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Endoplasmic reticulum (ER) dysfunction might have an important part to play in a range of neurological disorders, including cerebral ischaemia, sleep apnoea, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, the prion diseases, and familial encephalopathy with neuroserpin inclusion bodies. Protein misfolding in the ER initiates the well studied unfolded protein response in energy-starved neurons during stroke, which is relevant to the toxic effects of reperfusion. The toxic peptide amyloid β induces ER stress in Alzheimer's disease, which leads to activation of similar pathways, whereas the accumulation of polymeric neuroserpin in the neuronal ER triggers a poorly understood ER-overload response. In other neurological disorders, such as Parkinson's and Huntington's diseases, ER dysfunction is well recognised but the mechanisms by which it contributes to pathogenesis remain unclear. By targeting components of these signalling responses, amelioration of their toxic effects and so the treatment of a range of neurodegenerative disorders might become possible., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Accelerated lung function decline is a key COPD phenotype; however, its genetic control remains largely unknown. 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. 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. 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.

Rationale: Systemic glucocorticoids are used therapeutically to treat a variety of medical conditions. Epigenetic processes such as DNA methylation may reflect exposure to glucocorticoids and may be involved in mediating the responses and side effects associated with these medications., Objectives: To test the hypothesis that differences in DNA methylation are associated with current systemic steroid use., Methods: We obtained DNA methylation data at 27,578 CpG sites in 14,475 genes throughout the genome in two large, independent cohorts: the International COPD Genetics Network (n(discovery) = 1,085) and the Boston Early Onset COPD study (n(replication) = 369). Sites were tested for association with current systemic steroid use using generalized linear mixed models., Measurements and Main Results: A total of 511 sites demonstrated significant differential methylation by systemic corticosteroid use in all three of our primary models. Pyrosequencing validation confirmed robust differential methylation at CpG sites annotated to genes such as SLC22A18, LRP3, HIPK3, SCNN1A, FXYD1, IRF7, AZU1, SIT1, GPR97, ABHD16B, and RABGEF1. Functional annotation clustering demonstrated significant enrichment in intrinsic membrane components, hemostasis and coagulation, cellular ion homeostasis, leukocyte and lymphocyte activation and chemotaxis, protein transport, and responses to nutrients., Conclusions: Our analyses suggest that systemic steroid use is associated with site-specific differential methylation throughout the genome. Differentially methylated CpG sites were found in biologically plausible and previously unsuspected pathways; these genes and pathways may be relevant in the development of novel targeted therapies.

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).

Background: COPD, a leading cause of mortality is currently assessed by spirometry (forced expiratory volume in 1 second, FEV(1)). However FEV(1) does not correlate with patient mortality. ECLIPSE (Evaluation of Chronic obstructive pulmonary disease to Longitudinally Identify Predictive Surrogate Endpoints) aims to identify biomarkers that correlate with clinically relevant COPD subtypes, and to assess how these may predict disease progression. New methods were developed and validated to evaluate small molecules as potential diagnostic tools in patients with COPD, COPD related cachexia and cancer related cachexia., Methods and Findings: quantitative LC-MS/MS was developed to measure 34 amino acids and dipeptides for stratification of patient groups. Subsets of the ECLIPSE patients were used to assess biomarkers of lung obstruction; GOLD IV (n = 30) versus control (n = 30); emphysema (n = 38) versus airways disease (n = 21) and cachexia (n = 30) versus normal body mass index (n = 30). Serum from cachexic (n = 7) and non-cachexic (n = 5) pancreatic cancer patients were included as controls. Targeted LC-MS/MS distinguished GOLD IV patients from controls, patients with and without emphysema and patients with and without cachexia. Glutamine, aspartate and arginine were significantly increased (p < 0.05; FDR adjustment α < 0.1) in cachexia, emphysema and GOLD IV patients and aminoadipate was decreased. Several amino acid concentrations were significantly altered in patients with COPD but not patients with pancreatic cancer (serine, sarcosine, tryptophan, BCAAs and 3-methylhistdine). Increased γ-aminobutyrate (GABA, p < 0.01) levels were specific to cachexia in patients with pancreatic cancer. β-aminoisobutyrate, 1-methylhistidine and asparagine (p < 0.05) were common across the patients with cachexia from both the COPD and pancreatic cancer cohorts., Conclusion: these results demonstrate that a metabolomic fingerprint has potential to stratify patients for the treatment of COPD and may provide a means of assessing response to therapy. GOLD IV patients were distinguished from smoker control subjects, patients with emphysema were distinguished from those without emphysema and COPD patients displaying cachexia were distinguished from those not displaying cachexia. General markers of cachexia were discovered reflecting both COPD- and pancreatic cancer-related cachexia (increased glutamine, aspartate, arginine, and asparagine and decreased aminoadipate, β-aminoisobutyrate and 1-methylhistidine). Metabolomic biomarkers, particularly altered levels of GABA, could be exploited as a way of monitoring treatment efficacy and tumour recurrence for patients with pancreatic cancer experiencing cachexia.

Rationale: Genome-wide association studies (GWAS) have identified loci influencing lung function, but fewer genes influencing chronic obstructive pulmonary disease (COPD) are known., Objectives: Perform meta-analyses of GWAS for airflow obstruction, a key pathophysiologic characteristic of COPD assessed by spirometry, in population-based cohorts examining all participants, ever smokers, never smokers, asthma-free participants, and more severe cases., Methods: Fifteen cohorts were studied for discovery (3,368 affected; 29,507 unaffected), and a population-based family study and a meta-analysis of case-control studies were used for replication and regional follow-up (3,837 cases; 4,479 control subjects). Airflow obstruction was defined as FEV(1) and its ratio to FVC (FEV(1)/FVC) both less than their respective lower limits of normal as determined by published reference equations., Measurements and Main Results: The discovery meta-analyses identified one region on chromosome 15q25.1 meeting genome-wide significance in ever smokers that includes AGPHD1, IREB2, and CHRNA5/CHRNA3 genes. The region was also modestly associated among never smokers. Gene expression studies confirmed the presence of CHRNA5/3 in lung, airway smooth muscle, and bronchial epithelial cells. A single-nucleotide polymorphism in HTR4, a gene previously related to FEV(1)/FVC, achieved genome-wide statistical significance in combined meta-analysis. Top single-nucleotide polymorphisms in ADAM19, RARB, PPAP2B, and ADAMTS19 were nominally replicated in the COPD meta-analysis., Conclusions: These results suggest an important role for the CHRNA5/3 region as a genetic risk factor for airflow obstruction that may be independent of smoking and implicate the HTR4 gene in the etiology of airflow obstruction.

Palovarotene is an oral γ-selective retinoid agonist. In animal emphysema models, palovarotene reduced inflammation, promoted structural repair and functional improvement. REPAIR (Retinoid treatment of Emphysema in Patients on the α(1)-antitrypsin International Registry), was an investigator-initiated, double-blind, placebo-controlled randomised study to assess the safety and efficacy of 5 mg·day(-1) palovarotene given for 1 year to 262 patients with severe α(1)-antitrypsin deficiency and emphysema confirmed by computed tomography. Change in volume-adjusted 15th percentile point lung density from baseline in 1 year was the primary end-point; functional end-points were also regularly assessed. We randomly assigned 133 and 129 patients to placebo or palovarotene, respectively. Both groups were well matched for all baseline characteristics, including respiratory medications. 88% and 85% of patients completed 1 year of treatment with placebo and palovarotene, respectively. Palovarotene was generally well tolerated. In the study completers population, the placebo-corrected difference of lung density was -0.45 HU at week 28 (p=0.64) and -0.25 HU at week 52 (p=0.94). A nonsignificant treatment difference in most functional parameters of the lung in favour of the drug was observed over time suggesting potential pharmacological effects of palovarotene. Palovarotene 5 mg·day(-1) over 1 yr failed to show a significant benefit on lung density in moderate-to-severe emphysema secondary to severe α(1)-antitrypsin deficiency.

Background: Bronchodilator responsiveness is a potential phenotypic characteristic of chronic obstructive pulmonary disease (COPD). We studied whether change in lung function after a bronchodilator is abnormal in COPD, whether stable responder subgroups can be identified, and whether these subgroups experience different clinical outcomes., Methods: 1831 patients with COPD, 285 smoking (SC) and 228 non-smoking (NSC) controls from the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE) cohort. Spirometric reversibility to 400 μg inhaled salbutamol was assessed on four occasions over 1 year., Results: Forced expiratory volume in 1 s (FEV(1)) increase after salbutamol was similar in SC (mean 0.14 litres (SD 0.15)) and COPD (0.12 litres (0.15)) and was significantly greater than NSC (0.08 litres (0.14)). Reversibility status varied with repeated testing in parallel with the day-to-day variation in pre-bronchodilator FEV(1), which was similar in control subjects and patients with COPD. Absolute FEV(1) change decreased by Global initiative for chronic Obstructive Lung Disease (GOLD) stage in patients with COPD (GOLD II, mean 0.16 litres (SD 0.17); III, 0.10 litres (0.13); IV, 0.05 litres (0.08) as did chances of being classified as reversible. CT-defined emphysema was weakly related to the absolute change in FEV(1) post salbutamol. Consistently reversible patients (n=227) did not differ in mortality, hospitalisation or exacerbation experience from irreversible patients when allowing for differences in baseline FEV(1)., Limitations: Reversibility only assessed with salbutamol and defined by FEV(1) criteria. The COPD population was older than the control populations., Conclusions: Post-salbutamol FEV(1) change is similar in patients with COPD and smoking controls but is influenced by baseline lung function and the presence of emphysema. Bronchodilator reversibility status varies temporally and does not distinguish clinically relevant outcomes, making it an unreliable phenotype.

There is a paucity of biomarkers for chronic obstructive pulmonary disease (COPD). Metabolomics were applied to a defined COPD patient cohort from the ECLIPSE study (Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points). Results were correlated with accepted biomarkers for the disease. Baseline control serum (n=66) and Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage II (n=70), III (n=64) and IV (n=44) COPD patients were analysed by proton nuclear magnetic resonance ((1)H NMR). Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to confirm amino acid changes detected by (1)H NMR. Data were correlated with body composition, emphysema and systemic inflammation. (1)H NMR identified decreased lipoproteins, N,N-dimethylglycine, and increased glutamine, phenylalanine, 3-methylhistidine and ketone bodies in COPD patients with decreased branched-chain amino acids (BCAAs) observed in GOLD stage IV patients. BCAAs, their degradation products, 3-methylhistidine, ketone bodies, and triglycerides were correlated negatively with cachexia and positively with systemic inflammation. Emphysema patients also displayed decreased serum creatine, glycine and N,N-dimethylglycine. LC-MS/MS confirmed (1)H NMR findings relating to BCAAs, glutamine and 3-methylhistidine in GOLD stage IV patients. NMR-based metabolomics characterised COPD patients based on systemic effects and lung function parameters. Increased protein turnover occurred in all COPD patients with increased protein degradation in individuals with emphysema and cachexia.

The impact of cigarette smoking can persist for extended periods following smoking cessation and may involve epigenetic reprogramming. Changes in DNA methylation associated with smoking may help to identify molecular pathways that contribute to the latency between exposure and disease onset. Cross-sectional cohort data from subjects in the International COPD Genetics Network (n = 1085) and the Boston Early-Onset COPD study (n = 369) were analyzed as the discovery and replication cohorts, respectively. Genome-wide methylation data on 27 578 CpG sites in 14 475 genes were obtained on DNA from peripheral blood leukocytes using the Illumina HumanMethylation27K Beadchip in both cohorts. We identified 15 sites significantly associated with current smoking, 2 sites associated with cumulative smoke exposure, and, within the subset of former smokers, 3 sites associated with time since quitting cigarettes. Two loci, factor II receptor-like 3 (F2RL3) and G-protein-coupled receptor 15 (GPR15), were significantly associated in all three analyses and were validated by pyrosequencing. These findings (i) identify a novel locus (GPR15) associated with cigarette smoking and (ii) suggest the existence of dynamic, site-specific methylation changes in response to smoking which may contribute to the extended risks associated with cigarette smoking that persist after cessation.

Two primary chitinases have been identified in humans--acid mammalian chitinase (AMCase) and chitotriosidase (CHIT1). Mammalian chitinases have been observed to affect the host's immune response. The aim of this study was to test for association between genetic variation in the chitinases and phenotypes related to chronic obstructive pulmonary disease (COPD). Polymorphisms in the chitinase genes were selected based on previous associations with respiratory diseases. Polymorphisms that were associated with lung function level or rate of decline in the Lung Health Study (LHS) cohort were analyzed for association with COPD affection status in four other COPD case-control populations. Chitinase activity and protein levels were also related to genotypes. In the caucasian LHS population, the baseline forced expiratory volume in one second (FEV(1)) was significantly different between the AA and GG genotypic groups of the AMCase rs3818822 polymorphism. Subjects with the GG genotype had higher AMCase protein and chitinase activity compared with AA homozygotes. For CHIT1 rs2494303, a significant association was observed between rate of decline in FEV(1) and the different genotypes. In the African American LHS population, CHIT1 rs2494303 and AMCase G339T genotypes were associated with rate of decline in FEV(1). Although a significant effect of chitinase gene alleles was found on lung function level and decline in the LHS, we were unable to replicate the associations with COPD affection status in the other COPD study groups.

Rationale: Exacerbations of chronic obstructive pulmonary disease (COPD) and responses to treatment are heterogeneous., Objectives: Investigate the usefulness of blood eosinophils to direct corticosteroid therapy during exacerbations., Methods: Subjects with COPD exacerbations were entered into a randomized biomarker-directed double-blind corticosteroid versus standard therapy study. Subjects in the standard arm received prednisolone for 2 weeks, whereas in the biomarker-directed arm, prednisolone or matching placebo was given according to the blood eosinophil count biomarker. Both study groups received antibiotics. Blood eosinophils were measured in the biomarker-directed and standard therapy arms to define biomarker-positive and -negative exacerbations (blood eosinophil count > and ≤ 2%, respectively). The primary outcome was to determine noninferiority in health status using the chronic respiratory questionnaire (CRQ) and in the proportion of exacerbations associated with a treatment failure between subjects allocated to the biomarker-directed and standard therapy arms., Measurements and Main Results: There were 86 and 80 exacerbations in the biomarker-directed and standard treatment groups, respectively. In the biomarker-directed group, 49% of the exacerbations were not treated with prednisolone. CRQ improvement after treatment in the standard and biomarker-directed therapy groups was similar (0.8 vs. 1.1; mean difference, 0.3; 95% confidence interval, 0.0-0.6; P = 0.05). There was a greater improvement in CRQ in biomarker-negative exacerbations given placebo compared with those given prednisolone (mean difference, 0.45; 95% confidence interval, 0.01-0.90; P = 0.04). In biomarker-negative exacerbations, treatment failures occurred in 15% given prednisolone and 2% of those given placebo (P = 0.04)., Conclusions: The peripheral blood eosinophil count is a promising biomarker to direct corticosteroid therapy during COPD exacerbations, but larger studies are required.

The generation and subsequent aggregation of amyloid β (Aβ) peptides play a crucial initiating role in the pathogenesis of Alzheimer disease (AD). The two main isoforms of these peptides have 40 (Aβ(40)) or 42 residues (Aβ(42)), the latter having a higher propensity to aggregate in vitro and being the main component of the plaques observed in vivo in AD patients. We have designed a series of tandem dimeric constructs of these Aβ peptides to probe the manner in which changes in the aggregation kinetics of Aβ affect its deposition and toxicity in a Drosophila melanogaster model system. The levels of insoluble aggregates were found to be substantially elevated in flies expressing the tandem constructs of both Aβ(40) and Aβ(42) compared with the equivalent monomeric peptides, consistent with the higher effective concentration, and hence increased aggregation rate, of the peptides in the tandem repeat. A unique feature of the Aβ(42) constructs, however, is the appearance of high levels of soluble oligomeric aggregates and a corresponding dramatic increase in their in vivo toxicity. The toxic nature of the Aβ(42) peptide in vivo can therefore be attributed to the higher kinetic stability of the oligomeric intermediate states that it populates relative to those of Aβ(40) rather than simply to its higher rate of aggregation.

Rationale: Accurate prediction of mortality helps select patients for interventions aimed at improving outcome., Objectives: Because chronic obstructive pulmonary disease is characterized by low-grade systemic inflammation, we hypothesized that addition of inflammatory biomarkers to established predictive factors will improve accuracy., Methods: A total of 1,843 patients enrolled in the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints study were followed for 3 years. Kaplan-Meier curves, log-rank analysis, and Cox proportional hazards analyses determined the predictive value for mortality of clinical variables, while C statistics assessed the added discriminative power offered by addition of biomarkers., Measurements and Main Results: At recruitment we measured anthropometrics, spirometry, 6-minute walk distance, dyspnea, BODE index, history of hospitalization, comorbidities, and computed tomography scan emphysema. White blood cell and neutrophil counts, serum or plasma levels of fibrinogen, chemokine ligand 18, surfactant protein D, C-reactive protein, Clara cell secretory protein-16, IL-6 and -8, and tumor necrosis factor-α were determined at recruitment and subsequent visits. A total of 168 of the 1,843 patients (9.1%) died. Nonsurvivors were older and had more severe airflow limitation, increased dyspnea, higher BODE score, more emphysema, and higher rates of comorbidities and history of hospitalizations. The best predictive model for mortality using clinical variables included age, BODE, and hospitalization history (C statistic of 0.686; P < 0.001). One single biomarker (IL-6) significantly improved the C statistic to 0.708, but this was further improved to 0.726 (P = 0.003) by the addition of all biomarkers., Conclusions: The addition of a panel of selected biomarkers improves the ability of established clinical variables to predict mortality in chronic obstructive pulmonary disease. Clinical trial registered with www.clinicaltrials.gov (NCT00292552).

In conformational diseases, native protein conformers convert to pathological intermediates that polymerize. Structural characterization of these key intermediates is challenging. They are unstable and minimally populated in dynamic equilibria that may be perturbed by many analytical techniques. We have characterized a forme fruste deficiency variant of α(1)-antitrypsin (Lys154Asn) that forms polymers recapitulating the conformer-specific neo-epitope observed in polymers that form in vivo. Lys154Asn α(1)-antitrypsin populates an intermediate ensemble along the polymerization pathway at physiological temperatures. Nuclear magnetic resonance spectroscopy was used to report the structural and dynamic changes associated with this. Our data highlight an interaction network likely to regulate conformational change and do not support the recent contention that the disease-relevant intermediate is substantially unfolded. Conformational disease intermediates may best be defined using powerful but minimally perturbing techniques, mild disease mutants, and physiological conditions., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Exercise tolerance is an important clinical aspect of chronic obstructive pulmonary disease that can be easily and reliably measured with the 6-minute walking test (6MWT). To improve the utility of the 6MWT for patient and health care system management, the interpretation of the functional status measure in relation to death and hospitalization should be elucidated., Methods: Three-year, prospective, multicenter observational study to evaluate the predictive power of 6MWD for death or exacerbation-related hospitalization and to evaluate the factors that help determine 6MWD., Results: We measured 6MWD at baseline and annually in 2110 patients with clinically stable Global Initiative for Obstructive Lung Disease (GOLD) stage II-IV COPD and recorded exacerbation-related hospitalizations and all-cause mortality. During the study, 200 patients died and 650 were hospitalized. Using receiver operating characteristics, the best predictive thresholds of the 6MWD were 334 m for increased risk of death and 357 m for exacerbation-related hospitalization (area under the curve 0.67 and 0.60 respectively); however, the discriminatory thresholds, especially for mortality, were influenced by age. The mean (SE) 6MWD declined by 1.6 (1.2) m per year in GOLD II, 9.8 (1.3) m per year in GOLD III, and 8.5 (2.4) m per year in GOLD IV., Conclusion: The 6MWD provides prognostic information that may be useful for identifying high-risk patients with COPD., (Copyright © 2012 American Medical Directors Association, Inc. Published by Elsevier Inc. All rights reserved.)

The aims were to determine the effect of an oral inhibitor of the signaling mediator p38 mitogen-activated protein kinase (GW856553, losmapimod) on sputum neutrophils, pulmonary function, and blood biomarkers of inflammation in chronic obstructive pulmonary disease (COPD). Three hundred and two individuals with GOLD stage II COPD were randomized to oral losmapimod 7.5 mg twice daily, inhaled salmeterol/fluticasone propionate 50 µg/500 µg combination (SFC), or placebo in a 12-week, randomized, double-blind, double-dummy study (MKI102428/NCT00642148). Neither losmapimod nor SFC had an effect on the primary end point of sputum neutrophils. Losmapimod was well tolerated and reduced plasma fibrinogen by 11% (-0.4 g/L, ratio of effect of losmapimod/placebo 0.89; 95% confidence interval, 0.83-0.96; P = .002) with nonsignificant reductions in interleukin-6, interleukin-8, and C-reactive protein. There was evidence of improvement in hyperinflation with losmapimod compared with placebo (overall P = .02). Inhaled SFC significantly improved lung function and reduced serum CC-16 (ratio of effect of SFC/placebo 0.87; 95% confidence interval, 0.82-0.93; P < .001). It was concluded that oral losmapimod significantly reduced plasma fibrinogen in patients with COPD.

Rationale: Chronic obstructive pulmonary disease (COPD) is associated with local (lung) and systemic (blood) inflammation and manifestations. DNA methylation is an important regulator of gene transcription, and global and specific gene methylation marks may vary with cigarette smoke exposure., Objectives: To perform a comprehensive assessment of methylation marks in DNA from subjects well phenotyped for nonneoplastic lung disease., Methods: We conducted array-based methylation screens, using a test-replication approach, in two family-based cohorts (n = 1,085 and 369 subjects)., Measurements and Main Results: We observed 349 CpG sites significantly associated with the presence and severity of COPD in both cohorts. Seventy percent of the associated CpG sites were outside of CpG islands, with the majority of CpG sites relatively hypomethylated. Gene ontology analysis based on these 349 CpGs (330 genes) suggested the involvement of a number of genes responsible for immune and inflammatory system pathways, responses to stress and external stimuli, as well as wound healing and coagulation cascades. Interestingly, our observations include significant, replicable associations between SERPINA1 hypomethylation and COPD and lower average lung function phenotypes (combined P values: COPD, 1.5 × 10(-23); FEV(1)/FVC, 1.5 × 10(-35); FEV(1), 2.2 × 10(-40))., Conclusions: Genetic and epigenetic pathways may both contribute to COPD. Many of the top associations between COPD and DNA methylation occur in biologically plausible pathways. This large-scale analysis suggests that DNA methylation may be a biomarker of COPD and may highlight new pathways of COPD pathogenesis.

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 FEV(1) (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.

Human induced pluripotent stem cells offer the possibility of generating unlimited quantities of cells for autologous transplantation. By correcting the genetic defect underlying Z-allele α₁-antitrypsin deficiency, we recently provided the first proof of principle for application of human induced pluripotent stem cells in the treatment of inherited genetic disorders. Several important safety concerns will need to be addressed before this can be translated into clinical practice.

Background: Because chronic obstructive pulmonary disease (COPD) is a heterogeneous condition, the identification of specific clinical phenotypes is key to developing more effective therapies. To explore if the persistence of systemic inflammation is associated with poor clinical outcomes in COPD we assessed patients recruited to the well-characterized ECLIPSE cohort (NCT00292552)., Methods and Findings: Six inflammatory biomarkers in peripheral blood (white blood cells (WBC) count and CRP, IL-6, IL-8, fibrinogen and TNF-α levels) were quantified in 1,755 COPD patients, 297 smokers with normal spirometry and 202 non-smoker controls that were followed-up for three years. We found that, at baseline, 30% of COPD patients did not show evidence of systemic inflammation whereas 16% had persistent systemic inflammation. Even though pulmonary abnormalities were similar in these two groups, persistently inflamed patients during follow-up had significantly increased all-cause mortality (13% vs. 2%, p<0.001) and exacerbation frequency (1.5 (1.5) vs. 0.9 (1.1) per year, p<0.001) compared to non-inflamed ones. As a descriptive study our results show associations but do not prove causality. Besides this, the inflammatory response is complex and we studied only a limited panel of biomarkers, albeit they are those investigated by the majority of previous studies and are often and easily measured in clinical practice., Conclusions: Overall, these results identify a novel systemic inflammatory COPD phenotype that may be the target of specific research and treatment.

Background: The major concept behind augmentation therapy with human α(1)-antitrypsin (AAT) is to raise the levels of AAT in patients with protease inhibitor phenotype ZZ (Glu342Lys)-inherited AAT deficiency and to protect lung tissues from proteolysis and progression of emphysema., Objective: To evaluate the short-term effects of augmentation therapy (Prolastin) on plasma levels of AAT, C-reactive protein, and chemokines/cytokines., Materials and Methods: Serum and exhaled breath condensate were collected from individuals with protease inhibitor phenotype ZZ AAT deficiency-related emphysema (n = 12) on the first, third, and seventh day after the infusion of intravenous Prolastin. Concentrations of total and polymeric AAT, interleukin-8 (IL-8), monocyte chemotactic protein-1, IL-6, tumor necrosis factor-α, vascular endothelial growth factor, and C-reactive protein were determined. Blood neutrophils and primary epithelial cells were also exposed to Prolastin (1 mg/mL)., Results: There were significant fluctuations in serum (but not in exhaled breath condensate) levels of AAT polymers, IL-8, monocyte chemotactic protein-1, IL-6, tumor necrosis factor-α, and vascular endothelial growth factor within a week of augmentation therapy. In general, augmented individuals had higher AAT and lower serum levels of IL-8 than nonaugmented subjects. Prolastin added for 3 hours to neutrophils from protease inhibitor phenotype ZZ individuals in vitro reduced IL-8 release but showed no effect on cytokine/chemokine release from human bronchial epithelial cells., Conclusion: Within a week, augmentation with Prolastin induced fluctuations in serum levels of AAT polymers and cytokine/chemokines but specifically lowered IL-8 levels. It remains to be determined whether these effects are related to the Prolastin preparation per se or to the therapeutic efficacy of augmentation with AAT.

We have created a Drosophila model of lysozyme amyloidosis to investigate the in vivo behavior of disease-associated variants. To achieve this objective, wild-type (WT) protein and the amyloidogenic variants F57I and D67H were expressed in Drosophila melanogaster using the UAS-gal4 system and both the ubiquitous and retinal expression drivers Act5C-gal4 and gmr-gal4. The nontransgenic w(1118) Drosophila line was used as a control throughout. We utilized ELISA experiments to probe lysozyme protein levels, scanning electron microscopy for eye phenotype classification, and immunohistochemistry to detect the unfolded protein response (UPR) activation. We observed that expressing the destabilized F57I and D67H lysozymes triggers UPR activation, resulting in degradation of these variants, whereas the WT lysozyme is secreted into the fly hemolymph. Indeed, the level of WT was up to 17 times more abundant than the variant proteins. In addition, the F57I variant gave rise to a significant disruption of the eye development, and this correlated to pronounced UPR activation. These results support the concept that the onset of familial amyloid disease is linked to an inability of the UPR to degrade completely the amyloidogenic lysozymes prior to secretion, resulting in secretion of these destabilized variants, thereby leading to deposition and associated organ damage.

Rationale: Traditional genome-wide association studies (GWASs) of large cohorts of subjects with chronic obstructive pulmonary disease (COPD) have successfully identified novel candidate genes, but several other plausible loci do not meet strict criteria for genome-wide significance after correction for multiple testing., Objectives: The authors hypothesise that by applying unbiased weights derived from unique populations we can identify additional COPD susceptibility loci. Methods The authors performed a homozygosity haplotype analysis on a group of subjects with and without COPD to identify regions of conserved homozygosity haplotype (RCHHs). Weights were constructed based on the frequency of these RCHHs in case versus controls, and used to adjust the p values from a large collaborative GWAS of COPD., Results: The authors identified 2318 RCHHs, of which 576 were significantly (p<0.05) over-represented in cases. After applying the weights constructed from these regions to a collaborative GWAS of COPD, the authors identified two single nucleotide polymorphisms (SNPs) in a novel gene (fibroblast growth factor-7 (FGF7)) that gained genome-wide significance by the false discovery rate method. In a follow-up analysis, both SNPs (rs12591300 and rs4480740) were significantly associated with COPD in an independent population (combined p values of 7.9E-7 and 2.8E-6, respectively). In another independent population, increased lung tissue FGF7 expression was associated with worse measures of lung function., Conclusion: Weights constructed from a homozygosity haplotype analysis of an isolated population successfully identify novel genetic associations from a GWAS on a separate population. This method can be used to identify promising candidate genes that fail to meet strict correction for multiple testing.

Two recent metaanalyses of genome-wide association studies conducted by the CHARGE and SpiroMeta consortia identified novel loci yielding evidence of association at or near genome-wide significance (GWS) with FEV(1) and FEV(1)/FVC. We hypothesized that a subset of these markers would also be associated with chronic obstructive pulmonary disease (COPD) susceptibility. Thirty-two single-nucleotide polymorphisms (SNPs) in or near 17 genes in 11 previously identified GWS spirometric genomic regions were tested for association with COPD status in four COPD case-control study samples (NETT/NAS, the Norway case-control study, ECLIPSE, and the first 1,000 subjects in COPDGene; total sample size, 3,456 cases and 1,906 controls). In addition to testing the 32 spirometric GWS SNPs, we tested a dense panel of imputed HapMap2 SNP markers from the 17 genes located near the 32 GWS SNPs and in a set of 21 well studied COPD candidate genes. Of the previously identified GWS spirometric genomic regions, three loci harbored SNPs associated with COPD susceptibility at a 5% false discovery rate: the 4q24 locus including FLJ20184/INTS12/GSTCD/NPNT, the 6p21 locus including AGER and PPT2, and the 5q33 locus including ADAM19. In conclusion, markers previously associated at or near GWS with spirometric measures were tested for association with COPD status in data from four COPD case-control studies, and three loci showed evidence of association with COPD susceptibility at a 5% false discovery rate.

The intrinsic propensity of α(1)-antitrypsin to undergo conformational transitions from its metastable native state to hyperstable forms provides a motive force for its antiprotease function. However, aberrant conformational change can also occur via an intermolecular linkage that results in polymerization. This has both loss-of-function and gain-of-function effects that lead to deficiency of the protein in human circulation, emphysema and hepatic cirrhosis. One of the most promising therapeutic strategies being developed to treat this disease targets small molecules to an allosteric site in the α(1)-antitrypsin molecule. Partial filling of this site impedes polymerization without abolishing function. Drug development can be improved by optimizing data on the structure and dynamics of this site. A new 1.8 Å resolution structure of α(1)-antitrypsin demonstrates structural variability within this site, with associated fluctuations in its upper and lower entrance grooves and ligand-binding characteristics around the innermost stable enclosed hydrophobic recess. These data will allow a broader selection of chemotypes and derivatives to be tested in silico and in vitro when screening and developing compounds to modulate conformational change to block the pathological mechanism while preserving function.

Background: There is a need for biomarkers to better characterise individuals with COPD and to aid with the development of therapeutic interventions. A panel of putative blood biomarkers was assessed in a subgroup of the Evaluation of COPD Longitudinally to Identify Surrogate Endpoints (ECLIPSE) cohort., Methods: Thirty-four blood biomarkers were assessed in 201 subjects with COPD, 37 ex-smoker controls with normal lung function and 37 healthy non-smokers selected from the ECLIPSE cohort. Biomarker repeatability was assessed using baseline and 3-month samples. Intergroup comparisons were made using analysis of variance, repeatability was assessed through Bland-Altman plots, and correlations between biomarkers and clinical characteristics were assessed using Spearman correlation coefficients., Results: Fifteen biomarkers were significantly different in individuals with COPD when compared to former or non-smoker controls. Some biomarkers, including tumor necrosis factor-α and interferon-γ, were measurable in only a minority of subjects whilst others such as C-reactive protein showed wide variability over the 3-month replication period. Fibrinogen was the most repeatable biomarker and exhibited a weak correlation with 6-minute walk distance, exacerbation rate, BODE index and MRC dyspnoea score in COPD subjects. 33% (66/201) of the COPD subjects reported at least 1 exacerbation over the 3 month study with 18% (36/201) reporting the exacerbation within 30 days of the 3-month visit. CRP, fibrinogen interleukin-6 and surfactant protein-D were significantly elevated in those COPD subjects with exacerbations within 30 days of the 3-month visit compared with those individuals that did not exacerbate or whose exacerbations had resolved., Conclusions: Only a few of the biomarkers assessed may be useful in diagnosis or management of COPD where the diagnosis is based on airflow obstruction (GOLD). Further analysis of more promising biomarkers may reveal utility in subsets of patients. Fibrinogen in particular has emerged as a potentially useful biomarker from this cohort and requires further investigation., Trial Registration: SCO104960, clinicaltrials.gov identifier NCT00292552.

Human induced pluripotent stem cells (iPSCs) represent a unique opportunity for regenerative medicine because they offer the prospect of generating unlimited quantities of cells for autologous transplantation, with potential application in treatments for a broad range of disorders. However, the use of human iPSCs in the context of genetically inherited human disease will require the correction of disease-causing mutations in a manner that is fully compatible with clinical applications. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of human iPSCs. Here we show that a combination of zinc finger nucleases (ZFNs) and piggyBac technology in human iPSCs can achieve biallelic correction of a point mutation (Glu342Lys) in the α(1)-antitrypsin (A1AT, also known as SERPINA1) gene that is responsible for α(1)-antitrypsin deficiency. Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.

Background Cigarette smoking is a major risk factor for chronic obstructive pulmonary disease (COPD) and COPD severity. Previous genome-wide association studies (GWAS) have identified numerous single nucleotide polymorphisms (SNPs) associated with the number of cigarettes smoked per day (CPD) and a dopamine beta-hydroxylase (DBH) locus associated with smoking cessation in multiple populations. Objective To identify SNPs associated with lifetime average and current CPD, age at smoking initiation, and smoking cessation in patients with COPD. Methods GWAS were conducted in four independent cohorts encompassing 3441 ever-smoking patients with COPD (Global Initiative for Obstructive Lung Disease stage II or higher). Untyped SNPs were imputed using the HapMap (phase II) panel. Results from all cohorts were meta-analysed. Results Several SNPs near the HLA region on chromosome 6p21 and in an intergenic region on chromosome 2q21 showed associations with age at smoking initiation, both with the lowest p=2×10(-7). No SNPs were associated with lifetime average CPD, current CPD or smoking cessation with p<10(-6). Nominally significant associations with candidate SNPs within cholinergic receptors, nicotinic, alpha 3/5 (CHRNA3/CHRNA5; eg, p=0.00011 for SNP rs1051730) and cytochrome P450, family 2, subfamily A, polypeptide 6 (CYP2A6; eg, p=2.78×10(-5) for a non-synonymous SNP rs1801272) regions were observed for lifetime average CPD, however only CYP2A6 showed evidence of significant association with current CPD. A candidate SNP (rs3025343) in DBH was significantly (p=0.015) associated with smoking cessation. Conclusion The authors identified two candidate regions associated with age at smoking initiation in patients with COPD. Associations of CHRNA3/CHRNA5 and CYP2A6 loci with CPD and DBH with smoking cessation are also likely of importance in the smoking behaviours of patients with COPD.

Background: The identification of gene-by-environment interactions is important for understanding the genetic basis of chronic obstructive pulmonary disease (COPD). Many COPD genetic association analyses assume a linear relationship between pack-years of smoking exposure and forced expiratory volume in 1 s (FEV(1)); however, this assumption has not been evaluated empirically in cohorts with a wide spectrum of COPD severity., Methods: The relationship between FEV(1) and pack-years of smoking exposure was examined in four large cohorts assembled for the purpose of identifying genetic associations with COPD. Using data from the Alpha-1 Antitrypsin Genetic Modifiers Study, the accuracy and power of two different approaches to model smoking were compared by performing a simulation study of a genetic variant with a range of gene-by-smoking interaction effects., Results: Non-linear relationships between smoking and FEV(1) were identified in the four cohorts. It was found that, in most situations where the relationship between pack-years and FEV(1) is non-linear, a piecewise linear approach to model smoking and gene-by-smoking interactions is preferable to the commonly used total pack-years approach. The piecewise linear approach was applied to a genetic association analysis of the PI*Z allele in the Norway Case-Control cohort and a potential PI*Z-by-smoking interaction was identified (p=0.03 for FEV(1) analysis, p=0.01 for COPD susceptibility analysis)., Conclusion: In study samples of subjects with a wide range of COPD severity, a non-linear relationship between pack-years of smoking and FEV(1) is likely. In this setting, approaches that account for this non-linearity can be more powerful and less biased than the more common approach of using total pack-years to model the smoking effect.

Members of the serine protease inhibitor (serpin) superfamily are found in all branches of life and play an important role in the regulation of enzymes involved in proteolytic cascades. Mutants of the serpins result in a delay in folding, with unstable intermediates being cleared by endoplasmic reticulum-associated degradation. The remaining protein is either fully folded and secreted or retained as ordered polymers within the endoplasmic reticulum of the cell of synthesis. This results in a group of diseases termed the serpinopathies, which are typified by mutations of α(1)-antitrypsin and neuroserpin in association with cirrhosis and the dementia familial encephalopathy with neuroserpin inclusion bodies, respectively. Current evidence strongly suggests that polymers of mutants of α(1)-antitrypsin and neuroserpin are linked by the sequential insertion of the reactive loop of one molecule into β-sheet A of another. The ordered structure of the polymers within the endoplasmic reticulum stimulates nuclear factor-kappa B by a pathway that is independent of the unfolded protein response. This chronic activation of nuclear factor-kappa B may contribute to the cell toxicity associated with mutations of the serpins. We review the pathobiology of the serpinopathies and the development of novel therapeutic strategies for treating the inclusions that cause disease. These include the use of small molecules to block polymerization, stimulation of autophagy to clear inclusions and stem cell technology to correct the underlying molecular defect., (© 2011 The Authors Journal compilation © 2011 FEBS.)

Background: Small studies have linked α1 antitrypsin (α1AT) deficiency to patients with antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV)., Objective: To test the validity and the mechanism of this association between α1AT and AAV., Methods: The distribution of α1AT deficiency alleles Z and S was compared between 856 White Europeans with AAV and 1505 geographic and ethnically matched healthy controls. Genotyping was performed by allelic discrimination assay., Results: were compared between cases and controls using χ(2) tests. The serum and renal biopsies for α1AT polymers were compared using the polymer-specific 2C1 antibody. The role of α1AT polymers in promoting inflammation was investigated by examining their ability to prime neutrophils for ANCA activation as assessed by CD62L shedding, superoxide production and myeloperoxidase degranulation. Results The Z but not the S allele was over-represented in the patients compared with controls (HR=2.25, 95% CI 1.60 to 3.19). Higher concentrations of polymers of α1AT were detected in serum from patients carrying the Z allele than in those not carrying the Z allele (median (IQR) 1.40 (0.91-3.32) mg/dl vs 0.17 (0.06-0.28) mg/dl, p<0.001); polymers of α1AT were also seen in the renal biopsy of a patient with vasculitic glomerulonephritis. Polymers of α1AT primed neutrophils with CD62L shedding and increased superoxide production following ANCA activation. Carriage of the Z allele was not associated with disease severity, survival or relapse., Conclusions: The Z but not the S deficiency allele is associated with AAV. Polymers of α1AT are present in the serum and glomeruli of at least some patients with the Z allele, which may promote inflammation through priming of neutrophils.

Background: A key feature of chronic obstructive pulmonary disease (COPD) is an accelerated rate of decline in forced expiratory volume in 1 second (FEV(1)), but data on the variability and determinants of this change in patients who have established disease are scarce., Methods: We analyzed the changes in FEV(1) after administration of a bronchodilator over a 3-year period in 2163 patients. A random-coefficient model was used to evaluate possible predictors of both FEV(1) levels and their changes over time., Results: The mean (±SE) rate of change in FEV(1) was a decline of 33±2 ml per year, with significant variation among the patients studied. The between-patient standard deviation for the rate of decline was 59 ml per year. Over the 3-year study period, 38% of patients had an estimated decline in FEV(1) of more than 40 ml per year, 31% had a decline of 21 to 40 ml per year, 23% had a change in FEV(1) that ranged from a decrease of 20 ml per year to an increase of 20 ml per year, and 8% had an increase of more than 20 ml per year. The mean rate of decline in FEV(1) was 21±4 ml per year greater in current smokers than in current nonsmokers, 13±4 ml per year greater in patients with emphysema than in those without emphysema, and 17±4 ml per year greater in patients with bronchodilator reversibility than in those without reversibility., Conclusions: The rate of change in FEV(1) among patients with COPD is highly variable, with increased rates of decline among current smokers, patients with bronchodilator reversibility, and patients with emphysema.

Rationale: Exacerbations of chronic obstructive pulmonary disease (COPD) are heterogeneous with respect to inflammation and etiology., Objectives: Investigate biomarker expression in COPD exacerbations to identify biologic clusters and determine biomarkers that recognize clinical COPD exacerbation phenotypes, namely those associated with bacteria, viruses, or eosinophilic airway inflammation., Methods: Patients with COPD were observed for 1 year at stable and exacerbation visits. Biomarkers were measured in sputum and serum. Viruses and selected bacteria were assessed in sputum by polymerase chain reaction and routine diagnostic bacterial culture. Biologic phenotypes were explored using unbiased cluster analysis and biomarkers that differentiated clinical exacerbation phenotypes were investigated., Measurements and Main Results: A total of 145 patients (101 men and 44 women) entered the study. A total of 182 exacerbations were captured from 86 patients. Four distinct biologic exacerbation clusters were identified. These were bacterial-, viral-, or eosinophilic-predominant, and a fourth associated with limited changes in the inflammatory profile termed “pauciinflammatory.” Of all exacerbations, 55%, 29%, and 28% were associated with bacteria, virus, or a sputum eosinophilia. The biomarkers that best identified these clinical phenotypes were sputum IL-1β, 0.89 (area under receiver operating characteristic curve) (95% confidence interval [CI], 0.83–0.95); serum CXCL10, 0.83 (95% CI, 0.70–0.96); and percentage peripheral eosinophils, 0.85 (95% CI, 0.78–0.93), respectively., Conclusions: The heterogeneity of the biologic response of COPD exacerbations can be defined. Sputum IL-1β, serum CXCL10, and peripheral eosinophils are biomarkers of bacteria-, virus-, or eosinophil-associated exacerbations of COPD. Whether phenotype-specific biomarkers can be applied to direct therapy warrants further investigation.

Cachexia, whether assessed by body mass index (BMI) or fat-free mass index (FFMI), affects a significant proportion of patients with chronic obstructive pulmonary disease (COPD), and is an independent risk factor for increased mortality, increased emphysema, and more severe airflow obstruction. The variable development of cachexia among patients with COPD suggests a role for genetic susceptibility. The objective of the present study was to determine genetic susceptibility loci involved in the development of low BMI and FFMI in subjects with COPD. A genome-wide association study (GWAS) of BMI was conducted in three independent cohorts of European descent with Global Initiative for Chronic Obstructive Lung Disease stage II or higher COPD: Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-Points (ECLIPSE; n = 1,734); Norway-Bergen cohort (n = 851); and a subset of subjects from the National Emphysema Treatment Trial (NETT; n = 365). A genome-wide association of FFMI was conducted in two of the cohorts (ECLIPSE and Norway). In the combined analyses, a significant association was found between rs8050136, located in the first intron of the fat mass and obesity-associated (FTO) gene, and BMI (P = 4.97 × 10(-7)) and FFMI (P = 1.19 × 10(-7)). We replicated the association in a fourth, independent cohort consisting of 502 subjects with COPD from COPDGene (P = 6 × 10(-3)). Within the largest contributing cohort of our analysis, lung function, as assessed by forced expiratory volume at 1 second, varied significantly by FTO genotype. Our analysis suggests a potential role for the FTO locus in the determination of anthropomorphic measures associated with COPD.

The use of animal models in medical research provides insights into molecular and cellular mechanisms of human disease, and helps identify and test novel therapeutic strategies. Drosophila melanogaster--the common fruit fly--is one of the most well-established model organisms, as its study can be performed more readily and with far less expense than for other model animal systems, such as mice, fish, or primates. In the case of fruit flies, standard assays are based on the analysis of longevity and basic locomotor functions. Here we present the iFly tracking system, which enables to increase the amount of quantitative information that can be extracted from these studies, and to reduce significantly the duration and costs associated with them. The iFly system uses a single camera to simultaneously track the trajectories of up to 20 individual flies with about 100 μm spatial and 33 ms temporal resolution. The statistical analysis of fly movements recorded with such accuracy makes it possible to perform a rapid and fully automated quantitative analysis of locomotor changes in response to a range of different stimuli. We anticipate that the iFly method will reduce very considerably the costs and the duration of the testing of genetic and pharmacological interventions in Drosophila models, including an earlier detection of behavioural changes and a large increase in throughput compared to current longevity and locomotor assays., (This journal is © The Royal Society of Chemistry 2011)

Even in large-scale genome-wide association studies (GWASs), only a fraction of the true associations are detected at the genome-wide significance level. When few or no associations reach the significance threshold, one strategy is to follow up on the most promising candidates, i.e. the single nucleotide polymorphisms (SNPs) with the smallest association-test P-values, by genotyping them in additional studies. In this communication, we propose an overall test for GWASs that analyzes the SNPs with the most promising P-values simultaneously and therefore allows an early assessment of whether the follow-up of the selected SNPs is likely promising. We theoretically derive the properties of the proposed overall test under the null hypothesis and assess its power based on simulation studies. An application to a GWAS for chronic obstructive pulmonary disease suggests that there are true association signals among the top SNPs and that an additional follow-up study is promising., (© 2011 Wiley-Liss, Inc.)

Rationale: Chromosome 12p has been linked to chronic obstructive pulmonary disease (COPD) in the Boston Early-Onset COPD Study (BEOCOPD), but a susceptibility gene in that region has not been identified., Objectives: We used high-density single-nucleotide polymorphism (SNP) mapping to implicate a COPD susceptibility gene and an animal model to determine the potential role of SOX5 in lung development and COPD., Methods: On chromosome 12p, we genotyped 1,387 SNPs in 386 COPD cases from the National Emphysema Treatment Trial and 424 control smokers from the Normative Aging Study. SNPs with significant associations were then tested in the BEOCOPD study and the International COPD Genetics Network. Based on the human results, we assessed histology and gene expression in the lungs of Sox5(-/-) mice., Measurements and Main Results: In the case-control analysis, 27 SNPs were significant at P ≤ 0.01. The most significant SNP in the BEOCOPD replication was rs11046966 (National Emphysema Treatment Trial-Normative Aging Study P = 6.0 × 10(-4), BEOCOPD P = 1.5 × 10(-5), combined P = 1.7 × 10(-7)), located 3' to the gene SOX5. Association with rs11046966 was not replicated in the International COPD Genetics Network. Sox5(-/-) mice showed abnormal lung development, with a delay in maturation before the saccular stage, as early as E16.5. Lung pathology in Sox5(-/-) lungs was associated with a decrease in fibronectin expression, an extracellular matrix component critical for branching morphogenesis., Conclusions: Genetic variation in the transcription factor SOX5 is associated with COPD susceptibility. A mouse model suggests that the effect may be due, in part, to its effects on lung development and/or repair processes.

Background: Neuroserpin has shown neuroprotective effects in animal models of cerebral ischemia and has been associated with functional outcome after ischemic stroke. Our aim was to study whether neuroserpin serum levels could be associated to biomarkers of excitotoxicity, inflammation and blood brain barrier disruption., Methods: We prospectively included 129 patients with ischemic stroke (58.1% male; mean age, 72.4 ± 9.6 years) not treated with tPA within 12 hours (h) of symptoms onset (mean time, 4.7 ± 2.1 h). Poor functional outcome at 3 months was considered as a modified Rankin scale score >2. Serum levels of neuroserpin, Interleukin 6 (IL-6), Intercellular adhesion molecule-1 (ICAM-1), active Matrix metalloproteinase 9 (MMP-9), and cellular fibronectin (cFn) (determined by ELISA) and glutamate (determined by HPLC) were measured on admission, 24 and 72 h. The main variable was considered the decrease of neuroserpin levels within the first 24 h. ROC analysis was used to select the best predictive value for neuroserpin to predict poor functional outcome due to a lack of linearity., Results: The decrease of neuroserpin levels within the first 24 h was negatively correlated with serum levels at 24 hours of glutamate (r = -0.642), IL-6 (r = -0.678), ICAM-1 (r = -0.345), MMP-9 (r = -0.554) and cFn (r = -0.703) (all P < 0.0001). In the multivariate analysis, serum levels of glutamate (OR, 1.04; CI95%, 1.01-1.06, p = 0.001); IL-6 (OR, 1.4; CI95%, 1.1-1.7, p = 0.001); and cFn (OR, 1.3; CI95%, 1.1-1.6, p = 0.002) were independently associated with a decrease of neuroserpin levels <70 ng/mL at 24 h after adjusting for confounding factors., Conclusions: These findings suggest that neuroprotective properties of neuroserpin may be related to the inhibition of excitotoxicity, inflammation, as well as blood brain barrier disruption that occur after acute ischemic stroke.

Rationale: There are no accepted blood-based biomarkers in chronic obstructive pulmonary disease (COPD). Pulmonary and activation-regulated chemokine (PARC/CCL-18) is a lung-predominant inflammatory protein that is found in serum., Objectives: To determine whether PARC/CCL-18 levels are elevated and modifiable in COPD and to determine their relationship to clinical end points of hospitalization and mortality., Methods: PARC/CCL-18 was measured in serum samples from individuals who participated in the ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints) and LHS (Lung Health Study) studies and a prednisolone intervention study., Measurements and Main Results: Serum PARC/CCL-18 levels were higher in subjects with COPD than in smokers or lifetime nonsmokers without COPD (105 vs. 81 vs. 80 ng/ml, respectively; P < 0.0001). Elevated PARC/CCL-18 levels were associated with increased risk of cardiovascular hospitalization or mortality in the LHS cohort and with total mortality in the ECLIPSE cohort., Conclusions: Serum PARC/CCL-18 levels are elevated in COPD and track clinical outcomes. PARC/CCL-18, a lung-predominant chemokine, could be a useful blood biomarker in COPD.

Behavioural assays represent sensitive methods for detecting neuronal dysfunction in model organisms. A number of manual methods have been established for Drosophila, however these are time-consuming and generate parameter-poor phenotype descriptors. Here, we have developed an automated computer vision system to monitor accurately the three-dimensional locomotor trajectories of flies. This approach allows the quantitative description of fly trajectories, using small fly cohorts and short acquisition times. The application of this approach to a Drosophila model of Alzheimer's disease enables the early detection of progressive locomotor deficits and the quantitative assessment of phenotype severity. The approach can be widely applied to different disease models in a number of model organisms., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Chronic obstructive pulmonary disease (COPD) is a multicomponent condition that is characterized by partially reversible airflow obstruction. Serum surfactant protein D (SP-D) is synthesized by type II pneumocytes and Clara cells and participates in surfactant homeostasis and pulmonary host defense. Serum levels of SP-D are raised in individuals with COPD but there is no correlation between the serum level of SP-D and the severity of airflow obstruction. Serum SP-D is present in different forms that may have more utility as a biomarker for COPD. We report here the development of new monoclonal antibodies to full length and cleaved SP-D. We have assessed these and existing antibodies in 98 individuals with COPD recruited to the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE) cohort. Our data show that neither monoclonal antibodies to full length nor cleaved SP-D provide additional information over that obtained with a polyclonal antibody. Moreover, levels of serum nitrosylated-SP-D did not correlate with serum level of SP-D or any clinical phenotype of COPD. The measurement of modified SP-D is of limited value in characterising individuals with COPD.

α(1)-antitrypsin deficiency is an autosomal recessive disorder that results from point mutations in the SERPINA1 gene. The Z mutation (Glu342Lys) accounts for the majority of cases of severe α(1)-antitrypsin deficiency. It causes the protein to misfold into ordered polymers that accumulate within the endoplasmic reticulum of hepatocytes. It is these polymers that form the periodic acid Schiff positive inclusions that are characteristic of this condition. These inclusions are associated with neonatal hepatitis, cirrhosis and hepatocellular carcinoma. The lack of circulating α(1)-antitrypsin exposes the lungs to uncontrolled proteolytic attack and so can predispose the Z α(1)-antitrypsin homozygote to early-onset emphysema. α(1)-antitrypsin polymers can also form in extracellular tissues where they activate and sustain inflammatory cascades. This may provide an explanation for both progressive emphysema in individuals who receive adequate replacement therapy and the selective advantage associated with α(1)-antitrypsin deficiency. Therapeutic strategies are now being developed to block the aberrant conformational transitions of mutant α(1)-antitrypsin and so treat the associated disease.

Neuroserpin is a member of the serine protease inhibitor or serpin superfamily of proteins. It is secreted by neurones and plays an important role in the regulation of tissue plasminogen activator at the synapse. Point mutations in the neuroserpin gene cause the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. This is one of a group of disorders caused by mutations in the serpins that are collectively known as the serpinopathies. Others include α(1)-antitrypsin deficiency and deficiency of C1 inhibitor, antithrombin and α(1)-antichymotrypsin. The serpinopathies are characterised by delays in protein folding and the retention of ordered polymers of the mutant serpin within the cell of synthesis. The clinical phenotype results from either a toxic gain of function from the inclusions or a loss of function, as there is insufficient protease inhibitor to regulate important proteolytic cascades. We describe here the methods required to characterise the polymerisation of neuroserpin and draw parallels with the polymerisation of α(1)-antitrypsin. It is important to recognise that the conditions in which experiments are performed will have a major effect on the findings. For example, incubation of monomeric serpins with guanidine or urea will produce polymers that are not found in vivo. The characterisation of the pathological polymers requires heating of the folded protein or alternatively the assessment of ordered polymers from cell and animal models of disease or from the tissues of humans who carry the mutation., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Chronic obstructive pulmonary disease (COPD) is characterized by alveolar destruction and abnormal inflammatory responses to noxious stimuli. Surfactant protein-D (SFTPD) is immunomodulatory and essential to host defense. We hypothesized that polymorphisms in SFTPD could influence the susceptibility to COPD. We genotyped six single-nucleotide polymorphisms (SNPs) in surfactant protein D in 389 patients with COPD in the National Emphysema Treatment Trial (NETT) and 472 smoking control subjects from the Normative Aging Study (NAS). Case-control association analysis was performed using Cochran-Armitage trend tests and multivariate logistic regression. The replication of significant associations was attempted in the Boston Early-Onset COPD Study, the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE) Study, and the Bergen Cohort. We also correlated SFTPD genotypes with serum concentrations of surfactant protein-D (SP-D) in the ECLIPSE Study. In the NETT-NAS case-control analysis, four SFTPD SNPs were associated with susceptibility to COPD: rs2245121 (P = 0.01), rs911887 (P = 0.006), rs6413520 (P = 0.004), and rs721917 (P = 0.006). In the family-based analysis of the Boston Early-Onset COPD Study, rs911887 was associated with prebronchodilator and postbronchodilator FEV(1) (P = 0.003 and P = 0.02, respectively). An intronic SNP in SFTPD, rs7078012, was associated with COPD in the ECLIPSE Study and the Bergen Cohort. Multiple SFTPD SNPs were associated with serum SP-D concentrations in the ECLIPSE Study. We demonstrated an association of polymorphisms in SFTPD with COPD in multiple populations. We demonstrated a correlation between SFTPD SNPs and SP-D protein concentrations. The SNPs associated with COPD and SP-D concentrations differed, suggesting distinct genetic influences on susceptibility to COPD and SP-D concentrations.

Neuroserpin is a brain-derived natural inhibitor of tissue plasminogen activator (tPA) that has shown neuroprotective effects in animal models of brain ischaemia. Our aim was to investigate the association of neuroserpin levels in blood with functional outcome in patients with acute ischaemic stroke. Due to the potential effect of tPA treatment interfering on neuroserpin levels, we studied two different cohorts: 129 patients not treated with tPA and 80 patients treated with intravenous tPA within 3 hours (h) from symptoms onset. Neuroserpin levels were measured by ELISA. Good functional outcome at three months was defined as Rankin scale score ≤2. In the two cohorts, serum neuroserpin levels on admission were significantly higher than at 24 h, 72 h and in healthy subjects. In non tPA-treated patients, neuroserpin levels decrease at 24 h, but not levels at baseline, were associated with good outcome (for each quartile decrease, adjusted odds ratio [OR] 15.0; 95% confidence interval [CI], 3.5 to 66). In the tPA-treated cohort, high neuroserpin levels before tPA bolus had the stronger effect on favourable outcome (for each quartile, OR 13.5; 95%CI, 3.9 to 47). Furthermore, for each quartile in neuroserpin levels before tPA bolus there was a 80% (95%CI, 48 to 92) reduction in the probability of subsequent parenchymal haematoma. In summary, high serum neuroserpin levels before intravenous tPA and neuroserpin levels decrease at 24 h after ischaemic stroke, independently of tPA treatment, are associated with good functional outcome. These findings support the concept that neuroserpin might play an important role during cerebral ischaemia.

We have previously shown that overexpressing subunits of the iron-binding protein ferritin can rescue the toxicity of the amyloid β (Aβ) peptide in our Drosophila model system. These data point to an important pathogenic role for iron in Alzheimer disease. In this study, we have used an iron-selective chelating compound and RNAi-mediated knockdown of endogenous ferritin to further manipulate iron in the brain. We confirm that chelation of iron protects the fly from the harmful effects of Aβ. To understand the pathogenic mechanisms, we have used biophysical techniques to see how iron affects Aβ aggregation. We find that iron slows the progression of the Aβ peptide from an unstructured conformation to the ordered cross-β fibrils that are characteristic of amyloid. Finally, using mammalian cell culture systems, we have shown that iron specifically enhances Aβ toxicity but only if the metal is present throughout the aggregation process. These data support the hypothesis that iron delays the formation of well ordered aggregates of Aβ and so promotes its toxicity in Alzheimer disease.

Objective: High resolution computed tomography is widely used to investigate patients with suspected diffuse lung disease. Numerous studies have assessed the diagnostic performance of this investigation, but the diagnostic and therapeutic impacts have received little attention., Methods: The diagnostic and therapeutic impacts of high resolution computed tomography in routine clinical practice were evaluated prospectively. All 507 referrals for high-resolution computed tomography over 12 months in two centres were included. Requesting clinicians completed questionnaires before and after the investigation detailing clinical indications, working diagnoses, confidence level in each diagnosis, planned investigations and treatments., Results: Three hundred and fifty-four studies on 347 patients had complete data and were available for analysis. Following high-resolution computed tomography, a new leading diagnosis (the diagnosis with the highest confidence level) emerged in 204 (58%) studies; in 166 (47%) studies the new leading diagnosis was not in the original differential diagnosis. Mean confidence in the leading diagnosis increased from 6.7 to 8.5 out of 10 (p < 0.001). The invasiveness of planned investigations increased in 23 (7%) studies and decreased in 124 (35%) studies. The treatment plan was modified after 319 (90%) studies., Conclusions: Thoracic high-resolution computed tomography alters leading diagnosis, increases diagnostic confidence, and frequently changes investigation and management plans.