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1.  Silica-induced NLRP3 inflammasome activation in vitro and in rat lungs 
Rationale
Mineral particles in the lung cause inflammation and silicosis. In myeloid and bronchial epithelial cells the inflammasome plays a role in responses to crystalline silica. Thioredoxin (TRX) and its inhibitory protein TRX-interacting protein link oxidative stress with inflammasome activation. We investigated inflammasome activation by crystalline silica polymorphs and modulation by TRX in vitro, as well as its localization and the importance of silica surface reactivity in rats.
Methods
We exposed bronchial epithelial cells and differentiated macrophages to silica polymorphs quartz and cristobalite and measured caspase-1 activity as well as the release of IL-1β, bFGF and HMGB1; including after TRX overexpression or treatment with recombinant TRX. Rats were intratracheally instilled with vehicle control, Dörentruper quartz (DQ12) or DQ12 coated with polyvinylpyridine N-oxide. At days 3, 7, 28, 90, 180 and 360 five animals per treatment group were sacrificed. Hallmarks of silicosis were assessed with Haematoxylin-eosin and Sirius Red stainings. Caspase-1 activity in the bronchoalveolar lavage and caspase-1 and IL-1β localization in lung tissue were determined using Western blot and immunohistochemistry (IHC).
Results
Silica polymorphs triggered secretion of IL-1β, bFGF and HMGB1 in a surface reactivity dependent manner. Inflammasome readouts linked with caspase-1 enzymatic activity were attenuated by TRX overexpression or treatment. At day 3 and 7 increased caspase-1 activity was detected in BALF of the DQ12 group and increased levels of caspase-1 and IL-1β were observed with IHC in the DQ12 group compared to controls. DQ12 exposure revealed silicotic nodules at 180 and 360 days. Particle surface modification markedly attenuated the grade of inflammation and lymphocyte influx and attenuated the level of inflammasome activation, indicating that the development of silicosis and inflammasome activation is determined by crystalline silica surface reactivity.
Conclusion
Our novel data indicate the pivotal role of surface reactivity of crystalline silica to activate the inflammasome in cultures of both epithelial cells and macrophages. Inhibitory capacity of the antioxidant TRX to inflammasome activation was evidenced. DQ12 quartz exposure induced acute and chronic functional activation of the inflammasome in the heterogeneous cell populations of the lung in associated with its crystalline surface reactivity.
Electronic supplementary material
The online version of this article (doi:10.1186/s12989-014-0058-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12989-014-0058-0
PMCID: PMC4243278  PMID: 25406505
Crystalline silica; NLRP3 inflammasome; Caspase-1; IL-1β; HMGB1; bFGF; TRX; PVNO
2.  An observational, longitudinal study on the home environment of people with chronic obstructive pulmonary disease: the research protocol of the Home Sweet Home study 
BMJ Open  2014;4(11):e006098.
Introduction
Chronic obstructive pulmonary disease (COPD) represents an important public health challenge. Patients are confronted with limitations during activities of daily living (ADLs). Resident loved ones of patients with COPD may be uniquely positioned to witness these limitations. COPD may have an impact on not only the patients’ life, but also on the lives of the resident loved ones. Furthermore, COPD exacerbation-related hospital admissions often occur in patients with COPD. However, whether and to what extent these admissions influence resident loved ones’ burden and health status remains currently unknown. Therefore, the primary objectives of this study are to investigate the differences between patients with COPD and resident loved ones’ perceptions of patients’ health status and problematic ADLs and to study prospectively the effects of a COPD exacerbation on resident loved ones’ perceptions of patients’ health status and problematic ADLs.
Methods and analysis
An observational, longitudinal study will be performed in 192 patients with COPD and their 192 resident loved ones. Primary outcomes are daily functioning, ADL, disease-specific health status, generic health status and dyspnoea. These will be assessed during home visits at baseline and after 12 months. Additional home visits will be performed when a COPD exacerbation-related hospital admission occurs during the 12-month follow-up period.
Ethics and dissemination
This protocol was approved by the Medical Ethics Committee of the Catharina Hospital Eindhoven, the Netherlands (NL42721.060.12/M12-1280) and is registered in the Dutch Trial Register (NTR3941).
doi:10.1136/bmjopen-2014-006098
PMCID: PMC4244479  PMID: 25384686
Chronic Obstructive Pulmonary Disease; Home environment; Resident loved one; Family caregiver; Activities of Daily Living; Quality of Life
3.  Cigarette Smoke Extract Induces a Phenotypic Shift in Epithelial Cells; Involvement of HIF1α in Mesenchymal Transition 
PLoS ONE  2014;9(10):e107757.
In COPD, matrix remodeling contributes to airflow limitation. Recent evidence suggests that next to fibroblasts, the process of epithelial-mesenchymal transition can contribute to matrix remodeling. CSE has been shown to induce EMT in lung epithelial cells, but the signaling mechanisms involved are largely unknown and subject of this study. EMT was assessed in A549 and BEAS2B cells stimulated with CSE by qPCR, Western blotting and immunofluorescence for epithelial and mesenchymal markers, as were collagen production, cell adhesion and barrier integrity as functional endpoints. Involvement of TGF-β and HIF1α signaling pathways were investigated. In addition, mouse models were used to examine the effects of CS on hypoxia signaling and of hypoxia per se on mesenchymal expression. CSE induced EMT characteristics in A549 and BEAS2B cells, evidenced by decreased expression of epithelial markers and a concomitant increase in mesenchymal marker expression after CSE exposure. Furthermore cells that underwent EMT showed increased production of collagen, decreased adhesion and disrupted barrier integrity. The induction of EMT was found to be independent of TGF-β signaling. On the contrary, CS was able to induce hypoxic signaling in A549 and BEAS2B cells as well as in mice lung tissue. Importantly, HIF1α knock-down prevented induction of mesenchymal markers, increased collagen production and decreased adhesion after CSE exposure, data that are in line with the observed induction of mesenchymal marker expression by hypoxia in vitro and in vivo. Together these data provide evidence that both bronchial and alveolar epithelial cells undergo a functional phenotypic shift in response to CSE exposure which can contribute to increased collagen deposition in COPD lungs. Moreover, HIF1α signaling appears to play an important role in this process.
doi:10.1371/journal.pone.0107757
PMCID: PMC4199572  PMID: 25329389
4.  Metabolic load during strength training or NMES in individuals with COPD: results from the DICES trial 
BMC Pulmonary Medicine  2014;14:146.
Background
Strength training and neuromuscular electrical stimulation (NMES) are effective training modalities for improving muscle function, exercise performance and health status in individuals with COPD. The aim of the present study was to analyze the metabolic load of these training modalities at baseline, half-way, and at the end of an eight-week interdisciplinary pulmonary rehabilitation program in a subgroup of individuals with COPD of the DICES trial.
Methods
Of 24 individuals with COPD (FEV1: 34 ± 2% predicted, men: 58%, age: 66 (61–68) years), peak oxygen uptake (VO2), peak minute ventilation (VE), heart rate, oxygen saturation and symptom scores were assessed during HF-NMES (75 Hz), LF-NMES (15 Hz) and strength training at three moments during their pulmonary rehabilitation program.
Results
Intervention-related peak VO2 did not change over time during HF-NMES, LF-NMES or strength training. Intervention-related peak VE did not change over time during strength training or LF-NMES and increased slightly, but significantly over time during HF-NMES. Peak VO2 and VE were significantly higher during strength training compared to HF-NMES or LF-NMES. Oxygen saturation significantly decreased after the first measurements during HF-NMES and strength training group to baseline, while no significant changes in oxygen saturation were observed during the other measurements. Heart rate significantly increased compared to baseline in all groups at all moments and was significantly higher after strength training compared to HF-NMES or LF-NMES. Median end scores (points) for dyspnea, fatigue and muscle pain ranged from 1 to 3, from 0.5 to 2 and from 0 to 6 after HF-NMES, from 2 to 3, from 2 to 5 and from 0 to 9 after LF-NMES and from 2 to 5, from 1.5 to 4 and from 0 to 28 after strength training respectively.
Conclusions
To conclude, the metabolic load and symptom scores remain acceptable low over time with increasing training loads during HF-NMES, LF-NMES or strength training.
Trial registration
Trial registration:NTR2322
doi:10.1186/1471-2466-14-146
PMCID: PMC4236758  PMID: 25182377
Chronic obstructive pulmonary disease; Neuromuscular electrical stimulation; Pulmonary rehabilitation; Strength training
5.  Leptin as regulator of pulmonary immune responses: Involvement in respiratory diseases 
Leptin is an adipocyte-derived hormone, recognized as a critical mediator of the balance between food intake and energy expenditure by signalling through its functional receptor (Ob-Rb) in the hypothalamus. Structurally, leptin belongs to the long-chain helical cytokine family, and is now known to have pleiotropic functions in both innate and adaptive immunity. The presence of the functional leptin receptor in the lung together with evidence of increased airspace leptin levels arising during pulmonary inflammation, suggests an important role for leptin in lung development, respiratory immune responses and eventually pathogenesis of inflammatory respiratory diseases. The purpose of this article is to review our current understanding of leptin and its functional role on the different resident cell types of the lung in health as well as in the context of three major respiratory conditions being chronic obstructive pulmonary disease (COPD), asthma, and pneumonia.
doi:10.1016/j.pupt.2013.03.016
PMCID: PMC4122282  PMID: 23542720
Leptin; Asthma; COPD; Pneumonia; Pulmonary immunity
6.  Influence of diet and obesity on COPD development and outcomes 
The global increase in the prevalence and incidence of obesity has called serious attention to this issue as a major public health concern. Obesity is associated with many chronic diseases, including cardiovascular disease and diabetes, and recently the role of overweight and obesity in lung disease has received new interest. Independently of obesity, diet also plays a role as a risk factor for many chronic diseases, and evidence is accumulating to support a role for diet in the prevention and management of several lung diseases. Chronic obstructive lung disease is the third-leading cause of death globally, and both obesity and diet appear to play roles in its pathophysiology. Obesity has been associated with decreased lung-function measures in population-based studies, with increased prevalence of several lung diseases and with compromised pulmonary function. In contrast, obesity has a protective effect against mortality in severe chronic obstructive pulmonary disease (COPD). Nutrient intake and dietary patterns have also been associated with lung-function measures and the development and progression of COPD. Taken together, this suggests that a focus on obesity and diet should be part of public health campaigns to reduce the burden of lung disease, and could have important implications for clinicians in the management of their patients. Future research should also focus on elucidating these relationships in diverse populations and age-groups, and on understanding the complex interaction between behavior, environment, and genetics in the development and progression of COPD. The goal of this article is to review current evidence regarding the role that obesity and diet play in the development of COPD, and in COPD-related outcomes.
doi:10.2147/COPD.S50111
PMCID: PMC4130708  PMID: 25125974
diet; obesity; nutrition; lung function; COPD
7.  Prevalence of Metabolic Syndrome in COPD Patients and Its Consequences 
PLoS ONE  2014;9(6):e98013.
Background
The prevalence of metabolic syndrome in COPD patients and its impact on patient related outcomes has been little studied. We evaluated the prevalence of metabolic syndrome and clinical and functional characteristics in patients with COPD and healthy subjects.
Methods
228 COPD patients and 156 healthy subjects were included. Metabolic syndrome was defined using criteria of the IDF. In all patients spirometry, body composition, functional exercise performance, and mood and health status were assessed. Groups were stratified for BMI and gender.
Results
Metabolic syndrome was present in 57% of the COPD patients and 40% of the healthy subjects. After stratification for BMI, presence of metabolic syndrome in patients with a BMI ≥25 kg/m2 was higher than in healthy peers. Patients with metabolic syndrome and a BMI <25 kg/m2 had higher BMI, fat free mass index and bone mineral density, and a lower 6MWD than the BMI matched patients without metabolic syndrome. Spirometry, maximal ergometry, mood and health status, and blood gases were not different between those groups. In COPD patients with metabolic syndrome self-reported co-morbidities and medication use were higher than in those without.
Conclusion
Metabolic syndrome is more prevalent in overweight or obese COPD patients than in BMI matched healthy subjects. Metabolic syndrome did not additionally impact patients' functional outcomes, but did impact the prevalence of co-morbidities.
doi:10.1371/journal.pone.0098013
PMCID: PMC4064974  PMID: 24950070
8.  Early body weight loss during concurrent chemo-radiotherapy for non-small cell lung cancer 
Background and purpose
Radiation-esophagitis and weight loss are frequently observed toxicities in patients treated with concurrent chemo-radiotherapy (CT-RT) for non-small cell lung cancer (NSCLC) and might be related. The purpose was to investigate whether weight loss already starts early after initiation of CT-RT and precedes radiation-esophagitis.
Materials and methods
In a retrospective cohort, weight and esophagitis grade ≥2 were assessed during the first weeks of (CT-)RT in patients treated with concurrent (n = 102) or sequential (n = 92) therapy. In a prospective validation study, data on body weight, esophagitis grade ≥2, nutritional intake and muscle strength were obtained before, during and following CT-RT.
Results
In the retrospective cohort, early weight loss was observed in concurrently treated patients (p = 0.002), independent of esophagitis ≥ grade 2. Early weight loss was also observed in the prospective cohort (p = 0.003) and was not accompanied by decreases in nutritional intake. In addition lower limb muscle strength rapidly declined (p = 0.042). In the later weeks of treatment, further body weight loss occurred (p < 0.001) despite increased nutritional supplementation and body weight was only partly recovered after 4 weeks post CT-RT (p = 0.003).
Conclusions
Weight loss during concurrent CT-RT for NSCLC starts early and prior to onset of esophagitis, requiring timely and intense nutritional rehabilitation.
Electronic supplementary material
The online version of this article (doi:10.1007/s13539-013-0127-5) contains supplementary material.
doi:10.1007/s13539-013-0127-5
PMCID: PMC4053563  PMID: 24452446
Weight loss; Chemotherapy; Concurrent; Esophagitis; Non-small cell lung cancer; Radiotherapy
9.  Characteristics and determinants of endurance cycle ergometry and six-minute walk distance in patients with COPD 
Background
Exercise tolerance can be assessed by the cycle endurance test (CET) and six-minute walk test (6MWT) in patients with Chronic Obstructive Pulmonary Disease (COPD). We sought to investigate the characteristics of functional exercise performance and determinants of the CET and 6MWT in a large clinical cohort of COPD patients.
Methods
A dataset of 2053 COPD patients (43% female, age: 66.9 ± 9.5 years, FEV1% predicted: 48.2 ± 23.2) was analyzed retrospectively. Patients underwent, amongst others, respiratory function evaluation; medical tests and questionnaires, one maximal incremental cycle test where peak work rate was determined and two functional exercise tests: a CET at 75% of peak work rate and 6MWT. A stepwise multiple linear regression was used to assess determinants.
Results
On average, patients had impaired exercise tolerance (peak work rate: 56 ± 27% predicted, 6MWT: 69 ± 17% predicted). A total of 2002 patients had CET time of duration (CET-Tend) less than 20 min while only 51 (2.5%) of the patients achieved 20 min of CET-Tend . In former patients, the percent of predicted peak work rate achieved differed significantly between men (48 ± 21% predicted) and women (67 ± 31% predicted). In contrast, CET-Tend was longer in men (286 ± 174 s vs 250 ± 153 s, p < 0.001). Also, six minute walking distance (6MWD) was higher in men compared to women, both in absolute terms as in percent of predicted (443 m, 67%predicted vs 431 m, 72%predicted, p < 0.05). Gender was associated with the CET-Tend but BMI, FEV1 and FRC were related to the 6MWD highlighting the different determinants of exercise performance between CET and 6MWT.
Conclusions
CET-Tend is a valuable outcome of CET as it is related to multiple clinical aspects of disease severity in COPD. Gender difference should temper the interpretation of CET.
doi:10.1186/1471-2466-14-97
PMCID: PMC4229855  PMID: 24885117
Exercise; 6MWT; CET; CPET; COPD
10.  Similar matrix alterations in alveolar and small airway walls of COPD patients 
Background
Remodelling in COPD has at least two dimensions: small airway wall thickening and destruction of alveolar walls. Recent studies indicate that there is some similarity between alveolar and small airway wall matrix remodelling. The aim of this study was to characterise and assess similarities in alveolar and small airway wall matrix remodelling, and TGF-β signalling in COPD patients of different GOLD stages.
Methods
Lung tissue sections of 14 smoking controls, 16 GOLD II and 19 GOLD IV patients were included and stained for elastin and collagens as well as hyaluronan, a glycosaminoglycan matrix component and pSMAD2.
Results
Elastin was significantly decreased in COPD patients not only in alveolar, but also in small airway walls. Interestingly, both collagen and hyaluronan were increased in alveolar as well as small airway walls. The matrix changes were highly comparable between GOLD stages, with collagen content in the alveolar wall increasing further in GOLD IV. A calculated remodelling index, defined as elastin divided over collagen and hyaluronan, was decreased significantly in GOLD II and further lowered in GOLD IV patients, suggesting that matrix component alterations are involved in progressive airflow limitation. Interestingly, there was a positive correlation present between the alveolar and small airway wall stainings of the matrix components, as well as for pSMAD2. No differences in pSMAD2 staining between controls and COPD patients were found.
Conclusions
In conclusion, remodelling in the alveolar and small airway wall in COPD is markedly similar and already present in moderate COPD. Notably, alveolar collagen and a remodelling index relate to lung function.
doi:10.1186/1471-2466-14-90
PMCID: PMC4055380  PMID: 24886452
11.  COgnitive-Pulmonary Disease 
BioMed Research International  2014;2014:697825.
Over the past few decades, chronic obstructive lung disease (COPD) has been considered a disease of the lungs, often caused by smoking. Nowadays, COPD is regarded as a systemic disease. Both physical effects and effects on brains, including impaired psychological and cognitive functioning, have been demonstrated. Patients with COPD may have cognitive impairment, either globally or in single cognitive domains, such as information processing, attention and concentration, memory, executive functioning, and self-control. Possible causes are hypoxemia, hypercapnia, exacerbations, and decreased physical activity. Cognitive impairment in these patients may be related to structural brain abnormalities, such as gray-matter pathologic changes and the loss of white matter integrity which can be induced by smoking. Cognitive impairment can have a negative impact on health and daily life and may be associated with widespread consequences for disease management programs. It is important to assess cognitive functioning in patients with COPD in order to optimize patient-oriented treatment and to reduce personal discomfort, hospital admissions, and mortality. This paper will summarize the current knowledge about cognitive impairment as extrapulmonary feature of COPD. Hereby, the impact of smoking on cognitive functioning and the impact of cognitive impairment on smoking behaviour will be examined.
doi:10.1155/2014/697825
PMCID: PMC3971492  PMID: 24738069
12.  The COgnitive-Pulmonary Disease (COgnitive-PD) study: protocol of a longitudinal observational comparative study on neuropsychological functioning of patients with COPD 
BMJ Open  2014;4(3):e004495.
Introduction
Intact cognitive functioning is necessary for patients with chronic obstructive pulmonary disease (COPD) to understand the value of healthy lifestyle guidelines, to make informed decisions and subsequently act on it. Nevertheless, brain abnormalities and cognitive impairment have been found in patients with COPD. To date, it remains unknown which cognitive domains are affected and what the possible consequences are of cognitive impairment. Therefore, objectives of the study described are to determine neuropsychological functioning in patients with COPD, and its influence on health status, daily functioning and pulmonary rehabilitation outcome. Furthermore, structural and functional brain abnormalities and the relationship with cognitive and daily functioning will be explored.
Methods and analysis
A longitudinal observational comparative study will be performed in 183 patients with COPD referred for pulmonary rehabilitation and in 90 healthy control participants. Demographic and clinical characteristics, activities of daily living and knowledge about COPD will be assessed. Baseline cognitive functioning will be compared between patients and controls using a detailed neuropsychological testing battery. An MRI substudy will be performed to compare brain abnormalities between 35 patients with COPD with cognitive impairment and 35 patients with COPD without cognitive impairment. Patients will be recruited between November 2013 and November 2015.
Ethics and dissemination
The study has been approved by the Medical Ethics Committee of the University Hospital Maastricht and Maastricht University (NL45127.068.13/METC 13-3-035) and is registered in the Dutch trial register. All participants will provide written informed consent and can withdraw from the study at any point in time. Assessment and home visit data material will be managed anonymously. The results obtained can be used to optimise patient-oriented treatment for cognitively impaired patients with COPD. The findings will be disseminated in international peer-reviewed journals and through research conferences.
doi:10.1136/bmjopen-2013-004495
PMCID: PMC3948451  PMID: 24589828
Respiratory Medicine (see Thoracic Medicine); Neuropathology
13.  Association of plasma sRAGE, but not esRAGE with lung function impairment in COPD 
Respiratory Research  2014;15(1):24.
Rationale
Plasma soluble Receptor for Advanced Glycation End Product (sRAGE) is considered as a biomarker in COPD. The contribution of endogenous sRAGE (esRAGE) to the pool of plasma sRAGE and the implication of both markers in COPD pathogenesis is however not clear yet. The aim of the current study was therefore to measure plasma levels of esRAGE comparative to total sRAGE in patients with COPD and a control group. Further, we established the relations of esRAGE and total sRAGE with disease specific characteristics such as lung function and DLCO, and with different circulating AGEs.
Methods
Plasma levels of esRAGE and sRAGE were measured in an 88 patients with COPD and in 55 healthy controls. FEV1 (%predicted) and FEV1/VC (%) were measured in both groups; DLCO (%predicted) was measured in patients only. In this study population we previously reported that the AGE Nϵ-(carboxymethyl) lysine (CML) was decreased, Nϵ-(carboxyethyl) lysine (CEL) increased and pentosidine was not different in plasma of COPD patients compared to controls.
Results
Plasma esRAGE (COPD: 533.9 ± 412.4, Controls: 848.7 ± 690.3 pg/ml; p = 0.000) was decreased in COPD compared to controls. No significant correlations were observed between plasma esRAGE levels and lung function parameters or plasma AGEs. A positive correlation was present between esRAGE and total sRAGE levels in the circulation. Confirming previous findings, total sRAGE (COPD: 512.6 ± 403.8, Controls: 1834 ± 804.2 pg/ml; p < 0.001) was lower in patients compared to controls and was positively correlated FEV1 (r = 0.235, p = 0.032), FEV1/VC (r = 0.218, p = 0.047), and DLCO (r = 0.308, p = 0.006). sRAGE furthermore did show a significant positive association with CML (r = 0.321, p = 0.003).
Conclusion
Although plasma esRAGE is decreased in COPD patients compared to controls, only total sRAGE showed a significant and independent association with FEV1, FEV1/VC and DLCO, indicating that total sRAGE but not esRAGE may serve as marker of COPD disease state and severity.
doi:10.1186/1465-9921-15-24
PMCID: PMC3944004  PMID: 24564838
sRAGE; esRAGE; FEV1; COPD
15.  A randomised controlled trial on the efficacy of advance care planning on the quality of end-of-life care and communication in patients with COPD: the research protocol 
BMJ Open  2014;4(1):e004465.
Introduction
Recent research shows that advance care planning (ACP) for patients with chronic obstructive pulmonary disease (COPD) is uncommon and poorly carried out. The aim of the present study was to explore whether and to what extent structured ACP by a trained nurse, in collaboration with the chest physician, can improve outcomes in Dutch patients with COPD and their family.
Methods and analysis
A multicentre cluster randomised controlled trial in patients with COPD who are recently discharged after an exacerbation has been designed. Patients will be recruited from three Dutch hospitals and will be assigned to an intervention or control group, depending on the randomisation of their chest physician. Patients will be assessed at baseline and after 6 and 12 months. The intervention group will receive a structured ACP session by a trained nurse. The primary outcomes are quality of communication about end-of-life care, symptoms of anxiety and depression, quality of end-of-life care and quality of dying. Secondary outcomes include concordance between patient's preferences for end-of-life care and received end-of-life care, and psychological distress in bereaved family members of deceased patients. Intervention and control groups will be compared using univariate analyses and clustered regression analysis.
Ethics and dissemination
Ethical approval was received from the Medical Ethical Committee of the Catharina Hospital Eindhoven, the Netherlands (NL42437.060.12). The current project provides recommendations for guidelines on palliative care in COPD and supports implementation of ACP in the regular clinical care.
Clinical trial registration number
NTR3940.
doi:10.1136/bmjopen-2013-004465
PMCID: PMC3902375  PMID: 24384905
Advance Care Planning; End-of-life Care; Palliative Care; COPD
16.  Genome-Wide Association Analysis of Blood Biomarkers in Chronic Obstructive Pulmonary Disease 
Rationale: A genome-wide association study (GWAS) for circulating chronic obstructive pulmonary disease (COPD) biomarkers could identify genetic determinants of biomarker levels and COPD susceptibility.
Objectives: To identify genetic variants of circulating protein biomarkers and novel genetic determinants of COPD.
Methods: GWAS was performed for two pneumoproteins, Clara cell secretory protein (CC16) and surfactant protein D (SP-D), and five systemic inflammatory markers (C-reactive protein, fibrinogen, IL-6, IL-8, and tumor necrosis factor-α) in 1,951 subjects with COPD. For genome-wide significant single nucleotide polymorphisms (SNPs) (P < 1 × 10−8), association with COPD susceptibility was tested in 2,939 cases with COPD and 1,380 smoking control subjects. The association of candidate SNPs with mRNA expression in induced sputum was also elucidated.
Measurements and Main Results: Genome-wide significant susceptibility loci affecting biomarker levels were found only for the two pneumoproteins. Two discrete loci affecting CC16, one region near the CC16 coding gene (SCGB1A1) on chromosome 11 and another locus approximately 25 Mb away from SCGB1A1, were identified, whereas multiple SNPs on chromosomes 6 and 16, in addition to SNPs near SFTPD, had genome-wide significant associations with SP-D levels. Several SNPs affecting circulating CC16 levels were significantly associated with sputum mRNA expression of SCGB1A1 (P = 0.009–0.03). Several SNPs highly associated with CC16 or SP-D levels were nominally associated with COPD in a collaborative GWAS (P = 0.001–0.049), although these COPD associations were not replicated in two additional cohorts.
Conclusions: Distant genetic loci and biomarker-coding genes affect circulating levels of COPD-related pneumoproteins. A subset of these protein quantitative trait loci may influence their gene expression in the lung and/or COPD susceptibility.
Clinical trial registered with www.clinicaltrials.gov (NCT 00292552).
doi:10.1164/rccm.201206-1013OC
PMCID: PMC3622441  PMID: 23144326
biomarker; chronic obstructive pulmonary disease; genome-wide association study
17.  Associations between COPD related manifestations: a cross-sectional study 
Respiratory Research  2013;14(1):129.
Background
Cardiovascular disease, osteoporosis and emphysema are associated with COPD. Associations between these factors and whether they predict all-cause mortality in COPD patients are not well understood. Therefore, we examined associations between markers of cardiovascular disease (coronary artery calcification [CAC], thoracic aortic calcification [TAC] and arterial stiffness), bone density (bone attenuation of the thoracic vertebrae), emphysema (PI-950 and 15th percentile) and all-cause mortality in a COPD cohort.
Methods
We assessed CAC, TAC, bone attenuation of the thoracic vertebrae, PI-950 and 15th percentile on low-dose chest computed tomography in COPD subjects. We measured arterial stiffness as carotid-radial pulse wave velocity (PWV), and identified deaths from the national register.
Results
We studied 119 COPD subjects; aged 67.8 ±7.3, 66% were males and mean FEV1% predicted was 46.0 ±17.5. Subjects were classified into three pre-specificed groups: CAC = 0 (n = 14), 0 < CAC ≤ 400 (n = 41) and CAC > 400 (n = 64). Subjects with higher CAC were more likely to be older (p < 0.001) and male (p = 0.03), and more likely to have higher systolic blood pressure (p = 0.001) and a history of hypertension (p = 0.002) or ischemic heart disease (p = 0.003). Higher CAC was associated with higher PWV (OR 1.62, p = 0.04) and lower bone attenuation (OR 0.32, p = 0.02), but not with 15th percentile, after adjustment for age, sex and pack-years of smoking. In a Cox proportional hazards model, CAC, TAC and 15th percentile predicted all-cause mortality (HR 2.01, 2.09 and 0.66, respectively).
Conclusions
Increased CAC was associated with increased arterial stiffness and lower bone density in a COPD cohort. In addition, CAC, TAC and extent of emphysema predicted all-cause mortality.
Trial registration
Lothian NHS Board, Lothian Research Ethics Committee, LREC/2003/8/28.
doi:10.1186/1465-9921-14-129
PMCID: PMC3840707  PMID: 24251912
Arterial calcification; Arterial stiffness; Bone density; Cardiovascular disease; Co-morbidity; Computed tomography; COPD; Emphysema; Mortality; Osteoporosis
18.  Metabolic and Structural Changes in Lower-Limb Skeletal Muscle Following Neuromuscular Electrical Stimulation: A Systematic Review 
PLoS ONE  2013;8(9):e69391.
Background
Transcutaneous neuromuscular electrical stimulation (NMES) can be applied as a complementary intervention to regular exercise training programs. A distinction can be made between high-frequency (HF) NMES and low-frequency (LF) NMES. In order to increase understanding of the mechanisms of functional improvements following NMES, the purpose of this study was to systematically review changes in enzyme activity, muscle fiber type composition and muscle fiber size in human lower-limb skeletal muscles following only NMES.
Methods
Trials were collected up to march 2012 and were identified by searching the Medline/PubMed, EMBASE, Cochrane Central Register of Controlled Trials, CINAHL and The Physical Therapy Evidence Database (PEDro) databases and reference lists. 18 trials were reviewed in detail: 8 trials studied changes in enzyme activities, 7 trials studied changes in muscle fiber type composition and 14 trials studied changes in muscle fiber size following NMES.
Results
The methodological quality generally was poor, and the heterogeneity in study design, study population, NMES features and outcome parameters prohibited the use of meta-analysis. Most of the LF-NMES studies reported significant increases in oxidative enzyme activity, while the results concerning changes in muscle fiber composition and muscle size were conflicting. HF-NMES significantly increased muscle size in 50% of the studies.
Conclusion
NMES seems to be a training modality resulting in changes in oxidative enzyme activity, skeletal muscle fiber type and skeletal muscle fiber size. However, considering the small sample sizes, the variance in study populations, the non-randomized controlled study designs, the variance in primary outcomes, and the large heterogeneity in NMES protocols, it is difficult to draw definitive conclusions about the effects of stimulation frequencies on muscular changes.
doi:10.1371/journal.pone.0069391
PMCID: PMC3760845  PMID: 24019860
19.  Casein protein results in higher prandial and exercise induced whole body protein anabolism than whey protein in Chronic Obstructive Pulmonary Disease 
Objective
Exercise is known to improve physical functioning and health status in Chronic Obstructive Pulmonary Disease (COPD). Recently, disturbances in protein turnover and amino acid kinetics have been observed after exercise in COPD. The objective was to investigate which dairy protein is able to positively influence the protein metabolic response to exercise in COPD.
Materials and Methods
8 COPD patients and 8 healthy subjects performed a cycle test on two days while ingesting casein or whey protein. Whole body protein breakdown (WbPB), synthesis (WbPS), splanchnic amino acid extraction (SPE), and NetWbPS (=WbPS–WbPB) were measured using stable isotope methodology during 20 minutes of exercise (at 50% peak work load of COPD group). The controls performed a second exercise test at the same relative workload. Exercise was followed by 1 hour of recovery.
Results
In the healthy group, WbPS, SPE, and NetPS were higher during casein than during whey feeding (p<0.01). WbPS and NetPS were higher during exercise, independent of exercise intensity (p<0.01). NetPS was higher during casein feeding in COPD due to lower WbPB (p<0.05). Higher SPE was found during exercise during casein and whey feeding in COPD (p<0.05). Lactate levels during exercise were higher in COPD (p<0.05) independent of the protein. Post-exercise, lower NetPS values were found independent of protein type in both groups.
Conclusion
Casein resulted in more protein anabolism than whey protein which was maintained during and following exercise in COPD. Optimizing protein intake might be of importance for muscle maintenance during daily physical activities in COPD.
doi:10.1016/j.metabol.2012.03.001
PMCID: PMC3407276  PMID: 22512824
exercise; COPD; whole body protein metabolism; casein; whey
20.  A genome-wide association study of COPD identifies a susceptibility locus on chromosome 19q13 
Human Molecular Genetics  2011;21(4):947-957.
The genetic risk factors for chronic obstructive pulmonary disease (COPD) are still largely unknown. To date, genome-wide association studies (GWASs) of limited size have identified several novel risk loci for COPD at CHRNA3/CHRNA5/IREB2, HHIP and FAM13A; additional loci may be identified through larger studies. We performed a GWAS using a total of 3499 cases and 1922 control subjects from four cohorts: the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE); the Normative Aging Study (NAS) and National Emphysema Treatment Trial (NETT); Bergen, Norway (GenKOLS); and the COPDGene study. Genotyping was performed on Illumina platforms with additional markers imputed using 1000 Genomes data; results were summarized using fixed-effect meta-analysis. We identified a new genome-wide significant locus on chromosome 19q13 (rs7937, OR = 0.74, P = 2.9 × 10−9). Genotyping this single nucleotide polymorphism (SNP) and another nearby SNP in linkage disequilibrium (rs2604894) in 2859 subjects from the family-based International COPD Genetics Network study (ICGN) demonstrated supportive evidence for association for COPD (P = 0.28 and 0.11 for rs7937 and rs2604894), pre-bronchodilator FEV1 (P = 0.08 and 0.04) and severe (GOLD 3&4) COPD (P = 0.09 and 0.017). This region includes RAB4B, EGLN2, MIA and CYP2A6, and has previously been identified in association with cigarette smoking behavior.
doi:10.1093/hmg/ddr524
PMCID: PMC3298111  PMID: 22080838
21.  Silica induces NLRP3 inflammasome activation in human lung epithelial cells 
Background
In myeloid cells the inflammasome plays a crucial role in innate immune defenses against pathogen- and danger-associated patterns such as crystalline silica. Respirable mineral particles impinge upon the lung epithelium causing irreversible damage, sustained inflammation and silicosis. In this study we investigated lung epithelial cells as a target for silica-induced inflammasome activation.
Methods
A human bronchial epithelial cell line (BEAS-2B) and primary normal human bronchial epithelial cells (NHBE) were exposed to toxic but nonlethal doses of crystalline silica over time to perform functional characterization of NLRP3, caspase-1, IL-1β, bFGF and HMGB1. Quantitative RT-PCR, caspase-1 enzyme activity assay, Western blot techniques, cytokine-specific ELISA and fibroblast (MRC-5 cells) proliferation assays were performed.
Results
We were able to show transcriptional and translational upregulation of the components of the NLRP3 intracellular platform, as well as activation of caspase-1. NLRP3 activation led to maturation of pro-IL-1β to secreted IL-1β, and a significant increase in the unconventional release of the alarmins bFGF and HMGB1. Moreover, release of bFGF and HMGB1 was shown to be dependent on particle uptake. Small interfering RNA experiments using siNLRP3 revealed the pivotal role of the inflammasome in diminished release of pro-inflammatory cytokines, danger molecules and growth factors, and fibroblast proliferation.
Conclusion
Our novel data indicate the presence and functional activation of the NLRP3 inflammasome by crystalline silica in human lung epithelial cells, which prolongs an inflammatory signal and affects fibroblast proliferation, mediating a cadre of lung diseases.
doi:10.1186/1743-8977-10-3
PMCID: PMC3607900  PMID: 23402370
Silica; NLRP3 inflammasome; Caspase-1; IL-1β; HMGB1; bFGF
22.  Cigarette Smoke Targets Glutaredoxin 1, Increasing S-Glutathionylation and Epithelial Cell Death 
It is established that cigarette smoke (CS) causes irreversible oxidations in lung epithelial cells, and can lead to their death. However, its impact on reversible and physiologically relevant redox-dependent protein modifications remains to be investigated. Glutathione is an important antioxidant against inhaled reactive oxygen species as a direct scavenger, but it can also covalently bind protein thiols upon mild oxidative stress to protect them against irreversible oxidation. This posttranslational modification, known as S-glutathionylation, can be reversed under physiological conditions by the enzyme, glutaredoxin 1 (Grx1). The aim of this study was to investigate if CS modifies Grx1, and if this impacts on protein S-glutathionylation and epithelial cell death. Upon exposure of alveolar epithelial cells to CS extract (CSE), a decrease in Grx1 mRNA and protein expression was observed, in conjunction with decreased activity and increased protein S-glutathionylation. Using mass spectrometry, irreversible oxidation of recombinant Grx1 by CSE and acrolein was demonstrated, which was associated with attenuated enzyme activity. Furthermore, carbonylation of Grx1 in epithelial cells after exposure to CSE was shown. Overexpression of Grx1 attenuated CSE-induced increases in protein S-glutathionylation and increased survival. Conversely, primary tracheal epithelial cells of mice lacking Grx1 were more sensitive to CS-induced cell death, with corresponding increases in protein S-glutathionylation. These results show that CS can modulate Grx1, not only at the expression level, but can also directly modify Grx1 itself, decreasing its activity. These findings demonstrate a role for the Grx1/S-glutathionylation redox system in CS-induced lung epithelial cell death.
doi:10.1165/rcmb.2010-0249OC
PMCID: PMC3262689  PMID: 21454804
chronic obstructive pulmonary disease; cigarette smoke; cell death; glutaredoxin; protein S-glutathionylation
23.  Activation of the glutaredoxin-1 gene by Nuclear Factor kappa B enhances signaling 
Free radical biology & medicine  2011;51(6):1249-1257.
The transcription factor, Nuclear Factor kappa B (NF-κB) is a critical regulator of inflammation and immunity, and is negatively regulated via S-glutathionylation. The inhibitory effect of S-glutathionylation is overcome by glutaredoxin-1 (Grx1), which under physiological conditions catalyses deglutathionylation and enhances NF-κB activation. The mechanisms whereby expression of the Glrx1 gene is regulated remain unknown. Here we examined the role of NF-κB in regulating activation of Glrx1. Transgenic mice which express a doxycyclin-inducible constitutively active version of inhibitory kappa B kinase-beta (CA-IKKβ) demonstrate elevated expression of Grx1. Transient transfection of CA-IKKβ also resulted in significant induction of Grx1. A 2kb region Glrx1 promoter that contains two putative NF-κB binding sites was activated by CA-IKKβ, RelA/p50, and lipopolysaccharide (LPS). Chromatin immunoprecipitation experiments confirmed binding of RelA to the promoter of Glrx1 in response to LPS. Stimulation of C10 lung epithelial cells with LPS caused transient increases in Grx1 mRNA expression, and time-dependent increases in S-glutathionylation of IKKβ. Overexpression of Grx1 decreased S-glutathionylation of IKKβ, prolonged NF-κB activation, and increased levels of pro-inflammatory mediators. Collectively, this study demonstrates that the Glrx1 gene is positively regulated by NF-κB, and suggests a feed forward mechanism to promote NF-κB signaling by decreasing S-glutathionylation.
doi:10.1016/j.freeradbiomed.2011.06.025
PMCID: PMC3181077  PMID: 21762778
S-glutathionylation; Nuclear Factor kappa B; Glutaredoxin; Lung; Inhibitory kappa B kinase
24.  Examining fatigue in COPD: development, validity and reliability of a modified version of FACIT-F scale 
Introduction
Fatigue is a disruptive symptom that inhibits normal functional performance of COPD patients in daily activities. The availability of a short, simple, reliable and valid scale would improve assessment of the characteristics and influence of fatigue in COPD.
Methods
At baseline, 2107 COPD patients from the ECLIPSE cohort completed the Functional Assessment of Chronic Illness Therapy Fatigue (FACIT-F) scale. We used well-structured classic method, the principal components analysis (PCA) and Rasch analysis for structurally examining the 13-item FACIT-F.
Results
Four items were less able to capture fatigue characteristics in COPD and were deleted. PCA was applied to the remaining 9 items of the modified FACIT-F and resulted in three interpretable dimensions: i) general (5 items); ii) functional ability (2 items); and iii) psychosocial fatigue (2 items). The modified FACIT-F had high internal consistency (Cronbach's α = 0.91) and it did not fit a uni-dimensional Rasch model, confirming the prior output from the PCA. The correlations between total score and each dimension were ≥ 0.64 and within dimensions ≥0.43 (p < 0.001 for all).
The original and modified FACIT-F had significant convergent validity; its scores were associated with SGRQ total score (0.69 and 0.7) and mMRC dyspnoea scores (0.48 and 0.47), (p = <0.001 for all). The scale had meaningful discriminating ability in identifying patients with poor exercise performance and more depressive symptoms.
Conclusion
The original and modified FACIT-F are valid and reliable scales in COPD. The modified version is shorter and measures not only total fatigue but also its sub-components in COPD.
doi:10.1186/1477-7525-10-100
PMCID: PMC3491053  PMID: 22913289
Chronic obstructive pulmonary disease; Fatigue; Exercise capacity; Health status
25.  Correlations between disease-specific and generic health status questionnaires in patients with advanced COPD: a one-year observational study 
Background
Longitudinal studies analyzing the correlations between disease-specific and generic health status questionnaires at different time points in patients with advanced COPD are lacking. The aim of this study was to determine whether and to what extent a disease-specific health status questionnaire (Saint George’s Respiratory Questionnaire, SGRQ) correlates with generic health status questionnaires (EuroQol-5-Dimensions, EQ-5D; Assessment of Quality of Life instrument, AQoL; Medical Outcomes Study 36-Item Short-Form Health Survey, SF-36) at four different time points in patients with advanced COPD; and to determine the correlation between the changes in these questionnaires during one-year follow-up.
Methods
Demographic and clinical characteristics were assessed in 105 outpatients with advanced COPD at baseline. Disease-specific health status (SGRQ) and generic health status (EQ-5D, AQoL, SF-36) were assessed at baseline, four, eight, and 12 months. Correlations were determined between SGRQ and EQ-5D, AQoL, and SF-36 scores and changes in these scores. Agreement in direction of change was assessed.
Results
Eighty-four patients (80%) completed one-year follow-up and were included for analysis. SGRQ total score and EQ-5D index score, AQoL total score and SF-36 Physical Component Summary measure (SF-36 PCS) score were moderately to strongly correlated. The correlation of the changes between the SGRQ total score and EQ-5D index score, AQoL total score, SF-36 PCS, and SF-36 Mental Component Summary measure (SF-36 MCS) score were weak or absent. The direction of changes in SGRQ total scores agreed slightly with the direction of changes in EQ-5D index score, AQoL total score, and SF-36 PCS score.
Conclusions
At four, eight and 12 months after baseline, SGRQ total scores and EQ-5D index scores, AQoL total scores and SF-36 PCS scores were moderately to strongly correlated, while SGRQ total scores were weakly correlated with SF-36 MCS scores. The correlations between changes over time were weak or even absent. Disease-specific health status questionnaires and generic health status questionnaires should be used together to gain complete insight in health status and changes in health status over time in patients with advanced COPD.
doi:10.1186/1477-7525-10-98
PMCID: PMC3493349  PMID: 22909154
Chronic obstructive pulmonary disease; Health-related quality of life; St. George’s Respiratory Questionnaire; Health status; Disease-specific health status; Generic health status

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