Chronic Obstructive Pulmonary Disease Is Associated with Epigenome-Wide Differential Methylation in BAL Lung Cells.

DNA methylation patterns in chronic pulmonary obstructive disease (COPD) might offer new insights into disease pathogenesis. To assess methylation profiles in the main COPD target organ, we performed an epigenome-wide association study on BAL cells. Bronchoscopies were performed in 18 subjects with COPD and 15 control subjects (ex- and current smokers). DNA methylation was measured using the Illumina MethylationEPIC BeadChip Kit, covering more than 850,000 CpGs. Differentially methylated positions (DMPs) were examined for 1) enrichment in pathways and functional gene relationships using the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology, 2) accelerated aging using Horvath's epigenetic clock, 3) correlation with gene expression, and 4) colocalization with genetic variation. We found 1,155 Bonferroni-significant (P < 6.74 × 10-8) DMPs associated with COPD, many with large effect sizes. Functional analysis identified biologically plausible pathways and gene relationships, including enrichment for transcription factor activity. Strong correlation was found between DNA methylation and chronological age but not between COPD and accelerated aging. For 79 unique DMPs, DNA methylation correlated significantly with gene expression in BAL cells. Thirty-nine percent of DMPs were colocalized with COPD-associated SNPs. To the best of our knowledge, this is the first epigenome-wide association study of COPD on BAL cells, and our analyses revealed many differential methylation sites. Integration with mRNA data showed a strong functional readout for relevant genes, identifying sites where DNA methylation might directly affect expression. Almost half of DMPs were colocated with SNPs identified in previous genome-wide association studies of COPD, suggesting joint genetic and epigenetic pathways related to disease.

Birch pollen, air pollution and their interactive effects on airway symptoms and peak expiratory flow in allergic asthma during pollen season - a panel study in Northern and Southern Sweden.

BACKGROUND: Evidence of the role of interactions between air pollution and pollen exposure in subjects with allergic asthma is limited and need further exploration to promote adequate preventive measures. The objective of this study was to assess effects of exposure to ambient air pollution and birch pollen on exacerbation of respiratory symptoms in subjects with asthma and allergy to birch. METHODS: Thirty-seven subjects from two Swedish cities (Gothenburg and Umeå) with large variation in exposure to both birch-pollen and air pollutants, participated in the study. All subjects had confirmed allergy to birch and self-reported physician-diagnosed asthma. The subjects recorded respiratory symptoms such as rhinitis or eye irritation, dry cough, dyspnoea, the use of any asthma or allergy medication and peak respiratory flow (PEF), daily for five consecutive weeks during two separate pollen seasons and a control season without pollen. Nitrogen oxides (NOx), ozone (O3), particulate matter (PM2.5), birch pollen counts, and meteorological data were obtained from an urban background monitoring stations in the study city centres. The data were analysed using linear mixed effects models. RESULTS: During pollen seasons all symptoms and medication use were higher, and PEF was reduced in the subjects. In regression analysis, exposure to pollen at lags 0 to 2 days, and lags 0 to 6 days was associated with increased ORs of symptoms and decreased RRs for PEF. Pollen and air pollution interacted in some cases; during low pollen exposure, there were no associations between air pollution and symptoms, but during high pollen exposure, O3 concentrations were associated with increased OR of rhinitis or eye irritation, and PM2.5 concentrations were associated with increased ORs of rhinitis or eye irritation, dyspnea and increased use of allergy medication. CONCLUSIONS: Pollen and air pollutants interacted to increase the effect of air pollution on respiratory symptoms in allergic asthma. Implementing the results from this study, advisories for individuals with allergic asthma could be improved, minimizing the morbidities associated with the condition.

A gene expression-based single sample predictor of lung adenocarcinoma molecular subtype and prognosis.

Disease recurrence in surgically treated lung adenocarcinoma (AC) remains high. New approaches for risk stratification beyond tumor stage are needed. Gene expression-based AC subtypes such as the Cancer Genome Atlas Network (TCGA) terminal-respiratory unit (TRU), proximal-inflammatory (PI) and proximal-proliferative (PP) subtypes have been associated with prognosis, but show methodological limitations for robust clinical use. We aimed to derive a platform independent single sample predictor (SSP) for molecular subtype assignment and risk stratification that could function in a clinical setting. Two-class (TRU/nonTRU=SSP2) and three-class (TRU/PP/PI=SSP3) SSPs using the AIMS algorithm were trained in 1655 ACs (n = 9659 genes) from public repositories vs TCGA centroid subtypes. Validation and survival analysis were performed in 977 patients using overall survival (OS) and distant metastasis-free survival (DMFS) as endpoints. In the validation cohort, SSP2 and SSP3 showed accuracies of 0.85 and 0.81, respectively. SSPs captured relevant biology previously associated with the TCGA subtypes and were associated with prognosis. In survival analysis, OS and DMFS for cases discordantly classified between TCGA and SSP2 favored the SSP2 classification. In resected Stage I patients, SSP2 identified TRU-cases with better OS (hazard ratio [HR] = 0.30; 95% confidence interval [CI] = 0.18-0.49) and DMFS (TRU HR = 0.52; 95% CI = 0.33-0.83) independent of age, Stage IA/IB and gender. SSP2 was transformed into a NanoString nCounter assay and tested in 44 Stage I patients using RNA from formalin-fixed tissue, providing prognostic stratification (relapse-free interval, HR = 3.2; 95% CI = 1.2-8.8). In conclusion, gene expression-based SSPs can provide molecular subtype and independent prognostic information in early-stage lung ACs. SSPs may overcome critical limitations in the applicability of gene signatures in lung cancer.

Airway regulatory T cells are decreased in COPD with a rapid decline in lung function.

BACKGROUND: Differences in the expression of regulatory T cells (Tregs) have been suggested to explain why some smokers develop COPD and some do not. Upregulation of Tregs in response to smoking would restrain airway inflammation and thus the development of COPD; while the absense of such upregulation would over time lead to chronic inflammation and COPD. We hypothesized that-among COPD patients-the same mechanism would affect rate of decline in lung function; specifically, that a decreased expression of Tregs would be associated with a more rapid decline in FEV1. METHODS: Bronchoscopy with BAL was performed in 52 subjects recruited from the longitudinal OLIN COPD study; 12 with COPD and a rapid decline in lung function (loss of FEV1 ≥ 60 ml/year), 10 with COPD and a non-rapid decline in lung function (loss of FEV1 ≤ 30 ml/year), 15 current and ex-smokers and 15 non-smokers with normal lung function. BAL lymphocyte subsets were determined using flow cytometry. RESULTS: The proportions of Tregs with regulatory function (FoxP3+/CD4+CD25bright) were significantly lower in COPD subjects with a rapid decline in lung function compared to those with a non-rapid decline (p = 0.019). This result was confirmed in a mixed model regression analysis in which adjustments for inhaled corticosteroid usage, smoking, sex and age were evaluated. No significant difference was found between COPD subjects and smokers or non-smokers with normal lung function. CONCLUSIONS: COPD subjects with a rapid decline in lung function had lower proportions of T cells with regulatory function in BAL fluid, suggesting that an inability to suppress the inflammatory response following smoking might lead to a more rapid decline in FEV1. Trial registration Clinicaltrials.gov identifier NCT02729220.

Surfactant Protein A in particles in exhaled air (PExA), bronchial lavage and bronchial wash - a methodological comparison.

INTRODUCTION: At present, there are few methods available for monitoring respiratory diseases affecting distal airways. Bronchoscopy is the golden standard for sampling the lower airways. The recently developed method for collecting non-volatile material from exhaled air - PExA (Particles in Exhaled air) is a promising new tool, but no direct comparison between the two methods has yet been performed. The aim of the present study was to compare sampling using PExA with bronchial wash (BW) representing the larger more proximal airways and broncho-alveolar lavage (BAL) representing the distal airways. METHODS: 15 healthy non-smoking subjects (7 female/8 male), age 28 ± 4 years, with normal lung function were included in the study. PExA-sampling (2 × 250 ng particles) and bronchoscopy with BW (2 × 20 ml) and BAL (3 × 60 ml sterile saline) was performed. Albumin and Surfactant Protein A (SP-A) were analyzed with ELISA, and analyses of correlation were performed. RESULTS: A significant association was found between BAL-fluid albumin and PExA-albumin (rs:0.65 p = 0.01). There was also an association between SP-A in PExA and BAL, when corrected for albumin concentration (rs:0.61, p = 0.015). When correlating concentrations of albumin and SP-A in bronchial wash and PExA respectively, no associations were found. CONCLUSIONS: This is the first direct comparison between the bronchoscopy-based BW/BAL-fluids and material collected using the PExA methodology. Both albumin and albumin-corrected SP-A concentrations were significantly associated between BAL and PExA, however, no such association was found in either marker between BW and PExA. These results indicate that the PExA method samples the distal airways. PExA is thus considered a new promising non-invasive assessment for monitoring of the distal airways.

Proteolytic biomarkers are related to prognosis in COPD- report from a population-based cohort.

BACKGROUND: The imbalance between proteases and anti-proteases is considered to contribute to the development of COPD. Our aim was to evaluate the protease MMP-9, the antiprotease TIMP-1 and the MMP-9/TIMP-1-ratio as biomarkers in relation to prognosis. Prognosis was assessed as lung function decline and mortality. This was done among subjects with COPD in a population-based cohort. METHODS: In 2005, clinical examinations including spirometry and peripheral blood sampling, were made in a longitudinal population-based cohort. In total, 1542 individuals participated, whereof 594 with COPD. In 2010, 1031 subjects participated in clinical examinations, and 952 subjects underwent spirometry in both 2005 and 2010. Serum MMP-9 and TIMP-1 concentrations were measured with enzyme linked immunosorbent assay (ELISA). Mortality data were collected from the Swedish national mortality register from the date of examination in 2005 until 31st December 2010. RESULTS: The correlation between biomarkers and lung function decline was similar in non-COPD and COPD, but only significant for MMP-9 and MMP-9/TIMP-1-ratio in non-COPD. Mortality was higher in COPD than non-COPD (16% vs. 10%, p = 0.008). MMP-9 concentrations and MMP-9/TIMP-1 ratios in 2005 were higher among those who died during follow up, as well as among those alive but not participating in 2010, when compared to those participating in the 2010-examination. In non-COPD, male sex, age, burden of smoking, heart disease and MMP-9/TIMP-1 ratio were associated with increased risk for death, while increased TIMP-1 was protective. Among those with COPD, age, current smoking, increased MMP-9 and MMP-9/TIMP-1 ratio were associated with an increased risk for death. CONCLUSIONS: The expected association between these biomarkers and lung function decline in COPD was not confirmed in this population-based study, probably due to a healthy survivor effect. Still, it is suggested that increased proteolytic imbalance may be of greater prognostic importance in COPD than in non-COPD.

Cytotoxic lymphocytes in COPD airways: increased NK cells associated with disease, iNKT and NKT-like cells with current smoking.

BACKGROUND: Cytotoxic lymphocytes are increased in the airways of COPD patients. Whether this increase is driven primarily by the disease or by smoking is not clear, nor whether it correlates with the rate of decline in lung function. METHODS: Bronchoscopy with BAL was performed in 52 subjects recruited from the longitudinal OLIN COPD study according to pre-determined criteria; 12 with COPD and a rapid decline in lung function (loss of FEV1 ≥ 60 ml/year), 10 with COPD and a non-rapid decline in lung function (loss of FEV1 ≤ 30 ml/year), 15 current and ex-smokers and 15 non-smokers with normal lung function. BAL lymphocyte subsets were determined using flow cytometry. RESULTS: In BAL fluid, the proportions of NK, iNKT and NKT-like cells all increased with pack-years. Within the COPD group, NK cells - but not iNKT or NKT-like cells - were significantly elevated also in subjects that had quit smoking. In contrast, current smoking was associated with a marked increase in iNKT and NKT-like cells but not in NK cells. Rate of lung function decline did not significantly affect any of the results. CONCLUSIONS: In summary, increased proportions of NK cells in BAL fluid were associated with COPD; iNKT and NKT-like cells with current smoking but not with COPD. Interestingly, NK cell percentages did not normalize in COPD subjects that had quit smoking, indicating that these cells might play a role in the continued disease progression seen in COPD even after smoking cessation. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT02729220 .

Dendritic Cells and Monocytes with Distinct Inflammatory Responses Reside in Lung Mucosa of Healthy Humans.

Every breath we take contains potentially harmful pathogens or allergens. Dendritic cells (DCs), monocytes, and macrophages are essential in maintaining a delicate balance of initiating immunity without causing collateral damage to the lungs because of an exaggerated inflammatory response. To document the diversity of lung mononuclear phagocytes at steady-state, we performed bronchoscopies on 20 healthy subjects, sampling the proximal and distal airways (bronchial wash and bronchoalveolar lavage, respectively), as well as mucosal tissue (endobronchial biopsies). In addition to a substantial population of alveolar macrophages, we identified subpopulations of monocytes, myeloid DCs (MDCs), and plasmacytoid DCs in the lung mucosa. Intermediate monocytes and MDCs were highly frequent in the airways compared with peripheral blood. Strikingly, the density of mononuclear phagocytes increased upon descending the airways. Monocytes from blood and airways produced 10-fold more proinflammatory cytokines than MDCs upon ex vivo stimulation. However, airway monocytes were less inflammatory than blood monocytes, suggesting a more tolerant nature. The findings of this study establish how to identify human lung mononuclear phagocytes and how they function in normal conditions, so that dysregulations in patients with respiratory diseases can be detected to elucidate their contribution to immunity or pathogenesis.

Mass spectrometry profiling of oxylipins, endocannabinoids, and N-acylethanolamines in human lung lavage fluids reveals responsiveness of prostaglandin E2 and associated lipid metabolites to biodiesel exhaust exposure.

The adverse effects of petrodiesel exhaust exposure on the cardiovascular and respiratory systems are well recognized. While biofuels such as rapeseed methyl ester (RME) biodiesel may have ecological advantages, the exhaust generated may cause adverse health effects. In the current study, we investigated the responses of bioactive lipid mediators in human airways after biodiesel exhaust exposure using lipidomic profiling methods. Lipid mediator levels in lung lavage were assessed following 1-h biodiesel exhaust (average particulate matter concentration, 159 µg/m3) or filtered air exposure in 15 healthy individuals in a double-blinded, randomized, controlled, crossover study design. Bronchoscopy was performed 6 h post exposure and lung lavage fluids, i.e., bronchial wash (BW) and bronchoalveolar lavage (BAL), were sequentially collected. Mass spectrometry methods were used to detect a wide array of oxylipins (including eicosanoids), endocannabinoids, N-acylethanolamines, and related lipid metabolites in the collected BW and BAL samples. Six lipids in the human lung lavage samples were altered following biodiesel exhaust exposure, three from BAL samples and three from BW samples. Of these, elevated levels of PGE2, 12,13-DiHOME, and 13-HODE, all of which were found in BAL samples, reached Bonferroni-corrected significance. This is the first study in humans reporting responses of bioactive lipids following biodiesel exhaust exposure and the most pronounced responses were seen in the more peripheral and alveolar lung compartments, reflected by BAL collection. Since the responsiveness and diagnostic value of a subset of the studied lipid metabolites were established in lavage fluids, we conclude that our mass spectrometry profiling method is useful to assess effects of human exposure to vehicle exhaust.

Differences in the coronal proteome acquired by particles depositing in the lungs of asthmatic versus healthy humans.

Most inhaled nanomedicines in development are for the treatment of lung disease, yet little is known about their interaction with the respiratory tract lining fluids (RTLFs). Here we combined the use of nano-silica, as a protein concentrator, with label-free snapshot proteomics (LC-MS/MS; key findings confirmed by ELISA) to generate a quantitative profile of the RTLF proteome and provided insight into the evolved corona; information that may be used in future to improve drug targeting to the lungs by inhaled medicines. The asthmatic coronal proteome displayed a reduced contribution of surfactant proteins (SP-A and B) and a higher contribution of α1-antitrypsin. Pathway analysis suggested that asthmatic RTLFs may also be deficient in proteins related to metal handling (e.g. lactoferrin). This study demonstrates how the composition of the corona acquired by inhaled nanoparticles is modified in asthma and suggests depressed mucosal immunity even in mild airway disease.

Multi-platform metabolomics assays for human lung lavage fluids in an air pollution exposure study.

Metabolomics protocols are used to comprehensively characterize the metabolite content of biological samples by exploiting cutting-edge analytical platforms, such as gas chromatography (GC) or liquid chromatography (LC) coupled to mass spectrometry (MS) assays, as well as nuclear magnetic resonance (NMR) assays. We have developed novel sample preparation procedures combined with GC-MS, LC-MS, and NMR metabolomics profiling for analyzing bronchial wash (BW) and bronchoalveolar lavage (BAL) fluid from 15 healthy volunteers following exposure to biodiesel exhaust and filtered air. Our aim was to investigate the responsiveness of metabolite profiles in the human lung to air pollution exposure derived from combustion of biofuels, such as rapeseed methyl ester biodiesel, which are increasingly being promoted as alternatives to conventional fossil fuels. Our multi-platform approach enabled us to detect the greatest number of unique metabolites yet reported in BW and BAL fluid (82 in total). All of the metabolomics assays indicated that the metabolite profiles of the BW and BAL fluids differed appreciably, with 46 metabolites showing significantly different levels in the corresponding lung compartments. Furthermore, the GC-MS assay revealed an effect of biodiesel exhaust exposure on the levels of 1-monostearylglycerol, sucrose, inosine, nonanoic acid, and ethanolamine (in BAL) and pentadecanoic acid (in BW), whereas the LC-MS assay indicated a shift in the levels of niacinamide (in BAL). The NMR assay only identified lactic acid (in BW) as being responsive to biodiesel exhaust exposure. Our findings demonstrate that the proposed multi-platform approach is useful for wide metabolomics screening of BW and BAL fluids and can facilitate elucidation of metabolites responsive to biodiesel exhaust exposure. Graphical Abstract Graphical abstract illustrating the study workflow. NMR Nuclear Magnetic Resonance, LC-TOFMS Liquid chromatography-Time Of Flight Mass Spectrometry, GC Gas Chromatography-Mass spectrometry.

Enrichment of immunoregulatory proteins in the biomolecular corona of nanoparticles within human respiratory tract lining fluid.

When inhaled nanoparticles deposit in the lungs, they transit through respiratory tract lining fluid (RTLF) acquiring a biomolecular corona reflecting the interaction of the RTLF with the nanomaterial surface. Label-free snapshot proteomics was used to generate semi-quantitative profiles of corona proteins formed around silica (SiO2) and poly(vinyl) acetate (PVAc) nanoparticles in RTLF, the latter employed as an archetype drug delivery vehicle. The evolved PVAc corona was significantly enriched compared to that observed on SiO2 nanoparticles (698 vs. 429 proteins identified); however both coronas contained a substantial contribution from innate immunity proteins, including surfactant protein A, napsin A and complement (C1q and C3) proteins. Functional protein classification supports the hypothesis that corona formation in RTLF constitutes opsonisation, preparing particles for phagocytosis and clearance from the lungs. These data highlight how an understanding of the evolved corona is necessary for the design of inhaled nanomedicines with acceptable safety and tailored clearance profiles. FROM THE CLINICAL EDITOR: Inhaled nanoparticles often acquire a layer of protein corona while they go through the respiratory tract. Here, the authors investigated the identity of these proteins. The proper identification would improve the understanding of the use of inhaled nanoparticles in future therapeutics.

Relative and absolute reliability of measures of linoleic acid-derived oxylipins in human plasma.

Modern analytical techniques allow for the measurement of oxylipins derived from linoleic acid in biological samples. Most validatory work has concerned extraction techniques, repeated analysis of aliquots from the same biological sample, and the influence of external factors such as diet and heparin treatment upon their levels, whereas less is known about the relative and absolute reliability of measurements undertaken on different days. A cohort of nineteen healthy males were used, where samples were taken at the same time of day on two occasions, at least 7 days apart. Relative reliability was assessed using Lin's concordance correlation coefficients (CCC) and intraclass correlation coefficients (ICC). Absolute reliability was assessed by Bland-Altman analyses. Nine linoleic acid oxylipins were investigated. ICC and CCC values ranged from acceptable (0.56 [13-HODE]) to poor (near zero [9(10)- and 12(13)-EpOME]). Bland-Altman limits of agreement were in general quite wide, ranging from ±0.5 (12,13-DiHOME) to ±2 (9(10)-EpOME; log10 scale). It is concluded that relative reliability of linoleic acid-derived oxylipins varies between lipids with compounds such as the HODEs showing better relative reliability than compounds such as the EpOMEs. These differences should be kept in mind when designing and interpreting experiments correlating plasma levels of these lipids with factors such as age, body mass index, rating scales etc.

Effects of controlled diesel exhaust exposure on apoptosis and proliferation markers in bronchial epithelium - an in vivo bronchoscopy study on asthmatics, rhinitics and healthy subjects.

BACKGROUND: Epidemiological evidence demonstrates that exposure to traffic-derived pollution worsens respiratory symptoms in asthmatics, but controlled human exposure studies have failed to provide a mechanism for this effect. Here we investigated whether diesel exhaust (DE) would induce apoptosis or proliferation in the bronchial epithelium in vivo and thus contribute to respiratory symptoms. METHODS: Moderate (n = 16) and mild (n = 16) asthmatics, atopic non-asthmatic controls (rhinitics) (n = 13) and healthy controls (n = 21) were exposed to filtered air or DE (100 µg/m(3)) for 2 h, on two separate occasions. Bronchial biopsies were taken 18 h post-exposure and immunohistochemically analysed for pro-apoptotic and anti-apoptotic proteins (Bad, Bak, p85 PARP, Fas, Bcl-2) and a marker of proliferation (Ki67). Positive staining was assessed within the epithelium using computerized image analysis. RESULTS: No evidence of epithelial apoptosis or proliferation was observed in healthy, allergic or asthmatic airways following DE challenge. CONCLUSION: In the present study, we investigated whether DE exposure would affect markers of proliferation and apoptosis in the bronchial epithelium of asthmatics, rhinitics and healthy controls, providing a mechanistic basis for the reported increased airway sensitivity in asthmatics to air pollutants. In this first in vivo exposure investigation, we found no evidence of diesel exhaust-induced effects on these processes in the subject groups investigated.

Lipid mediator profiles differ between lung compartments in asthmatic and healthy humans.

Oxylipins are oxidised fatty acids that can exert lipid mediator functions in inflammation, and several oxylipins derived from arachidonic acid are linked to asthma. This study quantified oxylipin profiles in different regions of the lung to obtain a broad-scale characterisation of the allergic asthmatic inflammation in relation to healthy individuals. Bronchoalveolar lavage fluid (BALF), bronchial wash fluid and endobronchial mucosal biopsies were collected from 16 healthy and 16 mildly allergic asthmatic individuals. Inflammatory cell counts, immunohistochemical staining and oxylipin profiling were performed. Univariate and multivariate statistics were employed to evaluate compartment-dependent and diagnosis-dependent oxylipin profiles in relation to other measured parameters. Multivariate modelling showed significantly different bronchial wash fluid and BALF oxylipin profiles in both groups (R(2)Y[cum]=0.822 and Q(2)[cum]=0.759). Total oxylipin concentrations and five individual oxylipins, primarily from the lipoxygenase (LOX) pathway of arachidonic and linoleic acid, were elevated in bronchial wash fluid from asthmatics compared to that from healthy controls, supported by immunohistochemical staining of 15-LOX-1 in the bronchial epithelium. No difference between the groups was found among BALF oxylipins. In conclusion, bronchial wash fluid and BALF contain distinct oxylipin profiles, which may have ramifications for the study of respiratory diseases. Specific protocols for sampling proximal and distal airways separately should be employed for lipid mediator studies.

Assessment of the capacity of vehicle cabin air inlet filters to reduce diesel exhaust-induced symptoms in human volunteers.

BACKGROUND: Exposure to particulate matter (PM) air pollution especially derived from traffic is associated with increases in cardiorespiratory morbidity and mortality. In this study, we evaluated the ability of novel vehicle cabin air inlet filters to reduce diesel exhaust (DE)-induced symptoms and markers of inflammation in human subjects. METHODS: Thirty healthy subjects participated in a randomized double-blind controlled crossover study where they were exposed to filtered air, unfiltered DE and DE filtered through two selected particle filters, one with and one without active charcoal. Exposures lasted for one hour. Symptoms were assessed before and during exposures and lung function was measured before and after each exposure, with inflammation assessed in peripheral blood five hours after exposures. In parallel, PM were collected from unfiltered and filtered DE and assessed for their capacity to drive damaging oxidation reactions in a cell-free model, or promote inflammation in A549 cells. RESULTS: The standard particle filter employed in this study reduced PM10 mass concentrations within the exposure chamber by 46%, further reduced to 74% by the inclusion of an active charcoal component. In addition use of the active charcoal filter was associated by a 75% and 50% reduction in NO2 and hydrocarbon concentrations, respectively. As expected, subjects reported more subjective symptoms after exposure to unfiltered DE compared to filtered air, which was significantly reduced by the filter with an active charcoal component. There were no significant changes in lung function after exposures. Similarly diesel exhaust did not elicit significant increases in any of the inflammatory markers examined in the peripheral blood samples 5 hour post-exposure. Whilst the filters reduced chamber particle concentrations, the oxidative activity of the particles themselves, did not change following filtration with either filter. In contrast, diesel exhaust PM passed through the active charcoal combination filter appeared less inflammatory to A549 cells. CONCLUSIONS: A cabin air inlet particle filter including an active charcoal component was highly effective in reducing both DE particulate and gaseous components, with reduced exhaust-induced symptoms in healthy volunteers. These data demonstrate the effectiveness of cabin filters to protect subjects travelling in vehicles from diesel exhaust emissions.

Airway inflammatory responses to diesel exhaust in allergic rhinitics.

CONTEXT: Proximity to traffic, particularly to diesel-powered vehicles, has been associated with inducing and enhancing allergies. To investigate the basis for this association, we performed controlled exposures of allergic rhinitics to diesel exhaust (DE) at a dose known to be pro-inflammatory in healthy individuals. OBJECTIVE: We hypothesized that diesel-exhaust exposure would augment lower airway inflammation in allergic rhinitics. MATERIALS AND METHODS: Fourteen allergic rhinitics were exposed in a double-blinded, randomized trial to DE (100 µg/m³ PM10) and filtered air for 2 h on separate occasions. Bronchoscopy with endobronchial mucosal biopsies and airway lavage was performed 18 h post-exposure, and inflammatory markers were assessed. RESULTS: No evidence of neutrophilic airway inflammation was observed post-diesel, however, a small increase in myeloperoxidase was found in bronchoalveolar lavage (p = 0.032). We found no increases in allergic inflammatory cells. Reduced mast cell immunoreactivity for tryptase was observed in the epithelium (p = 0.013) parallel to a small decrease in bronchial wash stem cell factor (p = 0.033). DISCUSSION AND CONCLUSION: DE, at a dose previously shown to cause neutrophilic inflammation in healthy individuals, induced no neutrophilic inflammation in the lower airways of allergic rhinitics, consistent with previous reports in asthmatics. Although there was no increase in allergic inflammatory cell numbers, the reduction in tryptase in the epithelium may indicate mast cell degranulation. However, this occurred in the absence of allergic symptoms. These data do not provide a simplistic explanation of the sensitivity in rhinitics to traffic-related air pollution. The role of mast cells requires further investigation.

A cross-sectional study of inflammatory markers as determinants of circulating kynurenines in the Lung Cancer Cohort Consortium.

Circulating concentrations of metabolites (collectively called kynurenines) in the kynurenine pathway of tryptophan metabolism increase during inflammation, particularly in response to interferon-gamma (IFN-γ). Neopterin and the kynurenine/tryptophan ratio (KTR) are IFN-γ induced inflammatory markers, and together with C-reactive protein (CRP) and kynurenines they are associated with various diseases, but comprehensive data on the strength of associations of inflammatory markers with circulating concentrations of kynurenines are lacking. We measured circulating concentrations of neopterin, CRP, tryptophan and seven kynurenines in 5314 controls from 20 cohorts in the Lung Cancer Cohort Consortium (LC3). The associations of neopterin, KTR and CRP with kynurenines were investigated using regression models. In mixed models, one standard deviation (SD) higher KTR was associated with a 0.46 SD higher quinolinic acid (QA), and 0.31 SD higher 3-hydroxykynurenine (HK). One SD higher neopterin was associated with 0.48, 0.44, 0.36 and 0.28 SD higher KTR, QA, kynurenine and HK, respectively. KTR and neopterin respectively explained 24.1% and 16.7% of the variation in QA, and 11.4% and 7.5% of HK. CRP was only weakly associated with kynurenines in regression models. In summary, QA was the metabolite that was most strongly associated with the inflammatory markers. In general, the inflammatory markers were most strongly related to metabolites located along the tryptophan-NAD axis, which may support suggestions of increased production of NAD from tryptophan during inflammation.

Proinflammatory doses of diesel exhaust in healthy subjects fail to elicit equivalent or augmented airway inflammation in subjects with asthma.

BACKGROUND: Exposure to traffic-derived air pollutants, particularly diesel emissions, has been associated with adverse health effects, predominantly in individuals with pre-existing respiratory disease. Here the hypothesis that this heightened sensitivity reflects an augmentation of the transient inflammatory response previously reported in healthy adults exposed to diesel exhaust is examined. METHODS: 32 subjects with asthma (mild to moderate severity) and 23 healthy controls were exposed in a double-blinded crossover control fashion to both filtered air and diesel exhaust (100 µg/m(3) PM(10)) for 2 h. Airway inflammation was assessed by bronchoscopy 18 h postexposure. In addition, lung function, fraction of exhaled nitric oxide and bronchial reactivity to metacholine were examined in the subjects with asthma. RESULTS: In healthy control subjects a significant increase in submucosal neutrophils (p=0.004) was observed following the diesel challenge. Significant increases in neutrophil numbers (p=0.01), and in the concentrations of interleukin 6 (p=0.03) and myeloperoxidase (p=0.04), were also seen in bronchial wash after diesel, relative to the control air challenge. No evidence of enhanced airway inflammation was observed in the subjects with asthma following the diesel exposure. CONCLUSIONS: Exposure to diesel exhaust at concentrations consistent with roadside levels elicited an acute and active neutrophilic inflammation in the airways of healthy subjects. This response was absent in subjects with asthma, as was evidence supporting a worsening of allergic airway inflammation.

Expansion of CD4+CD25+ helper T cells without regulatory function in smoking and COPD.

BACKGROUND: Regulatory T cells have been implicated in the pathogenesis of COPD by the increased expression of CD25 on helper T cells along with enhanced intracellular expression of FoxP3 and low/absent CD127 expression on the cell surface. METHOD: Regulatory T cells were investigated in BALF from nine COPD subjects and compared to fourteen smokers with normal lung function and nine never-smokers. RESULTS: In smokers with normal lung function, the expression of CD25+CD4+ was increased, whereas the proportions of FoxP3+ and CD127+ were unchanged compared to never-smokers. Among CD4+ cells expressing high levels of CD25, the proportion of FoxP3+ cells was decreased and the percentage of CD127+ was increased in smokers with normal lung function. CD4+CD25+ cells with low/absent CD127 expression were increased in smokers with normal lung function, but not in COPD, when compared to never smokers. CONCLUSION: The reduction of FoxP3 expression in BALF from smokers with normal lung function indicates that the increase in CD25 expression is not associated with the expansion of regulatory T cells. Instead, the high CD127 and low FoxP3 expressions implicate a predominantly non-regulatory CD25+ helper T-cell population in smokers and stable COPD. Therefore, we suggest a smoking-induced expansion of predominantly activated airway helper T cells that seem to persist after COPD development.