A deep learning approach to the automatic detection of alignment errors in cryo-electron tomographic reconstructions.

Electron tomography is an imaging technique that allows for the elucidation of three-dimensional structural information of biological specimens in a very general context, including cellular in situ observations. The approach starts by collecting a set of images at different projection directions by tilting the specimen stage inside the microscope. Therefore, a crucial preliminary step is to precisely define the acquisition geometry by aligning all the tilt images to a common reference. Errors introduced in this step will lead to the appearance of artifacts in the tomographic reconstruction, rendering them unsuitable for the sample study. Focusing on fiducial-based acquisition strategies, this work proposes a deep-learning algorithm to detect misalignment artifacts in tomographic reconstructions by analyzing the characteristics of these fiducial markers in the tomogram. In addition, we propose an algorithm designed to detect fiducial markers in the tomogram with which to feed the classification algorithm in case the alignment algorithm does not provide the location of the markers. This open-source software is available as part of the Xmipp software package inside of the Scipion framework, and also through the command-line in the standalone version of Xmipp.

ScipionTomo: Towards cryo-electron tomography software integration, reproducibility, and validation.

Image processing in cryogenic electron tomography (cryoET) is currently at a similar state as Single Particle Analysis (SPA) in cryogenic electron microscopy (cryoEM) was a few years ago. Its data processing workflows are far from being well defined and the user experience is still not smooth. Moreover, file formats of different software packages and their associated metadata are not standardized, mainly since different packages are developed by different groups, focusing on different steps of the data processing pipeline. The Scipion framework, originally developed for SPA (de la Rosa-Trevín et al., 2016), has a generic python workflow engine that gives it the versatility to be extended to other fields, as demonstrated for model building (Martínez et al., 2020). In this article, we provide an extension of Scipion based on a set of tomography plugins (referred to as ScipionTomo hereafter), with a similar purpose: to allow users to be focused on the data processing and analysis instead of having to deal with multiple software installation issues and the inconvenience of switching from one to another, converting metadata files, managing possible incompatibilities, scripting (writing a simple program in a language that the computer must convert to machine language each time the program is run), etcetera. Additionally, having all the software available in an integrated platform allows comparing the results of different algorithms trying to solve the same problem. In this way, the commonalities and differences between estimated parameters shed light on which results can be more trusted than others. ScipionTomo is developed by a collaborative multidisciplinary team composed of Scipion team engineers, structural biologists, and in some cases, the developers whose software packages have been integrated. It is open to anyone in the field willing to contribute to this project. The result is a framework extension that combines the acquired knowledge of Scipion developers in close collaboration with third-party developers, and the on-demand design of functionalities requested by beta testers applying this solution to actual biological problems.

On bias, variance, overfitting, gold standard and consensus in single-particle analysis by cryo-electron microscopy.

Cryo-electron microscopy (cryoEM) has become a well established technique to elucidate the 3D structures of biological macromolecules. Projection images from thousands of macromolecules that are assumed to be structurally identical are combined into a single 3D map representing the Coulomb potential of the macromolecule under study. This article discusses possible caveats along the image-processing path and how to avoid them to obtain a reliable 3D structure. Some of these problems are very well known in the community. These may be referred to as sample-related (such as specimen denaturation at interfaces or non-uniform projection geometry leading to underrepresented projection directions). The rest are related to the algorithms used. While some have been discussed in depth in the literature, such as the use of an incorrect initial volume, others have received much less attention. However, they are fundamental in any data-analysis approach. Chiefly among them, instabilities in estimating many of the key parameters that are required for a correct 3D reconstruction that occur all along the processing workflow are referred to, which may significantly affect the reliability of the whole process. In the field, the term overfitting has been coined to refer to some particular kinds of artifacts. It is argued that overfitting is a statistical bias in key parameter-estimation steps in the 3D reconstruction process, including intrinsic algorithmic bias. It is also shown that common tools (Fourier shell correlation) and strategies (gold standard) that are normally used to detect or prevent overfitting do not fully protect against it. Alternatively, it is proposed that detecting the bias that leads to overfitting is much easier when addressed at the level of parameter estimation, rather than detecting it once the particle images have been combined into a 3D map. Comparing the results from multiple algorithms (or at least, independent executions of the same algorithm) can detect parameter bias. These multiple executions could then be averaged to give a lower variance estimate of the underlying parameters.

Cryo-EM and Single-Particle Analysis with Scipion.

Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at near-atomic resolution. In single-particle analysis, the vitrified sample is imaged by an electron beam and the detectors at the end of the microscope column produce movies of that sample. These movies contain thousands of images of identical particles in random orientations. The data need to go through an image processing workflow with multiple steps to obtain the final 3D reconstructed volume. The goal of the image processing workflow is to identify the acquisition parameters to be able to reconstruct the specimen under study. Scipion provides all the tools to create this workflow using several image processing packages in an integrative framework, also allowing the traceability of the results. In this article the whole image processing workflow in Scipion is presented and discussed with data coming from a real test case, giving all the details necessary to go from the movies obtained by the microscope to a high resolution final 3D reconstruction. Also, the power of using consensus tools that allow combining methods, and confirming results along every step of the workflow, improving the accuracy of the obtained results, is discussed.

La calidad de vida de pacientes asmáticos evaluada por el WHOQOL-BREF y el SGRQ / The quality of life of asthmatic patients evaluated by the WHOQOL-BREF and the SGRQ

ObjetivosLos objetivos del estudio fueron analizar la calidad de vida de pacientes asmáticos, evaluada mediante un instrumento genérico y otro específico, así como comprobar la correlación entre ambos.Material y métodoEstudio trasversal, descriptivo y observacional en el que participaron 51 asmáticos adultos (26 varones y 25 mujeres) con edad media de 54,46 años (SD 19,17 años), todos reclutados a partir de las consultas externas del Hospital Universitario de Salamanca, entre octubre de 2008 y marzo de 2009. Se realizó una evaluación clínica inicial que incluía una espirometría, formulario sociodemográfico y la aplicación de las versiones españolas del World Health Quality of Life-Bref (WHOQOL-BREF) y del Saint George Respiratory Questionnaire (SGRQ).ResultadosLa edad correlacionó de forma inversa y significativa con todos los dominios del WHOQOL-BREF y solo con el dominio actividad del SGRQ. La clasificación de la gravedad y el volumen espiratorio forzado (FEV1%pred) no presentaron relación con ninguno de los dominios de ambos instrumentos. Los dominios físico y psíquico del WHOQOL-BREF correlacionaron de forma inversa y significativa con todos los dominios del SGRQ.ConclusionesPese a la falta de relación entre la gravedad del asma y los distintos dominios del WHOQOL-BREF, el instrumento ha resultado ser sensible a la variable edad, y 2 de sus 4 dominios han correlacionado con todos los dominios del SGRQ(AU)

ObjectivesThis study has aimed to analyze the quality of life of asthmatic patients, evaluated by one generic and another specific instrument as well as to verify the correlations between them.Material and methodsA cross-sectional, descriptive and observational study with 51 adult asthmatic subjects (26 men and 25 women) with a mean age of 54.46 years (SD 19.17 years), all recruited from the out-patient clinic of the University Hospital of Salamanca, Spain, from October 2008 to March 2009. A clinical evaluation was performed, this including the spirometry, a socio-demographical form and the application of the Spanish version of the World Health Organization Quality of Life (WHOQOL-BREF) and the Saint George Respiratory Questionnaire (SGRQ).ResultsAge correlated inversely and significantly with all the WHOQOL-BREF domains and only with the activity domain of the SGRQ. The severity classification and forced expiratory volume (FEV1%pred) did not show any relationship with any of the domains of both instruments. The physical and psychic domains of the WHOQOL-BREF correlated inversely and significantly with all the SGRQ domains.ConclusionsIn spite of the lack of relation between asthma severity and the different domains of the WHOQOL-BREF, the instrument is sensitive to the age variable and two of its four domains have correlated with all of the SGRQ domains(AU)

[Lung cancer: how does it appear in our hospital?]. / Cáncer de pulmón: ¿cómo se presenta en nuestra consulta?

INTRODUCTION: Lung cancer is the most frequently diagnosed tumor in industrialized countries and that which causes the largest number of deaths. The aim of this study is to know the relevant clinical and epidemiological characteristics of lung cancer in the Health Care Area of Salamanca. PATIENTS AND METHODS: A cross-sectional descriptive epidemiological study conducted in 140 patients, in the Rapid Diagnosis of Lung Cancer Pneumology Department of the University Hospital of Salamanca. RESULTS: The percentage of men diagnosed is greater than for to women: 126 (90%) were men and 14 (10%) women. Ninety percent had had an active contact with the tobacco. The percentage of men who were ex-smokers is greater than that of those who smoke, there being a rate of packages per year in both groups at the time of diagnosis. Women continue to be mostly non-smokers, although there is a progressive increase of active smokers. The most frequent clinical manifestation on diagnosis is chest pain (36.4%), 46.4% of the patients having symptoms of the constitutional syndrome. The most frequent histological type among men is squamous cell carcinoma (35.7%), and among women adenocarcinoma (50%), the significant relationship (p < 0.05) between the histological stain and the principal risk factor, that is tobacco, standing out. CONCLUSIONS: The known tobacco-lung cancer relationship, corroborated in this study, makes it necessary to have a more effective fight against tobacco to reduce the incidence of lung cancer. It is also necessary to develop multidisciplinary protocols that help early diagnosis and make it possible to increase the percentage of curative treatments.

[Phenotypic characterization and course of chronic obstructive pulmonary disease in the PAC-COPD Study: design and methods]. / Caracterización fenotípica y evolución de la EPOC en el estudio PAC-COPD: diseño y metodología.

BACKGROUND AND OBJECTIVES: The Phenotype and Course of Chronic Obstructive Pulmonary Disease (PAC-COPD) study aims to improve our understanding of the phenotypic heterogeneity of this disease and the extent to which this heterogeneity is related to clinical course. The main objectives are a) to characterize the phenotypic variability in first-time hospitalizations for exacerbation of COPD and to propose a classification into subtypes and b) to ascertain the association between the defined subtypes and the clinical and functional course of COPD. PATIENTS AND METHODS: This is a cross-sectional and cohort study of 342 patients with COPD from 9 tertiary hospitals in 3 autonomous communities. The minimum follow-up period is 5 years. The main variables of interest are respiratory symptoms, smoking, alcohol use, physical activity, use of health care services, medical care, treatment received, activities of daily living, comorbid conditions, sleepiness, anxiety and depression, quality of life, forced spirometry and bronchodilation tests, lung volume and inspiratory capacity measured by body plethysmography, carbon monoxide diffusing capacity, baseline arterial blood gas values, respiratory and peripheral muscle function, electrocardiogram, body weight and composition measured by bioelectric impedance, chest radiograph, skin prick test, capacity for exercise measured in the 6-minute walk test and cardiopulmonary exercise test, induced sputum (for quantitative microbiological culture and determination of inflammatory markers), nighttime pulse oximetry, chest computed tomography scan, and echocardiography. Serum and plasma samples are also taken to measure levels of inflammatory markers and oxidative stress, for genetic analysis, and for other possible measurements that might be required in the future. The statistical analysis combines factor analysis and survival models such as Cox regression analysis. This project will enable us to reconsider the definition and classification of COPD and to better understand the factors associated with its natural history.

Caracterización fenotípica y evolución de la EPOC en el estudio PAC-COPD: diseño y metodología / Phenotypic Characterization and Course of Chronic Obstructive Pulmonary Disease in the PAC-COPD Study: Design and Methods

Introducción y objetivosel estudio Caracterización Fenotípica y Evolución de la Enfermedad Pulmonar Obstructiva Crónica (PAC-COPD) pretende mejorar la comprensión de la heterogeneidad fenotípica de esta enfermedad y el grado en que dicha heterogeneidad se relaciona con su evolución. Los principales objetivos son: a) caracterizar la variabilidad fenotípica de las personas que ingresan por primera vez por agudización de la enfermedad pulmonar obstructiva crónica (EPOC) y proponer una clasificación en subtipos, y b) estimar la asociación entre los subtipos definidos y la evolución clínica y funcional de la EPOC(AU)

Pacientes y métodoel diseño de investigación incluye un estudio transversal y un estudio de seguimiento de una cohorte de 342 pacientes con EPOC procedentes de 9 hospitales terciarios de 3 comunidades autónomas. El período de seguimiento abarca un mínimo de 5 años. Las principales medidas que se determinarán son las siguientes: síntomas respiratorios, tabaco, alcohol, actividad física, utilización de servicios sanitarios, atención médica, tratamientos recibidos, actividades de la vida diaria, comorbilidades, somnolencia, ansiedad y depresión, calidad de vida, espirometría forzada y prueba broncodilatadora, pletismografía corporal con volúmenes pulmonares y capacidad inspiratoria, capacidad de transferencia del monóxido de carbono, gasometría arterial basal, función de músculos respiratorios y periféricos, electrocardiograma, peso y composición nutricional mediante impedanciometría bioeléctrica, radiografía de tórax, pruebas cutáneas de alergia, capacidad de ejercicio según la prueba de la marcha de 6min y prueba de ejercicio cardiopulmonar, esputo inducido (para cultivo microbiológico cuantitativo y determinación de marcadores de inflamación), pulsioximetría nocturna, tomografía computarizada pulmonar y ecocardiografía. Además, se obtienen muestras de suero y plasma para la medición de marcadores inflamatorios y de estrés oxidativo, análisis genéticos y otras posibles mediciones que puedan plantearse en el futuro. Para el análisis estadístico de los resultados se combinará el análisis factorial con modelos de supervivencia como la regresión de Cox. El proyecto permitirá replantear la definición y clasificación de la EPOC, así como comprender mejor los factores asociados a su evolución(AU)

Background and ObjectivesThe Phenotype and Course of Chronic Obstructive Pulmonary Disease (PAC-COPD) study aims to improve our understanding of the phenotypic heterogeneity of this disease and the extent to which this heterogeneity is related to clinical course. The main objectives are a) to characterize the phenotypic variability in first-time hospitalizations for exacerbation of COPD and to propose a classification into subtypes and b) to ascertain the association between the defined subtypes and the clinical and functional course of COPD(AU)

Patients and MethodsThis is a cross-sectional and cohort study of 342 patients with COPD from 9 tertiary hospitals in 3 autonomous communities. The minimum follow-up period is 5 years. The main variables of interest are respiratory symptoms, smoking, alcohol use, physical activity, use of health care services, medical care, treatment received, activities of daily living, comorbid conditions, sleepiness, anxiety and depression, quality of life, forced spirometry and bronchodilation tests, lung volume and inspiratory capacity measured by body plethysmography, carbon monoxide diffusing capacity, baseline arterial blood gas values, respiratory and peripheral muscle function, electrocardiogram, body weight and composition measured by bioelectric impedance, chest radiograph, skin prick test, capacity for exercise measured in the 6-minute walk test and cardiopulmonary exercise test, induced sputum (for quantitative microbiological culture and determination of inflammatory markers), nighttime pulse oximetry, chest computed tomography scan, and echocardiography. Serum and plasma samples are also taken to measure levels of inflammatory markers and oxidative stress, for genetic analysis, and for other possible measurements that might be required in the future. The statistical analysis combines factor analysis and survival models such as Cox regression analysis. This project will enable us to reconsider the definition and classification of COPD and to better understand the factors associated with its natural history(AU)

Adenosine 5'-monophosphate in asthma: gas exchange and sputum cellular responses.

Adenosine 5'-monophosphate (AMP) bronchoprovocation reproduces the lung function abnormalities that occur spontaneously during acute asthma and detects peripheral airway inflammation better than direct bronchoconstrictive agents. Pulmonary gas exchange disturbances may reflect changes in small airways related to airway inflammation rather than bronchoconstriction alone. The present authors investigated whether AMP induced a greater imbalance in the ventilation/perfusion ratio than methacholine (MCh), at an equivalent degree of bronchoconstriction, with and without salbutamol pre-medication. In total, 36 asthmatics were studied in three randomised, double-blind, crossover studies: 1) before and after AMP or MCh; 2) before and 30 min after salbutamol or placebo, followed by AMP; or 3) MCh challenge. Sputum was collected before and 4 h post-challenge. Compared with MCh, AMP provoked similar pulmonary gas exchange abnormalities at an equivalent degree of intense bronchoconstriction (forced expiratory volume in one second decrease of 28-44%). While salbutamol blocked AMP- or MCh-induced bronchoconstriction, arterial oxygen tension (P(a,O(2))) and alveolar-arterial oxygen tension difference (P(A-a,O(2))) disturbances induced by AMP and MCh were only partially blocked (P(a,O(2)) by 46 and 42%, respectively; P(A-a,O(2)) by 58 and 57%, respectively). Compared with MCh, AMP increased the percentage of neutrophils (mean+/-se increased from 28+/-4% to 38+/-4%), but this increase did not occur after salbutamol pre-treatment. Both adenosine 5'-monophosphate and methacholine induced similar peripheral airway dysfunction. The fully inhibited adenosine 5'-monophosphate-induced neutrophilia with residual hypoxaemia observed after salbutamol treatment is probably related to beta(2)-agonists acting on both bronchial and pulmonary circulation.

Leukotriene D4-induced hypoxaemia in asthma is mediated by the cys-leukotriene1 receptor.

Bronchoprovocation with cysteinyl-leukotrienes (LTs) induces airflow obstruction and gas exchange abnormalities, namely ventilation-perfusion ratio (V'(A)/Q') imbalance. However, it is unknown which of the two different receptors for cysteinyl-LTs mediate these V'(A)/Q' disturbances. In a double-blinded, crossover design, 10 patients with mild asthma were randomised to receive an oral single dose of the selective cysteinyl-LT1 receptor antagonist montelukast (40 mg) or placebo before leukotriene (LT)D4 inhalation challenge. Gas exchange, including V'(A)/Q' descriptors were measured at baseline, 3 h after montelukast/placebo pretreatment and 5, 15 and 45 min after the LTD4 challenge. Compared with montelukast, inhalation of LTD(4) induced a marked fall in forced expiratory volume in one second (mean+/-se 33+/-2%) and profound V'(A)/Q' mismatching, reflected by a decreased arterial oxygen tension (from 100+/-4 to 75+/-3 mmHg) and an increased overall index of V'(A)/Q' heterogeneity dispersion of retention minus excretion inert gases corrected for dead space (from 4.9+/-1.2 to 8.4+/-1.1; normal< or =3.0; dimensionless), 5 min after placebo. Following montelukast, LTD4 produced no significant changes in any of the variables. In conclusion, these findings point to the view that leukotriene D4)-induced gas exchange disturbances and bronchoconstriction are both mediated by the cysteinyl-leukotriene1 receptor.

[Delays in the diagnosis of lung cancer]. / Demoras diagnósticas en el cáncer del pulmón.

OBJECTIVE: To study the clinical and demographic factors associated with delays in the diagnosis of lung cancer. PATIENTS AND METHODS: A 2-year prospective study of patients admitted to the respiratory medicine ward with a suspected diagnosis of lung cancer. We studied demographic factors, health care received, place of residence, and delays in carrying out diagnostic procedures. The following diagnostic time periods were defined: consultation (from first symptom to first medical visit), middle period (from first medical visit to hospital admission) and diagnostic (from hospital admission to histological diagnosis and clinical staging). RESULTS: One hundred thirteen patients with a mean age of 65 years (range, 36-90), 103 men and 10 women, were studied. The most frequent symptoms leading to consultation were coughing (10.6%), hemoptysis (19.5%), chest pain (26.5%), and shortness of breath (9.7%). First visits were to a primary care physician for 72%, to the hospital emergency room for 22%, or to a pulmonologist for 6%. Forty-four percent of the patients visited the doctor 2 or 3 times. The mean SD, numbers of days for the different time periods were as follows: consultation, 30.3 60; diagnosis, 18.6 19; middle period 37.9 63. The mean total time from first symptom to diagnosis was 85.7 87 days. The middle period, the time in hospital until diagnosis, and the total time were shorter when patients were referred by the primary care physician to the emergency room or were directly admitted to the hospital (P<.001). Only 25.7% of the staged lung cancers were operable. CONCLUSIONS: Delays in lung cancer diagnosis are long. The attitudes of primary care physicians and their relations with specialized care providers are crucial for reducing delays.

Demoras diagnósticas en el cáncer de pulmón / Delays in the Diagnosis of lung cancer

OBJETIVO: Estudio de los factores clínicos y demográficos asociados a las demoras diagnósticas del cáncer de pulmón (CP). PACIENTES Y MÉTODOS: Estudio prospectivo de dos años de evolución de pacientes ingresados en la planta de neumología por sospecha de CP. Se estudiaron variables demográficas, de atención sanitaria, hábitat y demoras en las exploraciones. Se definieron los tiempos diagnósticos siguientes: de consulta (desde el primer síntoma hasta la visita del primer médico), medio (el que va desde la visita médica hasta el ingreso) y diagnóstico (desde el ingreso hasta el diagnóstico histológico y la estadificación clínica). RESULTADOS: Se estudió a 113 pacientes con una edad media de 65 años (intervalo, 36-90), de los que 103 eran varones y 10, mujeres. Los síntomas más frecuentes por los que consultó el paciente fueron: tos (10,6 por ciento), hemoptisis (19,5 por ciento), dolor torácico (26,5 por ciento) y disnea (9,7 por ciento). El 72 por ciento de los pacientes consultaba por primera vez al médico de atención primaria (MAP), el 22 por ciento a urgencias del hospital y el 6 por ciento al neumólogo. El 44 por ciento realizó entre dos y tres visitas al médico. Las medias ñ desviaciones típicas, en días, de los diferentes tiempos fueron: consulta, 30,3 ñ 60; diagnóstico, 18,6 ñ 19; medio, 37,9 ñ 63, y total, 85,7 ñ 87. El tiempo medio, el tiempo hasta el diagnóstico y el tiempo total fueron inferiores cuando el paciente fue remitido, por el MAP, a urgencias o ingresado directamente en el hospital (p < 0,001). Sólo el 25,7 por ciento de los CP estadificados eran resecables quirúrgicamente. CONCLUSIONES: Los tiempos de demora diagnóstica en el CP son elevados. La actitud del MAP y su relación con la atención especializada es importante para su reducción (AU)

Pulmonary gas exchange responses to histamine and methacholine challenges in mild asthma.

Histamine (HIST) produces greater changes in bronchial and pulmonary vasculature, and so may produce more gas exchange abnormalities, than methacholine (MTH) after inhalational challenge. The goals of this study were to compare the effects of HIST and MTH challenge on pulmonary gas exchange in patients with mild asthma at an equivalent degree of bronchoconstriction. Eleven patients were studied (mean+/-SEM age, 22+/-1 yr; forced expiratory volume in one second (FEV1), 91+/-5% pred) using a randomized, double-blind cross-over design. Respiratory system resistance (Rrs), arterial blood gases, and ventilation-perfusion distributions were measured before and after HIST/MTH challenges when cumulative doses caused a 30% fall in FEV1. Compared with baseline, HIST and MTH provoked similar moderate to severe increases in Rrs (p<0.005 each), and mild to moderate decreases in arterial oxygen tension (Pa,O2) due to ventilation-perfusion abnormalities (dispersion of pulmonary blood flow -log SDQ-, 0.40+/-0.03-0.71+/-0.08 and 0.47+/-0.04-0.89+/-0.06; normal values <0.60-0.65), respectively, similar to those shown in mild to moderate acute asthma, without differences between them. For the same degree of airflow obstruction, both histamine and methacholine bronchoprovocations induce, in patients with mild asthma, very similar disturbances in ventilation-perfusion distribution and respiratory system resistance, suggesting similar mechanisms of airway narrowing.

Loss of phosphotyrosine phosphatase activity and changes in the tyrosine phosphorylation state of proteins after storage of sheep platelets in plasma or Seto solution at 4 degrees C.

BACKGROUND AND OBJECTIVES: During platelet storage an array of deleterious changes occur, through mechanisms not fully understood, which impair platelet haemostasis. Transfused platelets should maintain the integrated networks of signalling pathways that regulate platelet activation and functionality. We hypothesized that protein phosphorylation and dephosphorylation, which play a fundamental role in these pathways, might be affected by platelet storage. We therefore investigated whether the activity of phosphotyrosine phosphatase (PTP), which belongs to an oxidant-susceptible group of enzymes involved in the platelet signal-transduction pathways that ensure platelet functionality, is affected by platelet storage. MATERIALS AND METHODS: Using sheep platelet species as a model system, we conducted serial studies on the membranes of platelets and microparticles shed during platelet storage, in their own plasma or in a synthetic medium called Seto, for up to 5 days at 4 degrees C. RESULTS: A progressive decrease in both total and specific membrane-associated PTP activities from whole platelets (but not from microparticles) located within each platelet storage bag was observed from day 1 onwards in both types of storage media. These decreases could be partly avoided by the addition of vitamin E. Additionally, the observed decrease in PTP activity was accompanied with increases in the tyrosine phosphorylation of proteins from whole platelets or crude platelet membranes, the tyrosine phosphorylation state of proteins from microparticles remaining basically unchanged. CONCLUSION: Our findings suggest that alterations of at least the tyrosine phosphorylation balance might be one of the reasons for the decrease in the haemostatic function of stored platelets.

Platelet-activating factor antagonists: current status in asthma.

Platelet-activating factor (PAF) is a potent lipid-derived mediator of inflammation that is considered to have a potential role in the pathogenesis of asthma. PAF is produced by many cells associated with asthmatic inflammation and has the ability to evoke some of the clinical hallmarks of asthma, such as bronchoconstriction, mucus production and airway hyperresponsiveness (AHR). In addition, PAF has profound chemoattractant properties for eosinophils and neutrophils and it promotes an increase in microvascular permeability and oedema formation within the airways. Nevertheless, the definitive role of PAF in asthma remains elusive. PAF is formed as a result of the action of phospholipase A(2) and acetyltransferase on membrane phospholipids and it is degraded by a PAF-specific acetylhydrolase. The biological effects of PAF are mediated by the activation of specific receptors expressed on effector cell surfaces, although intracellular signalling and paracrine actions have been described. In addition, at least part of the pulmonary effects of PAF could be related to the secondary release of leukotrienes. In the clinical setting, different ways of modifying the activity of PAF have been explored, in particular the inhibitory actions of PAF receptor antagonists. Both natural and synthetic PAF receptor antagonists have shown conflicting results. Although second generation PAF antagonists (apafant, UK-74505, SR-27417A) appear to have a good protective effect against the systemic and pulmonary actions of inhaled PAF, the protective effects of these compounds on allergen-induced responses and AHR are more modest. In the treatment of asthma, PAF receptor antagonists have failed to produce a significant impact in either acute asthma attacks or the maintenance therapy of chronic forms. Other pharmacological interventions of proven efficacy in asthma, such as salbutamol or 5-lipoxygenase antagonists, have shown some anti-PAF effects. Whether the overall negative results with PAF receptor antagonists indicate that extracellular PAF is not a relevant mediator of airway inflammation or that the compounds explored are not capable of blocking the paracrine actions of PAF remains speculative. A PAF synthase inhibitor could be valuable in the elucidation of the role of PAF and it might be a promising and useful complementary therapeutic tool in the future.

Gas exchange response to a PAF receptor antagonist, SR 27417A, in acute asthma: a pilot study.

The pathogenic role of platelet-activating factor (PAF) in asthma has been questioned due to the limited or negative efficacy of PAF antagonists; however, in acute asthma (AA), where the endogenous release of PAF may be enhanced, the effects of PAF antagonist receptors have not been investigated. It was postulated that inhaled PAF provokes gas exchange defects in mild asthma likely to be related to airway vascular leakage. The response to a potent, selective PAF receptor antagonist, SR 27471A, on pulmonary gas exchange was studied, more specifically ventilation-perfusion (VA'/Q') distributions, in patients with AA within 48 h of hospitalization. A randomized, double-blind, placebo-controlled, parallel group (n=6, each) design was used. After baseline measurements, either placebo or SR 27417A (20 mg, orally) was administered and measurements were repeated 3 h later. Conventional anti-asthma medication was not interrupted. Despite a near-complete inhibition of the in vitro, platelet aggregation tests by 40 nM PAF (mean+/-SEM from 72+/-9 to 6+/-2%) and 80 nM PAF (from 81+/-7 to 6+/-3% both p<0.01) by SR 27471A indicating a good bioactivity of the compound, no significant changes in baseline forced expiratory volume in one second, (40+/-6%), respiratory system resistance (6.2+/-0.7 cmH2O x L(-1) x s), alveolar-arterial pressure difference for oxygen (5.2+/-0.4 kPa), arterial oxygen tension (9.0+/-0.5 kPa) or VA'/Q' distributions, as expressed by the dispersion of pulmonary blood flow (LogSD Q, 1.07+/-0.09; normal values <0.60), were observed. It is concluded that SR 27417A has limited value when added to the conventional treatment of acute asthma. These findings minimize the potential pathogenic role of endogenous platelet-activating factor as a relevant mediator of airway inflammation during acute asthma.

Effects of inhaled furosemide on platelet-activating factor challenge in mild asthma.

Furosemide (Fur) may have an anti-inflammatory effect on airways in patients with asthma although its intrinsic mechanism remains elusive. Platelet-activating factor (PAF) is a potent proinflammatory mediator that induces systemic and respiratory effects in normal control subjects and asthmatics. The aim of this study was to assess whether pretreatment with nebulized Fur (40 mg) was able to modulate PAF-induced systemic and respiratory effects in asthma. Eleven patients were studied (mean+/-sem 22+/-0.8 yrs) with mild asthma (forced expiratory volume in one second, 95+/-4%) in a randomized, double-blind, placebo-controlled, cross-over fashion, one week apart. PAF challenge (18 microg) was carried out 15 min after administration of Fur or placebo. Peripheral blood neutrophils, respiratory system resistance, and arterial blood gases were measured at baseline, and 5, 15 and 45 min after PAF; urinary cysteinyl leukotriene E4 (uLTE4) was also measured, at baseline and 120 min after PAF challenge. Although Fur did not alter PAF-induced systemic and respiratory effects, it did partially inhibit (63%; p<0.04) the increments of uLTE4 levels shown after PAF inhalation. It is concluded that furosemide is not effective in protecting against platelet-activating factor challenge in patients with asthma despite its potential inhibition of leukotriene synthesis. These findings reinforce the view that the pulmonary effects of platelet-activating factor are mediated through different pathways.

Comparison between in vitro lipid peroxidation in fresh sheep platelets and peroxidative processes during sheep platelet ageing under storage at 4 degrees C.

Incubation of sheep platelet crude membranes with xanthine oxidase (XO)/hypoxanthine/Fe(2+)-ADP revealed: (i) a fast peroxidative response - with a maximal linear rate of 14 nmol malondialdehyde (MDA) equivalents/mg protein, as evidenced by the thiobarbituric acid test - and a decrease in the polyunsaturated fatty acid (PUFA) content of the platelet crude membranes; (ii) a decrease in the lipid fluidity in the deep lipid core of the membranes but not at the membrane surface; (iii) a dramatic inhibitory effect on glucose 6-phosphatase (Glc-6-Pase) but not on acetylcholinesterase activity. Platelets were also aged by storage at 4 degrees C in their own plasma or in Seto additive solution. In these media, platelet aggregates were visible and the effects on platelet phospholipids, PUFA, lipid extract fluorescence, crude membrane fluidity and membrane-bound enzyme activities were assessed for comparison with those observed in in vitro lipid peroxidation. The sensitivity of membranes from stored platelets to lipid peroxidation was also assessed. Storage of platelets in plasma for 5 days was associated with different changes in their crude membranes such as decreases in arachidonic acid contents, the decrease not being avoided by the presence of phospholipase A(2) inhibitors, increases in MDA equivalents, conjugated dienes and lipid extract fluorescence, decreases in the amounts of MDA equivalents formed by platelet crude membranes treated with the oxidizing agents, changes in membrane fluidity and inhibition of Glc-6-Pase. All these alterations were less pronounced or even abolished after platelet storage in Seto. These findings suggest that platelet lipid peroxidation due to XO/hypoxanthine/Fe(2+)-ADP and platelet membrane alterations observed after platelet ageing under storage at 4 degrees C share common features. Also, as regards the prevention of peroxidative processes, Seto solution permits better storage of sheep platelets than plasma.

Effect of nitric oxide synthesis inhibition with nebulized L-NAME on ventilation-perfusion distributions in bronchial asthma.

Patients with clinically stable asthma may show ventilation-perfusion (V'A/Q') mismatch. Nitric oxide (NO), a potent endogenous vasodilator, is increased in exhaled air of asthmatics. Such an increased NO production may be detrimental for optimal V'A/Q' balance owing to the potential inhibition of hypoxic pulmonary vasoconstriction. This study was undertaken to investigate the relationship between the concentration of NO in exhaled air and the degree of gas-exchange impairment and to assess the effect of nebulized N(G)-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NO synthesis, on gas exchange in patients with asthma. Twelve patients (four females and eight males, aged 31+/-5 yrs) with clinically stable asthma (forced expiratory volume in one second (FEV1) 80+/-5%) not treated with glucocorticoids and increased exhaled NO (58+/-9 parts per billion (ppb)) were studied. Exhaled NO, respiratory system resistance (Rrs), arterial blood gases and V'A/Q' distributions were measured before and 30, 60, 90 and 120 min after placebo or L-NAME (10(-1) M) nebulization; in eight patients pulmonary haemodynamics were also measured. At baseline no relationships between exhaled NO and gas-exchange measurements were shown. Nebulized L-NAME induced a significant decrease in exhaled NO (p< 0.001), which was maximal at 90 min (-55+/-5%). However, after L-NAME no changes in Rrs, arterial oxygen tension, the alveolar-arterial pressure difference in oxygen or V'A/Q' distributions were shown and nebulized L-NAME did not modify pulmonary artery pressure. In conclusion, the degree of gas-exchange impairment in stable asthma is not related to nitric oxide concentration in exhaled air and nitric oxide synthesis inhibition with N(G)-nitro-L-arginine methyl ester does not alter gas exchange or pulmonary haemodynamics, such that ventilation-perfusion disturbances do not appear to be related to an increased synthesis of nitric oxide in the airways.

[Measurement of exhaled nitric oxide in healthy subjects]. / Medición del óxido nítrico exhalado en sujetos sanos.

BACKGROUND: Endogenously synthesized nitric oxide (NO) is present in exhaled air and its analysis could be used as a tool to monitor inflammatory airway diseases. The objective of the present study was to develop the methodology for the measurement of exhaled NO and to obtain reference values in a group of healthy subjects. SUBJECTS AND METHOD: Exhaled NO was measured in 40 healthy subjects and 22 asthmatic patients using a single breath manoeuvre and a chemiluminescence analyzer. Comparisons of exhaled NO while breathing both, room air and medical air, were performed in 20 subjects. In seven asthmatic patients we evaluated the effect of an inhibitor of NO-synthesis (L-NAME). RESULTS: Mean (SD) exhaled NO in healthy subjects was 18 (13) parts per billion (ppb). Intraindividual variability was 6.5 (6.5%). The concentration of exhaled NO could be overestimated when environmental NO was high (> 80 ppb). Smokers showed lower levels than nonsmokers (10 [7] vs 22 [13] ppb, respectively; p < 0.005), whereas asthmatic patients showed higher exhaled NO levels (62 [31] ppb; p < 0.001). In these patients nebulization of L-NAME induced a progressive fall in exhaled NO (maximal decrease, -68 [15%]; p < 0.01). CONCLUSIONS: The measurement of NO concentration in exhaled air is reproducible, not influenced by the usual levels of environmental NO, and sensible enough to detect changes induced by the administration of a specific inhibitor. Exhaled NO concentration decreases in smokers and increases in asthmatics.