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1.  Modules, networks and systems medicine for understanding disease and aiding diagnosis 
Genome Medicine  2014;6(10):82.
Many common diseases, such as asthma, diabetes or obesity, involve altered interactions between thousands of genes. High-throughput techniques (omics) allow identification of such genes and their products, but functional understanding is a formidable challenge. Network-based analyses of omics data have identified modules of disease-associated genes that have been used to obtain both a systems level and a molecular understanding of disease mechanisms. For example, in allergy a module was used to find a novel candidate gene that was validated by functional and clinical studies. Such analyses play important roles in systems medicine. This is an emerging discipline that aims to gain a translational understanding of the complex mechanisms underlying common diseases. In this review, we will explain and provide examples of how network-based analyses of omics data, in combination with functional and clinical studies, are aiding our understanding of disease, as well as helping to prioritize diagnostic markers or therapeutic candidate genes. Such analyses involve significant problems and limitations, which will be discussed. We also highlight the steps needed for clinical implementation.
doi:10.1186/s13073-014-0082-6
PMCID: PMC4254417  PMID: 25473422
2.  Glucose prediction by analysis of exhaled metabolites – a systematic review 
BMC Anesthesiology  2014;14:46.
Background
In critically ill patients, glucose control with insulin mandates time– and blood–consuming glucose monitoring. Blood glucose level fluctuations are accompanied by metabolomic changes that alter the composition of volatile organic compounds (VOC), which are detectable in exhaled breath. This review systematically summarizes the available data on the ability of changes in VOC composition to predict blood glucose levels and changes in blood glucose levels.
Methods
A systematic search was performed in PubMed. Studies were included when an association between blood glucose levels and VOCs in exhaled air was investigated, using a technique that allows for separation, quantification and identification of individual VOCs. Only studies on humans were included.
Results
Nine studies were included out of 1041 identified in the search. Authors of seven studies observed a significant correlation between blood glucose levels and selected VOCs in exhaled air. Authors of two studies did not observe a strong correlation. Blood glucose levels were associated with the following VOCs: ketone bodies (e.g., acetone), VOCs produced by gut flora (e.g., ethanol, methanol, and propane), exogenous compounds (e.g., ethyl benzene, o–xylene, and m/p–xylene) and markers of oxidative stress (e.g., methyl nitrate, 2–pentyl nitrate, and CO).
Conclusion
There is a relation between blood glucose levels and VOC composition in exhaled air. These results warrant clinical validation of exhaled breath analysis to monitor blood glucose levels.
doi:10.1186/1471-2253-14-46
PMCID: PMC4068184  PMID: 24963286
Glucose; Monitoring; Volatile organic compound; Breath
3.  A Dynamic Bronchial Airway Gene Expression Signature of Chronic Obstructive Pulmonary Disease and Lung Function Impairment 
Rationale: Molecular phenotyping of chronic obstructive pulmonary disease (COPD) has been impeded in part by the difficulty in obtaining lung tissue samples from individuals with impaired lung function.
Objectives: We sought to determine whether COPD-associated processes are reflected in gene expression profiles of bronchial airway epithelial cells obtained by bronchoscopy.
Methods: Gene expression profiling of bronchial brushings obtained from 238 current and former smokers with and without COPD was performed using Affymetrix Human Gene 1.0 ST Arrays.
Measurements and Main Results: We identified 98 genes whose expression levels were associated with COPD status, FEV1% predicted, and FEV1/FVC. In silico analysis identified activating transcription factor 4 (ATF4) as a potential transcriptional regulator of genes with COPD-associated airway expression, and ATF4 overexpression in airway epithelial cells in vitro recapitulates COPD-associated gene expression changes. Genes with COPD-associated expression in the bronchial airway epithelium had similarly altered expression profiles in prior studies performed on small-airway epithelium and lung parenchyma, suggesting that transcriptomic alterations in the bronchial airway epithelium reflect molecular events found at more distal sites of disease activity. Many of the airway COPD-associated gene expression changes revert toward baseline after therapy with the inhaled corticosteroid fluticasone in independent cohorts.
Conclusions: Our findings demonstrate a molecular field of injury throughout the bronchial airway of active and former smokers with COPD that may be driven in part by ATF4 and is modifiable with therapy. Bronchial airway epithelium may ultimately serve as a relatively accessible tissue in which to measure biomarkers of disease activity for guiding clinical management of COPD.
doi:10.1164/rccm.201208-1449OC
PMCID: PMC3707363  PMID: 23471465
chronic obstructive pulmonary disease; gene expression profiling; biologic markers
4.  Exhaled breath profiling for diagnosing acute respiratory distress syndrome 
Background
The acute respiratory distress syndrome (ARDS) is a common, devastating complication of critical illness that is characterized by pulmonary injury and inflammation. The clinical diagnosis may be improved by means of objective biological markers. Electronic nose (eNose) technology can rapidly and non–invasively provide breath prints, which are profiles of volatile metabolites in the exhaled breath. We hypothesized that breath prints could facilitate accurate diagnosis of ARDS in intubated and ventilated intensive care unit (ICU) patients.
Methods
Prospective single-center cohort study with training and temporal external validation cohort. Breath of newly intubated and mechanically ventilated ICU-patients was analyzed using an electronic nose within 24 hours after admission. ARDS was diagnosed and classified by the Berlin clinical consensus definition. The eNose was trained to recognize ARDS in a training cohort and the diagnostic performance was evaluated in a temporal external validation cohort.
Results
In the training cohort (40 patients with ARDS versus 66 controls) the diagnostic model for ARDS showed a moderate discrimination, with an area under the receiver–operator characteristic curve (AUC–ROC) of 0.72 (95%–confidence interval (CI): 0.63-0.82). In the external validation cohort (18 patients with ARDS versus 26 controls) the AUC–ROC was 0.71 [95%–CI: 0.54 – 0.87]. Restricting discrimination to patients with moderate or severe ARDS versus controls resulted in an AUC–ROC of 0.80 [95%–CI: 0.70 – 0.90]. The exhaled breath profile from patients with cardiopulmonary edema and pneumonia was different from that of patients with moderate/severe ARDS.
Conclusions
An electronic nose can rapidly and non–invasively discriminate between patients with and without ARDS with modest accuracy. Diagnostic accuracy increased when only moderate and severe ARDS patients were considered. This implicates that breath analysis may allow for rapid, bedside detection of ARDS, especially if our findings are reproduced using continuous exhaled breath profiling.
Trial registration
NTR2750, registered 11 February 2011.
doi:10.1186/1471-2466-14-72
PMCID: PMC4021554  PMID: 24767549
ARDS; Exhaled breath; Electronic nose; Volatile organic compound; Sensitivity and specificity
5.  How integration of global omics-data could help preparing for pandemics – a scent of influenza 
Pandemics caused by novel emerging or re-emerging infectious diseases could lead to high mortality and morbidity world-wide when left uncontrolled. In this perspective, we evaluate the possibility of integration of global omics-data in order to timely prepare for pandemics. Such an approach requires two major innovations. First, data that is obtained should be shared with the global community instantly. The strength of rapid integration of simple signals is exemplified by Google’sTM Flu Trend, which could predict the incidence of influenza-like illness based on online search engine queries. Second, omics technologies need to be fast and high-throughput. We postulate that analysis of the exhaled breath would be a simple, rapid and non-invasive alternative. Breath contains hundreds of volatile organic compounds that are altered by infection and inflammation. The molecular fingerprint of breath (breathprint) can be obtained using an electronic nose, which relies on sensor technology. These breathprints can be stored in an online database (a “breathcloud”) and coupled to clinical data. Comparison of the breathprint of a suspected subject to the breathcloud allows for a rapid decision on the presence or absence of a pathogen.
doi:10.3389/fgene.2014.00080
PMCID: PMC4000993  PMID: 24795745
pandemic; exhaled breath; systems biology; diagnosis; metabolomics; metabolite profiling
6.  Susceptibility to Chronic Mucus Hypersecretion, a Genome Wide Association Study 
PLoS ONE  2014;9(4):e91621.
Background
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.
Methods
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).
Results
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.
Conclusions
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.
doi:10.1371/journal.pone.0091621
PMCID: PMC3979657  PMID: 24714607
7.  Evaluation of coagulation activation after Rhinovirus infection in patients with asthma and healthy control subjects: an observational study 
Respiratory Research  2014;15(1):14.
Background
Asthma exacerbations are frequently triggered by rhinovirus infections. Both asthma and respiratory tract infection can activate haemostasis. Therefore we hypothesized that experimental rhinovirus-16 infection and asthmatic airway inflammation act in synergy on the haemostatic balance.
Methods
28 patients (14 patients with mild allergic asthma and 14 healthy non-allergic controls) were infected with low-dose rhinovirus type 16. Venous plasma and bronchoalveolar lavage fluid (BAL fluid) were obtained before and 6 days after infection to evaluate markers of coagulation activation, thrombin-antithrombin complexes, von Willebrand factor, plasmin-antiplasmin complexes, plasminogen activator inhibitor type-1, endogenous thrombin potential and tissue factor-exposing microparticles by fibrin generation test, in plasma and/or BAL fluid. Data were analysed by nonparametric tests (Wilcoxon, Mann Whitney and Spearman correlation).
Results
13 patients with mild asthma (6 females, 19-29 y) and 11 healthy controls (10 females, 19-31 y) had a documented Rhinovirus-16 infection. Rhinovirus-16 challenge resulted in a shortening of the fibrin generation test in BAL fluid of asthma patients (t = -1: 706 s vs. t = 6: 498 s; p = 0.02), but not of controls (t = -1: 693 s vs. t = 6: 636 s; p = 0.65). The fold change in tissue factor-exposing microparticles in BAL fluid inversely correlated with the fold changes in eosinophil cationic protein and myeloperoxidase in BAL fluid after virus infection (r = -0.517 and -0.528 resp., both p = 0.01).
Rhinovirus-16 challenge led to increased plasminogen activator inhibitor type-1 levels in plasma in patients with asthma (26.0 ng/mL vs. 11.5 ng/mL in healthy controls, p = 0.04). Rhinovirus-16 load in BAL showed a linear correlation with the fold change in endogenous thrombin potential, plasmin-antiplasmin complexes and plasminogen activator inhibitor type-1.
Conclusions
Experimental rhinovirus infection induces procoagulant changes in the airways of patients with asthma through increased activity of tissue factor-exposing microparticles. These microparticle-associated procoagulant changes are associated with both neutrophilic and eosinophilic inflammation. Systemic activation of haemostasis increases with Rhinoviral load.
Trial registration
This trial was registered at the Dutch trial registry (http://www.trialregister.nl): NTR1677.
doi:10.1186/1465-9921-15-14
PMCID: PMC3922343  PMID: 24502801
Rhinovirus; Coagulation; Fibrinolysis; Asthma; Microparticles; Inflammation
8.  Steroid Resistance in COPD? Overlap and Differential Anti-Inflammatory Effects in Smokers and Ex-Smokers 
PLoS ONE  2014;9(2):e87443.
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1371/journal.pone.0087443
PMCID: PMC3914834  PMID: 24505290
9.  dsRNA-induced changes in gene expression profiles of primary nasal and bronchial epithelial cells from patients with asthma, rhinitis and controls 
Respiratory Research  2014;15(1):9.
Background
Rhinovirus infections are the most common cause of asthma exacerbations. The complex responses by airway epithelium to rhinovirus can be captured by gene expression profiling. We hypothesized that: a) upper and lower airway epithelium exhibit differential responses to double-stranded RNA (dsRNA), and b) that this is modulated by the presence of asthma and allergic rhinitis.
Objectives
Identification of dsRNA-induced 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 study had a cross-sectional design including 18 subjects: 6 patients with allergic asthma with concomitant rhinitis, 6 patients with allergic rhinitis, and 6 healthy controls. Comparing 6 subjects per group, the estimated false discovery rate was approximately 5%. RNA was extracted from isolated and cultured primary epithelial cells from nasal biopsies and bronchial brushings stimulated with dsRNA (poly(I:C)), and analyzed by microarray (Affymetrix U133+ PM Genechip Array). Data were analysed using R and the Bioconductor Limma package. Overrepresentation of gene ontology groups were captured by GeneSpring GX12.
Results
In total, 17 subjects completed the study successfully (6 allergic asthma with rhinitis, 5 allergic rhinitis, 6 healthy controls). dsRNA-stimulated upper and lower airway epithelium from asthma patients demonstrated significantly fewer induced genes, exhibiting reduced down-regulation of mitochondrial genes. The majority of genes related to viral responses appeared to be similarly induced in upper and lower airways in all groups. However, the induction of several interferon-related genes (IRF3, IFNAR1, IFNB1, IFNGR1, IL28B) was impaired in patients with asthma.
Conclusions
dsRNA differentially changes transcriptional profiles of primary nasal and bronchial epithelial cells from patients with allergic rhinitis with or without asthma and controls. Our data suggest that respiratory viruses affect mitochondrial genes, and we identified disease-specific genes that provide potential targets for drug development.
doi:10.1186/1465-9921-15-9
PMCID: PMC3916078  PMID: 24475887
Asthma; Rhinitis; Epithelium; Gene expression; dsRNA
10.  The Impact of Allergic Rhinitis and Asthma on Human Nasal and Bronchial Epithelial Gene Expression 
PLoS ONE  2013;8(11):e80257.
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.
doi:10.1371/journal.pone.0080257
PMCID: PMC3839950  PMID: 24282527
11.  Electronic Nose Technology for Detection of Invasive Pulmonary Aspergillosis in Prolonged Chemotherapy-Induced Neutropenia: a Proof-of-Principle Study 
Journal of Clinical Microbiology  2013;51(5):1490-1495.
Although the high mortality rate of pulmonary invasive aspergillosis (IA) in patients with prolonged chemotherapy-induced neutropenia (PCIN) can be reduced by timely diagnosis, a diagnostic test that reliably detects IA at an early stage is lacking. We hypothesized that an electronic nose (eNose) could fulfill this need. An eNose can discriminate various lung diseases through the analysis of exhaled volatile organic compounds (VOCs). An eNose is cheap and noninvasive and yields results within minutes. In a single-center prospective cohort study, we included patients who were treated with chemotherapy expected to result in PCIN. Based on standardized indications, a full diagnostic workup was performed to confirm invasive aspergillosis or to rule it out. Patients with no aspergillosis were considered controls, and patients with probable or proven aspergillosis were considered index cases. Exhaled breath was examined with a Cyranose 320 (Smith Detections, Pasadena, CA). The resulting data were analyzed using principal component reduction. The primary endpoint was cross-validated diagnostic accuracy, defined as the percentage of patients correctly classified using the leave-one-out method. Accuracy was validated by 100,000 random classifications. We included 46 subjects who underwent 16 diagnostic workups, resulting in 6 cases and 5 controls. The cross-validated accuracy of the eNose in diagnosing IA was 90.9% (P = 0.022; sensitivity, 100%; specificity, 83.3%). Receiver operating characteristic analysis showed an area under the curve of 0.93. These preliminary data indicate that PCIN patients with IA have a distinct exhaled VOC profile that can be detected with eNose technology. The diagnostic accuracy of the eNose for invasive aspergillosis warrants validation.
doi:10.1128/JCM.02838-12
PMCID: PMC3647955  PMID: 23467602
12.  Toll-like receptors 2, 3 and 4 and thymic stromal lymphopoietin expression in fatal asthma 
Background
Airway inflammation in asthma involves innate immune responses. Toll-like receptors (TLRs) and thymic stromal lymphopoietin (TSLP) are thought to be involved in airway inflammation, but their expression in asthmatics’ both large and small airways has not been investigated.
Objective
To analyze the expression of TLR2, TLR3, TLR4 and TSLP in large and small airways of asthmatics and compare their expression in smoking and nonsmoking asthmatics; to investigate whether TLR expression is associated with eosinophilic or neutrophilic airway inflammation and with Mycoplasma pneumoniae and Chlamydophila pneumoniae infection.
Methods
Using immunohistochemistry and image analysis, we investigated TLR2, TLR3, TLR4 and TSLP expression in large and small airways of 24 victims of fatal asthma, FA, (13 nonsmokers, 11 smokers) and 9 deceased control subjects (DCtrl). TLRs were also measured in 18 mild asthmatics (MA) and 12 healthy controls (HCtrl). Mycoplasma pneumoniae and Chlamydophila pneumoniae in autopsy lung tissue was analyzed using real-time polymerase chain reaction. Airway eosinophils and neutrophils were measured in all subjects.
Results
Fatal asthma patients had higher TLR2 in the epithelial and outer layers of large and small airways compared with DCtrls. Smoking asthmatics had lower TLR2 levels in the inner and outer layers of the small airways than nonsmoking asthmatics. TSLP was increased in the epithelial and outer layers of the large airways of FA. FA patients had greater TLR3 expression in the outer layer of large airways and greater TLR4 expression in the outer layer of small airways. Eosinophilic airway inflammation was associated with TLR expression in the epithelium of FA. No bacterial DNA was detected in FA or DCtrls. MA and HCtrls had only a small difference in TLR3 expression.
Conclusions and Clinical Relevance
Increased expression of TLR 2, 3 and 4 and TSLP in fatal asthma may contribute to the acute inflammation surrounding asthma deaths.
doi:10.1111/j.1365-2222.2012.04047.x
PMCID: PMC3459227  PMID: 22994343
lung; innate immunity; immunohistochemistry
13.  Inflammatory phenotypes underlying uncontrolled childhood asthma despite inhaled corticosteroid treatment: rationale and design of the PACMAN2 study 
BMC Pediatrics  2013;13:94.
Background
The diagnosis of childhood asthma covers a broad spectrum of pathological mechanisms that can lead to similarly presenting clinical symptoms, but may nonetheless require different treatment approaches. Distinct underlying inflammatory patterns are thought to influence responsiveness to standard asthma medication.
Methods/design
The purpose of the PACMAN2 study is to identify inflammatory phenotypes that can discriminate uncontrolled childhood asthma from controlled childhood asthma by measures in peripheral blood and exhaled air. PACMAN2 is a nested, case–control follow-up study to the ongoing pharmacy-based “Pharmacogenetics of Asthma medication in Children: Medication with Anti-inflammatory effects” (PACMAN) study. The original PACMAN cohort consists of children aged 4–12 years with reported use of asthma medication. The PACMAN2 study will be conducted within the larger PACMAN cohort, and will focus on detailed phenotyping of a subset of the PACMAN children. The selected participants will be invited to a follow-up visit in a clinical setting at least six months after their baseline visit based on their adherence to usage of inhaled corticosteroids, their asthma symptoms in the past year, and their age (≥ 8 years). During the follow-up visit, current and long-term asthma symptoms, medication use, environmental factors, medication adherence and levels of exhaled nitric oxide will be reassessed. The following measures will also be examined: pulmonary function, exhaled volatile organic compounds, as well as inflammatory markers in peripheral blood and blood plasma. Comparative analysis and cluster-analyses will be used to identify markers that differentiate children with uncontrolled asthma despite their use of inhaled corticosteroids (ICS) (cases) from children whose asthma is controlled by the use of ICS (controls).
Discussion
Asthmatic children with distinct inflammatory phenotypes may respond differently to anti-inflammatory therapy. Therefore, by identifying inflammatory phenotypes in children with the PACMAN2 study, we may greatly impact future personalised treatment strategies, uncover new leads for therapeutic targets and improve the design of future clinical studies in the assessment of the efficacy of novel therapeutics.
doi:10.1186/1471-2431-13-94
PMCID: PMC3691827  PMID: 23768206
Asthma; Child; Phenotypes; Inflammation; Proteomics; Volatile organic compounds; Corticosteroids
14.  Volatile Metabolites of Pathogens: A Systematic Review 
PLoS Pathogens  2013;9(5):e1003311.
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.
doi:10.1371/journal.ppat.1003311
PMCID: PMC3649982  PMID: 23675295
15.  Inhaled Steroids Modulate Extracellular Matrix Composition in Bronchial Biopsies of COPD Patients: A Randomized, Controlled Trial 
PLoS ONE  2013;8(5):e63430.
Rationale
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.
Objective
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.
Methods
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.
Results
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.
Conclusions
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.
Trial Registration
ClinicalTrials.gov NCT00158847
doi:10.1371/journal.pone.0063430
PMCID: PMC3646783  PMID: 23667615
16.  Reduction in sputum neutrophil and eosinophil numbers by the PDE4 inhibitor roflumilast in patients with COPD 
Thorax  2007;62(12):1081-1087.
Background
Roflumilast is a targeted oral once‐daily administered phosphodiesterase 4 (PDE4) inhibitor with clinical efficacy in chronic obstructive pulmonary disease (COPD). Results from in vitro studies with roflumilast indicate that it has anti‐inflammatory properties that may be applicable for the treatment of COPD.
Methods
In a crossover study, 38 patients with COPD (mean (SD) age 63.1 (7.0) years, post‐bronchodilator forced expiratory volume in 1 s (FEV1) 61.0 (12.6)% predicted) received 500 μg roflumilast or placebo once daily for 4 weeks. Induced sputum samples were collected before and after 2 and 4 weeks of treatment. Differential and absolute cell counts were determined in whole sputum samples. Markers of inflammation were determined in sputum supernatants and blood. Spirometry was performed weekly.
Results
Roflumilast significantly reduced the absolute number of neutrophils and eosinophils/g sputum compared with placebo by 35.5% (95% CI 15.6% to 50.7%; p = 0.002) and 50.0% (95% CI 26.8% to 65.8%; p<0.001), respectively. The relative proportion of sputum neutrophils and eosinophils was not affected by treatment (p>0.05). Levels of soluble interleukin‐8, neutrophil elastase, eosinophil cationic protein and α2‐macroglobulin in sputum and the release of tumour necrosis factor α from blood cells were significantly reduced by roflumilast compared with placebo treatment (p<0.05 for all). Post‐bronchodilator FEV1 improved significantly during roflumilast compared with placebo treatment with a mean difference between treatments of 68.7 ml (95% CI 12.9 to 124.5; p = 0.018).
Conclusion
PDE4 inhibition by roflumilast treatment for 4 weeks reduced the number of neutrophils and eosinophils, as well as soluble markers of neutrophilic and eosinophilic inflammatory activity in induced sputum samples of patients with COPD. This anti‐inflammatory effect may in part explain the concomitant improvement in post‐bronchodilator FEV1.
doi:10.1136/thx.2006.075937
PMCID: PMC2094292  PMID: 17573446
17.  Differential distribution of inflammatory cells in large and small airways in smokers 
Journal of Clinical Pathology  2006;60(8):907-911.
Background
Smoking induces structural changes in the airways, and is considered a major factor in the development of airflow obstruction in chronic obstructive pulmonary disease. However, differences in inflammatory cell distribution between large airways (LA) and small airways (SA) have not been systematically explored in smokers.
Hypothesis
The content of cells infiltrating the airway wall differs between LA and SA.
Aims
To compare the content of neutrophils, macrophages, lymphocytes and mast cells infiltrating LA and SA in smokers who underwent surgery for lung cancer.
Methods
Lung tissue from 15 smokers was analysed. Inflammatory cells in the lamina propria were identified by immunohistochemical analysis, quantified by digital image analysis and expressed as number of cells per surface area.
Results
The number of neutrophils infiltrating the lamina propria of SA (median 225.3 cells/mm2) was higher than that in the lamina propria of LA (median 60.2 cells/mm2; p<0.001). Similar results were observed for mast cells: 313.3 and 133.7 cells/mm2 in the SA and LA, respectively (p<0.001). In contrast, the number of CD4 cells was higher in LA compared with SA (median 217.8 vs 80.5 cells/mm2; p = 0.042). Conclusions: These findings indicate a non‐uniform distribution of neutrophils and mast cells throughout the bronchial tree, and suggest that these cells may be involved in the development of smoking‐related peripheral lung injury.
doi:10.1136/jcp.2006.037002
PMCID: PMC1994511  PMID: 16917001
18.  Urinary Eosinophil Protein X in Childhood Asthma: Relation with Changes in Disease Control and Eosinophilic Airway Inflammation 
Mediators of Inflammation  2013;2013:532619.
The aim of this study was to assess cross-sectional and longitudinal correlations between uEPX and other markers of asthma control and eosinophilic airway inflammation. Methods. We measured uEPX at baseline, after 1 year and after 2 years in 205 atopic asthmatic children using inhaled fluticasone. At the same time points, we assessed symptom scores (2 weeks diary card), lung function (forced expiratory volume in one second (FEV1)), airway hyperresponsiveness (AHR), and percentage eosinophils in induced sputum (% eos). Results. We found negative correlations between uEPX and FEV1 at baseline (r = −0.18, P = 0.01), after 1 year (r = −0.25, P < 0.01) and after 2 years (r = −0.21, P = 0.02). Within-patient changes of uEPX showed a negative association with FEV1 changes (at 1 year: r = −0.24, P = 0.01; at 2 years: r = −0.21, P = 0.03). Within-patient changes from baseline of uEPX correlated with changes in % eos. No relations were found between uEPX and symptoms. Conclusion. In this population of children with atopic asthma, uEPX correlated with FEV1 and % eos, and within-subjects changes in uEPX correlated with changes in FEV1 and % eos. As the associations were weak and the scatter of uEPX wide, it seems unlikely that uEPX will be useful as a biomarker for monitoring asthma control in the individual child.
doi:10.1155/2013/532619
PMCID: PMC3557635  PMID: 23401643
19.  Publication Bias in Laboratory Animal Research: A Survey on Magnitude, Drivers, Consequences and Potential Solutions 
PLoS ONE  2012;7(9):e43404.
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.
doi:10.1371/journal.pone.0043404
PMCID: PMC3434185  PMID: 22957028
20.  Asthma control cost-utility randomized trial evaluation (ACCURATE): the goals of asthma treatment 
Background
Despite the availability of effective therapies, asthma remains a source of significant morbidity and use of health care resources. The central research question of the ACCURATE trial is whether maximal doses of (combination) therapy should be used for long periods in an attempt to achieve complete control of all features of asthma. An additional question is whether patients and society value the potential incremental benefit, if any, sufficiently to concur with such a treatment approach. We assessed patient preferences and cost-effectiveness of three treatment strategies aimed at achieving different levels of clinical control:
1. sufficiently controlled asthma
2. strictly controlled asthma
3. strictly controlled asthma based on exhaled nitric oxide as an additional disease marker
Design
720 Patients with mild to moderate persistent asthma from general practices with a practice nurse, age 18-50 yr, daily treatment with inhaled corticosteroids (more then 3 months usage of inhaled corticosteroids in the previous year), will be identified via patient registries of general practices in the Leiden, Nijmegen, and Amsterdam areas in The Netherlands. The design is a 12-month cluster-randomised parallel trial with 40 general practices in each of the three arms. The patients will visit the general practice at baseline, 3, 6, 9, and 12 months. At each planned and unplanned visit to the general practice treatment will be adjusted with support of an internet-based asthma monitoring system supervised by a central coordinating specialist nurse. Patient preferences and utilities will be assessed by questionnaire and interview. Data on asthma control, treatment step, adherence to treatment, utilities and costs will be obtained every 3 months and at each unplanned visit. Differences in societal costs (medication, other (health) care and productivity) will be compared to differences in the number of limited activity days and in quality adjusted life years (Dutch EQ5D, SF6D, e-TTO, VAS). This is the first study to assess patient preferences and cost-effectiveness of asthma treatment strategies driven by different target levels of asthma control.
Trial registration
Netherlands Trial Register (NTR): NTR1756
doi:10.1186/1471-2466-11-53
PMCID: PMC3295696  PMID: 22114896
21.  Cost-Effectiveness of Internet-Based Self-Management Compared with Usual Care in Asthma 
PLoS ONE  2011;6(11):e27108.
Background
Effectiveness 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 Findings
Cost-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 $50000 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.
Conclusions
Internet-based self-management of asthma can be as effective as current asthma care and costs are similar.
Trial Registration
Current Controlled Trials ISRCTN79864465
doi:10.1371/journal.pone.0027108
PMCID: PMC3214043  PMID: 22096523
22.  Systems medicine and integrated care to combat chronic noncommunicable diseases 
Genome Medicine  2011;3(7):43.
We propose an innovative, integrated, cost-effective health system to combat major non-communicable diseases (NCDs), including cardiovascular, chronic respiratory, metabolic, rheumatologic and neurologic disorders and cancers, which together are the predominant health problem of the 21st century. This proposed holistic strategy involves comprehensive patient-centered integrated care and multi-scale, multi-modal and multi-level systems approaches to tackle NCDs as a common group of diseases. Rather than studying each disease individually, it will take into account their intertwined gene-environment, socio-economic interactions and co-morbidities that lead to individual-specific complex phenotypes. It will implement a road map for predictive, preventive, personalized and participatory (P4) medicine based on a robust and extensive knowledge management infrastructure that contains individual patient information. It will be supported by strategic partnerships involving all stakeholders, including general practitioners associated with patient-centered care. This systems medicine strategy, which will take a holistic approach to disease, is designed to allow the results to be used globally, taking into account the needs and specificities of local economies and health systems.
doi:10.1186/gm259
PMCID: PMC3221551  PMID: 21745417
23.  In vivo imaging of the airway wall in asthma: fibered confocal fluorescence microscopy in relation to histology and lung function 
Respiratory Research  2011;12(1):85.
Background
Airway remodelling is a feature of asthma including fragmentation of elastic fibres observed in the superficial elastin network of the airway wall. Fibered confocal fluorescence microscopy (FCFM) is a new and non-invasive imaging technique performed during bronchoscopy that may visualize elastic fibres, as shown by in vitro spectral analysis of elastin powder. We hypothesized that FCFM images capture in vivo elastic fibre patterns within the airway wall and that such patterns correspond with airway histology. We aimed to establish the concordance between the bronchial elastic fibre pattern in histology and FCFM. Second, we examined whether elastic fibre patterns in histology and FCFM were different between asthmatic subjects and healthy controls. Finally, the association between these patterns and lung function parameters was investigated.
Methods
In a cross-sectional study comprising 16 subjects (8 atopic asthmatic patients with controlled disease and 8 healthy controls) spirometry and bronchoscopy were performed, with recording of FCFM images followed by endobronchial biopsy at the airway main carina. Elastic fibre patterns in histological sections and FCFM images were scored semi-quantitatively. Agreement between histology and FCFM was analysed using linearly weighted kappa κw.
Results
The patterns observed in histological sections and FCFM images could be divided into 3 distinct groups. There was good agreement between elastic fibre patterns in histology and FCFM patterns (κw 0.744). The semi-quantitative pattern scores were not different between asthmatic patients and controls. Notably, there was a significant difference in post-bronchodilator FEV1 %predicted between the different patterns by histology (p = 0.001) and FCFM (p = 0.048), regardless of asthma or atopy.
Conclusion
FCFM captures the elastic fibre pattern within the airway wall in humans in vivo. The association between post-bronchodilator FEV1 %predicted and both histological and FCFM elastic fibre patterns points towards a structure-function relationship between extracellular matrix in the airway wall and lung function.
Trial registration
Netherlands Trial Register NTR1306
doi:10.1186/1465-9921-12-85
PMCID: PMC3146829  PMID: 21699692
Asthma; Confocal Laser Scanning Microscopy; Extracellular Matrix; Respiratory Function Tests; Smooth muscle
24.  Smoking status and anti-inflammatory macrophages in bronchoalveolar lavage and induced sputum in COPD 
Respiratory Research  2011;12(1):34.
Background
Macrophages have been implicated in the pathogenesis of COPD. M1 and M2 macrophages constitute subpopulations displaying pro- and anti-inflammatory properties. We hypothesized that smoking cessation affects macrophage heterogeneity in the lung of patients with COPD. Our aim was to study macrophage heterogeneity using the M2-marker CD163 and selected pro- and anti-inflammatory mediators in bronchoalveolar lavage (BAL) fluid and induced sputum from current smokers and ex-smokers with COPD.
Methods
114 COPD patients (72 current smokers; 42 ex-smokers, median smoking cessation 3.5 years) were studied cross-sectionally and underwent sputum induction (M/F 99/15, age 62 ± 8 [mean ± SD] years, 42 (31-55) [median (range)] packyears, post-bronchodilator FEV1 63 ± 9% predicted, no steroids past 6 months). BAL was collected from 71 patients. CD163+ macrophages were quantified in BAL and sputum cytospins. Pro- and anti-inflammatory mediators were measured in BAL and sputum supernatants.
Results
Ex-smokers with COPD had a higher percentage, but lower number of CD163+ macrophages in BAL than current smokers (83.5% and 68.0%, p = 0.04; 5.6 and 20.1 ×104/ml, p = 0.001 respectively). The percentage CD163+ M2 macrophages was higher in BAL compared to sputum (74.0% and 30.3%, p < 0.001). BAL M-CSF levels were higher in smokers than ex-smokers (571 pg/ml and 150 pg/ml, p = 0.001) and correlated with the number of CD163+ BAL macrophages (Rs = 0.38, p = 0.003). No significant differences were found between smokers and ex-smokers in the levels of pro-inflammatory (IL-6 and IL-8), and anti-inflammatory (elafin, and Secretory Leukocyte Protease Inhibitor [SLPI]) mediators in BAL and sputum.
Conclusions
Our data suggest that smoking cessation partially changes the macrophage polarization in vivo in the periphery of the lung towards an anti-inflammatory phenotype, which is not accompanied by a decrease in inflammatory parameters.
doi:10.1186/1465-9921-12-34
PMCID: PMC3072953  PMID: 21426578
25.  Airway inflammation and mannitol challenge test in COPD 
Respiratory Research  2011;12(1):11.
Background
Eosinophilic airway inflammation has successfully been used to tailor anti-inflammatory therapy in chronic obstructive pulmonary disease (COPD). Airway hyperresponsiveness (AHR) by indirect challenges is associated with airway inflammation. We hypothesized that AHR to inhaled mannitol captures eosinophilia in induced sputum in COPD.
Methods
Twenty-eight patients (age 58 ± 7.8 yr, packyears 40 ± 15.5, post-bronchodilator FEV1 77 ± 14.0%predicted, no inhaled steroids ≥4 wks) with mild-moderate COPD (GOLD I-II) completed two randomized visits with hypertonic saline-induced sputum and mannitol challenge (including sputum collection). AHR to mannitol was expressed as response-dose-ratio (RDR) and related to cell counts, ECP, MPO and IL-8 levels in sputum.
Results
There was a positive correlation between RDR to mannitol and eosinophil numbers (r = 0.47, p = 0.03) and level of IL-8 (r = 0.46, p = 0.04) in hypertonic saline-induced sputum. Furthermore, significant correlations were found between RDR and eosinophil numbers (r = 0.71, p = 0.001), level of ECP (r = 0.72, p = 0.001), IL-8 (r = 0.57, p = 0.015) and MPO (r = 0.64, p = 0.007) in sputum collected after mannitol challenge. ROC-curves showed 60% sensitivity and 100% specificity of RDR for >2.5% eosinophils in mannitol-induced sputum.
Conclusions
In mild-moderate COPD mannitol hyperresponsiveness is associated with biomarkers of airway inflammation. The high specificity of mannitol challenge suggests that the test is particularly suitable to exclude eosinophilic airways inflammation, which may facilitate individualized treatment in COPD.
Trial registration
Netherlands Trial Register (NTR): NTR1283
doi:10.1186/1465-9921-12-11
PMCID: PMC3036630  PMID: 21241520

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