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1.
Bioanalysis ; 9(19): 1493-1508, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-29056059

RESUMEN

The analysis of biomarkers by ligand-binding assays offers significant challenges compared with the bioanalysis of small and large molecule drugs. The presence of endogenous analyte is a commonly cited issue. Also the sourcing and application of appropriate calibration or reference standards can present many issues. One of the main challenges is ensuring the continuity and validity of biomarker data when the source or lot number of calibration standard changes within or between studies. Several strategies exist in attempting to deal with this and standardize the biomarker data through the assay life or looking for ways to compare and normalize biomarker data. In this manuscript, the European Bioanalysis Forum view on dealing with calibration standards in biomarker assays is described.


Asunto(s)
Bioensayo/normas , Biomarcadores/metabolismo , Calibración , Europa (Continente) , Femenino , Humanos , Ligandos , Masculino , Estándares de Referencia
2.
Int J Infect Dis ; 46: 71-8, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-26923083

RESUMEN

OBJECTIVE: To evaluate non-inferiority of three doses of Quinvaxem in a compact prefilled auto-disabled (cPAD) injection system versus Quinvaxem in a single-dose vial administered with conventional syringe in terms of seroconversion/seroprotection rates for all antibodies (anti-hepatitis B (HB), anti-Haemophilus influenzae type b polyribosylribitol phosphate (Hib PRP), anti-diphtheria, anti-tetanus, anti-Bordetella pertussis) at 1 month after primary vaccination. METHODS: Four hundred healthy infants aged 42-65 days were randomized (1:1) to receive Quinvaxem in cPAD or single-dose vial at 6, 10, and 12 weeks of age. Blood samples were collected before vaccination and at 1 month after the third dose to determine seroconversion/seroprotection rates. Safety was assessed from solicited and unsolicited adverse events and serious adverse events (SAEs). RESULTS: Of the 400 infants randomized, 395 (98.8%) received all three vaccine doses. In the cPAD vs. single-dose vial groups, seroprotection rates against Hib PRP (both 98.5%), HB (92.9% vs. 93.4%), diphtheria (100% vs. 99%), and tetanus toxoids (both 100%), and seroconversion against B. pertussis (95.4% vs. 97%) were ≥92% at 1 month after the third vaccination (lower limits of 95% confidence intervals simultaneously greater than -10%). Geometric mean concentrations exceeded seroprotection/seroconversion thresholds by large margins. The incidences of induration and erythema were comparable between the groups; tenderness was slightly higher in the cPAD group (85.5% vs. 76.5%). No vaccine-related SAEs occurred. CONCLUSIONS: Quinvaxem in cPAD was non-inferior to single-dose vial with respect to seroprotection/seroconversion rates for all antibodies. Both presentations were well-tolerated.


Asunto(s)
Vacuna contra Difteria, Tétanos y Tos Ferina/inmunología , Difteria/prevención & control , Infecciones por Haemophilus/prevención & control , Vacunas contra Haemophilus/inmunología , Vacunas contra Hepatitis B/inmunología , Hepatitis B/prevención & control , Tétanos/prevención & control , Tos Ferina/prevención & control , Difteria/inmunología , Vacuna contra Difteria, Tétanos y Tos Ferina/administración & dosificación , Femenino , Infecciones por Haemophilus/inmunología , Vacunas contra Haemophilus/administración & dosificación , Hepatitis B/inmunología , Vacunas contra Hepatitis B/administración & dosificación , Humanos , Lactante , Masculino , Tétanos/inmunología , Vacunación/instrumentación , Vacunación/métodos , Vacunas Combinadas/administración & dosificación , Vacunas Combinadas/inmunología , Vacunas Conjugadas/administración & dosificación , Vacunas Conjugadas/inmunología , Tos Ferina/inmunología
3.
Hum Vaccin Immunother ; 10(7): 2089-97, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25424821

RESUMEN

As India is transitioning from high to intermediate hepatitis A endemicity, the need for hepatitis A vaccination programs increases. This study investigated the immunogenicity and safety of a virosomal hepatitis A vaccine (HAVpur Junior) compared with an aluminum-adsorbed hepatitis A vaccine (Havrix 720 Junior) in Indian children. Healthy children aged 18-47 months, stratified by age, were randomized to either HAVpur Junior or Havrix 720 Junior. The first dose of vaccine was administered on Day 1 and the second (booster) dose 6 months later. Antibodies against hepatitis A virus (HAV) were measured using a microparticle enzyme immunoassay. The primary objective assessed non-inferiority of HAVpur Junior to Havrix 720 Junior in terms of seroprotection rates (≥ 10 mIU/mL anti-HAV antibodies) at 1 month after the first vaccination. Non-inferiority was demonstrated if the lower limit of the 90% confidence interval of the group difference was greater than -10%. Local and systemic adverse events were recorded. The seroprotection rate at 1 month was 95.9% in the HAVpur Junior group and 96.6% in the Havrix 720 Junior group. As the lower limit of the 90% confidence interval of the group difference was greater than -10% (-4.7), non-inferiority of HAVpur Junior to Havrix 720 Junior was established. The overall incidence of adverse events (solicited and unsolicited) after each vaccination was similar in both groups. In conclusion, the aluminum-free virosomal vaccine HAVpur Junior induced a similar immune response to Havrix 720 Junior in healthy Indian children aged 18 to 47 months. Both vaccines were well tolerated. The study shows that the low-dose virosomal HAV vaccine is consistently efficacious and well tolerated in children of all age groups and is suitable for inclusion into Indian childhood vaccination schedules.


Asunto(s)
Anticuerpos de Hepatitis A/sangre , Vacunas contra la Hepatitis A/efectos adversos , Vacunas contra la Hepatitis A/inmunología , Hepatitis A/prevención & control , Adyuvantes Inmunológicos/administración & dosificación , Hidróxido de Aluminio/administración & dosificación , Preescolar , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/epidemiología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/patología , Femenino , Voluntarios Sanos , Vacunas contra la Hepatitis A/administración & dosificación , Humanos , Técnicas para Inmunoenzimas , India , Lactante , Masculino , Vacunas de Virosoma/administración & dosificación , Vacunas de Virosoma/efectos adversos , Vacunas de Virosoma/inmunología
4.
Bioanalysis ; 6(10): 1385-93, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24958122

RESUMEN

BACKGROUND: As most vaccines exert their protective capacity by eliciting pathogen-specific antibodies, antibody assays assessing immunogenicity of vaccines in development should be well characterized. Part of the validation of immunogenicity assays for vaccines is the study of stability of antibodies in serum. Materials & methods: Stability of antibodies in human serum was assessed by circumsporozoite-binding IgG ELISA designed for assessing the immunogenicity of a malaria vaccine under development, adenovirus neutralization assay, designed to assess neutralizing antibodies against adenovirus and commercially available test kits for hepatitis A and B. RESULTS: Stability studies indicated stability of serum-binding IgG antibodies and serum-neutralizing antibodies in: long-term storage below -65°C and -20°C; short-term storage; multiple freeze/thaw rounds; during shipment; and during heat inactivation. CONCLUSION: RESULTS have shown the stability of both binding and functional polyclonal antibodies in human serum under stable storage and common usage circumstances.


Asunto(s)
Anticuerpos Neutralizantes/sangre , Ensayo de Inmunoadsorción Enzimática , Vacunas/inmunología , Adenoviridae/inmunología , Congelación , Virus de la Hepatitis A/inmunología , Virus de la Hepatitis B/inmunología , Humanos , Estabilidad Proteica , Proteínas Protozoarias/inmunología , Análisis de Regresión , Temperatura , Factores de Tiempo
5.
Bioanalysis ; 4(15): 1883-94, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22943619

RESUMEN

Biomarkers have become increasingly important in drug development and many bioanalysts are getting involved. Consequently, different views on how to approach the bioanalysis of biomarkers have been published or are being developed. The European Bioanalysis Forum has intensively discussed this topic since 2010 and is ready with their recommendation on method establishment and bioanalysis of biomarkers. Acknowledging that the challenges step outside the bioanalytical laboratory is a cornerstone of our recommendation. The importance of integrating all scientific aspects, from purely analytical aspects, all the way to understanding the biology and effects of the biomarker, prior to embarking on method establishment or sample analysis, cannot be underestimated. Close and iterative interactions with the teams requesting the data is imperative to develop a bioanalytical strategy that combines science, analytical performance and regulations. The European Bioanalysis Forum developed a straightforward decision tree to help the scientific community in developing a bioanalytical strategy for any biomarker in drug development.


Asunto(s)
Biomarcadores Farmacológicos/análisis , Descubrimiento de Drogas/normas , Animales , Biomarcadores Farmacológicos/metabolismo , Europa (Continente) , Estudios de Evaluación como Asunto , Regulación Gubernamental , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
6.
Nephron Exp Nephrol ; 118(4): e79-86, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21311198

RESUMEN

AIM: To study whether microvascular leukocyte accumulation after rat renal ischemia and reperfusion (IR) is decreased by Rho kinase inhibition, independently of effects upon nitric oxide (NO) and renal blood flow. METHODS: Male Wistar rats were subjected to 60 min of ischemia by bilateral clamping and 60 min of reperfusion of the renal arteries, or a sham procedure. Haemodynamics were monitored and microsphere blood flow to the kidneys was measured. The infusion of the Rho kinase inhibitor (Y27632) was commenced before clamping and IR. The NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), was administered after the start of reperfusion whilst the dopamine-1 receptor agonist fenoldopam, a renal vasodilator, was infused during the reperfusion period. Digital imaging microscopy analysis of cryosections was done to determine leukocyte accumulation and vasodilator-stimulated phosphoprotein serine 239 phosphorylation (p-VASP ser 239), a marker of endothelial NO. RESULTS: Leukocytes (60-70% neutrophils) accumulated within blood vessels in the corticomedullary junction and medulla of the kidney. Leukocyte accumulation was markedly reduced by the Rho kinase inhibitor but not by fenoldopam. However, both drugs improved renal blood flow and microvascular expression of p-VASP ser 239 in the corticomedullary junction and medulla, which were decreased following IR. L-NAME treatment of IR animals pretreated with the Rho kinase inhibitor reduced blood flow and p-VASP ser 239 expression and increased leukocyte accumulation. CONCLUSION: Early microvascular leukocyte accumulation in the corticomedullary junction and medulla of the rat kidney after IR is ameliorated by Rho kinase inhibition. This effect is partly independent upon attenuation of decreased NO and renal blood flow.


Asunto(s)
Riñón/enzimología , Leucocitos/enzimología , Microcirculación/fisiología , Óxido Nítrico/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Daño por Reperfusión/enzimología , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/fisiología , Amidas/farmacología , Amidas/uso terapéutico , Animales , Riñón/irrigación sanguínea , Leucocitos/efectos de los fármacos , Leucocitos/patología , Masculino , Microcirculación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/farmacología , Piridinas/uso terapéutico , Ratas , Ratas Wistar , Circulación Renal/efectos de los fármacos , Circulación Renal/fisiología , Daño por Reperfusión/tratamiento farmacológico
7.
J Mol Cell Cardiol ; 50(3): 487-99, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21156182

RESUMEN

Previously we showed that left ventricular (LV) responsiveness to exercise-induced increases in noradrenaline was blunted in pigs with a recent myocardial infarction (MI) [van der Velden et al. Circ Res. 2004], consistent with perturbed ß-adrenergic receptor (ß-AR) signaling. Here we tested the hypothesis that abnormalities at the myofilament level underlie impaired LV responsiveness to catecholamines in MI. Myofilament function and protein composition were studied in remote LV biopsies taken at baseline and during dobutamine stimulation 3 weeks after MI or sham. Single permeabilized cardiomyocytes demonstrated reduced maximal force (F(max)) and higher Ca(2+)-sensitivity in MI compared to sham. F(max) did not change during dobutamine infusion in sham, but markedly increased in MI. Moreover, the dobutamine-induced decrease in Ca(2+)-sensitivity was significantly larger in MI than sham. Baseline phosphorylation assessed by phosphostaining of ß-AR target proteins myosin binding protein C (cMyBP-C) and troponin I (cTnI) in MI and sham was the same. However, the dobutamine-induced increase in overall cTnI phosphorylation and cTnI phosphorylation at protein kinase A (PKA)-sites (Ser23/24) was less in MI compared to sham. In contrast, the dobutamine-induced phosphorylation of cMyBP-C at Ser282 was preserved in MI, and coincided with increased autophosphorylation (at Thr282) of the cytosolic Ca(2+)-dependent calmodulin kinase II (CaMKII-δC). In conclusion, in post-infarct remodeled myocardium myofilament responsiveness to dobutamine is significantly enhanced despite the lower increase in PKA-mediated phosphorylation of cTnI. The increased myofilament responsiveness in MI may depend on the preserved cMyBP-C phosphorylation possibly resulting from increased CaMKII-δC activity and may help to maintain proper diastolic performance during exercise.


Asunto(s)
Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Agonistas de Receptores Adrenérgicos beta 1/farmacología , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Receptores Adrenérgicos beta/metabolismo , Remodelación Ventricular/efectos de los fármacos , Citoesqueleto de Actina/patología , Animales , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteínas Portadoras/metabolismo , Catecolaminas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Dobutamina/farmacología , Femenino , Ventrículos Cardíacos/metabolismo , Masculino , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/fisiología , Fosforilación/fisiología , Porcinos , Troponina I/metabolismo , Remodelación Ventricular/fisiología
8.
Nephron Exp Nephrol ; 114(1): e1-6, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-19816046

RESUMEN

BACKGROUND/AIMS: Decreased endothelium-dependent vasodilation and blood flow in renal ischemia-reperfusion (IR) may result in part from rho-kinase activation, and cyclooxygenase (COX) activation, and resultant reactive oxygen species (ROS) may be involved. METHODS: We tested this hypothesis in male Wistar rats, subjected to 60 min of bilateral clamping of the renal arteries and 60 min of reperfusion or a sham procedure, and treated by the rho-kinase inhibitor Y27632 (1 mg/kg) and/or the nonspecific COX inhibitor diclofenac (10 mg/kg). Renal blood flow was measured by fluorescent microspheres, and ROS in the arterial endothelium was quantified by dihydroethidium staining. Endothelium-dependent vasodilation was determined by an acetylcholine concentration-response curve in the presence or absence of diclofenac (10 microM). RESULTS: Y27632 increased renal blood flow and reduced ROS in vivo, and improved endothelium-dependent vasodilation in vitro, following IR with or without diclofenac. Following IR, diclofenac had no effect on renal blood flow and ROS in vivo, but improved endothelium-dependent vasodilation in vitro. CONCLUSION: Activation of rho-kinase impairs endothelium-dependent vasodilation and perfusion following renal IR, independently of COX and resultant ROS. In contrast, the vasodilatory effect of rho-kinase inhibition may be partly mediated by decreasing ROS, unrelated to COX and resultant vasoconstricting prostanoids.


Asunto(s)
Endotelio Vascular/metabolismo , Enfermedades Renales/metabolismo , Riñón/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/metabolismo , Quinasas Asociadas a rho/metabolismo , Animales , Velocidad del Flujo Sanguíneo , Riñón/irrigación sanguínea , Masculino , Ratas , Ratas Wistar , Transducción de Señal , Vasodilatación
9.
Circ Heart Fail ; 2(3): 233-42, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19808345

RESUMEN

BACKGROUND: Myofilament contractility of individual cardiomyocytes is depressed in remote noninfarcted myocardium and contributes to global left ventricular pump dysfunction after myocardial infarction (MI). Here, we investigated whether beta-blocker therapy could restore myofilament contractility. METHODS AND RESULTS: In pigs with a MI induced by ligation of the left circumflex coronary artery, beta-blocker therapy (bisoprolol, MI+beta) was initiated on the first day after MI. Remote left ventricular subendocardial biopsies were taken 3 weeks after sham or MI surgery. Isometric force was measured in single permeabilized cardiomyocytes. Maximal force (F(max)) was lower, whereas Ca(2+) sensitivity was higher in untreated MI compared with sham (both P<0.05). The difference in Ca(2+) sensitivity was abolished by treatment of cells with the beta-adrenergic kinase, protein kinase A. beta-blocker therapy partially reversed F(max) and Ca(2+) sensitivity to sham values and significantly reduced passive force. Despite the lower myofilament Ca(2+) sensitivity in MI+beta compared with untreated myocardium, the protein kinase A induced reduction in Ca(2+) sensitivity was largest in cardiomyocytes from myocardium treated with beta-blockers. Phosphorylation of beta-adrenergic target proteins (myosin binding protein C and troponin I) did not differ among groups, whereas myosin light chain 2 phosphorylation was reduced in MI, which coincided with increased expression of protein phosphatase 1. beta-blockade fully restored the latter alterations and significantly reduced expression of protein phosphatase 2a. CONCLUSIONS: beta-blockade reversed myofilament dysfunction and enhanced myofilament responsiveness to protein kinase A in remote myocardium after MI. These effects likely contribute to the beneficial effects of beta-blockade on global left ventricular function after MI.


Asunto(s)
Citoesqueleto de Actina/efectos de los fármacos , Antagonistas Adrenérgicos beta/farmacología , Bisoprolol/farmacología , Contracción Miocárdica/efectos de los fármacos , Infarto del Miocardio/tratamiento farmacológico , Miocitos Cardíacos/efectos de los fármacos , Disfunción Ventricular Izquierda/tratamiento farmacológico , Remodelación Ventricular/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Miosinas Cardíacas/metabolismo , Proteínas Portadoras/metabolismo , Conectina , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Femenino , Masculino , Proteínas Musculares/metabolismo , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Cadenas Ligeras de Miosina/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Proteína Fosfatasa 1/metabolismo , Proteína Fosfatasa 2/metabolismo , Recuperación de la Función , Porcinos , Factores de Tiempo , Troponina I/metabolismo , Disfunción Ventricular Izquierda/metabolismo , Disfunción Ventricular Izquierda/fisiopatología
10.
Am J Physiol Heart Circ Physiol ; 297(1): H200-7, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19395550

RESUMEN

Pulmonary arterial hypertension (PAH) is often treated with endothelin (ET) receptor blockade or phosphodiesterase-5 (PDE5) inhibition. Little is known about the specific effects on right ventricular (RV) function and metabolism. We determined the effects of single and combination treatment with Bosentan [an ET type A (ET(A))/type B (ET(B)) receptor blocker] and Sildenafil (a PDE5 inhibitor) on RV function and oxidative metabolism in monocrotaline (MCT)-induced PAH. Fourteen days after MCT injection, male Wistar rats were orally treated for 10 days with Bosentan, Sildenafil, or both. RV catheterization and echocardiography showed that MCT clearly induced PAH. This was evidenced by increased RV systolic pressure, reduced cardiac output, increased pulmonary vascular resistance (PVR), and reduced RV fractional shortening. Quantitative histochemistry showed marked RV hypertrophy and fibrosis. Monotreatment with Bosentan or Sildenafil had no effect on RV systolic pressure or cardiac function, but RV fibrosis was reduced and RV capillarization increased. Combination treatment did not reduce RV systolic pressure, but significantly lowered PVR, and normalized cardiac output, RV fractional shortening, and fibrosis. Only combination treatment increased the mitochondrial capacity of the RV, as reflected by increased succinate dehydrogenase and cytochrome c oxidase activities, associated with an activation of PKG, as indicated by increased VASP phosphorylation. Moreover, significant interactions were found between Bosentan and Sildenafil on PVR, cardiac output, RV contractility, PKG activity, and mitochondrial capacity. These data indicate that the combination of Bosentan and Sildenafil may beneficially contribute to RV adaptation in PAH, not only by reducing PVR but also by acting on the mitochondria in the heart.


Asunto(s)
Antagonistas de los Receptores de Endotelina , Hipertensión Pulmonar/fisiopatología , Mitocondrias Cardíacas/efectos de los fármacos , Inhibidores de Fosfodiesterasa 5 , Inhibidores de Fosfodiesterasa/farmacología , Arteria Pulmonar/fisiopatología , Animales , Peso Corporal/fisiología , Bosentán , Capilares/fisiología , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Difusión , Fibrosis , Hemodinámica/fisiología , Hipertensión Pulmonar/diagnóstico por imagen , Técnicas In Vitro , Masculino , Mitocondrias Cardíacas/enzimología , Contracción Miocárdica/efectos de los fármacos , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Mioglobina/metabolismo , Tamaño de los Órganos/fisiología , Consumo de Oxígeno , Piperazinas/farmacología , Purinas/farmacología , Ratas , Ratas Wistar , Citrato de Sildenafil , Sulfonamidas/farmacología , Sulfonas/farmacología , Ultrasonografía
11.
Circ Res ; 104(5): 679-87, 2009 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-19168443

RESUMEN

Contrast microbubbles in combination with ultrasound (US) are promising vehicles for local drug and gene delivery. However, the exact mechanisms behind intracellular delivery of therapeutic compounds remain to be resolved. We hypothesized that endocytosis and pore formation are involved during US and microbubble targeted delivery (UMTD) of therapeutic compounds. Therefore, primary endothelial cells were subjected to UMTD of fluorescent dextrans (4.4 to 500 kDa) using 1 MHz pulsed US with 0.22-MPa peak-negative pressure, during 30 seconds. Fluorescence microscopy showed homogeneous distribution of 4.4- and 70-kDa dextrans through the cytosol, and localization of 155- and 500-kDa dextrans in distinct vesicles after UMTD. After ATP depletion, reduced uptake of 4.4-kDa dextran and no uptake of 500-kDa dextran was observed after UMTD. Independently inhibiting clathrin- and caveolae-mediated endocytosis, as well as macropinocytosis significantly decreased intracellular delivery of 4.4- to 500-kDa dextrans. Furthermore, 3D fluorescence microscopy demonstrated dextran vesicles (500 kDa) to colocalize with caveolin-1 and especially clathrin. Finally, after UMTD of dextran (500 kDa) into rat femoral artery endothelium in vivo, dextran molecules were again localized in vesicles that partially colocalized with caveolin-1 and clathrin. Together, these data indicated uptake of molecules via endocytosis after UMTD. In addition to triggering endocytosis, UMTD also evoked transient pore formation, as demonstrated by the influx of calcium ions and cellular release of preloaded dextrans after US and microbubble exposure. In conclusion, these data demonstrate that endocytosis is a key mechanism in UMTD besides transient pore formation, with the contribution of endocytosis being dependent on molecular size.


Asunto(s)
Caveolas/metabolismo , Dextranos/metabolismo , Sistemas de Liberación de Medicamentos , Endocitosis , Células Endoteliales/metabolismo , Colorantes Fluorescentes/metabolismo , Microburbujas , Ultrasonido , Adenosina Trifosfato/metabolismo , Androstadienos/farmacología , Animales , Transporte Biológico , Bovinos , Caveolina 1/metabolismo , Células Cultivadas , Clorpromazina/farmacología , Clatrina/metabolismo , Medios de Contraste/administración & dosificación , Citosol/metabolismo , Dextranos/administración & dosificación , Dextranos/química , Endocitosis/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Arteria Femoral/metabolismo , Filipina/farmacología , Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/química , Imagenología Tridimensional , Infusiones Intravenosas , Microscopía Fluorescente , Peso Molecular , Fosfolípidos/administración & dosificación , Pinocitosis , Presión , Ratas , Ratas Wistar , Hexafluoruro de Azufre/administración & dosificación , Factores de Tiempo , Vesículas Transportadoras/metabolismo , Wortmanina
12.
Anesth Analg ; 107(4): 1276-83, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18806040

RESUMEN

INTRODUCTION: The mechanisms by which mechanical ventilation (MV) can injure remote organs, such as the kidney, remain poorly understood. We hypothesized that upregulation of systemic inflammation, as reflected by plasma interleukin-6 (IL-6) levels, in response to a lung-injurious ventilatory strategy, ultimately results in kidney dysfunction mediated by local endothelin-1 (ET-1) production and renal vasoconstriction. METHODS: Healthy, male Wistar rats were randomized to 1 of 2 MV settings (n=9 per group) and ventilated for 4 h. One group had a lung-protective setting using peak inspiratory pressure of 14 cm H2O and a positive end-expiratory pressure of 5 cm H2O; the other had a lung-injurious strategy using a peak inspiratory pressure of 20 cm H2O and positive end-expiratory pressure of 2 cm H2O. Nine randomly assigned rats served as nonventilated controls. We measured venous and arterial blood pressure and cardiac output (thermodilution method), renal blood flow (RBF) using fluorescent microspheres, and calculated creatinine clearance, urine flow, and fractional sodium excretion. Histological lung damage was assessed using hematoxylin-eosin staining. Renal ET-1 and plasma ET-1 and IL-6 concentrations were measured using enzyme-linked immunosorbent assays. RESULTS: Lung injury scores were higher after lung-injurious MV than after lung-protective ventilation or in sham controls. Lung-injurious MV resulted in significant production of renal ET-1 compared with the lung-protective and control groups. Simultaneously, RBF in the lung-injurious MV group was approximately 40% lower (P<0.05) than in the control group and 28% lower (P<0.05) than in the lung-protective group. Plasma ET-1 and IL-6 levels did not differ among the groups and systemic hemodynamics, such as cardiac output, were comparable. There was no effect on creatinine clearance, fractional sodium excretion, urine output, or kidney histology. CONCLUSIONS: Lung-injurious MV for 4 h in healthy rats results in significant production of renal ET-1 and in decreased RBF, independent of IL-6. Decreased RBF has no observable effect on kidney function or histology.


Asunto(s)
Endotelina-1/biosíntesis , Riñón/metabolismo , Lesión Pulmonar , Circulación Renal , Respiración Artificial/efectos adversos , Animales , Creatinina/metabolismo , Diuresis , Interleucina-6/sangre , Enfermedades Renales/etiología , Pulmón/patología , Masculino , Ratas , Ratas Wistar , Sodio/metabolismo
13.
Apoptosis ; 13(3): 404-12, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18165899

RESUMEN

Activation of cytoskeleton regulator Rho-kinase during ischemia-reperfusion (I/R) plays a major role in I/R injury and apoptosis. Since Rho-kinase is a negative regulator of the pro-survival phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway, we hypothesized that inhibition of Rho-kinase can prevent I/R-induced endothelial cell apoptosis by maintaining PI3-kinase/Akt activity and that protective effects of Rho-kinase inhibition are facilitated by prevention of F-actin rearrangement. Human umbilical vein endothelial cells were subjected to 1 h of simulated ischemia and 1 or 24 h of simulated reperfusion after treatment with Rho-kinase inhibitor Y-27632, PI3-kinase inhibitor wortmannin, F-actin depolymerizers cytochalasinD and latrunculinA and F-actin stabilizer jasplakinolide. Intracellular ATP levels decreased following I/R. Y-27632 treatment reduced I/R-induced apoptosis by 31% (P < 0.01) and maintained Akt activity. Both effects were blocked by co-treatment with wortmannin. Y-27632 treatment prevented the formation of F-actin bundles during I/R. Similar results were observed with cytochalasinD treatment. In contrast, latrunculinA and jasplakinolide treatment did not prevent the formation of F-actin bundles during I/R and had no effect on I/R-induced apoptosis. Apoptosis and Akt activity were inversely correlated (R (2) = 0.68, P < 0.05). In conclusion, prevention of F-actin rearrangement by Rho-kinase inhibition or by cytochalasinD treatment attenuated I/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity.


Asunto(s)
Actinas/química , Apoptosis/efectos de los fármacos , Endotelio Vascular/citología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Daño por Reperfusión , Quinasas Asociadas a rho/fisiología , Actinas/efectos de los fármacos , Amidas/farmacología , Androstadienos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Células Cultivadas , Citocalasina D/farmacología , Depsipéptidos/farmacología , Endotelio Vascular/efectos de los fármacos , Humanos , Piridinas/farmacología , Daño por Reperfusión/patología , Tiazolidinas/farmacología , Wortmanina , Quinasas Asociadas a rho/antagonistas & inhibidores
14.
Shock ; 29(2): 217-22, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17693923

RESUMEN

Acute renal failure during human sepsis is often nonoliguric. To study the underlying mechanisms, renal function was assessed in endotoxic and control male Wistar rats during and after saline loading and treatment with the selective V2 receptor agonist desmopressin. Escherichia coli endotoxin (dose, 8 mg/kg) was administered from time (t)=0 to t=60 min; saline loading (rate, 5 mL/100 g per hour) was administered from t=0 to t=120 min. Thereafter, half of each group received desmopressin (dose, 10 microg) for 1 h. The inner medullary (IM) osmolality, hematocrit, plasma, and urinary concentrations of sodium, potassium, urea, and osmolality were measured; then, aquaporin 2 (AQP2) immunohistochemistry was performed. Plasma vasopressin concentrations were measured at t=180 min. Saline loading increased urine volume in all rats. In the endotoxic group, mean arterial pressure decreased when saline loading was stopped. Despite increased hematocrit and vasopressin levels (>16 pg/mL), the endotoxin group had a low IM osmolality (mean +/- SEM, 412+/-0.04 mOsm/kg H2O) in comparison with the control group (mean +/- SEM, 1,094+/-0.17 mOsm/kg H2O) and was not able to either decrease urine volume or raise urine osmolality. Desmopressin treatment in endotoxin-treated rats maintained mean arterial pressure, increased sodium reabsorption, IM osmolality, and urine osmolality, and decreased urine flow. The AQP2 intensity decreased in the endotoxin group, and the apical localization disappeared; both were not affected by desmopressin. Our results indicate that endotoxemia in rats acutely diminishes renal urinary concentration capacity and is associated with a decreased IM osmolality and diminished apical AQP2 localization. These findings may help to explain nonoliguric acute renal failure in human septic shock.


Asunto(s)
Desamino Arginina Vasopresina/farmacología , Endotoxemia/tratamiento farmacológico , Animales , Acuaporina 2/análisis , Acuaporina 2/metabolismo , Desamino Arginina Vasopresina/administración & dosificación , Endotoxemia/inducido químicamente , Endotoxinas/administración & dosificación , Endotoxinas/farmacología , Hemodinámica/efectos de los fármacos , Inmunohistoquímica , Médula Renal/efectos de los fármacos , Médula Renal/metabolismo , Túbulos Renales Colectores/efectos de los fármacos , Túbulos Renales Colectores/metabolismo , Masculino , Concentración Osmolar , Potasio/metabolismo , Ratas , Ratas Wistar , Sodio/metabolismo , Vasopresinas/sangre
15.
J Muscle Res Cell Motil ; 29(6-8): 189-201, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-19140019

RESUMEN

In healthy human myocardium a tight balance exists between receptor-mediated kinases and phosphatases coordinating phosphorylation of regulatory proteins involved in cardiomyocyte contractility. During heart failure, when neurohumoral stimulation increases to compensate for reduced cardiac pump function, this balance is perturbed. The imbalance between kinases and phosphatases upon chronic neurohumoral stimulation is detrimental and initiates cardiac remodelling, and phosphorylation changes of regulatory proteins, which impair cardiomyocyte function. The main signalling pathway involved in enhanced cardiomyocyte contractility during increased cardiac load is the beta-adrenergic signalling route, which becomes desensitized upon chronic stimulation. At the myofilament level, activation of protein kinase A (PKA), the down-stream kinase of the beta-adrenergic receptors (beta-AR), phosphorylates troponin I, myosin binding protein C and titin, which all exert differential effects on myofilament function. As a consequence of beta-AR down-regulation and desensitization, phosphorylation of the PKA-target proteins within the cardiomyocyte may be decreased and alter myofilament function. Here we discuss involvement of altered PKA-mediated myofilament protein phosphorylation in different animal and human studies, and discuss the roles of troponin I, myosin binding protein C and titin in regulating myofilament dysfunction in cardiac disease. Data from the different animal and human studies emphasize the importance of careful biopsy procurement, and the need to investigate localization of kinases and phosphatases within the cardiomyocyte, in particular their co-localization with cardiac myofilaments upon receptor stimulation.


Asunto(s)
Citoesqueleto de Actina/patología , Cardiopatías/patología , Cardiopatías/fisiopatología , Miocitos Cardíacos/patología , Citoesqueleto de Actina/fisiología , Animales , Humanos , Ratones , Contracción Miocárdica/fisiología , Miocitos Cardíacos/fisiología , Especificidad de la Especie
16.
Cytometry A ; 69(9): 973-8, 2006 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-16680704

RESUMEN

BACKGROUND: Damaged and/or dysfunctional microvascular endothelium has been implicated in the pathogenesis of ischemic acute renal failure (ARF). Rapidly occurring changes in the endothelial F-actin cytoskeleton as observed in vitro might be responsible, but have been proven difficult to measure accurately in situ. Therefore, the purpose of this study was to examine several methods of digital image analysis in order to quantify the alterations of endothelial F-actin after renal ischemia and reperfusion (I/R), and to relate these to deterioration of renal function. METHODS: Frozen sections of Sham and I/R rat kidneys were fixed in 4% formaldehyde and stained with rhodamine-phalloïdin. Microvascular structures were captured using a 3i Marianastrade mark digital imaging fluorescence microscope workstation. Images were analyzed using 3i SlideBooktrade mark software, employing several masking techniques and line-scans. RESULTS: Digital image analysis demonstrated a decrease in the mean intensity of rhodamine-phalloïdin fluorescence after I/R from 1030 +/- 187 to 735 +/- 121 a.u. (arbitrary units, mean +/- SD, n = 7). The number of F-actin fragments per pixel increased from (15.8 +/- 4.9) x 10(-5) to (20.7 +/- 3.5) x 10(-5) (n = 7), indicating cytoskeletal fragmentation. In addition, line-scan analysis demonstrated a disturbed spatial orientation of the F-actin cytoskeleton after I/R. Finally, the loss of F-actin correlated with a rise in plasma creatinine. CONCLUSIONS: The methods of digital image analysis described in the present study demonstrate that renal I/R induces profound changes in the F-actin cytoskeletal structure of microvascular endothelial cells, implicating an injured and dysfunctional microvascular endothelium, which may contribute to acute renal failure (ARF).


Asunto(s)
Actinas/ultraestructura , Citoesqueleto/ultraestructura , Células Endoteliales/ultraestructura , Procesamiento de Imagen Asistido por Computador/métodos , Isquemia , Circulación Renal , Reperfusión , Animales , Masculino , Ratas , Ratas Wistar , Circulación Renal/fisiología
17.
Am J Physiol Renal Physiol ; 291(3): F606-11, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16525157

RESUMEN

Renal ischemia-reperfusion (I/R) results in vascular dysfunction characterized by a reduced endothelium-dependent vasodilatation and subsequently impaired blood flow. In this study, we investigated the role of Rho kinase in endothelial nitric oxide synthase (eNOS)-mediated regulation of renal blood flow and vasomotor tone in renal I/R. Male Wistar rats were subjected to 60-min bilateral clamping of the renal arteries or sham procedure. One hour before the clamping, the Rho kinase inhibitor Y27632 (1 mg/kg) was intravenously infused. After I/R, renal blood flow was measured using fluorescent microspheres. I/R resulted in a 62% decrease in renal blood flow. In contrast, the blood flow decrease in the group treated with the Rho kinase inhibitor (YI/R) was prevented. Endothelium-dependent vasodilatation of renal arcuate arteries to ACh was measured ex vivo in a pressure myograph. These experiments demonstrated that the in vivo treatment with the Rho kinase inhibitor prevented the decrease in the nitric oxide (NO)-mediated vasodilator response. In addition, after I/R renal interlobar arteries showed a decrease in phosphorylated eNOS and vasodilator-stimulated phosphoprotein, a marker for bioactive NO, which was attenuated by in vivo Rho kinase inhibition. These findings indicate that in vivo inhibition of Rho kinase in renal I/R preserves renal blood flow by improving eNOS function.


Asunto(s)
Riñón/irrigación sanguínea , Óxido Nítrico Sintasa de Tipo III/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Circulación Renal/fisiología , Daño por Reperfusión/enzimología , Acetilcolina/sangre , Animales , Moléculas de Adhesión Celular/metabolismo , Hemodinámica , Péptidos y Proteínas de Señalización Intracelular , Riñón/enzimología , Masculino , Proteínas de Microfilamentos/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Norepinefrina/sangre , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Ratas , Ratas Wistar , Arteria Renal/enzimología , Daño por Reperfusión/fisiopatología , Quinasas Asociadas a rho
18.
J Mol Cell Cardiol ; 39(2): 345-54, 2005 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-15978614

RESUMEN

Recent evidence suggests that oxidative stress is a common denominator in many aspects of cardiovascular pathogenesis. Free cellular iron plays a crucial catalytic role in the formation of highly toxic hydroxyl radicals, and thereby it may aggravate the contribution of oxidative stress to cardiovascular disease. Therefore, iron chelation may be an effective therapeutic approach, but the progress in this area is hindered by the lack of effective agents. In this study, using the rat heart myoblast-derived cell line H9c2, we aimed to investigate whether the novel lipophilic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) protects the cells against hydrogen peroxide (H2O2)-induced cytotoxicity. Exposure of cells to 100 micromol/l H2O2 has within 4 h induced a complete dissipation of their mitochondrial membrane potential (DeltaPsim). Co-treatment with SIH dose-dependently reduced (EC50=0.8 micromol/l) or even completely abolished (3 micromol/l) this collapse. Furthermore, the latter SIH concentration was capable to fully prevent alterations in cell morphology, and inhibited both apoptosis (annexin-V staining, nuclear chromatin shrinkage, TUNEL positivity) and necrosis (propidium iodide staining), even 24 h after the H2O2 exposure. In comparison, deferoxamin (a commercially available hydrophilic iron chelator used in clinical practice and most previous studies) was cytoprotective only at three-order higher and clinically unachievable concentrations (EC50=1300 micromol/l). Thus, in this study, we present iron chelation as a very powerful tool by which oxidative stress-induced myocardial damage can be prevented.


Asunto(s)
Aldehídos/farmacología , Citoprotección/efectos de los fármacos , Hidrazonas/farmacología , Quelantes del Hierro/farmacología , Mitocondrias/efectos de los fármacos , Mioblastos Cardíacos/citología , Mioblastos Cardíacos/efectos de los fármacos , Estrés Oxidativo , Aldehídos/química , Animales , Muerte Celular/efectos de los fármacos , Línea Celular , Forma de la Célula , Supervivencia Celular/efectos de los fármacos , Hidrazonas/química , Peróxido de Hidrógeno/farmacología , Quelantes del Hierro/química , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Mioblastos Cardíacos/metabolismo , Ratas
19.
Arterioscler Thromb Vasc Biol ; 23(2): 211-7, 2003 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-12588761

RESUMEN

OBJECTIVE: Growth factor-induced angiogenesis involves migration of endothelial cells (ECs) into perivascular areas and requires active remodeling of the endothelial F-actin cytoskeleton. The small GTPase RhoA previously has been implicated in vascular endothelial growth factor (VEGF)-induced signaling pathways, but its role has not been clarified. METHODS AND RESULTS: VEGF induced the activation of RhoA and recruited RhoA to the cell membrane of human ECs. This increase in RhoA activity is necessary for the VEGF-induced reorganization of the F-actin cytoskeleton, as demonstrated by adenoviral transfection of dominant-negative RhoA. Rho kinase mediated this effect of RhoA, as was demonstrated by the use of Y-27632, a specific inhibitor of Rho kinase. Inhibition of Rho kinase prevented the VEGF-enhanced EC migration in response to mechanical wounding but had no effect on basal EC migration. Furthermore, in an in vitro model for angiogenesis, inhibition of either RhoA or Rho kinase attenuated the VEGF-mediated ingrowth of ECs in a 3-dimensional fibrin matrix. CONCLUSIONS: VEGF-induced cytoskeletal changes in ECs require RhoA and Rho kinase, and activation of RhoA/Rho kinase signaling is involved in the VEGF-induced in vitro EC migration and angiogenesis.


Asunto(s)
Movimiento Celular/fisiología , Factores de Crecimiento Endotelial/fisiología , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Linfocinas/fisiología , Neovascularización Fisiológica/fisiología , Transducción de Señal/fisiología , Proteínas de Unión al GTP rho/fisiología , Proteína de Unión al GTP rhoA/fisiología , Amidas/farmacología , Capilares/enzimología , Capilares/metabolismo , Capilares/fisiología , Técnicas de Cultivo de Célula/métodos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Citoesqueleto/enzimología , Citoesqueleto/fisiología , Endotelio Vascular/enzimología , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Fibrina/metabolismo , Humanos , Masculino , Pene/citología , Pene/enzimología , Piridinas/farmacología , Venas Umbilicales/citología , Venas Umbilicales/enzimología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Proteínas de Unión al GTP rho/antagonistas & inhibidores
20.
Cardiovasc Res ; 57(2): 563-71, 2003 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-12566129

RESUMEN

OBJECTIVE: In a combination of in vivo and in vitro studies, we investigated mechanisms via which alpha-tocopherol, a lipid soluble form of vitamin E, can directly affect endothelial activation as induced by H(2)O(2) and TNFalpha. METHODS: We measured effects of alpha-tocopherol on H(2)O(2)-induced lipid peroxidation as determined with a fluorescent C-11 BODIPY(581/591) probe and on adhesion molecule expression in cultured endothelial cells. In 20 healthy volunteers treated with increasing doses of alpha-tocopherol up to 800 IU/ml for 12 weeks, plasma levels of soluble cell adhesion molecules (sCAMs) and C-reactive protein were measured. RESULTS: We showed that alpha-tocopherol protects cultured endothelial cells against H(2)O(2)-induced lipid peroxidation, while TNFalpha did not induce lipid peroxidation. Moreover, alpha-tocopherol attenuated H(2)O(2)-, but not TNFalpha-induced increases in adhesion molecule expression. In healthy persons, alpha-tocopherol decreased plasma levels of sE-selectin from 65+/-6 to 60+/-6 ng/ml (P=0.002), sVCAM from 893+/-31 to 853+/-23 ng/ml (P=0.022), and sICAM from 483+/-21 to 463+/-16 ng/ml (P=0.048). C-Reactive protein, as a sensitive marker of low grade inflammation, was not significantly affected. CONCLUSION: alpha-Tocopherol specifically inhibits lipid peroxidation-induced endothelial activation in vitro. The observed vitamin E-induced decrease in sCAMs in control subjects suggests that lipid peroxidation can take place in healthy individuals. Although vitamin E supplementation may be especially effective in specific groups of patients exposed to increased oxidative stress, our study suggests that vitamin E supplementation can be of benefit in healthy individuals as well.


Asunto(s)
Antioxidantes/farmacología , Moléculas de Adhesión Celular/metabolismo , Endotelio Vascular/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , alfa-Tocoferol/farmacología , Proteína C-Reactiva/metabolismo , Células Cultivadas , Endotelio Vascular/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Peroxidación de Lípido/fisiología , Estrés Oxidativo/efectos de los fármacos , Solubilidad , Factor de Necrosis Tumoral alfa/farmacología
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