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Bronchodilator responsiveness (BDR) in obstructive lung disease varies over time and may be associated with distinct clinical features.Is consistent BDR over time (always present) differentially associated with obstructive lung disease features relative to inconsistent (sometimes present) or never (never present) BDR in tobacco-exposed people with or without COPD?We retrospectively analyzed data from 2,269 tobacco-exposed participants in the Subpopulations and Intermediate Outcome Measures in COPD Study with or without COPD. We used various BDR definitions: change of ≥ 200 mL and ≥ 12% in FEVBoth consistent and inconsistent ATS-BDR were associated with asthma history and greater small airways disease (%parametric response mapping functional small airways disease) relative to never ATS-BDR in participants with GOLD stage 0 disease and the entire cohort. We observed similar findings using FEVDemonstration of BDR, even once, describes an obstructive lung disease phenotype with a history of asthma and greater small airways disease. Consistent demonstration of BDR indicated a high risk of lung function decline over time in the entire cohort and was associated with higher risk of progression to COPD in patients with GOLD stage 0 disease.

BACKGROUND: Improved understanding of the pathways associated with airway pathophysiologic features in COPD will identify new predictive biomarkers and novel therapeutic targets. RESEARCH QUESTION: Which physiologic pathways are altered in the airways of patients with COPD and will predict exacerbations? STUDY DESIGN AND METHODS: We applied a mass spectrometric panel of metabolomic biomarkers related to mucus hydration and inflammation to sputa from the multicenter Subpopulations and Intermediate Outcome Measures in COPD Study. Biomarkers elevated in sputa from patients with COPD were evaluated for relationships to measures of COPD disease severity and their ability to predict future exacerbations. RESULTS: Sputum supernatants from 980 patients were analyzed: 77 healthy nonsmokers, 341 smokers with preserved spirometry, and 562 patients with COPD (178 with Global Initiative on Chronic Obstructive Lung Disease [GOLD] stage 1 disease, 303 with GOLD stage 2 disease, and 81 with GOLD stage 3 disease) were analyzed. Biomarkers from multiple pathways were elevated in COPD and correlated with sputum neutrophil counts. Among the most significant analytes (false discovery rate, 0.1) were sialic acid, hypoxanthine, xanthine, methylthioadenosine, adenine, and glutathione. Sialic acid and hypoxanthine were associated strongly with measures of disease severity, and elevation of these biomarkers was associated with shorter time to exacerbation and improved prediction models of future exacerbations. INTERPRETATION: Biomarker evaluation implicated pathways involved in mucus hydration, adenosine metabolism, methionine salvage, and oxidative stress in COPD airway pathophysiologic characteristics. Therapies that target these pathways may be of benefit in COPD, and a simple model adding sputum-soluble phase biomarkers improves prediction of pulmonary exacerbations. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT01969344; URL: www.clinicaltrials.gov

Rationale: Individuals with Chronic Obstructive Pulmonary Disease (COPD) have a high prevalence of depression, which is associated with increased COPD hospitalizations and readmissions. Objectives:...

Rationale: Diagnosis of chronic obstructive pulmonary disease (COPD) relies on abnormal spirometry. However, spirometry may underestimate the effects of smoking, missing smokers with respiratory di...

Background The relationship between 25-hydroxyvitamin D (25-OH-vitamin D) and COPD outcomes remains unclear. Using the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS), we determined associations among baseline 25-OH-vitamin D and cross-sectional and longitudinal lung function and COPD exacerbations. Methods Serum 25-OH-vitamin D level was measured in stored samples from 1,609 SPIROMICS participants with COPD. 25-OH-vitamin D levels were modeled continuously and dichotomized as deficient ( Results Vitamin D deficiency was present in 21% of the cohort and was more prevalent in the younger, active smokers, and blacks. Vitamin D deficiency was independently associated with lower % predicted FEV1 (by 4.11%) at enrollment (95% CI, –6.90% to –1.34% predicted FEV1; P = .004), 1.27% predicted greater rate of FEV1 decline after 1 year (95% CI, –2.32% to –0.22% predicted/y; P = .02), and higher odds of any COPD exacerbation in the prior year (OR, 1.32; 95% CI, 1.00-1.74; P = .049). Each 10-ng/mL decrease in 25-OH-vitamin D was associated with lower baseline lung function (–1.04% predicted; 95% CI, –1.96% to –0.12% predicted; P = .03) and increased odds of any exacerbation in the year before enrollment (OR, 1.11; 95% CI, 1.01-1.22; P = .04). Conclusions Vitamin D deficiency is associated with worse cross-sectional and longitudinal lung function and increased odds of prior COPD exacerbations. These findings identify 25-OH-vitamin D levels as a potentially useful marker of adverse COPD-related outcomes.

Rationale: The role of PI (protease inhibitor) type Z heterozygotes and additional rare variant genotypes in the gene encoding alpha-1 antitrypsin, SERPINA1 (serpin peptidase inhibitor, clade A, me...

As SCUBA diving continues to rapidly grow in the United States and worldwide, physicians should have a fundamental working knowledge to provide care for an injured diver. SCUBA divers are faced with many hazards at depths that are normally well compensated for at sea level. Pressure gradients, changes in the partial pressure of inhaled gases and gas solubility can have disastrous effects to the diver if not managed properly. Many safety measures in SCUBA diving are governed by the laws of physics, but some have come under scrutiny. This has prompted increased research concerning in water recompression and flying after diving. This article will give physicians an understanding of the dangers divers encounter and the current treatment recommendations. We will also explore some controversies in diving medicine.

Background Aspirin use in COPD has been associated with reduced all-cause mortality in meta-regression analysis with few equivocal studies. However, the effect of aspirin on COPD morbidity is unknown. Methods Self-reported daily aspirin use was obtained at baseline from SPIROMICS participants with COPD (FEV1/FVC Results Among 1,698 participants, 45% reported daily aspirin use at baseline. Propensity score matching resulted in 503 participant pairs. Aspirin users had a lower incidence rate of total AECOPD (adjusted incidence rate ratio [IRR], 0.78; 95% CI, 0.65-0.94), with similar effect for moderate but not severe AECOPD (IRR, 0.86; 95% CI, 0.63-1.18). Aspirin use was associated with lower total St. George's Respiratory Questionnaire score (β, –2.2; 95% CI, –4.1 to –0.4), reduced odds of moderate-severe dyspnea (modified Medical Research Council questionnaire score ≥ 2; adjusted odds ratio, 0.69; 95% CI, 0.51-0.93), and COPD Assessment Test score (β, –1.1; 95% CI, –1.9 to –0.2) but not 6-min walk distance (β, 0.7 m; 95% CI, –14.3 to 15.6). Conclusions Daily aspirin use is associated with reduced rate of COPD exacerbations, less dyspnea, and better quality of life. Randomized clinical trials of aspirin use in COPD are warranted to account for unmeasured and residual confounding. Trial Registry ClinicalTrials.gov; No.: NCT01969344; URL: www.clinicaltrials.gov

Rationale: Rural residence is associated with poor outcomes in several chronic diseases. The association between rural residence and chronic obstructive pulmonary disease (COPD) exacerbations remains unclear.Objectives: In this work, we sought to determine the independent association between rural residence and COPD-related outcomes, including COPD exacerbations, airflow obstruction, and symptom burden.Methods: A total of 1,684 SPIROMICS (Subpopulations and Intermediate Outcome Measures in COPD Study) participants with forced expiratory volume in 1 second/forced vital capacity < 0.70 had geocoding-defined rural-urban residence status determined (N = 204 rural and N = 1,480 urban). Univariate and multivariate logistic and negative binomial regressions were performed to assess the independent association between rurality and COPD outcomes, including exacerbations, lung function, and symptom burden. The primary exposure of interest was rural residence, determined by geocoding of the home address to the block level at the time of study enrollment. Additional covariates of interest included demographic and clinical characteristics, occupation, and occupational exposures. The primary outcome measures were exacerbations determined over a 1-year course after enrollment by quarterly telephone calls and at an annual research clinic visit. The odds ratio (OR) and incidence rate ratio (IRR) of exacerbations that required treatment with medications, including steroids or antibiotics (total exacerbations), and exacerbations leading to hospitalization (severe exacerbations) were determined after adjusting for relevant covariates.Results: Rural residence was independently associated with a 70% increase in the odds of total exacerbations (OR, 1.70 [95% confidence interval (CI), 1.13-2.56]; P = 0.012) and a 46% higher incidence rate of total exacerbations (IRR 1.46 [95% CI, 1.02-2.10]; P = 0.039). There was no association between rural residence and severe exacerbations. Agricultural occupation was independently associated with increased odds and incidence of total and severe exacerbations. Inclusion of agricultural occupation in the analysis attenuated the association between rural residence and the odds and incidence rate of total exacerbations (OR, 1.52 [95% CI, 1.00-2.32]; P = 0.05 and IRR 1.39 [95% CI, 0.97-1.99]; P = 0.07). There was no difference in symptoms or airflow obstruction between rural and urban participants.Conclusions: Rural residence is independently associated with increased odds and incidence of total, but not severe, COPD exacerbations. These associations are not fully explained by agriculture-related exposures, highlighting the need for future research into potential mechanisms of the increased risk of COPD exacerbations in the rural population.

Summary Background Increased concentrations of eosinophils in blood and sputum in chronic obstructive pulmonary disease (COPD) have been associated with increased frequency of exacerbations, reduced lung function, and corticosteroid responsiveness. We aimed to assess whether high eosinophil concentrations in either sputum or blood are associated with a severe COPD phenotype, including greater exacerbation frequency, and whether blood eosinophils are predictive of sputum eosinophils. Methods We did a multicentre observational study analysing comprehensive baseline data from SPIROMICS in patients with COPD aged 40–80 years who had a smoking history of at least 20 pack-years, recruited from six clinical sites and additional subsites in the USA between Nov 12, 2010, and April 21, 2015. Inclusion criteria for this analysis were SPIROMICS baseline visit data with complete blood cell counts and, in a subset, acceptable sputum counts. We stratified patients on the basis of blood and sputum eosinophil concentrations and compared their demographic characteristics, as well as results from questionnaires, clinical assessments, and quantitative CT (QCT). We also analysed whether blood eosinophil concentrations reliably predicted sputum eosinophil concentrations. This study is registered with ClinicalTrials.gov (NCT01969344). Findings Of the 2737 patients recruited to SPIROMICS, 2499 patients were smokers and had available blood counts, and so were stratified by mean blood eosinophil count: 1262 patients with low ( 1 percentage predicted than the low sputum eosinophil group both before (65·7% [IQR 51·8–81·3] vs 75·7% [59·3–90·2], p vs 82·9% [67·8–95·9], p=0·001) bronchodilation. QCT density measures for emphysema and air trapping were significantly higher in the high sputum eosinophil group than the low sputum eosinophil group. Exacerbations requiring corticosteroids treatment were more common in the high versus low sputum eosinophil group (p=0·002). FEV 1 percentage predicted was significantly different between low and high blood eosinophil groups, but differences were less than those observed between the sputum groups. The high blood eosinophil group had slightly increased airway wall thickness (0·02 mm difference, p=0·032), higher St George Respiratory Questionnaire symptom scores (p=0·037), and increased wheezing (p=0·018), but no evidence of an association with COPD exacerbations (p=0·35) or the other indices of COPD severity, such as emphysema measured by CT density, COPD assessment test scores, Body-mass index, airflow Obstruction, Dyspnea, and Exercise index, or Global Initiative for Chronic Obstructive Lung Disease stage. Blood eosinophil counts showed a weak but significant association with sputum eosinophil counts (receiver operating characteristic area under the curve of 0·64, p Interpretation In a large, well characterised cohort of former and current smoking patients with a broad range of COPD severity, high concentrations of sputum eosinophils were a better biomarker than high concentrations of blood eosinophils to identify a patient subgroup with more severe disease, more frequent exacerbations, and increased emphysema by QCT. Blood eosinophils alone were not a reliable biomarker for COPD severity or exacerbations, or for sputum eosinophils. Clinical trials targeting eosinophilic inflammation in COPD should consider assessing sputum eosinophils. Funding National Institutes of Health, and National Heart, Lung, and Blood Institute.

Rationale: Despite awareness of chronic obstructive pulmonary disease (COPD) treatment recommendations, uptake is poor. The Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) spans 2010–2016, providing an opportunity to assess integration of 2011 Global Initiative for Obstructive Lung Disease (GOLD) treatment strategies over time in a large observational cohort study. Objectives: To evaluate how COPD treatment aligns with 2011 GOLD strategies and determine factors associated with failure to align with recommendations. Methods: Information on inhaled medication use collected via questionnaire annually for 4 years was compiled into therapeutic classes (long-acting antimuscarinic agent, long-acting β-agonist, inhaled corticosteroids [ICS], and combinations thereof). Medications were not modified by SPIROMICS investigators. 2011 GOLD COPD categories A, B, C, and D were assigned. Alignment of inhaler regimen with first-/second-line GOLD recommendations was determined, stratifying into recommendation aligned or nonaligned. Recommendation-nonaligned participants were further stratified into overuse and underuse categories. Results: Of 1,721 participants with COPD, at baseline, 52% of regimens aligned with GOLD recommendations. Among participants with nonaligned regimens, 46% reported underuse, predominately owing to lack of long-acting inhalers in GOLD category D. Of the 54% reporting overuse, 95% were treated with nonindicated ICS-containing regimens. Among 431 participants with 4 years of follow-up data, recommendation alignment did not change over time. When we compared 2011 and 2017 recommendations, we found that 47% did not align with either set of recommendations, whereas 35% were in alignment with both recommendations. Conclusions: Among SPIROMICS participants with COPD, nearly 50% reported inhaler regimens that did not align with GOLD recommendations. Nonalignment was driven largely by overuse of ICS regimens in milder disease and lack of long-acting inhalers in severe disease.

Summary Background Present treatment strategies to stratify exacerbation risk in patients with chronic obstructive pulmonary disease (COPD) rely on a history of two or more events in the previous year. We aimed to understand year to year variability in exacerbations and factors associated with consistent exacerbations over time. Methods In this longitudinal, prospective analysis of exacerbations in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) cohort, we analysed patients aged 40–80 years with COPD for whom 3 years of prospective data were available, identified through various means including care at academic and non-academic medical centres, word of mouth, and existing patient registries. Participants were enrolled in the study between Nov 12, 2010, and July 31, 2015. We classified patients according to yearly exacerbation frequency: no exacerbations in any year; one exacerbation in every year during 3 years of follow-up; and those with inconsistent exacerbations (individuals who had both years with exacerbations and years without during the 3 years of follow-up). Participants were characterised by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) spirometric category (1–4) on the basis of post-bronchodilator FEV 1 . Stepwise logistic regression was used to compare factors associated with one or more acute exacerbations of COPD every year for 3 years versus no exacerbations in the same timeframe. Additionally, a stepwise zero-inflated negative binomial model was used to assess predictors of exacerbation count during follow-up in all patients with available data. Baseline symptom burden was assessed with the COPD assessment test. This trial is registered with ClinicalTrials.gov, number NCT01969344. Findings 2981 patients were enrolled during the study. 1843 patients had COPD, of which 1105 patients had 3 years of complete, prospective follow-up data. 538 (49%) of 1105 patients had at least one acute exacerbation during the 3 years of follow-up, whereas 567 (51%) had none. 82 (7%) of 1105 patients had at least one acute exacerbation each year, whereas only 23 (2%) had two or more acute exacerbations in each year. An inconsistent pattern (both years with and without acute exacerbations) was common (456 [41%] of the group), particularly among GOLD stages 3 and 4 patients (256 [56%] of 456). In logistic regression, consistent acute exacerbations (≥1 event per year for 3 years) were associated with higher baseline symptom burden, previous exacerbations, greater evidence of small airway abnormality on CT, lower interleukin-15 concentrations, and higher interleukin-8 concentrations, than were no acute exacerbations. Interpretation Although acute exacerbations are common, the exacerbation status of most individuals varies markedly from year to year. Among patients who had any acute exacerbation over 3 years, very few repeatedly had two or more events per year. In addition to symptoms and history of exacerbations in the year before study enrolment, we identified several novel biomarkers associated with consistent exacerbations, including CT-defined small airway abnormality, and interleukin-15 and interleukin-8 concentrations. Funding National Institutes of Health, and National Heart, Lung, and Blood Institute.

Abnormalities in mucus production and qualitative properties such as mucus hydration are central to the pathophysiology of airway disease including cystic fibrosis, asthma, and chronic bronchitis. In vitro air–liquid interface epithelial cell cultures demonstrate direct relationships between mucociliary transport, periciliary liquid (PCL) height, and mucus concentration (expressed as percent solids or partial osmotic pressure). In health, the osmotic modulus/pressure of the PCL exceeds that of the mucus layer, resulting in efficient, low-friction movement of mucus. In disease, through multiple mechanisms, the osmotic pressure of the mucus begins to exceed basal PCL values, resulting in compression of the cilia and slowing of mucus transport. The in vivo data in both cystic fibrosis and chronic bronchitis parallel in vitro data demonstrating that when mucus osmotic pressure is increased, mucociliary clearance is decreased. In chronic bronchitis, there is a direct correlation between FEV1 and percent solids of mucus, demonstrating a strong relationship between disease progression and mucus abnormalities. Animal models, based mechanistically on raised sodium absorption (and therefore water absorption) from airway surfaces, mimic the pathophysiology of chronic obstructive pulmonary disease. Collectively, these data suggest the importance of mucus concentration in the pathogenesis of airway disease. It is important to understand the precise mechanisms that result in mucus hyperconcentration, for example, mucin overproduction versus abnormal regulation of ion/water transport, which may be unique to and characteristic of each disease phenotype. The measurement of mucus concentration may be a simple method to diagnose chronic bronchitis, monitor its progression, and serve as a biomarker for development of new therapies.

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 x 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.

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=2x10(-7). No SNPs were associated with lifetime average CPD, current CPD or smoking cessation with p

Rationale: Retrospective pharmacogenetic studies have questioned whether patients with asthma who are arginine homozygous at the β2-adrenergic receptor (position 16) should use long-acting β-agonists.Objectives: To examine whether the response to salmeterol alone or in combination with an inhaled corticosteroid is influenced by β- receptor polymorphisms.Methods: Subjects using only as-needed albuterol were screened and completed two sequential open-label run-in periods (8 wk on as-needed albuterol; 8 wk on as-needed ipratropium). Five hundred forty-four subjects were randomized by Arg16Gly genotype to salmeterol alone or with fluticasone propionate for 16 weeks. Change from baseline in morning peak expiratory flow was the primary endpoint.Measurements and Main Results: Lung function responses were sustained over treatment and no statistically significant changes from baseline between genotypes within treatments were observed. Overall mean changes in morning peak flow for salmeterol with fluticasone propio...

Aims: Typically, only a proportion of the patients suffering from common diseases respond to frequently prescribed drugs. Since the presence of drug nonresponders in pharmacogenetic studies can adversely affect statistical power we propose a method to restrict genetic tests to drug responders only. In this paper, we estimate drug nonresponse in a clinical trial for the asthma drug montelukast as either the result of an inactive genetic variant or the presence of subgroups of patients not responding to the drug. Materials & methods: We propose finite mixture models where unobserved (latent) categorical variables represent either a drug responder or nonresponder class. Analytical results show this method can substantially improve power by testing for genetic variants only in the drug-responder class. We also demonstrate how, if appropriate, placebo data can be used to further increase power to detect genetic effects. Results: It was estimated that only 25–30% of the subjects responded to the drug montelukast. Genetic-association tests confined to the responder group resulted in a substantial increase in explained genetic variance, between 10.3 and 13.2%, for four markers in the arachidonate 5-lipoxigenase (ALOX5) and cysteinyl leukotriene receptor 1 (CYSLTR1) genes. Conclusion: The presence of subgroups of patients that do not respond to the drug was an important reason for nonresponse. Additional analyses using finite mixture models in pharmacogenetic studies may provide insight into drug nonresponse and a better discrimination between true and false discoveries.

Quantitative phenotypes correlated with a complex disorder offer increased power to detect linkage in comparison to affected-unaffected classifications. Asthma is a complex disorder characterized by periods of bronchial obstruction and increased bronchial hyper reactivity. In childhood and early adulthood, asthma is frequently associated also with quantitative measures of atopy. Genome wide quantitative multipoint linkage analysis was conducted for serum IgE levels and percentage of positive skin prick test (SPT(per)) using three large groups of families originally ascertained for asthma. In this report, 438 and 429 asthma families were informative for linkage using IgE and SPT(per) which represents 690 independent families. Suggestive linkage (LOD > or = 2) was found on chromosomes 1, 3, and 8q with maximum LODs of 2.34 (IgE), 2.03 (SPT(per)), and 2.25 (IgE) near markers D1S1653, D3S2322-D3S1764, and D8S2324, respectively. The results from chromosomes 1 and 3 replicate previous reports of linkage. We also replicate linkage to 5q with peak LODs of 1.96 (SPT(per)) and 1.77 (IgE) at or near marker D5S1480. Our results provide further evidence implicating chromosomes 1, 3, and 5q. The current report represents one of the biggest genome scans so far reported for asthma related phenotypes. This study also demonstrates the utility of increased sample sizes and quantitative phenotypes in linkage analysis of complex disorders.

Proinflammatory and immunoregulatory products from C3 play a major role in phagocytosis, respiratory burst, and airways inflammation. C3 is critical in adaptive immunity; studies in mice deficient in C3 demonstrate that features of asthma are significantly attenuated in the absence of C3. To test the hypothesis that the C3 gene on chromosome 19p13.3-p13.2 contains variants associated with asthma and related phenotypes, we genotyped 25 single nucleotide polymorphism (SNP) markers distributed at intervals of approximately 1.9 kb within the C3 gene in 852 African Caribbean subjects from 125 nuclear and extended pedigrees. We used the multiallelic test in the family-based association test program to examine sliding windows comprised of 2-6 SNPs. A five-SNP window between markers rs10402876 and rs366510 provided strongest evidence for linkage in the presence of linkage disequilibrium for asthma, high log[total IgE], and high log[IL-13]/[log[IFN-gamma] in terms of global P-values (P = 0.00027, 0.00013, and 0.003, respectively). A three-SNP haplotype GGC for the first three of these markers showed best overall significance for the three phenotypes (P = 0.003, 0.007, 0.018, respectively) considering haplotype-specific tests. Taken together, these results implicate the C3 gene as a priority candidate controlling risk for asthma and allergic disease in this population of African descent.

Cysteinyl leukotrienes (CYSLTR) are potent biological mediators in the pathophysiology of asthma for which two receptors have been characterized, CYSLTR1 and CYSLTR2. The leukotriene modifying agents currently used to control bronchoconstriction and inflammation in asthmatic patients are CYSLTR1-specific leukotriene receptor antagonists. In this report, we investigated a possible role for therapeutic modulation of CYSLTR2 in asthma by investigating genetic association with asthma and further characterization of the pharmacology of a coding polymorphism.The association of CYSLTR2 polymorphisms with asthma was assessed by transmission disequilibrium test in two family-based collections (359 families from Denmark and Minnesota, USA and 384 families from the Genetics of Asthma International Network).A significant association of the coding polymorphism, 601AG, with asthma was observed (P = 0.003). We replicated these findings in a collection of 384 families from the Genetics of Asthma International Network (P = 0.04). The G allele is significantly under-transmitted to asthmatics, indicating a possible role for this receptor in resistance to asthma. The potency of cysteinyl leukotrienes at the wild-type CYSLTR2 and the coding polymorphism 601AG were assessed using a calcium mobilization assay. The potency of LTC4 and LTE4 was similar for both forms of the receptor and LTB4 was inactive, however, LTD4 was approximately five-fold less potent on 601AG compared to wild-type CYSLTR2.Since 601AG alters the potency of LTD4 and this variant allele may be associated with resistance to asthma, it is possible that modulation of the CYSLTR2 may be useful in asthma pharmacotherapy.

Alcohol abuse is a risk factor for cancers of the gastrointestinal tract, and it also can precipitate psoriasis characterized by hyperproliferation of epidermal cells. Because these effects of alcohol may involve stimulation of cell growth, and ethanol (EtOH) was shown to enhance DNA synthesis in mouse fibroblasts and epidermal cells, we conducted a study to determine whether EtOH can also stimulate mitogenesis in human fibroblasts and keratinocytes. In keratinocytes, EtOH had no effects on mitogenesis after shorter (17-hr) treatments, but it partially prevented inhibition of DNA synthesis elicited by longer treatments (3-4 days) with 2 mM calcium (Ca 2+ ), a differentiation-inducing agent. In contrast, treatment of serum-starved zinc-treated (40 μM) human skin fibroblasts with 50-60 mM EtOH for 17 hr resulted in increased DNA synthesis. EtOH-induced DNA synthesis was blocked by I mM EGTA, a specific Ca 2+ chelator. Despite thc presence of 1.8 mM Ca 2+ in the cell culture medium, the addition of 1 mM extra Ca 2+ (final concentration, 2.8 mM) for 17 hr induced DNA synthesis, presumably mediated by Ca 2+ receptors. In eight independent human skin fibroblast lines examined, treatment with EtOH for 46 hr, but not for 17 hr, invariably enhanced the effects of Ca 2+ on DNA synthesis, consistent with synergistic stimulation of cell proliferation by EtOH and Ca 2+ . Neomycin, a Ca 2+ receptor agonist, and EtOH also exerted synergistic effects on DNA synthesis after longer (46-hr) treatments. In mousc NIH 3T3 fibroblasts, both EtOH- and Ca 2+ -enhanced DNA synthesis after 17-hr treatment, but they stimulated cell proliferation only in combination. The results indicate that in human fibroblasts, EtOH can potentiate the longer-term effects of high concentrations of Ca 2+ on DNA synthesis whereas, in keratinocytes, EtOH may inhibit Ca 2+ -induced differentiation.

The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) stimulates both the synthesis and phospholipase D (PLD)-mediated hydrolysis of phosphatidylcholine (PtdCho). Here, attached and suspended NIH 3T3 fibroblasts as well as variants of the MCF-7 human breast carcinoma cell line expressing PKC-alpha and a PtdCho-specific PLD activity at widely different levels were used to determine the possible role of PKC-alpha, PtdCho hydrolysis, and choline uptake in the mediation of PMA effect on PtdCho synthesis. In wild-type MCF-7 cells, which express both PKC-alpha and PLD activities at very low levels, PMA had little effects on the uptake or incorporation [14C]choline into PtdCho. In multidrug resistant MCF-7/MDR1 cells, which highly express PKC-alpha but lack the PtdCho-specific PLD activity, 100-nM PMA had relatively small stimulatory effects on the uptake of [14C]choline (approximately 1.5-fold) and [14C]PtdCho synthesis (1.5- to 2-fold). In NIH 3T3 fibroblasts and MCF-7/PKC-alpha cells, both expressing PKC-alpha and PLD activities at high levels, 10-100-nM PMA enhanced [14C]choline uptake only slightly (1.7- to 2.2-fold), while it had much greater (approximately 4-9-fold) stimulatory effects on PtdCho synthesis. PMA significantly enhanced the formation of phosphatidic acid (PtdOH) in MCF-7/PKC-alpha cells (2.8-fold increase), but not in MCF-7/MDR1 cells (1.4-fold increase), while in both cell lines it had only small (1.3-1.5-fold) stimulatory effects on 1,2-diacylglycerol (1, 2-DAG) formation. In suspended NIH 3T3 cells, 200-300-mM ethanol blocked the stimulatory effect of PMA on PtdOH formation without affecting PtdCho synthesis indicating that neither PtdOH nor 1,2-DAG derived from it is a mediator of PMA effect on PtdCho synthesis. In attached NIH 3T3 cells, dimethylbenz[a]anthracene enhanced phosphocholine formation and, thus, choline uptake without increasing PtdCho synthesis or modifying the effect of PMA. While the results indicate that the stimulatory effect of PMA on PtdCho synthesis requires the expression of both PKC-alpha and a PtdCho-specific PLD, they do not support a role for 1,2-DAG, PtdOH or choline in the mediation of PMA effect.

In serum-starved NIH 3T3 clone 7 fibroblasts, choline phosphate (ChoP) (0.5-1 mM) and insulin synergistically stimulate DNA synthesis. Here we report that ATP also greatly enhanced the mitogenic effects of ChoP (0.1-1 mM) both in the absence and presence of insulin; maximal potentiating effects required 50-100 microM ATP. The co-mitogenic effects of ATP were mimicked by adenosine 5'-O-(3-thiotriphosphate), adenosine 5'-O-(2-thiodiphosphate), ADP, and UTP, but not by AMP or adenosine, indicating the mediatory role of a purinergic P2 receptor. Externally added ChoP acted on DNA synthesis without its detectable uptake into fibroblasts, indicating that ChoP can be a mitogen only if it is released from cells. Extracellular ATP (10-100 microM) induced extensive release of ChoP from fibroblasts. ChoP had negligible effects, even in the presence of ATP or insulin, on the activity state of p42/p44 mitogen-activated protein kinases, while in combination these agents stimulated the activity of phosphatidylinositol 3'-kinase (PI 3'-kinase). Expression of a dominant negative mutant of the p85 subunit of PI 3'-kinase or treatments with the PI 3'-kinase inhibitor wortmannin only partially (approximately 40-50%) reduced the combined effects of ChoP, ATP, and insulin on DNA synthesis; in contrast, the pp70 S6 kinase inhibitor rapamycin almost completely inhibited these effects. ATP and insulin also potentiated, while rapamycin strongly inhibited, the mitogenic effects of sphingosine 1-phosphate (S1P). Furthermore, even maximally effective concentrations of ChoP and S1P synergistically stimulated DNA synthesis. The results indicate that in the presence of extracellular ATP and/or S1P, ChoP induces mitogenesis through an extracellular site by mechanisms involving the activation of pp70 S6 kinase and, to a lesser extent, PI 3'-kinase.

The molecular basis of bilayer tubule formation in hydrated galactosylceramide (GalCer) dispersions has been investigated by synthesizing different chain-pure GalCers and examining their aqueous mesomorphic phase structure by freeze fracture and negative-stain electron microscopy. Thermotropic characterization of the GalCer species by differential scanning calorimetry provided supplementary information that verified the phase state under which morphological observations were carried out. Under aqueous conditions and at room temperature, N-24:1 delta 15(cis) GalSph, the predominant monounsaturated, nonhydroxy acyl species of bovine brain GalCer (NFA-GalCer), formed cylindrical mesomorphic self-assemblies consisting almost exclusively of "nanotubes," i.e., lipid bilayer tubules of relatively uniform length and diameter (length, 250–400 nm; diameter, 25–30 nm). In contrast, N-24:0 GalSph, the major saturated, nonhydroxy acyl species of bovine brain GalCer, displayed no tendency to form these relatively small "nanotubes." Rather, N-24:0 GalSph formed larger, variable-length ribbon-like structures (length, 5,000–10,000 nm) that often appeared to undulate and, occasionally, appeared to be helically twisted. Interestingly, bovine brain GalCer, which contains high levels of the N-24:1 delta 15(cis) and N-24:0 species as well as 2-hydroxy acyl chains, formed multilamellar liposomes of variable size and showed little tendency to form cylindrical structures. This result suggested that changes to the polar interface/headgroup region imparted by the 2-hydroxy acyl species strongly influenced bilayer tubule and cylinder formation in GalCer. To define this influence more clearly, other sphingoid-based and glycerol-based lipids were investigated. Morphological characterization of N-24:1 delta 15(cis) sphingosylphosphorylcholine (24:1 SM) revealed no evidence of bilayer cylinder or tubule formation. Similar results were obtained with aqueous dispersions of 1-palmitoyl-2-nervonoyl phosphatidylcholine (16:0, 24:1 PC). Hence, the bulkier, more hydrated, zwitterionic phosphocholine headgroup inhibited the formation of bilayer nanotubes and cylinders under physiological saline conditions.

As determined by freeze fracture electron microscopy, increasing levels of bovine brain galactosylceramide (GalCer) altered the surface structure of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayers by inducing a striking "macro-ripple" phase in the larger, multilamellar lipid vesicles at GalCer mole fractions between 0.4 and 0.8. The term "macro-ripple" phase was used to distinguish it from the P beta' ripple phase observed in saturated, symmetric-chain length phosphatidylcholines. Whereas the P beta' ripple phase displays two types of corrugations, one with a wavelength of 12–15 nm and the other with a wavelength of 25–35 nm, the macro-ripple phase occurring in GalCer/POPC dispersions was of one type with a wavelength of 100–110 nm. Also, in contrast to the extended linear arrays of adjacent ripples observed in the P beta' ripple phase, the macro-ripple phase of GalCer/POPC dispersions was interrupted frequently by packing defects resulting from double dislocations and various disclinations and, thus, appeared to be continuously twisting and turning. Control experiments verified that the macro-ripple phase was not an artifact of incomplete lipid mixing or demixing during preparation. Three different methods of lipid mixing were compared: a spray method of rapid solvent evaporation, a sublimation method of solvent removal, and solvent removal using a rotary evaporation apparatus. Control experiments also revealed that the macro-ripple phase was observed regardless of whether lipid specimens were prepared by either ultra-rapid or manual plunge freezing methods as well as either in the presence or absence of the cryo-protectant glycerol. The macro-ripple phase was always observed in mixtures that were fully annealed by incubation above the main thermal transition of both POPC and bovine brain GalCer before rapid freezing. If the GalCer mixed with POPC contained only nonhydroxy acyl chains or only 2-hydroxy acyl chains, then the occurrence of macro-ripple phase decreased dramatically.

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; FEV1/FVC, 1.5 × 10−35; FEV1, 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.

A major goal of this work was to determine in NIH 3T3 fibroblasts whether the recently described effects of H2O2 on phospholipase D-mediated hydrolysis of phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho) are mediated by similar or different mechanisms. While exposure of NIH 3T3 fibroblasts to H2O2 stimulated the hydrolysis of both PtdEtn and PtdCho, the following important differences were noted: (i) prolonged (24 h) treatment of fibroblasts with 400 nM phorbol 12-myristate 13-acetate (PMA) blocked the stimulatory effect of H2O2 on PtdEtn, but not on PtdCho, hydrolysis; (ii) PMA-induced hydrolysis of PtdEtn, but not PtdCho, was inhibited by H2O2; (iii) the stimulatory effect of H2O2 was additive with that of sphingosine or staurosporine, inhibitors of protein kinase C, on the hydrolysis of PtdCho, but not PtdEtn; (iv) with membranes isolated from H2O2-treated fibroblasts, the hydrolysis of PtdCho, but not PtdEtn, was increased compared to values obtained with control membranes. These results imply that H2O2 regulates PtdEtn and PtdCho hydrolysis by different mechanisms. Stimulation of PtdEtn hydrolysis by H2O2, sphingosine, and staurosporine may commonly involve, at least in part, neutralization of an inhibitory protein kinase C isozyme.

The severity of insulin-dependent diabetes mellitus had a marked effect upon the development of myocardial sequelae in the rat. Even with the same degree of hyperglycemia, glycosuria, polydipsia, and polyuria, moderately diabetic animals did not develop the degenerative ultrastructural changes seen in myocardium from more severely diabetic rats. These included decreased cardiocyte size, loss and disorganization of myofibrils, and loss of sarcoplasmic reticulum and transverse tubules. Since hyperglycemia and glycosuria are frequently used as the primary, and often sole, criteria for identifying diabetes in experimental animals, this study demonstrates the need to more specifically define the severity of the disease in studies of the heart.

Secretory leukocyte proteinase inhibitor (SLPI) is a potent elastase, trypsin, and chymotrypsin inhibitor occurring in all mucous secretions. Its inhibitory potency and profile suggested that it may become a therapeutic adjuvant in diseases where proteinases play a pathogenetic role. In the course of developing recombinant SLPI for therapeutic purposes, we studied its pharmacokinetics after intravenous, intraperitoneal, and intratracheal application to rats. In plasma, SLPI was determined with an ELISA or by following a radiotracer [( 35S]SLPI). In bronchoalveolar lavage fluid (BALF), SLPI was determined additionally by a functional assay (elastase inhibitory capacity). Intravenously applied SLPI (2 mg/kg) was rapidly cleared, with half-times of distribution of 6 min and half-times of elimination of 50 min. Very little (less than 5%) appeared in the urine even after 24 h. Approximately 80% of intraperitoneally injected SLPI (12 mg/kg) was absorbed and generated maximal plasma concentration of 6 to 10 micrograms/ml 30 to 120 min after administration. When given intratracheally (8.6 mg/kg), SLPI disappeared from the lungs, with a half-time of 4 to 5 h. This value was the same whether the remaining SLPI in BALF was determined radiometrically, by ELISA or by the functional assay, indicating minimal metabolism in the lung. As in the case of intraperitoneal application, SLPI was absorbed systemically, resulting in a maximal plasma level of about 2 micrograms/ml 1 to 2 h after application. In contrast to the measurements in BALF, the ELISA and radiotracer measurements in plasma correlated only for the first 2 h after application and diverged progressively after that, suggesting breakdown of the molecule once it reaches the plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Asthma is a complex disease and the intricate interplay between genetic and environmental factors underlies the overall phenotype of the disease. Families with at least two siblings with asthma were collected from Europe, Australia and the US. A genome scan using a set of 364 families with a panel of 396 microsatellite markers was conducted. Nonparametric linkage analyses were conducted for asthma and three asthma-related phenotypes: bronchial hyper-reactivity (BHR), strict definition of asthma and atopic asthma. Nine chromosomal regions with LOD scores greater than 1.5 were identified ( chromosomes 1q, 2p, 3q, 4p, 4q, 6q, 12q, 20p and 21). Linkage refinement analysis was performed for three BHR loci by genotyping single nucleotide polymorphisms at an average marker density of 1 cM. The LOD scores increased to 3.07 at chromosome 4p and 4.58 at chromosome 2p, while the chromosome 6p locus did not refine. The LOD score at the chromosome 2p locus is highly significant on a genome-wide basis. The refined locus covers a region with a physical size of 12.2 Mb. Taken together, these results provide evidence for a major asthma susceptibility locus on chromosome 2p.

In fibroblasts, the protein ]kinase C (PKC) activator phorbol 12-myristate (PMA) either inhibits or stimulates phospholipase C-mediated hydrolysis of phosphatidylethanolamine in the absence or presence of ethanol, respectively. Here, we demonstrate that the specific PKC inhibitor bisindolylmaleimide GF 109203X prevents only the inhibitory, but not the stimulatory, PMA effect.

Advances in technologies and the availability of a single nucleotide polymorphism (SNP) map are beginning to show the true potential for the human genome project to affect patient healthcare. A whole genome scan, the use of 100,000–300,000 SNPs across the genome, is now possible. Use of traditional approaches and the whole genome scan will result in identification of disease susceptibility genes and development of many new treatments in the longer term. In the shorter term, the goal will be to predict those patients at risk to experience an adverse reaction or those with a high probability for improved efficacy (i.e. pharmacogenetics). As progress is made in the area of disease genetics and pharmacogenetics, our understanding of disease susceptibility and its interrelationship with drug response will improve, making targeted therapy (i.e. the right drug to the right patient) a reality.

The present study was undertaken to address the question whether lipid differentiation occurs in junctional domains which could imply a functional requirement for specific lipids in junctional structures. Junction enriched membranes were isolated from bovine lens fiber cells using Tris and urea treatment, and the presence of junctional structures was ascertained by electron microscopy. Enrichment in major intrinsic protein (MIP, MP26) was monitored by SDS polyacrylamide gel electrophoresis. Junctional lipids were extracted by a modified Folch procedure, to quantitatively recover cholesterol, and lipid classes were analyzed. While 99.5% of total lens protein was solubilized in the course of junction isolation, 43.9% of cell phospholipids (PL) and 64.1% of cell cholesterol (Chol) were conserved. Cholesterol was by far the predominant lipid in the junction enriched lens fiber cell membranes (833 nmol/mg protein) and was more abundant than all phospholipids combined (682 nmol/mg protein). In isolating the junctional membranes, cholesterol levels increased 144-fold, and average phospholipid levels increased 99-fold, which resulted in an increase in Chol/PL ratio from 0.84 to 1.22. Different phospholipids showed substantially different degrees of enrichment with highest enrichments seen for the phosphatidylethanolamine fraction (152-fold) and sphingomyelin (101-fold). Thus, the phospholipids of the junction enriched membranes consisted mainly of ethanolamine glycerophospholipids (37.3%) and sphingomyelin (28.6%), with lesser amounts of choline glycerophospholipids (23.5%) and phosphatidylserine (9.2%) present. Our data suggest that the MP26 junction enriched membranes of bovine lens fiber cells contain differentiated lipid domains, and that cholesterol, ethanolamine glycerophospholipids and sphingomyelin are the prevalent boundary lipids of the major intrinsic protein in these domains.

Many human tumors contain high concentrations of ethanolamine phosphate (EtnP). An important question is whether increased formation of EtnP is merely the consequence of cell transformation, or is it associated with the process of carcinogenesis. Here we show that in C3H/10T1/2 embryonic fibroblasts, an established cellular model for the study of carcinogenesis, the environmental carcinogens, 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P) (0.1-1 microgram/ml concentration; 24 h treatment), stimulate phosphorylation of ethanolamine derived from increased hydrolysis of phosphatidylethanolamine. The results suggest that increased formation of EtnP is associated with the early stages of carcinogenesis. This observation may have prognostic value.

The biological significance of the sphingomyelins (SM) and monoglycosylated sphingolipids like galactosylceramides (GalCer) are well documented Our recent investigation showed tubular bilayers in the aqueous dispersions of N-nervonoyl GalCer [N-(24:lΔ15,cls) GalCer] (a major fatty acyl moiety of natural GalCer). To determine the influence of lipid head groups on the resulting mesophasic morphology, we investigated microstructural self-assemblies of N-nervonoyl-SM [N-(24:1 Δ15,cls) SM; the second most abundant sphingomyelin in mammalian cell membranes], 1- palmitoyl-2-nervonoyl phosphatidylcholine [PNPC] (the lipid species with the same acyl chain configuration as in N-(24: 1) GalCer) and also compared it with egg-SM by freeze-fracture EM.Procedures for synthesizing and purifying N-(24:1) GalCer, N-(24:1) SM, and PNPC have been reported . Egg-SM was purchased from Avanti Polar Lipids, Alabaster AL. All lipids were >99% pure as checked by thin layer chromatography. Lipid dispersions were prepared by hydrating dry lipid with phosphate buffer (pH 6.6) at 80-90°C (3-5 min), vigorously vortexing (1 min) and repeating this procedure for three times prior to three freeze-thaw cycles.

In isolated canine tracheal smooth muscle, repeated administrations of histamine result in a rapid reduction in contractile response to about 15% of the initial contraction (tachyphylaxis). Development of this tachyphylaxis is specific inasmuch as: 1) it does not develop to acetylcholine (10−6 M or 10−4 M), or serotonin (10−5 M); and 2) maximally developed histamine tachyphylaxis is not associated with a parallel reduction in response to acetylcholine. Pretreatment with propranolol (10−5 M) or phentolamine (10−4 M) does not prevent tachyphylaxis: however, pretreatment with atropine (10−4 M) does prevent tachyphylaxis in about 50% of the animals tested. Tachyphylaxis to histamine can be reversed in a dose- and time-dependent fashion with prostaglandin synthesis inhibiting agents. The order of potency obtained with such compounds (indomethacin > mefenamic acid > oxyphenbutazone > acetylsalicylic acid) is consistent with potencies for inhibition of prostaglandin synthesis found in the literature. Also, in indomethacin pretreated strips in which tachyphylaxis to histamine was prevented, exogenous addition of PGE2 (1.42×10−10 M to 2.84×10−9 M) and PGA2 in a high concentration (2.9×10−9 M) are capable of selectively reducing the response to histamine without an effect on acetylcholine-induced contractions. These data suggest that the mechanism of histamine tachyphylaxis in the canine tracheal smooth muscle preparation involves prostaglandin synthesis.

An efficient procedure for preparing phytenic acid methyl ester, free of isomers, from phytol is reported. Phytyl phytenate and other isoprenoid wax esters were synthesized. Gas liquid chromatography of these wax esters and other compounds related to phytol and phytenic acid is described. The alkyl constituents of isoprenoid wax esters can be analyzed after alkaline methanolysis and the acyl constituents after acidic methanolysis. The applicability of these methods to natural mixtures was demonstrated with wax esters from mosses which contained both types of isoprenoids and with wax esters from healthy and frost damaged grass which contained phytol, but not phytenic acid.