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Background: Lung cancer has the highest mortality of all cancers. The aim of this study was to examine DNA hypermethylation in sputum and validate its diagnostic accuracy for lung cancer. Methods: DNA hypermethylation of RASSF1A, APC , cytoglobin, 3OST2, PRDM14, FAM19A4 and PHACTR3 was analysed in sputum samples from symptomatic lung cancer patients and controls (learning set: 73 cases, 86 controls; validation set: 159 cases, 154 controls) by quantitative methylation-specific PCR. Three statistical models were used: (i) cutoff based on Youden’s J index, (ii) cutoff based on fixed specificity per marker of 96% and (iii) risk classification of post-test probabilities. Results: In the learning set, approach (i) showed that RASSF1A was best able to distinguish cases from controls (sensitivity 42.5%, specificity 96.5%). RASSF1A, 3OST2 and PRDM14 combined demonstrated a sensitivity of 82.2% with a specificity of 66.3%. Approach (ii) yielded a combination rule of RASSF1A, 3OST2 and PHACTR3 (sensitivity 67.1%, specificity 89.5%). The risk model (approach iii) distributed the cases over all risk categories. All methods displayed similar and consistent results in the validation set. Conclusions: Our findings underscore the impact of DNA methylation markers in symptomatic lung cancer diagnosis. RASSF1A is validated as diagnostic marker in lung cancer.

Background: Chronic obstructive pulmonary disease (COPD) is associated with airway inflammation. Although smoking cessation improves symptoms and the decline in lung function in COPD, it is unknown whether bronchial inflammation in patients with established COPD varies with the duration of smoking cessation. Methods: 114 patients (99 men) with COPD of mean (SD) age 62 (8) years, a median (IQR) smoking history of 42 (31–55) pack years, no inhaled or oral corticosteroids, all current or ex-smokers (n = 42, quit >1 month, median cessation duration 3.5 years), post-bronchodilator FEV1 63 (9)% predicted, and FEV1/IVC 48 (9)% were studied cross sectionally. The numbers of subepithelial T lymphocytes (CD3, CD4, CD8), neutrophils, macrophages, eosinophils, mast cells, and plasma cells were measured in bronchial biopsy specimens (median (IQR)/0.1 mm2) using fully automated image analysis. Results: Ex-smokers with COPD had higher CD3+, CD4+, and plasma cell numbers than current smokers with COPD (149 (88–225) v 108 (61–164), p = 0.036; 58 (32–90) v 40 (25–66), p = 0.023; and 9.0 (5.5–20) v 7.5 (3.1–14), p = 0.044, respectively), but no difference in other inflammatory cells. Short term ex-smokers (<3.5 years) had higher CD4+ and CD8+ cell numbers than current smokers (p = 0.017, p = 0.023; respectively). Conversely, long term ex-smokers (quit ⩾3.5 years) had lower CD8+ cell numbers than short term ex-smokers (p = 0.009), lower CD8/CD3 ratios than both current smokers and short-term ex-smokers (p = 0.012, p = 0.003; respectively), and higher plasma cell numbers than current smokers (p = 0.003). Conclusions: With longer duration of smoking cessation, CD8 cell numbers decrease and plasma cell numbers increase. This indicates that bronchial T lymphocyte and plasma cell counts, but not other inflammatory cells, are related to duration of smoking cessation in patients with COPD.

Background Human Rhinovirus (HRV) is responsible for the majority of common colds and is frequently accompanied by secondary bacterial infections through poorly understood mechanisms. We investigated the effects of experimental human HRV serotype 16 infection on the upper respiratory tract microbiota. Methods Six healthy volunteers were infected with HRV16. We performed 16S ribosomal RNA-targeted pyrosequencing on throat swabs taken prior, during and after infection. We compared overall community diversity, phylogenetic structure of the ecosystem and relative abundances of the different bacteria between time points. Results During acute infection strong trends towards increases in the relative abundances of Haemophilus parainfluenzae and Neisseria subflava were observed, as well as a weaker trend towards increases of Staphylococcus aureus . No major differences were observed between day-1 and day 60, whereas differences between subjects were very high. Conclusions HRV16 infection is associated with the increase of three genera known to be associated with secondary infections following HRV infections. The observed changes of upper respiratory tract microbiota could help explain why HRV infection predisposes to bacterial otitis media, sinusitis and pneumonia.

Background: Ongoing inflammatory processes resulting in airway and vascular remodelling characterise chronic obstructive pulmonary disease (COPD). Vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) could play a role in tissue remodelling and angiogenesis in COPD. Methods: The cellular expression pattern of VEGF, Flt-1, and KDR/Flk-1 was examined by immunohistochemistry in central and peripheral lung tissues obtained from ex-smokers with COPD (forced expiratory volume in 1 second (FEV1) <75% predicted; n = 14) or without COPD (FEV1 >85% predicted; n = 14). The immunohistochemical staining of each molecule was quantified using a visual scoring method with grades ranging from 0 (no) to 3 (intense). Results: VEGF, Flt-1, and KDR/Flk-1 immunostaining was localised in vascular and airway smooth muscle (VSM and ASM) cells, bronchial, bronchiolar and alveolar epithelium, and macrophages. Pulmonary endothelial cells expressed Flt-1 and KDR/Flk-1 abundantly but not VEGF. Bronchial VEGF expression was higher in microvascular VSM cells and ASM cells of patients with COPD than in patients without COPD (1.7 and 1.6-fold, p<0.01, respectively). VEGF expression in intimal and medial VSM (1.7 and 1.3-fold, p<0.05) of peripheral pulmonary arteries associated with the bronchiolar airways was more intense in COPD, as was VEGF expression in the small pulmonary vessels in the alveolar region (1.5 and 1.7-fold, p<0.02). In patients with COPD, KDR/Flk-1 expression was enhanced in endothelial cells and in intimal and medial VSM (1.3, 1.9 and 1.5-fold, p<0.02) while endothelial Flt-1 expression was 1.7 times higher (p<0.03). VEGF expression was significantly increased in bronchiolar and alveolar epithelium as well as in bronchiolar macrophages (1.5-fold, p<0.001). The expression of VEGF in bronchial VSM and mucosal microvessels as well as bronchiolar epithelium was inversely correlated with FEV1 (r<−0.45; p<0.01). Conclusions: VEGF and its receptors Flt-1 and KDR/Flk-1 may be involved in peripheral vascular and airway remodelling processes in an autocrine and/or paracrine manner. This system may also be associated with epithelial cell viability during airway wall remodelling in COPD.

Background Monitoring sputum eosinophils in asthma predicts exacerbations and improves management of asthma. Thus far, blood eosinophils and FENO show contradictory results in predicting eosinophilic airway inflammation. More recently, serum periostin was proposed as a novel biomarker for eosinophilic inflammation. Objectives Quantifying the mutual relationships of blood eosinophils, FENO, and serum periostin with sputum eosinophils by external validation in two independent cohorts across various severities of asthma. Methods The first cohort consisted of 110 patients with mild to moderate asthma (external validation cohort). The replication cohort consisted of 37 patients with moderate to severe asthma. Both cohorts were evaluated cross-sectionally. Sputum was induced for the assessment of eosinophils. In parallel, blood eosinophil counts, serum periostin concentrations and FENO were assessed. The diagnostic accuracy of these markers to identify eosinophilic asthma (sputum eosinophils ≥3%) was calculated using receiver operating characteristics area under the curve (ROC AUC). Results In the external validation cohort, ROC AUC for blood eosinophils was 89% (p<0.001) and for FENO level 78% (p<0.001) to detect sputum eosinophilia ≥3%. Serum periostin was not able to distinguish eosinophilic from non-eosinophilic airway inflammation (ROC AUC=55%, p=0.44). When combining these three variables, no improvement was seen. The diagnostic value of blood eosinophils was confirmed in the replication cohort (ROC AUC 85%, p<0.001). Conclusions In patients with mild to moderate asthma, as well as patients with more severe asthma, blood eosinophils had the highest accuracy in the identification of sputum eosinophilia in asthma. The use of blood eosinophils can facilitate individualised treatment and management of asthma. Trial registration NTR1846 and NTR2364.

Background Macrophages constitute a heterogeneous cell population with pro- (MΦ1) and anti-inflammatory (MΦ2) cells. The soluble chitinase-like-protein YKL-40 is expressed in macrophages and various other cell types, and has been linked to a variety of inflammatory diseases, including COPD. Dexamethasone strongly reduces YKL-40 expression in peripheral blood mononuclear cells (PBMC) in vitro. We hypothesized that: a) YKL-40 is differentially expressed by MΦ1 and MΦ2, b) is decreased by corticosteroids and c) that long-term treatment with inhaled corticosteroids (ICS) affects YKL-40 levels in serum and sputum of COPD patients. Methods Monocytes of healthy subjects were cultured in vitro for 7 days with either GM-CSF or M-CSF (for MΦ1 and MΦ2, respectively) and stimulated for 24 h with LPS, TNFα, or oncostatin M (OSM). MΦ1 and MΦ2 differentiation was assessed by measuring secretion of IL-12p40 and IL-10, respectively. YKL-40 expression in macrophages was measured by quantitative RT-PCR (qPCR) and ELISA; serum and sputum YKL-40 levels were analyzed by ELISA. Results Pro-inflammatory MΦ1 cells secreted significantly more YKL-40 than MΦ2, which was independent of stimulation with LPS, TNFα or OSM ( p  < 0.001) and confirmed by qPCR. Dexamethasone dose-dependently and significantly inhibited YKL-40 protein and mRNA levels in MΦ1. Serum YKL-40 levels of COPD patients were significantly higher than sputum YKL-40 levels but were not significantly changed by ICS treatment. Conclusions YKL-40 secretion from MΦ1 cells is higher than from MΦ2 cells and is unaffected by further stimulation with pro-inflammatory agents. Furthermore, YKL-40 release from cultured monocyte-derived macrophages is inhibited by dexamethasone especially in MΦ1, but ICS treatment did not change YKL-40 serum and sputum levels in COPD. These results indicate that YKL-40 expression could be used as a marker for MΦ1 macrophages in vitro, but not for monitoring the effect of ICS in COPD. Trial registration ClinicalTrials.gov, registration number: NCT00158847

Background: Several studies suggest that the periphery of the lung is the major site of inflammation in asthma. Fractional exhaled nitric oxide (Feno) and 8-isoprostane have been proposed as biomarkers of inflammation and oxidative stress. We therefore hypothesised that small airway dysfunction in asthma is of inflammatory origin that can be detected by molecular markers in exhaled air. To test this hypothesis, we examined the relationship of Feno and 8-isoprostane in exhaled air with small airways function as assessed by the single breath nitrogen test. Methods: Sixteen patients (14 women) with mild atopic asthma (forced expiratory volume in 1 second >80% predicted) of mean (SD) age 23.0 (5.5) years participated in a cross sectional study. Feno was recorded by chemiluminescence and 8-isoprostane was measured by ELISA in concentrated exhaled breath condensate. The slope of phase III (δN2) and the closing volume (CV) were assessed from the single breath washout curve. Results: The median Feno level was 30.4 ppb (range 10.1–82.8), the median 8-isoprostane concentration in exhaled breath condensate was 2.2 pg/ml (range 1.6–2.7), and the mean (SD) δN2 value was 1.1 (0.4)% N2/l. Feno was positively associated with δN2 (rs = 0.54, p = 0.032) while 8-isoprostane was inversely correlated with FEV1% predicted (rs = −0.58; p = 0.017) and CV as a percentage of vital capacity (rs = 0.58; p = 0.019). Conclusions: Feno and 8-isoprostane in exhaled air are associated with small airways function in mild asthma. This suggests that these markers reflect small airway inflammation and favours a role for them as disease markers that is complementary to spirometry in the monitoring of patients with asthma.

Interleukin-5 (IL-5) is essential for the formation of eosinophils, which are thought to have a major role in the pathogenesis of asthma and other allergic diseases. We aimed to assess the effects of monoclonal antibody to IL-5 on blood and sputum eosinophils, airway hyperresponsiveness, and the late asthmatic reaction to inhaled allergen in patients with mild asthma. We did a double-blind randomised placebo-controlled trial, in which a single intravenous infusion of humanised (IgG-k) monoclonal antibody to IL-5 (SB-240563) was given at doses of 2·5 mg/kg (n=8) or 10·0 mg/kg (n=8). The effects of treatment on responses to inhaled allergen challenge, sputum eosinophils, and airway hyper-responsiveness to histamine were measured at weeks 1 and 4 with monitoring of blood eosinophil counts for up to 16 weeks. Monoclonal antibody against IL-5 lowered the mean blood eosinophil count at day 29 from 0·25x10 9 /L (95% CI 0·16-0·34) in the placebo group to 0·04x10 9 /L (0·00-0·07) in the 10 mg/kg group (p<0·0001), and prevented the blood eosinophilia that follows allergen challenge. After inhaled allergen challenge, 9 days after treatment, the percentage sputum eosinophils were 12·2% in the placebo group and lowered to 0·9% (−1·2 to 3·0; p=0·0076) in the 10 mg/kg group, and this effect persisted at day 30 after the dose. There was no significant effect of monoclonal antibody to IL-5 on the late asthmatic response or on airway hyperresponsiveness to histamine. A single dose of monoclonal antibody to IL-5 decreased blood eosinophils for up to 16 weeks and sputum eosinophils at 4 weeks, which has considerable therapeutic potential for asthma and allergy. However, our findings question the role of eosinophils in mediating the late asthmatic response and causing airway hyper-responsiveness.

Background Clinical studies investigating medicinal products need to comply with laws concerning good clinical practice (GCP) and good manufacturing practice (GMP) to guarantee the quality and safety of the product, to protect the health of the participating individual and to assure proper performance of the study. However, there are no specific regulations or guidelines for non-Medicinal Investigational Products (non-MIPs) such as allergens, enriched food supplements, and air pollution components. As a consequence, investigators will avoid clinical research and prefer preclinical models or in vitro testing for e.g. toxicology studies. The aim of this article is to 1) briefly review the current guidelines and regulations for Investigational Medicinal Products; 2) present a standardised approach to ensure the quality and safety of non-MIPs in human in vivo research; and 3) discuss some lessons we have learned. Methods and results We propose a practical line of approach to compose a clarifying product dossier (PD), comprising the description of the production process, the analysis of the raw and final product, toxicological studies, and a thorough risk-benefit-analysis. This is illustrated by an example from a human in vivo research model to study exposure to air pollutants, by challenging volunteers with a suspension of carbon nanoparticles (the component of ink cartridges for laser printers). Conclusion With this novel risk-based approach, the members of competent authorities are provided with standardised information on the quality of the product in relation to the safety of the participants, and the scientific goal of the study.

Genetic variation may partly explain asthma treatment response heterogeneity. We aimed to identify common and rare genetic variants associated with asthma that was not well controlled despite inhaled corticosteroid (ICS) treatment. Data of 110 children was collected in the Children Asthma Therapy Optimal trial. Associations of genetic variation with measures of lung function (FEV 1 %pred), airway hyperresponsiveness (AHR) to methacholine (Mch PD20) and treatment response outcomes were analyzed using the exome chip. The 17q12-21 locus (containing ORMDL3 and GSMDB) previously associated with childhood asthma was investigated separately. Single-nucleotide polymorphisms (SNPs) in the 17q12-21 locus were found nominally associated with the outcomes. The strongest association in this region was found for rs72821893 in KRT25 with FEV 1 %pred ( P =3.75*10 −5 ), Mch PD20 ( P =0.00095) and Mch PD20-based treatment outcome ( P =0.006). No novel single SNPs or burden tests were significantly associated with the outcomes. The 17q12-21 region was associated with FEV 1 %pred and AHR, and additionally with ICS treatment response.