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Oxidative stress is believed to be a major driver of inflammation in smoking asthmatics. The U-BIOPRED project recruited a cohort of Severe Asthma smokers/ex-smokers (SAs/ex) and non-smokers (SAn) with extensive clinical and biomarker information enabling characterization of these subjects. We investigated oxidative stress in severe asthma subjects by analysing urinary 8-iso-PGF2α and the mRNA-expression of the main pro-oxidant (NOX2; NOSs) and anti-oxidant (SODs; CAT; GPX1) enzymes in the airways of SAs/ex and SAn. All the severe asthma U-BIOPRED subjects were further divided into current smokers with severe asthma (CSA), ex-smokers with severe asthma (ESA) and non-smokers with severe asthma (NSA) to deepen the effect of active smoking. Clinical data, urine and sputum were obtained from severe asthma subjects. A bronchoscopy to obtain bronchial biopsy and brushing was performed in a subset of subjects. The main clinical data were analysed for each subset of subjects (urine-8-iso-PGF2α; IS-transcriptomics; BB-transcriptomics; BBr-transcriptomics). Urinary 8-iso-PGF2α was quantified using mass spectrometry. Sputum, bronchial biopsy and bronchial brushing were processed for mRNA expression microarray analysis. Urinary 8-iso-PGF2α was increased in SAs/ex, median (IQR) = 31.7 (24.5-44.7) ng/mmol creatinine, compared to SAn, median (IQR) = 26.6 (19.6-36.6) ng/mmol creatinine (p< 0.001), and in CSA, median (IQR) = 34.25 (24.4-47.7), vs. ESA, median (IQR) = 29.4 (22.3-40.5), and NSA, median (IQR) = 26.5 (19.6-16.6) ng/mmol creatinine (p = 0.004). Sputum mRNA expression of NOX2 was increased in SAs/ex compared to SAn (probe sets 203922_PM_s_at fold-change = 1.05 p = 0.006; 203923_PM_s_at fold-change = 1.06, p = 0.003; 233538_PM_s_at fold-change = 1.06, p = 0.014). The mRNA expression of antioxidant enzymes were similar between the two severe asthma cohorts in all airway samples. NOS2 mRNA expression was decreased in bronchial brushing of SAs/ex compared to SAn (fold-change = -1.10; p = 0.029). NOS2 mRNA expression in bronchial brushing correlated with FeNO (Kendal's Tau = 0.535; p< 0.001). From clinical and inflammatory analysis, FeNO was lower in CSA than in ESA in all the analysed subject subsets (p< 0.01) indicating an effect of active smoking. Results about FeNO suggest its clinical limitation, as inflammation biomarker, in severe asthma active smokers. These data provide evidence of greater systemic oxidative stress in severe asthma smokers as reflected by a significant changes of NOX2 mRNA expression in the airways, together with elevated urinary 8-iso-PGF2α in the smokers/ex-smokers group. Trial registration ClinicalTrials.gov-Identifier: NCT01976767.

OBJECTIVE:To investigate whether exhaled breath analysis using an electronic nose can identify differences between inflammatory joint diseases and healthy controls. METHODS:In a cross-sectional study, the exhaled breath of 21 rheumatoid arthritis (RA) and 18 psoriatic arthritis (PsA) patients with active disease was compared to 21 healthy controls using an electronic nose (Cyranose 320; Smiths Detection, Pasadena, CA, USA). Breathprints were analyzed with principal component analysis, discriminant analysis, and area under curve (AUC) of receiver operating characteristics (ROC) curves. Volatile organic compounds (VOCs) were identified by gas chromatography and mass spectrometry (GC-MS), and relationships between breathprints and markers of disease activity were explored. RESULTS:Breathprints of RA patients could be distinguished from controls with an accuracy of 71% (AUC 0.75, 95% CI 0.60-0.90, sensitivity 76%, specificity 67%). Breathprints from PsA patients were separated from controls with 69% accuracy (AUC 0.77, 95% CI 0.61-0.92, sensitivity 72%, specificity 71%). Distinction between exhaled breath of RA and PsA patients exhibited an accuracy of 69% (AUC 0.72, 95% CI 0.55-0.89, sensitivity 71%, specificity 72%). There was a positive correlation in RA patients of exhaled breathprints with disease activity score (DAS28) and number of painful joints. GC-MS identified seven key VOCs that significantly differed between the groups. CONCLUSIONS:Exhaled breath analysis by an electronic nose may play a role in differential diagnosis of inflammatory joint diseases. Data from this study warrant external validation.

BACKGROUND:The decline in lung function can be reduced by long-term inhaled corticosteroid (ICS) treatment in subsets of patients with chronic obstructive pulmonary disease (COPD). We aimed to identify which clinical, physiological and non-invasive inflammatory characteristics predict the benefits of ICS on lung function decline in COPD. METHODS:Analysis was performed in 50 steroid-naive compliant patients with moderate to severe COPD (postbronchodilator forced expiratory volume in one second (FEV1), 30-80% of predicted, compatible with GOLD stages II-III), age 45-75 years, >10 packyears smoking and without asthma. Patients were treated with fluticasone propionate (500 μg bid) or placebo for 2.5 years. Postbronchodilator FEV1, dyspnea and health status were measured every 3 months; lung volumes, airway hyperresponsiveness (PC20), and induced sputum at 0, 6 and 30 months. A linear mixed effect model was used for analysis of this hypothesis generating study. RESULTS:Significant predictors of attenuated FEV1-decline by fluticasone treatment compared to placebo were: fewer packyears smoking, preserved diffusion capacity, limited hyperinflation and lower inflammatory cell counts in induced sputum (p

Chronic mucus hypersecretion (CMH) is associated with an increased frequency of respiratory infections, excess lung function decline, and increased hospitalisation and mortality rates in the general population. It is associated with smoking, but it is unknown why only a minority of smokers develops CMH. A plausible explanation for this phenomenon is a predisposing genetic constitution. Therefore, we performed a genome wide association (GWA) study of CMH in Caucasian populations.GWA analysis was performed in the NELSON-study using the Illumina 610 array, followed by replication and meta-analysis in 11 additional cohorts. In total 2,704 subjects with, and 7,624 subjects without CMH were included, all current or former heavy smokers (≥20 pack-years). Additional studies were performed to test the functional relevance of the most significant single nucleotide polymorphism (SNP).A strong association with CMH, consistent across all cohorts, was observed with rs6577641 (p = 4.25×10(-6), OR = 1.17), located in intron 9 of the special AT-rich sequence-binding protein 1 locus (SATB1) on chromosome 3. The risk allele (G) was associated with higher mRNA expression of SATB1 (4.3×10(-9)) in lung tissue. Presence of CMH was associated with increased SATB1 mRNA expression in bronchial biopsies from COPD patients. SATB1 expression was induced during differentiation of primary human bronchial epithelial cells in culture.Our findings, that SNP rs6577641 is associated with CMH in multiple cohorts and is a cis-eQTL for SATB1, together with our additional observation that SATB1 expression increases during epithelial differentiation provide suggestive evidence that SATB1 is a gene that affects CMH.

Inhaled corticosteroids (ICS) reduce exacerbation rates and improve health status but can increase the risk of pneumonia in COPD. The GLUCOLD study, investigating patients with mild-to-moderate COPD, has shown that long-term (2.5-year) ICS therapy induces anti-inflammatory effects. The literature suggests that cigarette smoking causes ICS insensitivity. The aim of this study is to compare anti-inflammatory effects of ICS in persistent smokers and persistent ex-smokers in a post-hoc analysis of the GLUCOLD study.Persistent smokers (n = 41) and persistent ex-smokers (n = 31) from the GLUCOLD cohort were investigated. Effects of ICS treatment compared with placebo were estimated by analysing changes in lung function, hyperresponsiveness, and inflammatory cells in sputum and bronchial biopsies during short-term (0-6 months) and long-term (6-30 months) treatment using multiple regression analyses.Bronchial mast cells were reduced by short-term and long-term ICS treatment in both smokers and ex-smokers. In contrast, CD3⁺, CD4⁺, and CD8⁺ cells were reduced by short-term ICS treatment in smokers only. In addition, sputum neutrophils and lymphocytes, and bronchial CD8⁺ cells were reduced after long-term treatment in ex-smokers only. No significant interactions existed between smoking and ICS treatment.Even in the presence of smoking, long-term ICS treatment may lead to anti-inflammatory effects in the lung. Some anti-inflammatory ICS effects are comparable in smokers and ex-smokers with COPD, other effects are cell-specific. The clinical relevance of these findings, however, are uncertain.

Ideally, invading bacteria are detected as early as possible in critically ill patients: the strain of morbific pathogens is identified rapidly, and antimicrobial sensitivity is known well before the start of new antimicrobial therapy. Bacteria have a distinct metabolism, part of which results in the production of bacteria-specific volatile organic compounds (VOCs), which might be used for diagnostic purposes. Volatile metabolites can be investigated directly in exhaled air, allowing for noninvasive monitoring. The aim of this review is to provide an overview of VOCs produced by the six most abundant and pathogenic bacteria in sepsis, including Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli. Such VOCs could be used as biological markers in the diagnostic approach of critically ill patients. A systematic review of existing literature revealed 31 articles. All six bacteria of interest produce isopentanol, formaldehyde, methyl mercaptan, and trimethylamine. Since humans do not produce these VOCs, they could serve as biological markers for presence of these pathogens. The following volatile biomarkers were found for identification of specific strains: isovaleric acid and 2-methyl-butanal for Staphylococcus aureus; 1-undecene, 2,4-dimethyl-1-heptane, 2-butanone, 4-methyl-quinazoline, hydrogen cyanide, and methyl thiocyanide for Pseudomonas aeruginosa; and methanol, pentanol, ethyl acetate, and indole for Escherichia coli. Notably, several factors that may effect VOC production were not controlled for, including used culture media, bacterial growth phase, and genomic variation within bacterial strains. In conclusion, VOCs produced by bacteria may serve as biological markers for their presence. Goal-targeted studies should be performed to identify potential sets of volatile biological markers and evaluate the diagnostic accuracy of these markers in critically ill patients.

BACKGROUND: The link between upper and lower airways in patients with both asthma and allergic rhinitis is still poorly understood. As the biological complexity of these disorders can be captured by gene expression profiling we hypothesized that the clinical expression of rhinitis and/or asthma is related to differential gene expression between upper and lower airways epithelium. OBJECTIVE: Defining gene expression profiles of primary nasal and bronchial epithelial cells from the same individuals and examining the impact of allergic rhinitis with and without concomitant allergic asthma on expression profiles. METHODS: This cross-sectional study included 18 subjects (6 allergic asthma and allergic rhinitis; 6 allergic rhinitis; 6 healthy controls). The estimated false discovery rate comparing 6 subjects per group was approximately 5%. RNA was extracted from isolated and cultured epithelial cells from bronchial brushings and nasal biopsies, and analyzed by microarray (Affymetrix U133+ PM Genechip Array). Data were analysed using R and Bioconductor Limma package. For gene ontology GeneSpring GX12 was used. RESULTS: The study was successfully completed by 17 subjects (6 allergic asthma and allergic rhinitis; 5 allergic rhinitis; 6 healthy controls). Using correction for multiple testing, 1988 genes were differentially expressed between healthy lower and upper airway epithelium, whereas in allergic rhinitis with or without asthma this was only 40 and 301 genes, respectively. Genes influenced by allergic rhinitis with or without asthma were linked to lung development, remodeling, regulation of peptidases and normal epithelial barrier functions. CONCLUSIONS: Differences in epithelial gene expression between the upper and lower airway epithelium, as observed in healthy subjects, largely disappear in patients with allergic rhinitis with or without asthma, whilst new differences emerge. The present data identify several pathways and genes that might be potential targets for future drug development.

Smoking and inflammation contribute to the pathogenesis of chronic obstructive pulmonary disease (COPD), which involves changes in extracellular matrix. This is thought to contribute to airway remodeling and airflow obstruction. We have previously observed that long-term treatment with inhaled corticosteroids can not only reduce bronchial inflammation, but can also attenuate lung function decline in moderate-severe COPD. We hypothesized that inhaled corticosteroids and current smoking modulate bronchial extracellular matrix components in COPD.To compare major extracellular matrix components (elastic fibers; proteoglycans [versican, decorin]; collagens type I and III) in bronchial biopsies 1) after 30-months inhaled steroids treatment or placebo; and 2) between current and ex-smokers with COPD.We included 64 moderate-severe, steroid-naive COPD patients (24/40 (ex)-smokers, 62±7 years, 46 (31-54) packyears, post-bronchodilator forced expiratory volume in one second (FEV1) 62±9% predicted) at baseline in this randomized, controlled trial. 19 and 13 patients received 30-months treatment with fluticasone or placebo, respectively. Bronchial biopsies collected at baseline and after 30 months were studied using (immuno)histochemistry to evaluate extracellular matrix content. Percentage and density of stained area were calculated by digital image analysis.30-Months inhaled steroids increased the percentage stained area of versican (9.6% [CI 0.9 to 18.3%]; p = 0.03) and collagen III (20.6% [CI 3.8 to 37.4%]; p = 0.02) compared to placebo. Increased collagen I staining density correlated with increased post-bronchodilator FEV1 after inhaled steroids treatment (Rs = 0.45, p = 0.04). There were no differences between smokers and ex-smokers with COPD in percentages and densities for all extracellular matrix proteins.These data show that long-term inhaled corticosteroids treatment partially changes the composition of extracellular matrix in moderate-severe COPD. This is associated with increased lung function, suggesting that long-term inhaled steroids modulate airway remodeling thereby potentially preventing airway collapse in COPD. Smoking status is not associated with bronchial extracellular matrix proteins.ClinicalTrials.gov NCT00158847.

CONTEXT: Publication bias jeopardizes evidence-based medicine, mainly through biased literature syntheses. Publication bias may also affect laboratory animal research, but evidence is scarce. OBJECTIVES: To assess the opinion of laboratory animal researchers on the magnitude, drivers, consequences and potential solutions for publication bias. And to explore the impact of size of the animals used, seniority of the respondent, working in a for-profit organization and type of research (fundamental, pre-clinical, or both) on those opinions. DESIGN: Internet-based survey. SETTING: All animal laboratories in The Netherlands. PARTICIPANTS: Laboratory animal researchers. MAIN OUTCOME MEASURE(S): Median (interquartile ranges) strengths of beliefs on 5 and 10-point scales (1: totally unimportant to 5 or 10: extremely important). RESULTS: Overall, 454 researchers participated. They considered publication bias a problem in animal research (7 (5 to 8)) and thought that about 50% (32-70) of animal experiments are published. Employees (n = 21) of for-profit organizations estimated that 10% (5 to 50) are published. Lack of statistical significance (4 (4 to 5)), technical problems (4 (3 to 4)), supervisors (4 (3 to 5)) and peer reviewers (4 (3 to 5)) were considered important reasons for non-publication (all on 5-point scales). Respondents thought that mandatory publication of study protocols and results, or the reasons why no results were obtained, may increase scientific progress but expected increased bureaucracy. These opinions did not depend on size of the animal used, seniority of the respondent or type of research. CONCLUSIONS: Non-publication of "negative" results appears to be prevalent in laboratory animal research. If statistical significance is indeed a main driver of publication, the collective literature on animal experimentation will be biased. This will impede the performance of valid literature syntheses. Effective, yet efficient systems should be explored to counteract selective reporting of laboratory animal research.

BackgroundEffectiveness of Internet-based self-management in patients with asthma has been shown, but its cost-effectiveness is unknown. We conducted a cost-effectiveness analysis of Internet-based asthma self-management compared with usual care.Methodology and principal findingsCost-effectiveness analysis alongside a randomized controlled trial, with 12 months follow-up. Patients were aged 18 to 50 year and had physician diagnosed asthma. The Internet-based self-management program involved weekly on-line monitoring of asthma control with self-treatment advice, remote Web communications, and Internet-based information. We determined quality adjusted life years (QALYs) as measured by the EuroQol-5D and costs for health care use and absenteeism. We performed a detailed cost price analysis for the primary intervention. QALYs did not statistically significantly differ between the Internet group and usual care: difference 0.024 (95% CI, -0.016 to 0.065). Costs of the Internet-based intervention were $254 (95% CI, $243 to $265) during the period of 1 year. From a societal perspective, the cost difference was $641 (95% CI, $-1957 to $3240). From a health care perspective, the cost difference was $37 (95% CI, $-874 to $950). At a willingness-to-pay of $50,000 per QALY, the probability that Internet-based self-management was cost-effective compared to usual care was 62% and 82% from a societal and health care perspective, respectively.ConclusionsInternet-based self-management of asthma can be as effective as current asthma care and costs are similar.Trial registrationCurrent Controlled Trials ISRCTN79864465.