Feasibility of automated slope III and Scond analysis in children.

BACKGROUND Multiple breath washout (MBW) derived Scond is an established index of ventilation inhomogeneity. Time-consuming post hoc calculations of the expirogram's slope of alveolar phase III (SIII) and the lack of available software hampered widespread application of Scond. METHODS Seventy-two school-aged children (45 with cystic fibrosis; CF) performed 3 nitrogen MBW. We tested a new automated algorithm for Scond analysis (Scondauto ) which comprised breath selection for SIII detection, calculation and reporting of test quality. We compared Scondauto to (i) standard Scond analysis (Scondmanual ) with manual breath selection and to (ii) pragmatic Scond analysis including all breaths (Scondall ). Primary outcomes were success rate and agreement between different Scond protocols, and Scond fitting quality (linear regression R(2) ). RESULTS Average Scondauto (0.06 for CF and 0.01 for controls) was not different from Scondmanual (0.06 for CF and 0.01 for controls) and showed comparable fitting quality (R(2) 0.53 for CF and 0.13 for controls vs. R(2) 0.54 for CF and 0.13 for controls). Scondall was similar in CF and controls but with inferior fitting quality compared to Scondauto and Scondmanual . CONCLUSIONS Automated Scond calculation is feasible and produces robust results comparable to the standard manual way of Scond calculation. This algorithm provides a valid, fast and objective tool for regular use, even in children. Pediatr Pulmonol. © 2014 Wiley Periodicals, Inc.

Alternative outcomes for the multiple breath washout in children with CF

BACKGROUND The lung clearance index (LCI) measured by multiple-breath washout (MBW) has been proposed as an outcome for clinical trials; however, MBW is time consuming and LCI can be affected by breathing pattern. We aimed to evaluate moment ratios and abbreviated LCI in school-aged children with mild-to-moderate CF lung disease. METHODS Using existing data from three studies we assessed the sensitivity of moment ratios and abbreviated LCIs to (i) detect mild-to-moderate CF lung disease and (ii) detect treatment effects after 4weeks of hypertonic saline or dornase alfa inhalation. MBW was measured by respiratory mass spectrometry using 4% "sulphur hexafluoride as a tracer gas. RESULTS Compared to the traditional LCI, moment ratios and abbreviated LCIs were similarly sensitive to detect CF lung disease. Moment ratios consistently demonstrated treatment effects, whereas abbreviated LCIs were less sensitive. CONCLUSIONS Both moment ratios and abbreviated LCI are suitable to differentiate health from disease. Sensitivity is decreased for abbreviated LCIs in interventional studies in mild CF lung disease.

A numerical model for inert gas transport in the lung based on fractal airway morphology

Patients suffering from cystic fibrosis (CF) show thick secretions, mucus plugging and bronchiectasis in bronchial and alveolar ducts. This results in substantial structural changes of the airway morphology and heterogeneous ventilation. Disease progression and treatment effects are monitored by so-called gas washout tests, where the change in concentration of an inert gas is measured over a single or multiple breaths. The result of the tests based on the profile of the measured concentration is a marker for the severity of the ventilation inhomogeneity strongly affected by the airway morphology. However, it is hard to localize underlying obstructions to specific parts of the airways, especially if occurring in the lung periphery. In order to support the analysis of lung function tests (e.g. multi-breath washout), we developed a numerical model of the entire airway tree, coupling a lumped parameter model for the lung ventilation with a 4th-order accurate finite difference model of a 1D advection-diffusion equation for the transport of an inert gas. The boundary conditions for the flow problem comprise the pressure and flow profile at the mouth, which is typically known from clinical washout tests. The natural asymmetry of the lung morphology is approximated by a generic, fractal, asymmetric branching scheme which we applied for the conducting airways. A conducting airway ends when its dimension falls below a predefined limit. A model acinus is then connected to each terminal airway. The morphology of an acinus unit comprises a network of expandable cells. A regional, linear constitutive law describes the pressure-volume relation between the pleural gap and the acinus. The cyclic expansion (breathing) of each acinus unit depends on the resistance of the feeding airway and on the flow resistance and stiffness of the cells themselves. Special care was taken in the development of a conservative numerical scheme for the gas transport across bifurcations, handling spatially and temporally varying advective and diffusive fluxes over a wide range of scales. Implicit time integration was applied to account for the numerical stiffness resulting from the discretized transport equation. Local or regional modification of the airway dimension, resistance or tissue stiffness are introduced to mimic pathological airway restrictions typical for CF. This leads to a more heterogeneous ventilation of the model lung. As a result the concentration in some distal parts of the lung model remains increased for a longer duration. The inert gas concentration at the mouth towards the end of the expirations is composed of gas from regions with very different washout efficiency. This results in a steeper slope of the corresponding part of the washout profile.

Toxicity of aged gasoline exhaust particles to normal and diseased airway epithelia

Particulate matter (PM) pollution is a leading cause of premature death, particularly in those with pre-existing lung disease. A causative link between particle properties and adverse health effects remains unestablished mainly due to complex and variable physico-chemical PM parameters. Controlled laboratory experiments are required. Generating atmospherically realistic Aerosols and performing cell-exposure studies at relevant particle-doses are challenging. Here we examine gasoline-exhaust particle toxicity from a Euro-5 passenger car in a uniquely realistic exposure scenario, combining a smog chamber simulating atmospheric ageing, an aerosol enrichment System varying particle number concentration independent of particle chemistry, and an aerosol Deposition chamber physiologically delivering particles on air-liquid interface (ALI) cultures reproducing normal and susceptible health status. Gasoline-exhaust is an important PM source with largely unknown health effects. We investigated acute responses of fully-differentiated normal, distressed (antibiotics treated) normal, and cystic fibrosis human bronchial epithelia (HBE), and a proliferating, single-cell type bronchial epithelial cell-line (BEAS-2B). We show that a single, short-term exposure to realistic doses of atmospherically-aged gasoline-exhaust particles impairs epithelial key-defence mechanisms, rendering it more vulnerable to subsequent hazards. We establish dose-response curves at realistic particle-concentration levels. Significant differences between cell models suggest the use of fully differentiated HBE is most appropriate in future toxicity studies.

Multivalency effects in Pseudomonas aeruginosa biofilm inhibition and dispersal by glycopeptide dendrimers targeting lectin LecA

The galactose specific lectin LecA partly mediates the formation of antibiotic resistant biofilms by Pseudomonas aeruginosa, an opportunistic pathogen causing lethal airways infections in immunocompromised and cystic fibrosis patients, suggesting that preventing LecA binding to natural saccharides might provide new opportunities for treatment. Here 8-fold (G3) and 16-fold (G4) galactosylated analogs of GalAG2, a tetravalent G2 glycopeptide dendrimer LecA ligand and P. aeruginosa biofilm inhibitor, were obtained by convergent chloroacetyl thioether (ClAc) ligation between 4-fold or 8-fold chloroacetylated dendrimer cores and digalactosylated dendritic arms. Hemagglutination inhibition, isothermal titration calorimetry and biofilm inhibition assays showed that G3 dendrimers bind LecA slightly better than their parent G2 dendrimers and induce complete biofilm inhibition and dispersal of P. aeruginosa biofilms, while G4 dendrimers show reduced binding and no biofilm inhibition. A binding model accounting for the observed saturation of glycopeptide dendrimer galactosyl groups and LecA binding sites is proposed based on the crystal structure of a G3 dendrimer LecA complex.

Retraction

Vareille M, Kieninger E, Alves MP, et al. Impaired type I and type III interferon induction and rhinovirus control in human cystic fibrosis airway epithelial cells. Thorax 2012;67:517-25. This article has been retracted. In our article recently published in Thorax, we described a novel mechanism explaining the increased susceptibility of patients with cystic fibrosis (CF) to rhinovirus infections, namely defective interferon type I and III production by CF airway epithelial cells. In experiments performed after publication of the article we were unable to consistently replicate our findings of deficient interferon type I and III production by CF airway epithelial cells upon rhinovirus infection. In the light of these results, we carried out detailed investigations of the data reported in the above manuscript and regrettably found evidence of deliberate manipulation of experimental data by the first author Dr M. Vareille. This manipulation was accompanied in some instances by absence of original data files. The manipulation/original data absence involved data presented in most, if not all of the figures, thus we wish to fully retract the paper and apologise to the readers of Thorax and to the scientific community for the inconvenience this has caused. We also checked data published by our group in manuscripts on which Dr Vareille was a co-author and found that data published in these manuscripts had not been manipulated. These two manuscripts, whose data and conclusions we stand by are: Edwards MR, Regamey N, Vareille M, et al. Impaired innate interferon induction in severe therapy resistant atopic asthmatic children. Mucosal Immunol 2013;6:797–806. doi: 10.1038/mi.2012.118. and Kieninger E, Vareille M, Kopf BS, et al. Lack of an exaggerated inflammatory response on virus infection in cystic fibrosis. Eur Respir J 2012;39:297–304. doi: 10.1183/09031936.00054511. Dr. Vareille has received a letter from the Secretary General of the University of Bern condemning her scientific misconduct as a severe offence against the rules of scientific integrity. Her current employers have also been informed. All co-authors of the publication including Dr. Vareille concur with the retraction statement.

BIOCHEMICAL CHARACTERIZATION OF BINDING PARTNERS OF TWO HSP70 CO-CHAPERONES IN SACCHAROMYCES CEREVISIAE

Cells are exposed to a variety of environmental and physiological changes including temperature, pH and nutrient availability. These changes cause stress to cells, which results in protein misfolding and altered cellular protein homeostasis. How proteins fold into their three-dimensional functional structure is a fundamental biological process with important relevance to human health. Misfolded and aggregated proteins are linked to multiple neurodegenerative diseases, cardiovascular disease and cystic fibrosis. To combat proteotoxic stress, cells deploy an array of molecular chaperones that assist in the repair or removal of misfolded proteins. Hsp70, an evolutionarily conserved molecular chaperone, promotes protein folding and helps maintain them in a functional state. Requisite co-chaperones, including nucleotide exchange factors (NEFs) strictly regulate and serve to recruit Hsp70 to distinct cellular processes or locations. In yeast and human cells, three structurally non-related cytosolic NEFs are present: Sse1 (Hsp110), Fes1 (HspBP1) and Snl1 (Bag-1). Snl1 is unique among the cytosolic NEFs as it is localized at the ER membrane with its Hsp70 binding (BAG) domain exposed to the cytosol. I discovered that Snl1 distinctly interacts with assembled ribosomes and several lines of evidence indicate that this interaction is both independent of and concurrent with binding to Hsp70 and is not dependent on membrane localization. The ribosome-binding site is identified as a short lysine-rich motif within the amino terminus of the Snl1 BAG domain distinct from the Hsp70 interaction region. In addition, I demonstrate ribosome association with the Snl1 homolog in the pathogenic fungus, Candida albicans and localize this putative NEF to a perinuclear/ER membrane, suggesting functional conservation in fungal BAG domain-containing proteins. As a first step in determining specific domain architecture in fungal BAG proteins, I present the preliminary steps of protein purification and analysis of the minimal Hsp70 binding region in in both S.cerevisiae and C. albicans Snl1. Contrary to previous in vitro evidence which showed the Fes1 NEF to interact with both cytosolic Hsp70s, Ssa and Ssb, Fes1 is shown to interact specifically with Ssa when expressed under normal cellular conditions in S. cerevisiae. This is the first reported evidence of Hsp70 binding selectivity for a cytosolic NEF, and suggests a possible mechanism to achieve specificity in Hsp70-dependent functions. Taken together, the work presented in this dissertation highlights the striking divergence among Hsp70 co-chaperones in selecting binding partners, which may correlate with their specific roles in the cell.

Análisis sobre la eficacia de niveles moderados de actividad física para reducir el riesgo de padecer enfermedades cardiovasculares : Un Meta-análisis

Objetivos: a. Determinar la eficacia de la actividad física en la reducción del riesgo de enfermedades cardiovasculares; b. Analizar los cambios relativos en los niveles de riesgo a padecer enfermedades cardiovasculares de acuerdo a diferentes intensidades (baja-moderada) de actividad física. Metodología: Se realizó un Meta-análisisde los estudios encontrados en la base de datos PUBMED. Se calculó el tamaño del efecto medio y se aplicó el test de Eggerpara descartar un posible sesgo de publicación. Al detectarse heterogeneidad, se procedió a realizar un análisis de las variables moderadoras. Resultados: Se obtuvo un tamaño del efecto medio de 0.762 (0.678-0857; 95 por ciento IC). El test de Egger arrojó un p-valor de 0.67 (I.C. 0.95), de manera tal que se confirmó la ausencia de sesgo de publicación. El análisis moderador determinó que los años de seguimiento (p=0.000; 95 por ciento IC) y los países de estudio (p=0.0096; 95 por ciento IC) son significativos. Conclusión: Se puede concluir en que la práctica de actividad física a intensidades moderadas ofrece un efecto protector sobre los individuos que la realizan disminuyendo el riesgo a padecer enfermedades cardiovasculares

Efectos de un programa de actividad física en escolares

Objetivos: Comprobar el efecto que tiene sobre el alumnado de Educación Primaria un programa de actividad física durante los recreos escolares mediante juegos pre-deportivos, en la composición corporal y en la condición física de los mismos. Metodología: Se realizó un análisis de las diferencias antes-después de muestras independientes de las pruebas de condición física y de las medidas antropométricas (grupo control y experimental), así como el valor de p de los contrastes, realizado mediante un análisis estadístico con el programa IBM SPSS 20.0. Aplicación a 54 alumnos y alumnas de Educación Primaria, de entre 9 y 11 años. Instrumentos: test de los 500 metros (resistencia); test de lanzamiento de balón medicinal (fuerza tren superior); test del salto horizontal (fuerza tren inferior); test de los 50 metros (velocidad); test de flexión profunda de tronco (flexibilidad). Resultados: Un estilo de vida activo, practicando actividad física diaria, se relaciona con mejoras en la condición física y en la composición corporal de sus practicantes. Conclusiones: Un programa de actividad física realizado en los recreos escolares con una duración de 30 minutos durante tres días a la semana, produce mejoras en los valores del IMC y la condición física de quien lo practica.

Análisis sobre la eficacia de niveles moderados de actividad física para reducir el riesgo de padecer enfermedades cardiovasculares : Un Meta-análisis

Objetivos: a. Determinar la eficacia de la actividad física en la reducción del riesgo de enfermedades cardiovasculares; b. Analizar los cambios relativos en los niveles de riesgo a padecer enfermedades cardiovasculares de acuerdo a diferentes intensidades (baja-moderada) de actividad física. Metodología: Se realizó un Meta-análisisde los estudios encontrados en la base de datos PUBMED. Se calculó el tamaño del efecto medio y se aplicó el test de Eggerpara descartar un posible sesgo de publicación. Al detectarse heterogeneidad, se procedió a realizar un análisis de las variables moderadoras. Resultados: Se obtuvo un tamaño del efecto medio de 0.762 (0.678-0857; 95 por ciento IC). El test de Egger arrojó un p-valor de 0.67 (I.C. 0.95), de manera tal que se confirmó la ausencia de sesgo de publicación. El análisis moderador determinó que los años de seguimiento (p=0.000; 95 por ciento IC) y los países de estudio (p=0.0096; 95 por ciento IC) son significativos. Conclusión: Se puede concluir en que la práctica de actividad física a intensidades moderadas ofrece un efecto protector sobre los individuos que la realizan disminuyendo el riesgo a padecer enfermedades cardiovasculares

Efectos de un programa de actividad física en escolares

Objetivos: Comprobar el efecto que tiene sobre el alumnado de Educación Primaria un programa de actividad física durante los recreos escolares mediante juegos pre-deportivos, en la composición corporal y en la condición física de los mismos. Metodología: Se realizó un análisis de las diferencias antes-después de muestras independientes de las pruebas de condición física y de las medidas antropométricas (grupo control y experimental), así como el valor de p de los contrastes, realizado mediante un análisis estadístico con el programa IBM SPSS 20.0. Aplicación a 54 alumnos y alumnas de Educación Primaria, de entre 9 y 11 años. Instrumentos: test de los 500 metros (resistencia); test de lanzamiento de balón medicinal (fuerza tren superior); test del salto horizontal (fuerza tren inferior); test de los 50 metros (velocidad); test de flexión profunda de tronco (flexibilidad). Resultados: Un estilo de vida activo, practicando actividad física diaria, se relaciona con mejoras en la condición física y en la composición corporal de sus practicantes. Conclusiones: Un programa de actividad física realizado en los recreos escolares con una duración de 30 minutos durante tres días a la semana, produce mejoras en los valores del IMC y la condición física de quien lo practica.

Análisis sobre la eficacia de niveles moderados de actividad física para reducir el riesgo de padecer enfermedades cardiovasculares : Un Meta-análisis

Objetivos: a. Determinar la eficacia de la actividad física en la reducción del riesgo de enfermedades cardiovasculares; b. Analizar los cambios relativos en los niveles de riesgo a padecer enfermedades cardiovasculares de acuerdo a diferentes intensidades (baja-moderada) de actividad física. Metodología: Se realizó un Meta-análisisde los estudios encontrados en la base de datos PUBMED. Se calculó el tamaño del efecto medio y se aplicó el test de Eggerpara descartar un posible sesgo de publicación. Al detectarse heterogeneidad, se procedió a realizar un análisis de las variables moderadoras. Resultados: Se obtuvo un tamaño del efecto medio de 0.762 (0.678-0857; 95 por ciento IC). El test de Egger arrojó un p-valor de 0.67 (I.C. 0.95), de manera tal que se confirmó la ausencia de sesgo de publicación. El análisis moderador determinó que los años de seguimiento (p=0.000; 95 por ciento IC) y los países de estudio (p=0.0096; 95 por ciento IC) son significativos. Conclusión: Se puede concluir en que la práctica de actividad física a intensidades moderadas ofrece un efecto protector sobre los individuos que la realizan disminuyendo el riesgo a padecer enfermedades cardiovasculares

Efectos de un programa de actividad física en escolares

Objetivos: Comprobar el efecto que tiene sobre el alumnado de Educación Primaria un programa de actividad física durante los recreos escolares mediante juegos pre-deportivos, en la composición corporal y en la condición física de los mismos. Metodología: Se realizó un análisis de las diferencias antes-después de muestras independientes de las pruebas de condición física y de las medidas antropométricas (grupo control y experimental), así como el valor de p de los contrastes, realizado mediante un análisis estadístico con el programa IBM SPSS 20.0. Aplicación a 54 alumnos y alumnas de Educación Primaria, de entre 9 y 11 años. Instrumentos: test de los 500 metros (resistencia); test de lanzamiento de balón medicinal (fuerza tren superior); test del salto horizontal (fuerza tren inferior); test de los 50 metros (velocidad); test de flexión profunda de tronco (flexibilidad). Resultados: Un estilo de vida activo, practicando actividad física diaria, se relaciona con mejoras en la condición física y en la composición corporal de sus practicantes. Conclusiones: Un programa de actividad física realizado en los recreos escolares con una duración de 30 minutos durante tres días a la semana, produce mejoras en los valores del IMC y la condición física de quien lo practica.

Fluorescence energy transfer detection as a homogeneous DNA diagnostic method

A homogeneous DNA diagnostic assay based on template-directed primer extension detected by fluorescence resonance energy transfer, named template-directed dye-terminator incorporation (TDI) assay, has been developed for mutation detection and high throughput genome analysis. Here, we report the successful application of the TDI assay to detect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the human leukocyte antigen H (HLA-H) gene, and the receptor tyrosin kinase (RET) protooncogene that are associated with cystic fibrosis, hemochromatosis, and multiple endocrine neoplasia, type 2, respectively. Starting with total human DNA, the samples are amplified by the PCR followed by enzymatic degradation of excess primers and deoxyribonucleoside triphosphates before the primer extension reaction is performed. All these standardized steps are performed in the same tube, and the fluorescence changes are monitored in real time, making it a useful clinical DNA diagnostic method.

Development of an epithelium-specific expression cassette with human DNA regulatory elements for transgene expression in lung airways

The efficient expression of therapeutic genes in target cells or tissues is an important component of efficient and safe gene therapy. Utilizing regulatory elements from the human cytokeratin 18 (K18) gene, including 5′ genomic sequences and one of its introns, we have developed a novel expression cassette that can efficiently express reporter genes, as well as the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, in cultured lung epithelial cells. CFTR transcripts expressed from the native K18 enhancer/promoter include two alternative splicing products, due to the activation of two cryptic splice sites in the CFTR coding region. Modification of the K18 intron and CFTR cDNA sequences eliminated the cryptic splice sites without changing the CFTR amino acid sequence, and led to enhanced CFTR mRNA and protein expression as well as biological function. Transgenic expression analysis in mice showed that the modified expression cassette can direct efficient and epithelium-specific expression of the Escherichia coli LacZ gene in the airways of fetal lungs, with no detectable expression in lung fibroblasts or endothelial cells. This is the first expression cassette which selectively directs lung transgene expression for CFTR gene therapy to airway epithelia.