are zymogens of plasma serine proteases required for normal hemostasis. The purpose of this work was to evaluate FXI and FIX as potential therapeutic targets by means of a refined ferric chloride (FeCl(3))-induced arterial injury model in factor-deficient mice. Various concentrations of FeCl(3) were used to establish the arterial thrombosis model in C57BL/6 mice. Carotid artery blood flow was completely blocked within 10 min in C57BL/6 mice by application of 3.5% FeCl(3). In contrast, FXI- and FIX-deficient mice were fully protected from occlusion induced by 5% FeCl(3), and were partially protected against the effect of 7.5% FeCl(3). The protective effect was comparable to very high doses of heparin (1000 units kg(-1)) and substantially more effective than aspirin. While FXI and FIX deficiencies were indistinguishable in the carotid artery injury model, there was a marked difference in a tail-bleeding-time assay. FXI-deficient and wild-type mice have similar bleeding times, while FIX deficiency was associated with severely prolonged bleeding times (>5.8-fold increase, P < 0.01). Given the relatively mild bleeding diathesis associated with FXI deficiency, therapeutic inhibition of FXI may be a reasonable strategy for treating or preventing thrombus formation.
Asunto(s)
Arterias Carótidas/efectos de los fármacos , Factor IX/fisiología , Deficiencia del Factor XI/patología , Factor XI/fisiología , Compuestos Férricos/farmacología , Hemofilia B/patología , Animales , Arterias/efectos de los fármacos , Arterias/lesiones , Aspirina/farmacología , Tiempo de Sangría , Velocidad del Flujo Sanguíneo , Enfermedades de las Arterias Carótidas/patología , Cloruros , Relación Dosis-Respuesta a Droga , Genotipo , Heparina/química , Heparina/farmacología , Homocigoto , Ratones , Ratones Endogámicos C57BL , Agregación Plaquetaria , Flujo Sanguíneo Regional/efectos de los fármacos , Trombosis/patología , Trombosis/terapia , Factores de TiempoRESUMEN
We determined the renal and depressor activities of 10, 50, and 100 pmol/kg per minute i.v. of human atrial natriuretic peptide-(99-126) in conscious one-kidney, one clip dogs with chronic hypertension and modest renal dysfunction, as indicated by mild proteinuria. Atrial natriuretic peptide increased fractional sodium excretion by 0.009 +/- 0.002, 0.042 +/- 0.005, and 0.049 +/- 0.007, respectively; urinary excretion of atrial natriuretic peptide by -0.4 +/- 0.8, 3.3 +/- 1.4, and 15.8 +/- 7.4 fmol/min; and cGMP excretion by 0.65 +/- 0.06, 1.65 +/- 0.08, and 4.88 +/- 0.85 nmol/min in one-kidney shams. The changes in fractional sodium excretion were significantly attenuated in the hypertensive dogs (0.005 +/- 0.002, 0.018 +/- 0.003, and 0.022 +/- 0.004, respectively) despite exaggerated increases in atrial natriuretic peptide excretion (3.3 +/- 1.6, 22.0 +/- 5.0, and 46.6 +/- 10.8 fmol/min) and cGMP excretion (0.96 +/- 0.47, 4.51 +/- 1.27, and 7.06 +/- 1.38 nmol/min). The slope of the line relating urinary atrial natriuretic peptide to cGMP was significantly suppressed in the hypertensive dogs, suggesting a downregulation of the guanylate cyclase-linked receptors. The slope of the relationship between cGMP excretion and the natriuretic response was also depressed in the hypertensive dogs, indicating possible impairment of cGMP signal transduction. The differences between sham and hypertensive dogs were diminished when urinary levels of atrial natriuretic peptide were maximized by prior treatment with SQ 28603, an inhibitor of neutral endopeptidase EC 3.4.24.11. Atrial natriuretic peptide caused comparable decreases in mean arterial pressure and increases in glomerular filtration rate in sham and hypertensive dogs, suggesting similar vascular reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)