Assessment of wood smoke induced pulmonary toxicity in normal- and chronic bronchitis-like bronchial and alveolar lung mucosa models at air-liquid interface.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) has the highest increased risk due to household air pollution arising from biomass fuel burning. However, knowledge on COPD patho-mechanisms is mainly limited to tobacco smoke exposure. In this study, a repeated direct wood smoke (WS) exposure was performed using normal- (bro-ALI) and chronic bronchitis-like bronchial (bro-ALI-CB), and alveolar (alv-ALI) lung mucosa models at air-liquid interface (ALI) to assess broad toxicological end points. METHODS: The bro-ALI and bro-ALI-CB models were developed using human primary bronchial epithelial cells and the alv-ALI model was developed using a representative type-II pneumocyte cell line. The lung models were exposed to WS (10 min/exposure; 5-exposures over 3-days; n = 6-7 independent experiments). Sham exposed samples served as control. WS composition was analyzed following passive sampling. Cytotoxicity, total cellular reactive oxygen species (ROS) and stress responsive NFkB were assessed by flow cytometry. WS exposure induced changes in gene expression were evaluated by RNA-seq (p ≤ 0.01) followed by pathway enrichment analysis. Secreted levels of proinflammatory cytokines were assessed in the basal media. Non-parametric statistical analysis was performed. RESULTS: 147 unique compounds were annotated in WS of which 42 compounds have inhalation toxicity (9 very high). WS exposure resulted in significantly increased ROS in bro-ALI (11.2%) and bro-ALI-CB (25.7%) along with correspondingly increased NFkB levels (bro-ALI: 35.6%; bro-ALI-CB: 18.1%). A total of 1262 (817-up and 445-down), 329 (141-up and 188-down), and 102 (33-up and 69-down) genes were differentially regulated in the WS-exposed bro-ALI, bro-ALI-CB, and alv-ALI models respectively. The enriched pathways included the terms acute phase response, mitochondrial dysfunction, inflammation, oxidative stress, NFkB, ROS, xenobiotic metabolism of AHR, and chronic respiratory disorder. The enrichment of the 'cilium' related genes was predominant in the WS-exposed bro-ALI (180-up and 7-down). The pathways primary ciliary dyskinesia, ciliopathy, and ciliary movement were enriched in both WS-exposed bro-ALI and bro-ALI-CB. Interleukin-6 and tumor necrosis factor-α were reduced (p < 0.05) in WS-exposed bro-ALI and bro-ALI-CB. CONCLUSION: Findings of this study indicate differential response to WS-exposure in different lung regions and in chronic bronchitis, a condition commonly associated with COPD. Further, the data suggests ciliopathy as a candidate pathway in relation to WS-exposure.

MiR-29b level-mediated regulation of Klotho methylation via DNMT3A targeting in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a chronic lung disease characterized by chronic bronchitis and emphysema. Cigarette smoke extract (CSE) is the predominant cause of COPD. This study aimed to investigate the effects of miR-29b and their underlying mechanisms in a COPD cell model. MiR-29b and DNMT3A expression in lung tissue samples (taken at least 5 cm away from the tumor lesion) of NSCLC cases with smoking (n = 30), without smoking (n = 30), and with COPD (with smoking) (n = 30) was researched by qRT-PCR. A medium containing 10 % CSE was employed to induce murine alveolar macrophage MH-S cells to establish COPD cells. 5-Aza-cdr (5-AZA-2'-deoxycytidine) was used to block DNMT3A. The relationship and interaction between miR-29b and DNMT3A were validated through the dual luciferase reporter assay. The expression levels of macrophage M1 polarization marker proteins iNOS and TNF-α, DNMT3A, and Klotho protein were monitored using western blotting. The methylation levels of the miR-29b precursor gene and Klotho promoter were detected by quantitative methylation-specific PCR (MS-qPCR). The levels of IL-1ß, IL-6, and TNF-α in cell culture medium were detected via ELISA. It was found that the expression of miR-29b was downregulated, as a result of increased DNA methylation, and that of DNMT3A was upregulated in the lung tissues of NSCLC cases with COPD (with smoking). DNMT3A expression was negatively correlated with miR-29b expression in the lung tissues of NSCLC cases with COPD (with smoking). In addition, miR-29b expression was distinctly downregulated in CSE-induced MH-S cells and inhibited CSE-induced M1 polarization and inflammation. Importantly, DNMT3A was identified as a direct target gene of miR-29b. MiR-29b is negatively regulated by DNMT3A-mediated DNA methylation. Moreover, Klotho expression was downregulated and the Klotho promoter methylation level was increased in lung tissues of NSCLC cases with COPD (with smoking). The negative feedback between miR-29b and DNMT3A modulates CSE-induced M1 polarization and inflammation in macrophages as well as Klotho promoter methylation in CSE-mediated MH-S. Collectively, these findings indicate that the miR-29b level in COPD controls Klotho methylation via DNMT3, which maybe a promising target for the treatment of COPD.

Saliva as a non-invasive specimen for COPD assessment.

BACKGROUND: People with COPD have been reported to bear a distinct airway microbiota from healthy individuals based on bronchoalveolar lavage (BAL) and sputum samples. Unfortunately, the collection of these samples involves relatively invasive procedures and is resource-demanding, limiting its regular use. Non-invasive samples from the upper airways could constitute an interesting alternative, but its relationship with COPD is still underexplored. We examined the merits of saliva to identify the typical profile of COPD oral bacteria and test its association with the disease. METHODS: Outpatients with COPD and age-sex matched healthy controls were recruited and characterised based on clinical parameters and 16S rRNA profiling of oral bacteria. A clustering analysis based on patients' oral bacteria beta-diversity and logistic regressions were performed to evaluate the association between oral bacteria composition and COPD. RESULTS: 128 individuals participated (70 patients and 58 controls). Differential abundance analyses showed differences in patients comparable to the ones previously observed in samples from the lower respiratory tract, i.e., an increase in Proteobacteria (particularly Haemophilus) and loss of microbiota diversity. An unsupervised clustering analysis separated patients in two groups based on microbiota composition differing significantly in the frequency of patients hospitalized due to severe acute exacerbation of COPD (AECOPD) and in the frequency of GOLD D patients. Furthermore, a low frequency of Prevotella was associated with a significantly higher risk of recent severe AECOPD and of being GOLD D. CONCLUSION: Salivary bacteria showed an association with COPD, particularly with severe exacerbations, supporting the use of this non-invasive specimen for future studies of heterogeneous respiratory diseases like COPD.

Association of resting energy expenditure and nutritional substrate oxidation with COPD stage and prediction indexes.

While increase in resting energy expenditure (REE) of COPD patients is generally accepted, there is a lack of information about nutritional substrates oxidation (NSO) in this specific population. The aim of this study was comparison of REE and NSO from indirect calorimetry between COPD patients and control subjects and to evaluate possible associations with the disease stage and prediction indexes. In this observational study, 50 consecutive outpatients with stable COPD (COPD group) were examined and compared with 25 volunteers without respiratory problems (control group). Body composition, REE and NSO were determined in all study participants. All COPD subjects underwent a comprehensive examination to determine COPD severity and prognostic scales. Measured REE values adjusted for body weight, fat-free mass (FFM), and body surface were approximately 10% higher in COPD patients than in the control group. Respiratory quotient (RQ) and non-protein RQ (nRQ) values were respectively 5% and 10% higher in the COPD group. Adjusted carbohydrate oxidation was almost two times higher in comparison with the control group. We found no differences in absolute values of lipid and protein oxidation between the groups. Correlation analysis proved a positive association of relatively expressed REE and oxidation of lipids, and a negative association of RQ, nRQ and oxidation of carbohydrates with the value of prediction indexes. In conclusion, our study demonstrated metabolic changes in COPD patients leading to increased values of REE and changes in NSO which were associated with the disease stage, and which can be applied for nutritional support in clinical practice.

Uric acid and uric acid to creatinine ratio in the assessment of chronic obstructive pulmonary disease: Potential biomarkers in multicomponent models comprising IL-1beta.

Chronic obstructive pulmonary disease (COPD) is a complex and heterogeneous disease, with oxidative stress and inflammation implicated in its development. Uric acid (UA) could exert anti-oxidative, pro-oxidative or pro-inflammatory effects, depending on the specific context. It was recently shown that soluble UA, and not just its crystals, could activate the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, leading to interleukin (IL)-1ß secretion. We aimed to assess the differences in blood levels of UA and its ratio with creatinine (UCR) between COPD patients and healthy subjects, as well as their association with disease severity, smoking status, common COPD comorbidities and therapy regimes. The diagnostic characteristics of UA and UCR were also explored. This study included 109 stable COPD patients and 95 controls and measured white blood cells (WBC), C-reactive protein (CRP), fibrinogen (Fbg), IL-1ß, creatinine (CREAT) and UA. All of the parameters were increased in COPD patients, except for CREAT. UA and UCR were positively associated with WBC, CRP and IL-1ß. COPD smokers had lower UA and UCR values. Common COPD therapy did not affect UA or UCR, while patients with cardiovascular diseases (CVD) had higher UA, but not UCR, levels. Patients with higher UCR values showed worse disease-related outcomes (lung function, symptoms, quality of life, history of exacerbations, BODCAT and BODEx). Also, UCR differentiated patients with different severity of airflow limitation as well as symptoms and exacerbations. The great individual predictive potential of UCR and IL-1ß was observed with their odds ratios (OR) being 2.09 and 5.53, respectively. Multiparameter models of UA and UCR that included IL-1ß were able to correctly classify 86% and 90% of cases, respectively. We suggest that UA might be a useful biomarker when combined with IL-1ß, while UCR might be even more informative and useful in overall COPD assessments.

Comparison of Proteomic Assessment Methods in Multiple Cohort Studies.

Novel proteomics platforms, such as the aptamer-based SOMAscan platform, can quantify large numbers of proteins efficiently and cost-effectively and are rapidly growing in popularity. However, comparisons to conventional immunoassays remain underexplored, leaving investigators unsure when cross-assay comparisons are appropriate. The correlation of results from immunoassays with relative protein quantification is explored by SOMAscan. For 63 proteins assessed in two chronic obstructive pulmonary disease (COPD) cohorts, subpopulations and intermediate outcome measures in COPD Study (SPIROMICS), and COPDGene, using myriad rules based medicine multiplex immunoassays and SOMAscan, Spearman correlation coefficients range from -0.13 to 0.97, with a median correlation coefficient of ≈0.5 and consistent results across cohorts. A similar range is observed for immunoassays in the population-based Multi-Ethnic Study of Atherosclerosis and for other assays in COPDGene and SPIROMICS. Comparisons of relative quantification from the antibody-based Olink platform and SOMAscan in a small cohort of myocardial infarction patients also show a wide correlation range. Finally, cis pQTL data, mass spectrometry aptamer confirmation, and other publicly available data are integrated to assess relationships with observed correlations. Correlation between proteomics assays shows a wide range and should be carefully considered when comparing and meta-analyzing proteomics data across assays and studies.

Bayesian inference of networks across multiple sample groups and data types.

In this article, we develop a graphical modeling framework for the inference of networks across multiple sample groups and data types. In medical studies, this setting arises whenever a set of subjects, which may be heterogeneous due to differing disease stage or subtype, is profiled across multiple platforms, such as metabolomics, proteomics, or transcriptomics data. Our proposed Bayesian hierarchical model first links the network structures within each platform using a Markov random field prior to relate edge selection across sample groups, and then links the network similarity parameters across platforms. This enables joint estimation in a flexible manner, as we make no assumptions on the directionality of influence across the data types or the extent of network similarity across the sample groups and platforms. In addition, our model formulation allows the number of variables and number of subjects to differ across the data types, and only requires that we have data for the same set of groups. We illustrate the proposed approach through both simulation studies and an application to gene expression levels and metabolite abundances on subjects with varying severity levels of chronic obstructive pulmonary disease. Bayesian inference; Chronic obstructive pulmonary disease (COPD); Data integration; Gaussian graphical model; Markov random field prior; Spike and slab prior.

Cigarette smoke-induced HMGB1 translocation and release contribute to migration and NF-κB activation through inducing autophagy in lung macrophages.

High-mobility group box 1 (HMGB1) shows pro-inflammatory activity in various inflammatory diseases and has been found up-regulated in chronic obstructive pulmonary disease (COPD). Lung macrophages play an important role in airway inflammation and lung destruction in COPD, yet whether HMGB1 is involved in cigarette smoke (CS)-induced lung macrophage dysfunction is unknown. We sought to evaluate the intracellular localization and release of HMGB1 in lung macrophages from COPD patients and CS-exposed mice, and to investigate the role of HMGB1 in regulating autophagy in CS extract (CSE)-treated lung macrophages (MH-S cells). Our results showed that HMGB1 was highly expressed in lung tissues and sera of COPD patients and CS-exposed mice, along with predominantly cytoplasmic exporting from nuclei in lung macrophages. In vitro experiments revealed that CSE promoted the expression, nucleocytoplasmic translocation and release of HMGB1 partly via the nicotinic acetylcholine receptor (nAChR). Blockade of HMGB1 with chicken anti-HMGB1 polyclonal antibody (anti-HMGB1) or glycyrrhizin (Gly) attenuated the increase of LC3B-II and Beclin1, migration and p65 phosphorylation, suggesting the involvement of HMGB1 in autophagy, migration and NF-κB activation of lung macrophages. Hydroxychloroquine (CQ), an autophagy inhibitor, enhanced the increase of LC3B-II but not Beclin1 in CSE or rHMGB1-treated MH-S cells, and inhibition of autophagy by CQ and 3-methyladenine (3-MA) abrogated the migration and p65 phosphorylation of CSE-treated cells. These results indicate that CS-induced HMGB1 translocation and release contribute to migration and NF-κB activation through inducing autophagy in lung macrophages, providing novel evidence for HMGB1 as a potential target of intervention in COPD.

Establishment of rapid risk assessment model for cigarette smoke extract exposure in chronic obstructive pulmonary disease.

Rapid risk assessment models for different types of cigarette smoke extract (CSE) exposure are critical to understanding the etiology of chronic obstructive pulmonary disease. The present study investigated inflammation of cultured tracheal tissues with CSE exposure. Rat trachea rings were isolated, cultured, then exposed to various concentrations of CSE from 3R4 F reference cigarettes for 4 h. Tissue/cellular morphology, ultrastructure, viability and damage, inflammatory cell infiltration, and inflammatory protein levels were measured and compared to untreated controls. Human bronchial epithelial cells (BEAS-2B) exposed to 0 or 300 µg/mL CSE were cocultured with macrophages to assess extent of mobilization and phagocytosis. Endotracheal epithelium cilia densities were significantly reduced with increasing CSE concentrations, while mucous membranes became increasingly disordered; both eventually disappeared. Macrophages became larger as the CSE concentration increased, with microvilli and extended pseudopodium covering their surface, and many primary and secondary lysosomes present in the cytoplasm. Inflammatory cell infiltration also increased with increasing CSE dose, as did intracellular adhesion molecule-1(ICAM-1), interleukin-6(IL-6). The method described here may be useful to qualitatively characterized the effects of the compound under study. Then, we use BEAS-2B cell line system to strength the observation made in the cultured tissues. Probably, an approach to integrate results from both experiments will facilitate its application. These results demonstrate that cultured rat tracheal rings have a whole-tissue structure that undergoes inflammatory processes similar to in vivo tissues upon CSE exposure.

COPD assessment test and FEV1: do they predict oxygen uptake in COPD?

BACKGROUND: Chronic obstructive pulmonary disease (COPD) manifests itself in complex ways, with local and systemic effects; because of this, a multifactorial approach is needed for disease evaluation, in order to understand its severity and impact on each individual. Thus, our objective was to study the correlation between easily accessible variables, usually available in clinical practice, and maximum aerobic capacity, and to determine models for peak oxygen uptake (VO2peak) estimation in COPD patients. SUBJECTS AND METHODS: Individuals with COPD were selected for the study. At the first visit, clinical evaluation was performed. During the second visit, the volunteers were subjected to the cardiopulmonary exercise test. To determine the correlation coefficient of VO2peak with forced expiratory volume in 1 second (FEV1) (% pred.) and the COPD Assessment Test score (CATs), Pearson or Spearman tests were performed. VO2 at the peak of the exercise was estimated from the clinical variables by simple and multiple linear regression analyses. RESULTS: A total of 249 subjects were selected, 27 of whom were included after screening (gender: 21M/5F; age: 65.0±7.3 years; body mass index: 26.6±5.0 kg/m2; FEV1 (% pred.): 56.4±15.7, CAT: 12.4±7.4). Mean VO2 peak was 12.8±3.0 mL⋅kg-1⋅min-1 and VO2peak (% pred.) was 62.1%±14.9%. VO2peak presented a strong positive correlation with FEV1 (% pred.), r: 0.70, and a moderate negative correlation with the CATs, r: -0.54. In the VO2peak estimation model based on the CAT (estimated VO2peak =15.148- [0.185× CATs]), the index explained 20% of the variance, with estimated error of 2.826 mL⋅kg-1⋅min-1. In the VO2peak estimation model based on FEV1 (estimated VO2peak =6.490+ [0.113× FEV1]), the variable explained 50% of the variance, with an estimated error of 2.231 mL⋅kg-1⋅min-1. In the VO2peak estimation model based on CATs and FEV1 (estimated VO2peak =8.441- [0.0999× CAT] + [0.1000× FEV1]), the variables explained 55% of the variance, with an estimated error of 2.156 mL⋅kg-1⋅min-1. CONCLUSION: COPD patients' maximum aerobic capacity has a significant correlation with easily accessible and widely used clinical variables, such as the CATs and FEV1, which can be used to estimate peak VO2.

Development of a Neo-Epitope Specific Assay for Serological Assessment of Type VII Collagen Turnover and Its Relevance in Fibroproliferative Disorders.

Type VII collagen is the main component of the anchoring fibrils connecting the basement membrane to the underlying interstitial matrix. Mutations in the type VII collagen gene cause dystrophic epidermolysis bullosa. Increased levels of type VII collagen in the skin have been reported in patients with systemic sclerosis (SSc), whereas reduced levels in the airways have been related to asthma. This indicates that type VII collagen plays an important part in upholding tissue integrity and that its remodeling may lead to pathological states. The aim of this study was to investigate the role of type VII collagen remodeling in fibroproliferative disorders. We produced monoclonal antibody targeting a specific fragment of type VII collagen (C7M) released to the systemic circulation and developed a neo-epitope specific competitive enzyme-linked immunosorbent assay (ELISA). Biological relevance was evaluated in serum from patients with SSc or chronic obstructive pulmonary disease (COPD). The C7M ELISA was technically robust and specific for the C7M neo-epitope. Serum C7M levels were significantly elevated in two cohorts of patients with SSc and in patients with COPD as compared with healthy individuals (P < 0.0001). The C7M ELISA enabled quantification of type VII collagen turnover in serum. Elevated serum C7M levels indicated that the turnover rate of type VII collagen was significantly increased in patients with SSc or COPD, suggesting a pathological role. Thus, the C7M ELISA may become useful in future investigations of type VII collagen turnover in fibroproliferative disorders, and it may prove a valuable tool for evaluating novel anti-fibrotic drugs.

Drug Delivery from an Innovative LAMA/LABA Co-suspension Delivery Technology Fixed-Dose Combination MDI: Evidence of Consistency, Robustness, and Reliability.

To ensure consistency of clinical outcomes, orally inhaled therapies must exhibit consistent delivered dose and aerosol properties at the time of manufacturing, throughout storage, and during various patient-use conditions. Achieving consistency across these scenarios has presented a significant challenge, especially for combination products that contain more than one drug. This study characterized the delivered dose and aerosol properties of glycopyrrolate/formoterol fumarate metered dose inhaler (GFF MDI; Bevespi Aerosphere™). GFF MDI, a fixed-dose combination (FDC) of a long-acting muscarinic antagonist, glycopyrrolate (18 µg, equivalent to glycopyrronium 14.4 µg), and a long-acting ß2-agonist, formoterol fumarate (9.6 µg; equivalent to formoterol fumarate dihydrate 10 µg), is formulated using innovative co-suspension delivery technology, which suspends micronized drug crystals with spray-dried phospholipid porous particles in hydrofluoroalkane propellant. In this study, delivered dose uniformity was assessed through the labeled number of doses, and aerosol properties, such as percent fine particle fraction (FPF) and mass median aerodynamic diameter, were determined by cascade impaction. GFF MDI achieved reproducible dose delivery and an FPF greater than 55%, whether formulated and delivered as a monocomponent or dual FDC. The performance of GFF MDI was maintained across various manufacturing batches, under extended storage, and with variations in flow rate. Furthermore, unlike a GFF drug crystal-only suspension, drug delivery remained consistent for GFF MDI when simulated patient-handling errors were applied, such as reduced shake energy and delays between shaking and actuation. These results demonstrate that co-suspension delivery technology overcomes well-known sources of variability in MDI drug delivery.

Procalcitonin Biomarker Algorithm Reduces Antibiotic Prescriptions, Duration of Therapy, and Costs in Chronic Obstructive Pulmonary Disease: A Comparison in the Netherlands, Germany, and the United Kingdom.

Antibiotics are often recommended as treatment for patients with chronic obstructive pulmonary disease (COPD) exacerbations. However, not all COPD exacerbations are caused by bacterial infections and there is consequently considerable misuse and overuse of antibiotics among patients with COPD. This poses a severe burden on healthcare resources such as increased risk of developing antibiotic resistance. The biomarker procalcitonin (PCT) displays specificity to distinguish bacterial inflammations from nonbacterial inflammations and may therefore help to rationalize antibiotic prescriptions. We report in this study, a three-country comparison of the health and economic consequences of a PCT biomarker-guided prescription and clinical decision-making strategy compared to current practice in hospitalized patients with COPD exacerbations. A decision tree was developed, comparing the expected costs and effects of the PCT algorithm to current practice in the Netherlands, Germany, and the United Kingdom. The time horizon of the model captured the length of hospital stay and a societal perspective was also adopted. The primary health outcome was the duration of antibiotic therapy. The incremental cost-effectiveness ratio was defined as the incremental costs per antibiotic day avoided. The incremental cost savings per day on antibiotic therapy avoided were (in Euros) €90 in the Netherlands, €125 in Germany, and €52 in the United Kingdom. Probabilistic sensitivity analyses showed that in the majority of simulations, the PCT biomarker strategy was superior to current practice (the Netherlands: 58%, Germany: 58%, and the United Kingdom: 57%). In conclusion, the PCT biomarker algorithm to optimize antibiotic prescriptions in COPD is likely to be cost-effective compared to current practice. Both the percentage of patients who start with antibiotic treatment as well as the duration of antibiotic therapy are reduced with the PCT decision algorithm, leading to a decrease in total costs per patient. Economic analysis based on real-life data is recommended for further research. Biomarker-driven prescription algorithms are important instruments for personalized medicine in COPD. This also attests to the emerging convergence of biomarker innovations and the broader field of Health Technology Assessment (HTA).

Personalised Medicine for Asthma and Chronic Obstructive Pulmonary Disease.

Asthma and chronic obstructive pulmonary disease (COPD) are prevalent conditions, and despite recent advances and multiple available therapies and interventions, there remains a significant unmet clinical need. In recent years, it has become clear that there is both significant heterogeneity within each of these conditions and additionally significant overlap in many of the clinical and inflammatory features. In parallel, useful clinical and immunological biomarkers which inform about prognosis and response to therapy have emerged in both asthma and COPD. These biomarkers will allow both better targeting of existing treatments and the identification of those patients who will respond to novel therapies which are now becoming available. Biomarkers will also facilitate the identification of novel therapeutic targets for future development. Delivery of precision medicine in airways disease is now feasible and is a core component of a personalised healthcare delivery in asthma and COPD.

Biomarker Development in COPD: Moving From P Values to Products to Impact Patient Care.

There is a great interest in developing biomarkers to enable precision medicine and improve health outcomes of patients with COPD. However, biomarker development is extremely challenging and expensive, and translation of research endeavors to date has been largely unsuccessful. In most cases, biomarkers fail because of poor replication of initial promising results in independent cohorts and/or inability to transfer the biomarker from a discovery platform to a clinical assay. Ultimately, new biomarker assays must address 5 questions for optimal clinical translation. They include the following: is the biomarker likely to be (1) superior (will the test outperform current standards?); (2) actionable (will the test change patient management?); (3) valuable (will the test improve patient outcomes?); (4) economical (will the implementation of the biomarker in the target population be cost-saving or cost-effective?); and (5) clinically deployable (is there a pathway for the biomarker and analytical technology to be implemented in a clinical laboratory?)? In this article we review some of the major barriers to biomarker development in COPD and provide possible solutions to overcome these limitations, enabling translation of promising biomarkers from discovery experiments to clinical implementation.

Reproducibility of NIRS assessment of muscle oxidative capacity in smokers with and without COPD.

Low muscle oxidative capacity contributes to exercise intolerance in chronic obstructive pulmonary disease (COPD). Near-infrared spectroscopy (NIRS) allows non-invasive determination of the muscle oxygen consumption (mV̇O2) recovery rate constant (k), which is proportional to oxidative capacity assuming two conditions are met: 1) exercise intensity is sufficient to fully-activate mitochondrial oxidative enzymes; 2) sufficient O2 availability. We aimed to determine reproducibility (coefficient of variation, CV; intraclass correlation coefficient, ICC) of NIRS k assessment in the gastrocnemius of 64 participants with (FEV1 64±23%predicted) or without COPD (FEV1 98±14%predicted). 10-15s dynamic contractions preceded 6min of intermittent arterial occlusions (5-10s each, ∼250mmHg) for k measurement. k was lower (P<0.05) in COPD (1.43±0.4min-1; CV=9.8±5.9%, ICC=0.88) than controls (1.74±0.69min-1; CV=9.9±8.4%; ICC=0.93). Poor k reproducibility was more common when post-contraction mV̇O2 and deoxygenation were low, suggesting insufficient exercise intensity for mitochondrial activation and/or the NIRS signal contained little light reflected from active muscle. The NIRS assessment was well tolerated and reproducible for muscle dysfunction evaluation in COPD.

Exhaled and non-exhaled non-invasive markers for assessment of respiratory inflammation in patients with stable COPD and healthy smokers.

We aimed at comparing exhaled and non-exhaled non-invasive markers of respiratory inflammation in patients with chronic obstructive pulmonary disease (COPD) and healthy subjects and define their relationships with smoking habit. Forty-eight patients with stable COPD who were ex-smokers, 17 patients with stable COPD who were current smokers, 12 healthy current smokers and 12 healthy ex-smokers were included in a cross-sectional, observational study. Inflammatory outcomes, including prostaglandin (PG) E2 and 15-F2t-isoprostane (15-F2t-IsoP) concentrations in exhaled breath condensate (EBC) and sputum supernatants, fraction of exhaled nitric oxide (FENO) and sputum cell counts, and functional (spirometry) outcomes were measured. Sputum PGE2 was elevated in both groups of smokers compared with ex-smoker counterpart (COPD: P < 0.02; healthy subjects: P < 0.03), whereas EBC PGE2 was elevated in current (P = 0.0065) and ex-smokers with COPD (P = 0.0029) versus healthy ex-smokers. EBC 15-F2t-IsoP, a marker of oxidative stress, was increased in current and ex-smokers with COPD (P < 0.0001 for both) compared with healthy ex-smokers, whereas urinary 15-F2t-IsoP was elevated in both smoker groups (COPD: P < 0.01; healthy subjects: P < 0.02) versus healthy ex-smokers. FENO was elevated in ex-smokers with COPD versus smoker groups (P = 0.0001 for both). These data suggest that the biological meaning of these inflammatory markers depends on type of marker and biological matrix in which is measured. An approach combining different types of outcomes can be used for assessing respiratory inflammation in patients with COPD. Large studies are required to establish the clinical utility of this strategy.

Assessment of exhaled carbon monoxide in exacerbations of chronic obstructive pulmonary disease.

Introduction Exhaled carbon monoxide (eCO) has been widely implicated as a pulmonary biomarker in respiratory diseases. The aim of this study was to investigate whether the treatment of patients with severe acute exacerbation of chronic obstructive pulmonary disease (AECOPD) could be aided by monitoring the changes in eCO. Methods The levels of eCO along with routine clinical parameters were analyzed in 29 current smoker and 33 ex-smoker COPD patients, first at the time of hospital admission, and again at discharge following the standard treatment. Patients with AECOPD were also stratified according to sputum bacteria. Results At exacerbation, the levels of eCO were increased in current smokers compared to ex-smokers (6.0 [2.0-9.5] versus 1.0 [1.0-2.0] ppm, p < 0.001). Similarly, eCO levels were higher in smokers after treatment (7.0 [2.0-12.5] versus 1.0 [1.0-2.0] ppm, p < 0.001). Treatment of AECOPD did not affect eCO concentrations. The levels of eCO were not statistically different between bacterial and non-bacterial AECOPD either. Investigating a subgroup of current smoker patients (n = 15), there was a significant correlation between the levels of eCO and blood carboxyhemoglobin concentrations both at exacerbation and discharge. No associations were found between eCO and lung function or blood gas parameters. Conclusion Our results suggest that monitoring eCO during the treatment of AECOPD is of limited clinical value.

Klotho Reduction in Alveolar Macrophages Contributes to Cigarette Smoke Extract-induced Inflammation in Chronic Obstructive Pulmonary Disease.

Abnormal inflammation and accelerated decline in lung function occur in patients with chronic obstructive pulmonary disease (COPD). Klotho, an anti-aging protein, has an anti-inflammatory function. However, the role of Klotho has never been investigated in COPD. The aim of this study is to investigate the possible role of Klotho by alveolar macrophages in airway inflammation in COPD. Klotho levels were assessed in the lung samples and peripheral blood mononuclear cells of non-smokers, smokers, and patients with COPD. The regulation of Klotho expression by cigarette smoke extract (CSE) was studied in vitro, and small interfering RNA (siRNA) and recombinant Klotho were employed to investigate the role of Klotho on CSE-induced inflammation. Klotho expression was reduced in alveolar macrophages in the lungs and peripheral blood mononuclear cells of COPD patients. CSE decreased Klotho expression and release from MH-S cells. Knockdown of endogenous Klotho augmented the expression of the inflammatory mediators, such as MMP-9, IL-6, and TNF-α, by MH-S cells. Exogenous Klotho inhibited the expression of CSE-induced inflammatory mediators. Furthermore, we showed that Klotho interacts with IκBα of the NF-κB pathway. Dexamethasone treatment increased the expression and release level of Klotho in MH-S cells. Our findings suggest that Klotho plays a role in sustained inflammation of the lungs, which in turn may have therapeutic implications in COPD.