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1.  Pro–Surfactant Protein B As a Biomarker for Lung Cancer Prediction 
Journal of Clinical Oncology  2013;31(36):4536-4543.
Purpose
Preliminary studies have identified pro–surfactant protein B (pro-SFTPB) to be a promising blood biomarker for non–small-cell lung cancer. We conducted a study to determine the independent predictive potential of pro-SFTPB in identifying individuals who are subsequently diagnosed with lung cancer.
Patients and Methods
Pro-SFTPB levels were measured in 2,485 individuals, who enrolled onto the Pan-Canadian Early Detection of Lung Cancer Study by using plasma sample collected at the baseline visit. Multivariable logistic regression models were used to evaluate the predictive ability of pro-SFTPB in addition to known lung cancer risk factors. Calibration and discrimination were evaluated, the latter by an area under the receiver operating characteristic curve (AUC). External validation was performed with samples collected in the Carotene and Retinol Efficacy Trial (CARET) participants using a case-control study design.
Results
Adjusted for age, sex, body mass index, personal history of cancer, family history of lung cancer, forced expiratory volume in one second percent predicted, average number of cigarettes smoked per day, and smoking duration, pro-SFTPB (log transformed) had an odds ratio of 2.220 (95% CI, 1.727 to 2.853; P < .001). The AUCs of the full model with and without pro-SFTPB were 0.741 (95% CI, 0.696 to 0.783) and 0.669 (95% CI, 0.620 to 0.717; difference in AUC P < .001). In the CARET Study, the use of pro-SFPTB yielded an AUC of 0.683 (95% CI, 0.604 to 0.761).
Conclusion
Pro-SFTPB in plasma is an independent predictor of lung cancer and may be a valuable addition to existing lung cancer risk prediction models.
doi:10.1200/JCO.2013.50.6105
PMCID: PMC3871515  PMID: 24248694
2.  Club Cell Protein 16 and Disease Progression in Chronic Obstructive Pulmonary Disease 
Rationale: Club (Clara) cell protein 16 (CC-16) is a protein that is synthesized predominantly in the lungs and is detectable in serum. Its expression decreases with lung injury and smoking, and is thus a marker of bronchial cell dysfunction.
Objectives: To evaluate the possibility of using serum CC-16 as a biomarker for disease progression in chronic obstructive pulmonary disease (COPD).
Methods: We measured serum CC-16 levels from 4,724 subjects with mild-to-moderate airflow limitation in the Lung Health Study. Using a linear regression model, we determined the relationship of serum CC-16 concentrations to decline in lung function over 9 years. In addition, to determine whether CC-16 plays a major role in the pathogenesis of mild COPD, we exposed CC-16–deficient (−/−) mice to 6 months of cigarette smoke.
Measurements and Main Results: Reduced serum concentrations of CC-16 were associated with accelerated decline in FEV1 over 9 years (P < 0.0001), and this association persisted after adjustments for age, sex, race, smoking status, airway reactivity, body mass index, and baseline FEV1 (P = 0.0002). However, CC-16−/− mice did not demonstrate an enhanced risk of emphysema or small airway remodeling in response to cigarette smoke.
Conclusions: Serum CC-16 is associated with disease progression, and may assist in the identification of “rapid progressors.” However, the absence of CC-16 does not appear to modify the risk of cigarette-related COPD in mice.
doi:10.1164/rccm.201305-0892OC
PMCID: PMC3917377  PMID: 24245748
biomarker; chronic obstructive pulmonary disease; disease progression; smoking
4.  Immunoglobulin G4-related lung disease: A disease with many different faces 
Immunoglobulin (Ig) G4-related lung disease is a fibroinflammatory entity that presents in protean ways. Diagnostically, IgG4-related lung disease requires a high clinical index of suspicion complemented by elevated serum IgG4 levels and/or biopsy that shows the characteristic pathological features. The disease is almost always responsive to systemic corticosteroids. However, relapse is common following their discontinuation. The authors present three cases to highlight the diverse clinical features, and to illustrate the diagnostic and therapeutic approaches to this disease.
PMCID: PMC3810041  PMID: 24093112
Corticosteroids; IgG4-related lung disease; Immunoglobulins
5.  Genes related to emphysema are enriched for ubiquitination pathways 
BMC Pulmonary Medicine  2014;14(1):187.
Background
Increased small airway resistance and decreased lung elasticity contribute to the airflow limitation in chronic obstructive pulmonary disease (COPD). The lesion that corresponds to loss of lung elasticity is emphysema; the small airway obstruction is due to inflammatory narrowing and obliteration. Despite their convergence in altered physiology, different mechanisms contribute to these processes. The relationships between gene expression and these specific phenotypes may be more revealing than comparison with lung function.
Methods
We measured the ratio of alveolar surface area to lung volume (SA/V) in lung tissue from 43 smokers. Two samples from 21 subjects, in which SA/V differed by >49 cm2/mL were profiled to select genes whose expression correlated with SA/V. Significant genes were tested for replication in the 22 remaining subjects.
Results
The level of expression of 181 transcripts was related to SA/V ( p < 0.05). When these genes were tested in the 22 remaining subjects as a replication, thirty of the 181 genes remained significantly associated with SA/V (P < 0.05) and the direction of association was the same in 164/181. Pathway and network analysis revealed enrichment of genes involved in protein ubiquitination, and western blotting showed altered expression of genes involved in protein ubiquitination in obstructed individuals.
Conclusion
This study implicates modified protein ubiquitination and degradation as a potentially important pathway in the pathogenesis of emphysema.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2466-14-187) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2466-14-187
PMCID: PMC4280711  PMID: 25432663
Pulmonary emphysema; Surface area to lung volume ratio; Gene expression; Transcriptional analysis; mRNA; Cigarette smoking; Protein ubquitination
6.  Burden of subclinical heart and lung disease detected on thoracic CT scans of HIV patients on HAART 
Journal of the International AIDS Society  2014;17(4Suppl 3):19716.
Introduction
The aim was to determine the prevalence of lung and heart abnormalities on thoracic CT scans in HIV-infected patients who were treated with antiretroviral therapy (ART).
Material and Methods
Thoracic CT scans of 903 patients infected with HIV (mean age 48±7 yrs, 29% females) were reviewed by three radiologists by consensus. Patients were phenotyped according to smoking status, pack years and years since cessation for ex-smokers. Individuals known to have active lung or heart disease at the time of CT scanning were excluded. Multimorbidity lung and heart disease (MLHD) was defined by the presence of >2 lung or heart abnormalities on the CT scan.
Results
Prevalence of lung abnormalities were: 326 patients (36.1%) with emphysema, 271 (30.0%) with bronchiolitis, 44 (4.9%) with non-calcified lung nodules, 568 (63%) with significant bronchial wall thickening, 150 (16.7%) with bronchiectasis, 9 (1%) with interstitial lung disease. Overall, 445 patients (49.3%) had >2 lung abnormalities. Imaging findings suggestive of prior myocardial infarction (MI) were found in 1.4% (13 patients); 26.6% (240 patients) had CAC scores of 1 to 100, and 9.8% (89 patients) had CAC>100. 13.6% (123 patients) of the patients had CAC>100 and/or previous MI. MLHD was present in 484 patients (53.6%) and among 78 patients (16%) who never smoked. Table 1 describes CT findings according to pack year and stop smoking groups vs never smokers.
MLHD increased proportional to cumulative smoking history (p for trend <0.001) and decreased in proportion to the number of years since smoking cessation (p for trend=0.017). Independent predictors for MLHD were: age (OR=1.07, CI 1.05–1.10), sex (OR=1.59, CI 1.15–2.19), current smoking (OR=1.76, CI 1.08–2.89), and pack-years history of smoking (OR=1.03, CI 1.02–1.05). In patients who never smoked, nadir CD4<200 was significantly associated with MLHD after adjustment for age and sex (OR=1.98, CI 1.98–3.63).
Conclusions
MLHD is common in HIV-infected individuals even in non-smokers. Reduced CD4 count (hence severity of HIV infection) may be an important risk factor for chronic lung and heart disease. Thoracic CT scans may provide an excellent screening tool to detect MLHD in HIV-infected patients.
doi:10.7448/IAS.17.4.19716
PMCID: PMC4225346  PMID: 25397462
7.  The Burden of Image Based Emphysema and Bronchiolitis in HIV-Infected Individuals on Antiretroviral Therapy 
PLoS ONE  2014;9(10):e109027.
Background
With the widespread use of anti-retroviral therapy (ART), individuals infected with human immune deficiency virus (HIV) are increasingly experiencing morbidity and mortality from respiratory disorders. However, the prevalence or the risk factors associated with emphysema and bronchiolitis are largely unknown.
Methods
Thoracic computed tomography (CT) scans were performed in 1,446 patients infected with HIV who were on ART and who attended a tertiary care metabolic clinic (average age 48 years and 29% females). Detailed history and physical examination including anthropometric measurements were performed. Complete pulmonary function tests were performed in a subset of these patients (n = 364). No subjects were acutely ill with a respiratory condition at the time of CT scanning.
Findings
Nearly 50% of the subjects had CT evidence for emphysema, bronchiolitis or both with 13% (n = 195) showing bronchiolitis, 19% (n = 274) showing emphysema and 16% (n = 238) revealing both. These phenotypes were synergistically associated with reduced regular physical activity (p for interaction <.0001). The most significant risk factors for both phenotypes were cigarette smoking, intravenous drug use and peripheral leucocytosis. Together, the area-under-the curve statistics was 0.713 (p = 0.0037) for discriminating those with and without these phenotypes. There were no significant changes in lung volumes or flow rates related to these phenotypes, though the carbon monoxide diffusion capacity was reduced for the emphysema phenotype.
Interpretation
Emphysema and bronchiolitis are extremely common in HIV-infected patients who are treated with ART and can be identified by use of thoracic CT scanning.
doi:10.1371/journal.pone.0109027
PMCID: PMC4212912  PMID: 25354261
8.  Bacterial microbiome of lungs in COPD 
Chronic obstructive pulmonary disease (COPD) is currently the third leading cause of death in the world. Although smoking is the main risk factor for this disease, only a minority of smokers develop COPD. Why this happens is largely unknown. Recent discoveries by the human microbiome project have shed new light on the importance and richness of the bacterial microbiota at different body sites in human beings. The microbiota plays a particularly important role in the development and functional integrity of the immune system. Shifts or perturbations in the microbiota can lead to disease. COPD is in part mediated by dysregulated immune responses to cigarette smoke and other environmental insults. Although traditionally the lung has been viewed as a sterile organ, by using highly sensitive genomic techniques, recent reports have identified diverse bacterial communities in the human lung that may change in COPD. This review summarizes the current knowledge concerning the lung microbiota in COPD and its potential implications for pathogenesis of the disease.
doi:10.2147/COPD.S38932
PMCID: PMC3937108  PMID: 24591822
chronic obstructive pulmonary disease; bacterial microbiome; lungs
9.  Changes in the Bacterial Microbiota in Gut, Blood, and Lungs following Acute LPS Instillation into Mice Lungs 
PLoS ONE  2014;9(10):e111228.
Introduction
Previous reports have shown that the gastrointestinal (GI) bacterial microbiota can have profound effects on the lungs, which has been described as the “gut-lung axis”. However, whether a “lung-gut” axis exists wherein acute lung inflammation perturbs the gut and blood microbiota is unknown.
Methods
Adult C57/Bl6 mice were exposed to one dose of LPS or PBS instillation (n = 3 for each group) directly into lungs. Bacterial microbiota of the bronchoalveolar lavage fluid, blood, and cecum were determined using 454 pyrotag sequencing and quantitative polymerase chain reaction (qPCR) at 4 through 168 hours post-instillation. We then investigated the effects of oral neomycin and streptomycin (n = 8) on the microbiota at 4 and 24 hours post LPS instillation versus control treatment (n = 5 at baseline and 4 hours, n = 7 at 24 hours).
Results
At 24 hours post LPS instillation, the total bacterial count was significantly increased in the cecum (P<0.05); whereas the total bacterial count in blood was increased at 4, 48, and 72 hours (P<0.05). Antibiotic treatment reduced the total bacteria in blood but not in the cecum. The increase in total bacteria in the blood correlated with Phyllobacteriaceae OTU 40 and was significantly reduced in the blood for both antibiotic groups (P<0.05).
Conclusion
LPS instillation in lungs leads to acute changes in the bacterial microbiota in the blood and cecum, which can be modulated with antibiotics.
doi:10.1371/journal.pone.0111228
PMCID: PMC4205020  PMID: 25333938
10.  A Comparison between Droplet Digital and Quantitative PCR in the Analysis of Bacterial 16S Load in Lung Tissue Samples from Control and COPD GOLD 2 
PLoS ONE  2014;9(10):e110351.
Background
Low biomass in the bacterial lung tissue microbiome utilizes quantitative PCR (qPCR) 16S bacterial assays at their limit of detection. New technology like droplet digital PCR (ddPCR) could allow for higher sensitivity and accuracy of quantification. These attributes are needed if specific bacteria within the bacterial lung tissue microbiome are to be evaluated as potential contributors to diseases such as chronic obstructive pulmonary disease (COPD). We hypothesize that ddPCR is better at quantifying the total bacterial load in lung tissue versus qPCR.
Methods
Control (n = 16) and COPD GOLD 2 (n = 16) tissue samples were obtained from patients who underwent lung resection surgery, were cut on a cryotome, and sections were assigned for use in quantitative histology or for DNA extraction. qPCR and ddPCR were performed on these samples using primers spanning the V2 region on the 16S rRNA gene along with negative controls. Total 16S counts were compared between the two methods. Both methods were assessed for correlations with quantitative histology measurements of the tissue.
Results
There was no difference in the average total 16S counts (P>0.05) between the two methods. However, the negative controls contained significantly lower counts in the ddPCR (0.55 ± 0.28 16S/uL) than in the qPCR assay (1.00 ± 0.70 16S copies) (P <0.05). The coefficient of variation was significantly lower for the ddPCR assay (0.18 ± 0.14) versus the qPCR assay (0.62 ± 0.29) (P<0.05).
Conclusion
Overall the ddPCR 16S assay performed better by reducing the background noise in 16S of the negative controls compared with 16S qPCR assay.
doi:10.1371/journal.pone.0110351
PMCID: PMC4199711  PMID: 25329701
11.  Estradiol Increases Mucus Synthesis in Bronchial Epithelial Cells 
PLoS ONE  2014;9(6):e100633.
Airway epithelial mucus hypersecretion and mucus plugging are prominent pathologic features of chronic inflammatory conditions of the airway (e.g. asthma and cystic fibrosis) and in most of these conditions, women have worse prognosis compared with male patients. We thus investigated the effects of estradiol on mucus expression in primary normal human bronchial epithelial cells from female donors grown at an air liquid interface (ALI). Treatment with estradiol in physiological ranges for 2 weeks caused a concentration-dependent increase in the number of PAS-positive cells (confirmed to be goblet cells by MUC5AC immunostaining) in ALI cultures, and this action was attenuated by estrogen receptor beta (ER-β) antagonist. Protein microarray data showed that nuclear factor of activated T-cell (NFAT) in the nuclear fraction of NHBE cells was increased with estradiol treatment. Estradiol increased NFATc1 mRNA and protein in ALI cultures. In a human airway epithelial (1HAE0) cell line, NFATc1 was required for the regulation of MUC5AC mRNA and protein. Estradiol also induced post-translational modification of mucins by increasing total fucose residues and fucosyltransferase (FUT-4, -5, -6) mRNA expression. Together, these data indicate a novel mechanism by which estradiol increases mucus synthesis in the human bronchial epithelium.
doi:10.1371/journal.pone.0100633
PMCID: PMC4070981  PMID: 24964096
12.  Bronchodilator Responsiveness and Reported Respiratory Symptoms in an Adult Population 
PLoS ONE  2013;8(3):e58932.
Background
The relationship between patient-reported symptoms and objective measures of lung function is poorly understood.
Aim
To determine the association between responsiveness to bronchodilator and respiratory symptoms in random population samples.
Methods
4669 people aged 40 years and older from 8 sites in Canada completed interviewer-administered respiratory questionnaires and performed spirometry before and after administration of 200 ug of inhaled salbutamol. The effect of anthropometric variables, smoking exposure and doctor-diagnosed asthma (DDA) on bronchodilator responsiveness in forced expiratory volume in 1 second (FEV1) and in forced vital capacity (FVC) were evaluated. Multiple logistic regression was used to test for association between quintiles of increasing changes in FEV1 and in FVC after bronchodilator and several respiratory symptoms.
Results
Determinants of bronchodilator change in FEV1 and FVC included age, DDA, smoking, respiratory drug use and female gender [p<0.005 to p<0.0001 ]. In subjects without doctor-diagnosed asthma or COPD, bronchodilator response in FEV1 was associated with wheezing [p for trend<0.0001], while bronchodilator response for FVC was associated with breathlessness. [p for trend <0.0001].
Conclusions
Bronchodilator responsiveness in FEV1 or FVC are associated with different respiratory symptoms in the community. Both flow and volume bronchodilator responses are useful parameters which together can be predictive of both wheezing and breathlessness in the general population.
doi:10.1371/journal.pone.0058932
PMCID: PMC3598856  PMID: 23554960
13.  Biomarkers in airway diseases 
The inherent limitations of spirometry and clinical history have prompted clinicians and scientists to search for surrogate markers of airway diseases. Although few biomarkers have been widely accepted into the clinical armamentarium, the authors explore three sources of biomarkers that have shown promise as indicators of disease severity and treatment response. In asthma, exhaled nitric oxide measurements can predict steroid responsiveness and sputum eosinophil counts have been used to titrate anti-inflammatory therapies. In chronic obstructive pulmonary disease, inflammatory plasma biomarkers, such as fibrinogen, club cell secretory protein-16 and surfactant protein D, can denote greater severity and predict the risk of exacerbations. While the multitude of disease phenotypes in respiratory medicine make biomarker development especially challenging, these three may soon play key roles in the diagnosis and management of airway diseases.
PMCID: PMC3814266  PMID: 23762888
Airway disease; Asthma; Biomarkers; Chronic obstructive pulmonary disease
14.  Whole Exome Re-Sequencing Implicates CCDC38 and Cilia Structure and Function in Resistance to Smoking Related Airflow Obstruction 
PLoS Genetics  2014;10(5):e1004314.
Chronic obstructive pulmonary disease (COPD) is a leading cause of global morbidity and mortality and, whilst smoking remains the single most important risk factor, COPD risk is heritable. Of 26 independent genomic regions showing association with lung function in genome-wide association studies, eleven have been reported to show association with airflow obstruction. Although the main risk factor for COPD is smoking, some individuals are observed to have a high forced expired volume in 1 second (FEV1) despite many years of heavy smoking. We hypothesised that these “resistant smokers” may harbour variants which protect against lung function decline caused by smoking and provide insight into the genetic determinants of lung health. We undertook whole exome re-sequencing of 100 heavy smokers who had healthy lung function given their age, sex, height and smoking history and applied three complementary approaches to explore the genetic architecture of smoking resistance. Firstly, we identified novel functional variants in the “resistant smokers” and looked for enrichment of these novel variants within biological pathways. Secondly, we undertook association testing of all exonic variants individually with two independent control sets. Thirdly, we undertook gene-based association testing of all exonic variants. Our strongest signal of association with smoking resistance for a non-synonymous SNP was for rs10859974 (P = 2.34×10−4) in CCDC38, a gene which has previously been reported to show association with FEV1/FVC, and we demonstrate moderate expression of CCDC38 in bronchial epithelial cells. We identified an enrichment of novel putatively functional variants in genes related to cilia structure and function in resistant smokers. Ciliary function abnormalities are known to be associated with both smoking and reduced mucociliary clearance in patients with COPD. We suggest that genetic influences on the development or function of cilia in the bronchial epithelium may affect growth of cilia or the extent of damage caused by tobacco smoke.
Author Summary
Very large genome-wide association studies in general population cohorts have successfully identified at least 26 genes or gene regions associated with lung function and a number of these also show association with chronic obstructive pulmonary disease (COPD). However, these findings explain a small proportion of the heritability of lung function. Although the main risk factor for COPD is smoking, some individuals have normal or good lung function despite many years of heavy smoking. We hypothesised that studying these individuals might tell us more about the genetics of lung health. Re-sequencing of exomes, where all of the variation in the protein-coding portion of the genome can be measured, is a recent approach for the study of low frequency and rare variants. We undertook re-sequencing of the exomes of “resistant smokers” and used publicly available exome data for comparisons. Our findings implicate CCDC38, a gene which has previously shown association with lung function in the general population, and genes involved in cilia structure and lung function as having a role in resistance to smoking.
doi:10.1371/journal.pgen.1004314
PMCID: PMC4006731  PMID: 24786987
15.  Longer Telomere Length in COPD Patients with α1-Antitrypsin Deficiency Independent of Lung Function 
PLoS ONE  2014;9(4):e95600.
Oxidative stress is involved in the pathogenesis of airway obstruction in α1-antitrypsin deficient patients. This may result in a shortening of telomere length, resulting in cellular senescence. To test whether telomere length differs in α1-antitrypsin deficient patients compared with controls, we measured telomere length in DNA from peripheral blood cells of 217 α1-antitrypsin deficient patients and 217 control COPD patients. We also tested for differences in telomere length between DNA from blood and DNA from lung tissue in a subset of 51 controls. We found that telomere length in the blood was significantly longer in α1-antitrypsin deficient COPD patients compared with control COPD patients (p = 1×10−29). Telomere length was not related to lung function in α1-antitrypsin deficient patients (p = 0.3122) or in COPD controls (p = 0.1430). Although mean telomere length was significantly shorter in the blood when compared with the lungs (p = 0.0078), telomere length was correlated between the two tissue types (p = 0.0122). Our results indicate that telomere length is better preserved in α1-antitrypsin deficient COPD patients than in non-deficient patients. In addition, measurement of telomere length in the blood may be a suitable surrogate for measurement in the lung.
doi:10.1371/journal.pone.0095600
PMCID: PMC3998943  PMID: 24763308
16.  Effects of inhaled corticosteroids on airway inflammation in chronic obstructive pulmonary disease: a systematic review and meta-analysis 
Background:
Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation in the small airways. The effect of inhaled corticosteroids (ICS) on lung inflammation in COPD remains uncertain. We sought to determine the effects of ICS on inflammatory indices in bronchial biopsies and bronchoalveolar lavage fluid of patients with COPD.
Methods:
We searched Medline, Embase, Cinahl, and the Cochrane database for randomized, controlled clinical trials that used bronchial biopsies and bronchoalveolar lavage to evaluate the effects of ICS in stable COPD. For each chosen study, we calculated the mean differences in the concentrations of inflammatory cells before and after treatment in both intervention and control groups. These values were then converted into standardized mean differences (SMD) to accommodate the differences in patient selection, clinical treatment, and biochemical procedures that were employed across the original studies. If significant heterogeneity was present (P < 0.1), then a random effects model was used to pool the original data; otherwise, a fixed effects model was used.
Results:
We identified eight original studies that met the inclusion criteria. Four studies used bronchial biopsies (n =102 participants) and showed that ICS were effective in reducing CD4 and CD8 cell counts (SMD, −0.52 units and −0.66 units, 95% confidence interval). The five studies used bronchoalveolar lavage fluid (n =309), which together showed that ICS reduced neutrophil and lymphocyte counts (SMD, −0.64 units and −0.64 units, 95% confidence interval). ICS on the other hand significantly increased macrophage counts (SMD, 0.68 units, 95% confidence interval) in bronchoalveolar lavage fluid.
Conclusion:
ICS has important immunomodulatory effects in airways with COPD that may explain its beneficial effect on exacerbations and enhanced risk of pneumonia.
doi:10.2147/COPD.S32765
PMCID: PMC3459653  PMID: 23055709
chronic obstructive pulmonary disease; bronchial biopsies; bronchoalveolar lavage; inhaled corticosteroids; inflammation; inflammatory markers; meta-analysis
17.  Plasma sCD14 as a Biomarker to Predict Pulmonary Exacerbations in Cystic Fibrosis 
PLoS ONE  2014;9(2):e89341.
Background
One in four cystic fibrosis (CF) patients diagnosed with a pulmonary exacerbation will not recover their baseline lung function despite standard treatment. This highlights the importance of preventing such events. Clinical decision-making can be improved through a simple blood test that predicts individuals at elevated short-term risk of an exacerbation.
Methods
We obtained plasma samples from 30 stable CF patients from the St. Paul’s Hospital Adult CF Clinic (Vancouver, Canada). For 15 patients, an additional plasma sample was obtained during an exacerbation. Soluble CD14 (sCD14) and C-reactive protein (CRP) were quantified using ELISA kits. Myeloperoxidase (MPO), interleukin(IL)-6, IL-1β, monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), and granulocyte colony-stimulating factor (G-CSF) were quantified using Luminex™ immunoassays. Stable state biomarker levels were examined in their ability to predict individuals that would experience a pulmonary exacerbation requiring intravenous (IV) antibiotics within 4 months. Paired stable and exacerbation plasma biomarker levels were also compared.
Results
sCD14 levels were significantly higher in patients that experienced a pulmonary exacerbation requiring IV antibiotics within 4 months (p = 0.001). sCD14 cut-off value of 1450 ng/mL was associated with an area under the curve of 0.91 (95% CI 0.83–0.99) for predicting an exacerbation within 4 months of a stable visit, with a sensitivity of 100% and specificity of 82%. Plasma sCD14 levels were significantly higher during exacerbations than during periods of clinical stability (p = 0.03).
Conclusions
Plasma sCD14 is a promising biomarker for identifying CF patients who will exacerbate within 4 months of a stable visit but requires further study in larger, independent cohorts.
doi:10.1371/journal.pone.0089341
PMCID: PMC3930718  PMID: 24586701
18.  Genome-wide study identifies two loci associated with lung function decline in mild to moderate COPD 
Human genetics  2012;132(1):79-90.
Rationale
Accelerated lung function decline is a key COPD phenotype; however its genetic control remains largely unknown.
Methods
We performed a genome-wide association study using the Illumina Human660W-Quad v.1_A BeadChip. Generalized estimation equations were used to assess genetic contributions to lung function decline over a 5-year period in 4,048 European-American Lung Health Study participants with largely mild COPD. Genotype imputation was performed using reference HapMap II data. To validate regions meeting genome-wide significance, replication of top SNPs was attempted in independent cohorts. Three genes (TMEM26, ANK3 and FOXA1) within the regions of interest were selected for tissue expression studies using immunohistochemistry.
Measurements and Main Results
Two intergenic SNPs (rs10761570, rs7911302) on chromosome 10 and one SNP on chromosome 14 (rs177852) met genome-wide significance after Bonferroni. Further support for the chromosome 10 region was obtained by imputation, the most significantly associated imputed SNPs (rs10761571, rs7896712) being flanked by observed markers rs10761570 and rs7911302. Results were not replicated in four general population cohorts or a smaller cohort of subjects with moderate to severe COPD; however, we show novel expression of genes near regions of significantly associated SNPS, including TMEM26 and FOXA1 in airway epithelium and lung parenchyma, and ANK3 in alveolar macrophages. Levels of expression were associated with lung function and COPD status.
Conclusions
We identified two novel regions associated with lung function decline in mild COPD. Genes within these regions were expressed in relevant lung cells and their expression related to airflow limitation suggesting they may represent novel candidate genes for COPD susceptibility.
doi:10.1007/s00439-012-1219-6
PMCID: PMC3536920  PMID: 22986903
COPD; lung function decline; GWAS; genome wide association; genes; polymorphisms
20.  Budesonide/Formoterol Enhances the Expression of Pro Surfactant Protein-B in Lungs of COPD Patients 
PLoS ONE  2013;8(12):e83881.
Rationale & Aim
Pulmonary surfactants are essential components of lung homeostasis. In chronic obstructive pulmonary disease (COPD), surfactant expression decreases in lungs whereas, there is a paradoxical increase in protein expression in plasma. The latter has been associated with poor health outcomes in COPD. The purpose of this study was to determine the relationship of surfactants and other pneumoproteins in bronchoalveolar lavage (BAL) fluid and plasma to airflow limitation and the effects of budesonide/formoterol on this relationship.
Methods
We recruited (clinical trials.gov identifier: NCT00569712) 7 smokers without COPD and 30 ex and current smokers with COPD who were free of exacerbations for at least 4 weeks. All subjects were treated with budesonide/formoterol 400/12 µg twice a day for 4 weeks. BAL fluid and plasma samples were obtained at baseline and the end of the 4 weeks. We measured lung-predominant pneumoproteins: pro-Surfactant Protein-B (pro-SFTPB), Surfactant Protein-D (SP-D), Club Cell Secretory Protein-16 (CCSP-16) and Pulmonary and Activation-Regulated Chemokine (PARC/CCL-18) in BAL fluid and plasma.
Results
BAL Pro-SFTPB concentrations had the strongest relationship with airflow limitation as measured by FEV1/FVC (Spearman rho = 0.509; p = 0.001) and FEV1% of predicted (Spearman rho =  0.362; p = 0.028). Plasma CCSP-16 concentrations were also significantly related to airflow limitation (Spearman rho = 0.362; p = 0.028 for FEV1% of predicted). The other biomarkers in BAL fluid or plasma were not significantly associated with airflow limitation. In COPD subjects, budesonide/formoterol significantly increased the BAL concentrations of pro-SFTPB by a median of 62.46 ng/ml (p = 0.022) or 48.7% from baseline median value.
Conclusion
Increased severity of COPD is associated with reduced Pro-SFTPB levels in BAL fluid. Short-term treatment with budesonide/formoterol increases these levels in BAL fluid. Long term studies will be needed to determine the clinical relevance of this observation.
doi:10.1371/journal.pone.0083881
PMCID: PMC3873417  PMID: 24386300
21.  Improved patient outcome with smoking cessation: when is it too late? 
Smoking is the leading modifiable risk factor for chronic obstructive pulmonary disease (COPD), cardiovascular disease (CVD), and lung cancer. Smoking cessation is the only proven way of modifying the natural course of COPD. It is also the most effective way of reducing the risk for myocardial infarction and lung cancer. However, the full benefits of tobacco treatment may not be realized until many years of abstinence. All patients with COPD, regardless of severity, appear to benefit from tobacco treatment. Similarly, patients with recent CVD events also benefit from tobacco treatment. The risk of total mortality and rate of recurrence of lung cancer is substantially lower in smokers who manage to quit smoking following the diagnosis of early stage lung cancer or small cell lung cancer. Together, these data suggest that tobacco treatment is effective both as a primary and a secondary intervention in reducing total morbidity and mortality related to COPD, CVD, and lung cancer. In this paper, we summarize the evidence for tobacco treatment and the methods by which smoking cessation can be promoted in smokers with lung disease.
doi:10.2147/COPD.S10771
PMCID: PMC3144846  PMID: 21814462
COPD; lung cancer; tobacco treatment; smoking cessation
22.  Aging does not Enhance Experimental Cigarette Smoke-Induced COPD in the Mouse 
PLoS ONE  2013;8(8):e71410.
It has been proposed that the development of COPD is driven by premature aging/premature senescence of lung parenchyma cells. There are data suggesting that old mice develop a greater inflammatory and lower anti-oxidant response after cigarette smoke compared to young mice, but whether these differences actually translate into greater levels of disease is unknown. We exposed C57Bl/6 female mice to daily cigarette smoke for 6 months starting at age 3 months (Ayoung@) or age 12 months (Aold@), with air-exposed controls. There were no differences in measures of airspace size between the two control groups and cigarette smoke induced exactly the same amount of emphysema in young and old. The severity of smoke-induced small airway remodeling using various measures was identical in both groups. Smoke increased numbers of tissue macrophages and neutrophils and levels of 8-hydroxyguanosine, a marker of oxidant damage, but there were no differences between young and old. Gene expression studies using laser capture microdissected airways and parenchyma overall showed a trend to lower levels in older animals and a somewhat lesser response to cigarette smoke in both airways and parenchyma but the differences were usually not marked. Telomere length was greatest in young control mice and was decreased by both smoking and age. The senescence marker p21Waf1 was equally upregulated by smoke in young and old, but p16INK4a, another senescence marker, was not upregulated at all. We conclude, in this model, animal age does not affect the development of emphysema and small airway remodeling.
doi:10.1371/journal.pone.0071410
PMCID: PMC3732225  PMID: 23936505
23.  Refining Susceptibility Loci of Chronic Obstructive Pulmonary Disease with Lung eqtls 
PLoS ONE  2013;8(7):e70220.
Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of mortality worldwide. Recent genome-wide association studies (GWAS) have identified robust susceptibility loci associated with COPD. However, the mechanisms mediating the risk conferred by these loci remain to be found. The goal of this study was to identify causal genes/variants within susceptibility loci associated with COPD. In the discovery cohort, genome-wide gene expression profiles of 500 non-tumor lung specimens were obtained from patients undergoing lung surgery. Blood-DNA from the same patients were genotyped for 1,2 million SNPs. Following genotyping and gene expression quality control filters, 409 samples were analyzed. Lung expression quantitative trait loci (eQTLs) were identified and overlaid onto three COPD susceptibility loci derived from GWAS; 4q31 (HHIP), 4q22 (FAM13A), and 19q13 (RAB4B, EGLN2, MIA, CYP2A6). Significant eQTLs were replicated in two independent datasets (n = 363 and 339). SNPs previously associated with COPD and lung function on 4q31 (rs1828591, rs13118928) were associated with the mRNA expression of HHIP. An association between mRNA expression level of FAM13A and SNP rs2045517 was detected at 4q22, but did not reach statistical significance. At 19q13, significant eQTLs were detected with EGLN2. In summary, this study supports HHIP, FAM13A, and EGLN2 as the most likely causal COPD genes on 4q31, 4q22, and 19q13, respectively. Strong lung eQTL SNPs identified in this study will need to be tested for association with COPD in case-control studies. Further functional studies will also be needed to understand the role of genes regulated by disease-related variants in COPD.
doi:10.1371/journal.pone.0070220
PMCID: PMC3728203  PMID: 23936167
24.  The Lung Tissue Microbiome in Chronic Obstructive Pulmonary Disease 
Rationale: Based on surface brushings and bronchoalveolar lavage fluid, Hilty and coworkers demonstrated microbiomes in the human lung characteristic of asthma and chronic obstructive pulmonary disease (COPD), which have now been confirmed by others.
Objectives: To extend these findings to human lung tissue samples.
Methods: DNA from lung tissue samples was obtained from nonsmokers (n = 8); smokers without COPD (n = 8); patients with very severe COPD (Global Initiative for COPD [GOLD] 4) (n = 8); and patients with cystic fibrosis (CF) (n = 8). The latter served as a positive control, with sterile water as a negative control. All bacterial community analyses were based on polymerase chain reaction amplifying 16S rRNA gene fragments. Total bacterial populations were measured by quantitative polymerase chain reaction and bacterial community composition was assessed by terminal restriction fragment length polymorphism analysis and pyrotag sequencing.
Measurement and Main Results: Total bacterial populations within lung tissue were small (20–1,252 bacterial cells per 1,000 human cells) but greater in all four sample groups versus the negative control group (P < 0.001). Terminal restriction fragment length polymorphism analysis and sequencing distinguished three distinct bacterial community compositions: one common to the nonsmoker and smoker groups, a second to the GOLD 4 group, and the third to the CF-positive control group. Pyrotag sequencing identified greater than 1,400 unique bacterial sequences and showed an increase in the Firmicutes phylum in GOLD 4 patients versus all other groups (P < 0.003) attributable to an increase in the Lactobacillus genus (P < 0.0007).
Conclusions: There is a detectable bacterial community within human lung tissue that changes in patients with very severe COPD.
doi:10.1164/rccm.201111-2075OC
PMCID: PMC3359894  PMID: 22427533
COPD; bacteria; microbiome

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