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1.  Superoxide Dismutase Expression Attenuates Cigarette Smoke– or Elastase-generated Emphysema in Mice 
Rationale
Oxidants are believed to play a major role in the development of emphysema.
Objectives
This study aimed to determine if the expression of human copper–zinc superoxide dismutase (CuZnSOD) within the lungs of mice protects against the development of emphysema.
Methods
Transgenic CuZnSOD and littermate mice were exposed to cigarette smoke (6 h/d, 5 d/wk, for 1 yr) and compared with nonexposed mice. A second group was treated with intratracheal elastase to induce emphysema.
Measurements
Lung inflammation was measured by cell counts and myeloperoxidase levels. Oxidative damage was assessed by immunofluorescence for 3-nitrotyrosine and 8-hydroxydeoxyguanosine and lipid peroxidation levels. The development of emphysema was determined by measuring the mean linear intercept (Lm).
Main Results
Smoke exposure caused a fourfold increase in neutrophilic inflammation and doubled lung myeloperoxidase activity. This inflammatory response did not occur in the smoke-exposed CuZnSOD mice. Similarly, CuZnSOD expression prevented the 58% increase in lung lipid peroxidation products that occurred after smoke exposure. Most important, CuZnSOD prevented the onset of emphysema in both the smoke-induced model (Lm, 68 exposed control vs. 58 exposed transgenic; p < 0.04) and elastase-generated model (Lm, 80 exposed control vs. 63 exposed transgenic; p < 0.03). These results demonstrate for the first time that antioxidants can prevent smoke-induced inflammation and can counteract the proteolytic cascade that leads to emphysema formation in two separate animal models of the disease.
Conclusions
These findings indicate that strategies aimed at enhancing or supplementing lung antioxidants could be effective for the prevention and treatment of this disease.
doi:10.1164/rccm.200506-850OC
PMCID: PMC3982860  PMID: 16387805
emphysema; inflammation; oxidants
2.  Leukotrienes in Acute Lung Injury 
doi:10.1164/rccm.2505008
PMCID: PMC2718469  PMID: 16040787
3.  ABCA3 Mutations Associated with Pediatric Interstitial Lung Disease 
Rationale: ABCA3 is a member of the ATP-binding cassette family of proteins that mediate the translocation of a wide variety of substrates, including lipids, across cellular membranes. Mutations in the gene encoding ABCA3 were recently identified in full-term neonates with fatal surfactant deficiency.
Objective: To test the hypothesis that ABCA3 mutations are not always associated with fatal neonatal lung disease but are a cause of pediatric interstitial lung disease.
Methods: DNA samples were obtained from 195 children with chronic lung disease of unknown etiology. The 30 coding exons of the ABCA3 gene were sequenced in four unrelated children with a referring diagnosis of desquamative interstitial pneumonitis and who were older than 10 years at the time of enrollment.
Results: Three of four patients (ages 16, 23, and 11 years) with desquamative interstitial pneumonitis had ABCA3 mutations identified on both alleles. All three had the same missense mutation (E292V) and a second unique mutation. The E292V mutation was not found on 200 control alleles from adults without lung disease, but seven additional patients of the remaining study patients had the E292V mutation on one allele. Immunohistochemical analysis of surfactant protein expression in three patients revealed a specific staining pattern for surfactant protein-B, which was the same pattern observed in several infants with fatal lung disease due to ABCA3 mutations.
Conclusion: ABCA3 mutations cause some types of interstitial lung disease in pediatric patients.
doi:10.1164/rccm.200503-504OC
PMCID: PMC1403838  PMID: 15976379
desquamative interstitial pneumonitis; pulmonary alveolar proteinosis; surfactant
4.  Negative regulation of myofibroblast differentiation by phosphatase and tensin homologue deleted on chromosome ten 
Rationale: Myofibroblasts are primary effector cells in idiopathic pulmonary fibrosis. Defining mechanisms of myofibroblast differentiation may be critical to the development of novel therapeutic agents. Objective: To show that myofibroblast differentiation is regulated by phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activity in vivo and to identify a potential mechanism by which this occurs. Methods: We utilized tissue sections of surgical lung biopsies from patients with idiopathic pulmonary fibrosis to localize expression of PTEN and α-SMA. We utilized cell culture of pten-/- and wild-type fibroblasts as well as adenoviral strategies and pharmacologic inhibitors to determine the mechanism by which PTEN inhibits α-SMA, fibroblast proliferation, and collagen production. Results: In human lung specimens of idiopathic pulmonary fibrosis, myofibroblasts within fibroblastic foci demonstrate diminished PTEN expression. Further, inhibition of PTEN in mice worsened bleomycin-induced fibrosis. In pten-/- fibroblasts, and in normal fibroblasts in which PTEN is inhibited, α-SMA, proliferation, and collagen production is upregulated. Addition of transforming growth factor-β to wild-type cells, but not pten-/- cells, results in increased α-SMA expression in a time-dependent fashion. In pten-/- cells, reconstitution of PTEN decreases α-SMA expression, proliferation, and collagen production, whereas overexpression of PTEN in wild-type cells inhibits transforming growth factor-β-induced myofibroblast differentiation. Both the protein and lipid phosphatase actions of PTEN are capable of modulating the myofibroblast phenotype. Conclusions: The results indicate that in idiopathic pulmonary fibrosis, myofibroblasts have diminished PTEN expression. Inhibition of PTEN in vivo promotes fibrosis, and PTEN inhibits myofibroblast differentiation in vitro.
doi:10.1164/rccm.200507-1058OC
PMCID: PMC1434700  PMID: 16179636
myofibroblast; fibrosis; PTEN; phosphatase; smooth muscle actin
5.  Prospective study of dietary patterns and persistent cough with phlegm among Chinese Singaporeans 
Rationale
Using principal components analysis to examine dietary patterns complements the evaluation of individual food and nutrient intake in relation to health outcomes, but has not yet been applied to nonmalignant respiratory disease or symptoms.
Objective
To examine the relation between patterns of dietary intake at baseline and new-onset of persistent cough with phlegm in a population-based cohort of Singapore Chinese.
Methods
A 165-item validated food frequency questionnaire was administered in-person at baseline in 1993. We identified 623 cases of incident cough with phlegm among 52,325 subjects by telephone interview from 1999 through 2004. We identified two distinct food patterns: a “meat-dim sum” pattern characterized by pork and chicken dim sum foods and noodle dishes, and a “vegetable-fruit-soy” pattern characterized by vegetables, fruit and soyfood items.
Main Results
The meat-dim sum pattern was positively associated with new-onset cough with phlegm (odds ratio=1.43; 95% confidence interval: 1.08, 1.89; comparing fourth to first quartile, P for trend=0.02,), after adjustment for age, gender, total energy intake, smoking, education and non-starch polysaccharide intake, a protective factor for cough with phlegm in this cohort. Weaker associations were seen for more chronic symptoms and for incident asthma. A weak inverse association for the vegetable-fruit-soy pattern disappeared after adjustment for non-starch polysaccharide intake.
Conclusion
A diet rich in meats, sodium plus refined carbohydrates may increase risk of developing cough with phlegm, independently of the apparent beneficial effects of a diet high in non-starch polysaccharides in this Singapore Chinese cohort.
doi:10.1164/rccm.200506-901OC
PMCID: PMC1447591  PMID: 16239624
Asthma; chronic bronchitis; COPD; diet; signs and symptoms; respiratory
6.  ENDOTOXIN EXPOSURE IS A RISK FACTOR FOR ASTHMA: THE NATIONAL SURVEY OF ENDOTOXIN IN U.S. HOUSING 
Background: While research has shown that early life exposure to household endotoxin protects against development of allergies, studies are less clear on the relationship between household endotoxin exposure and prevalence of wheezing and asthma. We assayed 2552 house dust samples in a representative nationwide sample to explore relationships between endotoxin exposures and risk factors for asthma, asthma symptoms and medication use.
Methods: House dust was vacuum-sampled from five locations within homes and assayed for endotoxin. Health, demographic and housing information was assessed through questionnaire and on-site evaluation of 2456 residents of 831 homes selected to represent the demographics of the U.S.
Results: Endotoxin concentration (EU/mg) and load (EU/m2) were highly correlated (r=0.73-0.79). Geometric mean endotoxin concentrations were (in EU/mg): bedroom floors: 35.3 (5th-95thpercentile: 5.0-260); bedding: 18.7 (2.0-142); family room floors: 63.9 (11.5-331); sofas: 44.8 (6.4-240); kitchen floors: 80.5 (9.8-512). Multivariate analysis demonstrated significant relationships between increasing endotoxin levels and diagnosed asthma, asthma symptoms in the past year, current use of asthma medications, and wheezing among residents of the homes. These relationships were strongest for bedroom floor and bedding dust and were observed in adults only. Modeling the joint effect of bedding and bedroom floor endotoxin on recent asthma symptoms yielded an adjusted odds ratio of 2.83 (95%CI: 1.01-7.87). When stratified by allergy status, allergic subjects with higher endotoxin exposure were no more likely to have diagnosed asthma or asthma symptoms than non-allergic subjects.
Conclusion: This study demonstrates that household endotoxin exposure is a significant risk factor for increased asthma prevalence.
doi:10.1164/rccm.200505-758OC
PMCID: PMC1379232  PMID: 16141442
Wheeze; Airways Inflammation; House Dust; Lipopolysaccharide
7.  Inflammatory Basis of Exercise-induced Bronchoconstriction 
Rationale
Exercise-induced bronchoconstriction (EIB) is a highly prevalent condition with unclear pathogenesis. Two competing theories of the pathogenesis of EIB differ regarding the inflammatory basis of this condition.
Objectives
Our goals were to establish whether epithelial cell and mast cell activation with release of inflammatory mediators occurs during EIB and how histamine and cysteinyl leukotriene antagonists alter the airway events occurring during EIB.
Methods
Induced sputum was used to measure mast cell mediators and eicosanoids at baseline and 30 minutes after exercise challenge in 25 individuals with asthma with EIB. In a randomized, double-blind crossover study, the cysteinyl leukotriene antagonist montelukast and antihistamine loratadine or two matched placebos were administered for two doses before exercise challenge.
Main Results
The percentage of columnar epithelial cells in induced sputum at baseline was associated with the severity of EIB. After exercise challenge, histamine, tryptase, and cysteinyl leukotrienes significantly increased and prostaglandin E2 and thromboxane B2 significantly decreased in the airways, and there was an increase in columnar epithelial cells in the airways. The concentration of columnar epithelial cells was associated with the levels of histamine and cysteinyl leukotrienes in the airways. Treatment with montelukast and loratadine inhibited the release of cysteinyl leukotrienes and histamine into the airways, but did not inhibit the release of columnar epithelial cells into the airways.
Conclusions
These data indicate that epithelial cells, mast cell mediators, and eicosanoids are released into the airways during EIB, supporting an inflammatory basis for EIB.
doi:10.1164/rccm.200412-1667OC
PMCID: PMC2041799  PMID: 15947280
asthma; eicosanoid; epithelial cell; exercise-induced bronchoconstriction; mast cell
8.  Rifapentine, Moxifloxacin, or DNA Vaccine Improves Treatment of Latent Tuberculosis in a Mouse Model 
Rationale
Priorities for developing improved regimens for treatment of latent tuberculosis (TB) infection include (1) developing shorter and/or more intermittently administered regimens that are easier to supervise and (2) developing and evaluating regimens that are active against multidrug-resistant organisms.
Objectives and Methods
By using a previously validated murine model that involves immunizing mice with Mycobacterium bovis bacillus Calmette-Guérin to augment host immunity before infection with virulent Mycobacterium tuberculosis, we evaluated new treatment regimens including rifapentine and moxifloxacin, and assessed the potential of the Mycobacterium leprae heat shock protein-65 DNA vaccine to augment the activity of moxifloxacin.
Measurements
Quantitative spleen colony-forming unit counts, and the proportion of mice with culture-positive relapse after treatment, were determined.
Main Results
Three-month, once-weekly regimens of rifapentine combined with either isoniazid or moxifloxacin were as active as daily isoniazid for 6–9 mo. Six-month daily combinations of moxifloxacin with pyrazinamide, ethionamide, or ethambutol were more active than pyrazinamide plus ethambutol, a regimen recommended for latent TB infection after exposure to multidrug-resistant TB. The combination of moxifloxacin with the experimental nitroimidazopyran PA-824 was especially active. Finally, the heat shock protein-65 DNA vaccine had no effect on colony-forming unit counts when given alone, but augmented the bactericidal activity of moxifloxacin.
Conclusions
Together, these findings suggest that rifapentine, moxifloxacin, and, perhaps, therapeutic DNA vaccination have the potential to improve on the current treatment of latent TB infection.
doi:10.1164/rccm.200507-1047OC
PMCID: PMC1853317  PMID: 16151038
DNA vaccine; latency; moxifloxacin; rifapentine; tuberculosis
9.  Prospective Study of Dietary Patterns and Persistent Cough with Phlegm among Chinese Singaporeans 
Rationale: Using principal components analysis to examine dietary patterns complements the evaluation of individual food and nutrient intake in relation to health outcomes, but has not yet been applied to nonmalignant respiratory disease or symptoms. Objective: To examine the relation between patterns of dietary intake at baseline and new onset of persistent cough with phlegm in a population-based cohort of Singapore Chinese. Methods: A 165-item validated food frequency questionnaire was administered in-person at baseline in 1993. We identified 623 cases of incident cough with phlegm among 52,325 subjects by telephone interview from 1999 through 2004. We identified two distinct food patterns: a “meat-dim sum” pattern characterized by pork and chicken dim sum foods and noodle dishes, and a “vegetable-fruit-soy” pattern characterized by vegetables, fruit, and soyfood items. Main Results: The meat-dim sum pattern was positively associated with new-onset cough with phlegm (odds ratio, 1.43; 95% confidence interval, 1.08, 1.89; comparing fourth to first quartile, p for trend = 0.02), after adjustment for age, sex, total energy intake, smoking, education, and nonstarch polysaccharide intake, a protective factor for cough with phlegm in this cohort. Weaker associations were seen for more chronic symptoms and for incident asthma. A weak inverse association for the vegetable-fruit-soy pattern disappeared after adjustment for nonstarch polysaccharide intake. Conclusion: A diet rich in meats, sodium, and refined carbohydrates may increase risk of developing cough with phlegm, independently of the apparent beneficial effects of a diet high in fiber in this Singapore Chinese cohort.
doi:10.1164/rccm.200506-901OC
PMCID: PMC1447591  PMID: 16239624
asthma; chronic bronchitis; chronic obstructive pulmonary disease; diet; respiratory, signs and symptoms
10.  Negative Regulation of Myofibroblast Differentiation by PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome 10) 
Rationale: Myofibroblasts are primary effector cells in idiopathic pulmonary fibrosis (IPF). Defining mechanisms of myofibroblast differentiation may be critical to the development of novel therapeutic agents.
Objective: To show that myofibroblast differentiation is regulated by phosphatase and tensin homolog deleted on chromosome 10 (PTEN) activity in vivo, and to identify a potential mechanism by which this occurs.
Methods: We used tissue sections of surgical lung biopsies from patients with IPF to localize expression of PTEN and α–smooth muscle actin (α-SMA). We used cell culture of pten−/− and wild-type fibroblasts, as well as adenoviral strategies and pharmacologic inhibitors, to determine the mechanism by which PTEN inhibits α-SMA, fibroblast proliferation, and collagen production.
Results: In human lung specimens of IPF, myofibroblasts within fibroblastic foci demonstrated diminished PTEN expression. Furthermore, inhibition of PTEN in mice worsened bleomycin-induced fibrosis. In pten−/− fibroblasts, and in normal fibroblasts in which PTEN was inhibited, α-SMA, proliferation, and collagen production was upregulated. Addition of transforming growth factor-β to wild-type cells, but not pten−/− cells, resulted in increased α-SMA expression in a time-dependent fashion. In pten−/− cells, reconstitution of PTEN decreased α-SMA expression, proliferation, and collagen production, whereas overexpression of PTEN in wild-type cells inhibited transforming growth factor-β–induced myofibroblast differentiation. It was observed that both the protein and lipid phosphatase actions of PTEN were capable of modulating the myofibroblast phenotype.
Conclusions: The results indicate that in IPF, myofibroblasts have diminished PTEN expression. Inhibition of PTEN in vivo promotes fibrosis, and PTEN inhibits myofibroblast differentiation in vitro.
doi:10.1164/rccm.200507-1058OC
PMCID: PMC1434700  PMID: 16179636
fibrosis; myofibroblast; phosphatase; PTEN; smooth muscle actin
11.  Rifapentine, Moxifloxacin, or DNA Vaccine Improves Treatment of Latent Tuberculosis in a Mouse Model 
Rationale: Priorities for developing improved regimens for treatment of latent tuberculosis (TB) infection include (1) developing shorter and/or more intermittently administered regimens that are easier to supervise and (2) developing and evaluating regimens that are active against multidrug-resistant organisms.
Objectives and Methods: By using a previously validated murine model that involves immunizing mice with Mycobacterium bovis bacillus Calmette-Guérin to augment host immunity before infection with virulent Mycobacterium tuberculosis, we evaluated new treatment regimens including rifapentine and moxifloxacin, and assessed the potential of the Mycobacterium leprae heat shock protein-65 DNA vaccine to augment the activity of moxifloxacin.
Measurements: Quantitative spleen colony-forming unit counts, and the proportion of mice with culture-positive relapse after treatment, were determined.
Main Results: Three-month, once-weekly regimens of rifapentine combined with either isoniazid or moxifloxacin were as active as daily isoniazid for 6–9 mo. Six-month daily combinations of moxifloxacin with pyrazinamide, ethionamide, or ethambutol were more active than pyrazinamide plus ethambutol, a regimen recommended for latent TB infection after exposure to multidrug-resistant TB. The combination of moxifloxacin with the experimental nitroimidazopyran PA-824 was especially active. Finally, the heat shock protein-65 DNA vaccine had no effect on colony-forming unit counts when given alone, but augmented the bactericidal activity of moxifloxacin.
Conclusions: Together, these findings suggest that rifapentine, moxifloxacin, and, perhaps, therapeutic DNA vaccination have the potential to improve on the current treatment of latent TB infection.
doi:10.1164/rccm.200507-1047OC
PMCID: PMC1853317  PMID: 16151038
DNA vaccine; latency; moxifloxacin; rifapentine; tuberculosis
12.  Endotoxin Exposure Is a Risk Factor for Asthma 
Background: Although research has shown that early life exposure to household endotoxin protects against development of allergies, studies are less clear on the relationship between household endotoxin exposure and prevalence of wheezing and asthma. We as- sayed 2,552 house dust samples in a representative nationwide sam- ple to explore relationships between endotoxin exposures and risk factors for asthma, asthma symptoms, and medication use.
Methods: House dust was vacuum-sampled from five locations within homes and assayed for endotoxin. Health, demographic, and housing information was assessed through questionnaire and on-site evaluation of 2,456 residents of 831 homes selected to represent the demographics of the United States.
Results: Endotoxin concentration (EU/mg) and load (EU/m2) were highly correlated (r = 0.73–0.79). Geometric mean endotoxin concentrations were as follows (in EU/mg): bedroom floors, 35.3 (5th–95th percentile, 5.0–260); bedding, 18.7 (2.0–142); family room floors, 63.9 (11.5–331); sofas, 44.8 (6.4–240); and kitchen floors, 80.5 (9.8–512). Multivariate analysis demonstrated significant relationships between increasing endotoxin levels and diagnosed asthma, asthma symptoms in the past year, current use of asthma medications, and wheezing among residents of the homes. These relationships were strongest for bedroom floor and bedding dust and were observed in adults only. Modeling the joint effect of bedding and bedroom floor endotoxin on recent asthma symptoms yielded an adjusted odds ratio of 2.83 (95% confidence interval, 1.01–7.87). When stratified by allergy status, allergic subjects with higher endotoxin exposure were no more likely to have diagnosed asthma or asthma symptoms than nonallergic subjects.
Conclusion: This study demonstrates that household endotoxin exposure is a significant risk factor for increased asthma prevalence.
doi:10.1164/rccm.200505-758OC
PMCID: PMC1379232  PMID: 16141442
airway inflammation; house dust; lipopolysaccharide; wheeze
13.  ABCA3 Mutations Associated with Pediatric Interstitial Lung Disease 
Rationale: ABCA3 is a member of the ATP-binding cassette family of proteins that mediate the translocation of a wide variety of substrates, including lipids, across cellular membranes. Mutations in the gene encoding ABCA3 were recently identified in full-term neonates with fatal surfactant deficiency.
Objective: To test the hypothesis that ABCA3 mutations are not always associated with fatal neonatal lung disease but are a cause of pediatric interstitial lung disease.
Methods: DNA samples were obtained from 195 children with chronic lung disease of unknown etiology. The 30 coding exons of the ABCA3 gene were sequenced in four unrelated children with a referring diagnosis of desquamative interstitial pneumonitis and who were older than 10 years at the time of enrollment.
Results: Three of four patients (ages 16, 23, and 11 years) with desquamative interstitial pneumonitis had ABCA3 mutations identified on both alleles. All three had the same missense mutation (E292V) and a second unique mutation. The E292V mutation was not found on 200 control alleles from adults without lung disease, but seven additional patients of the remaining study patients had the E292V mutation on one allele. Immunohistochemical analysis of surfactant protein expression in three patients revealed a specific staining pattern for surfactant protein-B, which was the same pattern observed in several infants with fatal lung disease due to ABCA3 mutations.
Conclusion: ABCA3 mutations cause some types of interstitial lung disease in pediatric patients.
doi:10.1164/rccm.200503-504OC
PMCID: PMC1403838  PMID: 15976379
desquamative interstitial pneumonitis; pulmonary alveolar proteinosis; surfactant
14.  Inflammatory Basis of Exercise-induced Bronchoconstriction 
Rationale: Exercise-induced bronchoconstriction (EIB) is a highly prevalent condition with unclear pathogenesis. Two competing theories of the pathogenesis of EIB differ regarding the inflammatory basis of this condition. Objectives: Our goals were to establish whether epithelial cell and mast cell activation with release of inflammatory mediators occurs during EIB and how histamine and cysteinyl leukotriene antagonists alter the airway events occurring during EIB. Methods: Induced sputum was used to measure mast cell mediators and eicosanoids at baseline and 30 minutes after exercise challenge in 25 individuals with asthma with EIB. In a randomized, double-blind crossover study, the cysteinyl leukotriene antagonist montelukast and antihistamine loratadine or two matched placebos were administered for two doses before exercise challenge. Main Results: The percentage of columnar epithelial cells in induced sputum at baseline was associated with the severity of EIB. After exercise challenge, histamine, tryptase, and cysteinyl leukotrienes significantly increased and prostaglandin E2 and thromboxane B2 significantly decreased in the airways, and there was an increase in columnar epithelial cells in the airways. The concentration of columnar epithelial cells was associated with the levels of histamine and cysteinyl leukotrienes in the airways. Treatment with montelukast and loratadine inhibited the release of cysteinyl leukotrienes and histamine into the airways, but did not inhibit the release of columnar epithelial cells into the airways. Conclusions: These data indicate that epithelial cells, mast cell mediators, and eicosanoids are released into the airways during EIB, supporting an inflammatory basis for EIB.
doi:10.1164/rccm.200412-1667OC
PMCID: PMC2041799  PMID: 15947280
asthma; eicosanoid; epithelial cell; exercise-induced bronchoconstriction; mast cell
15.  Reversal of Allergen-induced Airway Remodeling by CysLT1 Receptor Blockade 
Rationale: Airway inflammation in asthma is accompanied by structural changes, including goblet cell metaplasia, smooth muscle cell layer thickening, and subepithelial fibrosis. This allergen-induced airway remodeling can be replicated in a mouse asthma model.
Objectives: The study goal was to determine whether established airway remodeling in a mouse asthma model is reversible by administration of the cysteinyl leukotriene (CysLT)1 receptor antagonist montelukast, the corticosteroid dexamethasone, or the combination montelukast + dexamethasone.
Methods: BALB/c mice, sensitized by intraperitoneal ovalbumin (OVA) as allergen, received intranasal OVA periodically Days 14–73 and montelukast or dexamethasone or placebo from Days 73–163.
Measurements and Main Results: Allergen-induced trafficking of eosinophils into the bronchoalveolar lavage fluid and lung interstitium and airway goblet cell metaplasia, smooth muscle cell layer thickening, and subepithelial fibrosis present on Day 73 persisted at Day 163, 3 mo after the last allergen challenge. Airway hyperreactivity to methacholine observed on Day 73 in OVA-treated mice was absent on Day 163. In OVA-treated mice, airway eosinophil infiltration and goblet cell metaplasia were reduced by either montelukast or dexamethasone alone. Montelukast, but not dexamethasone, reversed the established increase in airway smooth muscle mass and subepithelial collagen deposition. By immunocytochemistry, CysLT1 receptor expression was significantly increased in airway smooth muscle cells in allergen-treated mice compared with saline-treated controls and was reduced by montelukast, but not dexamethasone, administration.
Conclusions: These data indicate that established airway smooth muscle cell layer thickening and subepithelial fibrosis, key allergen-induced airway structural changes not modulated by corticosteroids, are reversible by CysLT1 receptor blockade therapy.
doi:10.1164/rccm.200501-088OC
PMCID: PMC2662952  PMID: 16387808
eosinophils; fibrosis; mucus; smooth muscle
16.  Serotonin Transporter Polymorphisms in Familial and Idiopathic Pulmonary Arterial Hypertension 
Rationale: Serotonin is a pulmonary vasoconstrictor and smooth muscle cell mitogen. The serotonin transporter (SERT) is abundant in pulmonary vascular smooth muscle. Compared with the short (S) allele, the long (L) SERT promoter allele is associated with increased SERT transcription and more severe pulmonary hypertension in a cohort of patients with chronic obstructive pulmonary disease, and was more prevalent in a cohort with idiopathic pulmonary arterial hypertension (IPAH), compared with control subjects.
Objective: We hypothesized that the SERT L allele would associate with an earlier age at diagnosis and/or shorter survival interval in pulmonary arterial hypertension (PAH) than the S allele.
Methods: SERT promoters from 166 familial PAH (FPAH), 83 IPAH, and 125 control subjects were sequenced. One hundred twenty-seven of the patients with FPAH had a known mutation in bone morphogenetic protein receptor 2 (BMPR2).
Results: The mean age at diagnosis was 35.8 yr in patients with FPAH and 41.1 yr in patients with IPAH (p = 0.02). There were no significant differences in distribution of the LL, LS, or SS genotypes in IPAH, FPAH, or unaffected BMPR2 mutation carriers. In FPAH, the LL genotype was associated with an earlier age at diagnosis (p < 0.02).
Conclusions: In patients with IPAH, these SERT genotypes do not correlate with age at diagnosis or survival interval. In patients with FPAH, the LL genotype correlates with an earlier age at diagnosis than SL or SS, although survival among the groups was similar. The correlation of the SERT promoter polymorphism with age at diagnosis in FPAH suggests a possible relationship between the SERT and BMPR2.
doi:10.1164/rccm.200509-1361OC
PMCID: PMC2662954  PMID: 16339917
familial pulmonary arterial hypertension; 5-HT; 5-HTT; idiopathic pulmonary arterial hypertension; primary pulmonary hypertension; serotonin transporter
17.  Metalworking Fluid with Mycobacteria and Endotoxin Induces Hypersensitivity Pneumonitis in Mice 
Background: Human cases of hypersensitivity pneumonitis (HP) have been reported among machinists for over 10 yr. Although mycobacteria have been implicated as causal agents, this has not been established in experimental studies and the mechanisms remain unclear. Other constituents of in-use metalworking fluids (MWFs) may also contribute to the development of lung disease. We investigated the potential for Mycobacterium immunogenum (MI) in MWFs to induce HP.
Methods: Mice were exposed intranasally for 3 wk to MI (isolated from MWFs), Saccharopolyspora rectivirgula (positive control), saline, endotoxin, MWFs spiked with endotoxin and/or MI, used MWFs, and particulate-fortified used MWFs. Responses were assessed 96 h after the last exposure.
Results: Mice exposed to MI in MWFs developed lung pathology consistent with HP along with significantly more monocytes and neutrophils in lung lavage, increased CD4+/CD8+ T-lymphocyte ratio, and marked pulmonary lymphocytosis on histologic examination when compared with saline-treated control mice. Mice with Grade 2 or higher pathology (0–4 point scale) exhibited significantly elevated macrophage inflammatory protein–1α and IL-10 and a trend toward higher RANTES 96 h after the final dose. Endotoxin coexposure augmented lung pathology.
Conclusion: MWFs containing mycobacteria induced granulomatous lung lesions, peribronchiolar lymphocytosis, increased cell concentrations in lavage, and up-regulation of several cytokines. These findings are consistent with HP.
doi:10.1164/rccm.200405-627OC
PMCID: PMC2662953  PMID: 16387809
endotoxin; hypersensitivity pneumonitis; metalworking fluids; mycobacteria; Saccharopolyspora rectivirgula
18.  Increased Glucocorticoid Receptor β Alters Steroid Response in Glucocorticoid-insensitive Asthma 
Rationale: Glucocorticoids (GCs) are highly effective in the treatment of asthma. However, some individuals have GC-insensitive asthma.
Objectives: To evaluate the functional response to steroids of bronchoalveolar lavage (BAL) cells from sites of airway inflammation from patients with GC-insensitive versus GC-sensitive asthma. As well, to attempt to define the functional role of glucocorticoid receptor (GCR)β (a splicing variant, and dominant negative inhibitor of, the classic GCRα) in controlling GCRα nuclear translocation and transactivation at a molecular level.
Methods and Measurements: Fiberoptic bronchoscopy with collection of BAL fluid was performed on seven patients with GC-sensitive asthma and eight patients with GC-insensitive asthma. GCRα cellular shuttling in response to 10−6 M dexamethasone treatment and GCRβ expression were analyzed in BAL cells by immunofluorescence staining. The effects of overexpression and silencing of GCRβ mRNA on GCRα function were assessed.
Main Results: Significantly reduced nuclear translocation of GCRα in response to steroids was found in BAL cells from patients with GC-insensitive asthma. BAL macrophages from patients with GC-insensitive asthma had significantly increased levels of cytoplasmic and nuclear GCRβ. It was demonstrated that GCRα nuclear translocation and its transactivation properties were proportionately reduced by level of viral transduction of the GCRβ gene into the DO-11.10 cell line. RNA silencing of GCRβ mRNA in human BAL macrophages from patients with GC-insensitive asthma resulted in enhanced dexamethasone-induced GCRα transactivation.
Conclusions: GC insensitivity is associated with loss of GCRα nuclear translocation in BAL cells and elevated GCRβ, which may inhibit GCRα transactivation in response to steroids.
doi:10.1164/rccm.200507-1046OC
PMCID: PMC2662945  PMID: 16387802
asthma; bronchoalveolar lavage cells; glucocorticoid insensitivity; glucocorticoid receptor
19.  Risk Factors for Death of Patients with Cystic Fibrosis Awaiting Lung Transplantation 
Rationale: The optimal timing for listing of cystic fibrosis patients for lung transplantation is controversial.
Objectives: We conducted a retrospective cohort study of 343 patients listed for lung transplantation at four academic medical centers to identify risk factors for death while awaiting transplantation.
Methods: Data on possible risk factors were abstracted from medical records.
Measurements: Time to death, patient demographic characteristics, and risk factors for death while awaiting transplantation were assessed. Univariate and multivariate survival analyses were performed using Cox regression.
Results: By univariate analyses, FEV1 ⩽ 30% predicted (HR, 3.8; 95% CI, 2.0–7.5), PaCO2 ⩾ 50 mm Hg (HR, 1.85; 95% CI, 1.1–3.0), and shorter height (HR, 1.8; 95% CI, 1.1–3.0) were associated with a higher risk of death. Referral from an accredited cystic fibrosis center was associated with a lower risk (HR, 0.53; 95% CI, 0.30–0.92). The final multivariate model included referral from an accredited cystic fibrosis center (HR, 0.5; 95% CI, 0.3–1.0) and listing year after 1996 (HR, 0.4; 95% CI, 0.2–0.7); both were associated with a lower risk of death. FEV1 ⩽ 30% predicted (HR, 6.8; 95% CI, 2.4–19.3), PaCO2 ⩾ 50 mm Hg (HR, 6.9; 95% CI, 1.5–32.1), and use of a nutritional intervention (HR, 2.3; 95% CI, 1.3–4.1) were associated with increased risk. Patients with FEV1 > 30% predicted had a higher risk of death only when their PaCO2 was ⩾ 50 mm Hg (HR, 7.0; 95% CI, 1.5–32), while the increased risk of death with FEV1 ⩽ 30% was not further influenced by the presence of hypercapnia.
Conclusions: We identified risk factors for waiting list mortality that could impact on transplant listing and allocation guidelines.
doi:10.1164/rccm.200410-1369OC
PMCID: PMC2662949  PMID: 16387803
cystic fibrosis; lung transplantation; survival
20.  Leflunomide Prevents Alveolar Fluid Clearance Inhibition by Respiratory Syncytial Virus 
Rationale: Previously, we demonstrated that intranasal infection of BALB/c mice with respiratory syncytial virus (RSV) resulted in an early 40% reduction in alveolar fluid clearance (AFC), an effect mediated via P2Y purinergic receptors.
Objectives: To confirm that RSV-induced inhibition of AFC is mediated by uridine triphosphate (UTP), and to demonstrate that inhibition of de novo pyrimidine synthesis with leflunomide prevents increased UTP release after RSV infection, and thereby also prevents inhibition of AFC by RSV.
Methods: BALB/c mice were infected intranasally with RSV strain A2. AFC was measured in anesthetized, ventilated mice by instillation of 5% bovine serum albumin into the dependent lung. Some mice were pretreated with leflunomide or 6-mercaptopurine.
Measurements and Main Results: RSV-mediated inhibition of AFC is associated temporally with a 20-nM increase in UTP and ATP content of bronchoalveolar lavage fluid, hypoxemia, and altered nasal potential difference. RSV-mediated nucleotide release, AFC inhibition, and physiologic sequelae thereof can be prevented by pretreatment of mice with the de novo pyrimidine synthesis inhibitor leflunomide, which is not toxic to the mice, and which does not affect RSV replication in the lungs. In contrast, pretreatment of mice with 6-mercaptopurine, an inhibitor of de novo purine synthesis, has no beneficial effect on AFC or other indicators of disease progression. Finally, RSV-mediated inhibition of AFC is prevented by volume-regulated anion channel inhibitors.
Conclusion: Pyrimidine synthesis or release pathways may provide novel therapeutic targets to counter the pathophysiologic sequelae of impaired AFC in RSV disease.
doi:10.1164/rccm.200508-1200OC
PMCID: PMC2662951  PMID: 16387801
ion transport; paramyxovirus infections; pneumonia, viral; pulmonary edema
21.  Detection of Respiratory Syncytial Virus in Adults with Chronic Obstructive Pulmonary Disease 
Rationale: Recently, respiratory syncytial virus (RSV) RNA has been identified by reverse transcriptase–polymerase chain reaction (RT-PCR) from a high percentage of patients with stable chronic obstructive pulmonary disease (COPD). These data raise the possibility of persistent low-grade infection in this population, which could have implications in COPD pathogenesis.
Objectives: RSV persistence was investigated by testing respiratory secretions from subjects with COPD during illness and at regular intervals over 1 yr.
Methods: Nasal and sputum samples from subjects with COPD were tested by one-tube nested RT-PCR for RSV every 2 mo and during respiratory illnesses for 1 yr. Subjects positive for RSV were evaluated weekly until negative in two consecutive samples. Nasal secretions and serum were tested for RSV antibody. A rise of fourfold or greater was defined as evidence of RSV infection.
Results: A total of 112 patients were enrolled and the illnesses of 92 patients were evaluated. RSV was detected by RT-PCR in 6/92 (6.5%) illness nasal samples versus 0/685 routine nasal samples and in 5/69 (7.2%) illness sputum samples versus 3 /315 (0.9%) routine. Four additional RSV infections were identified by serum antibody responses. Of the RSV infections 86% were associated with serum or nasal antibody responses and 73% had symptoms of acute respiratory illness.
Conclusions: Most RSV infections in patients with COPD are associated with symptomatic respiratory illnesses and measurable immune responses. Our data do not support the concept of RSV persistence in this population.
doi:10.1164/rccm.200510-1681OC
PMCID: PMC2662947  PMID: 16387798
chronic obstructive pulmonary disease exacerbation; persistent infection; viral infection
22.  How Well Designed Is the Human Lung? 
doi:10.1164/rccm.200510-1682OE
PMCID: PMC2662944  PMID: 16387797
23.  Resident Pleural Macrophages Are Key Orchestrators of Neutrophil Recruitment in Pleural Inflammation 
Rationale: The role played by resident pleural macrophages in the initiation of pleural inflammation is currently unclear.
Objective: To evaluate the role of resident pleural macrophages in the initiation of inflammation.
Methods: We have used a conditional macrophage ablation strategy to determine the role of resident pleural macrophages in the regulation of neutrophil recruitment in a murine model of experimental pleurisy induced by the administration of carrageenan and formalin- fixed Staphylococcus aureus.
Measurements and Main Results: Conditional macrophage ablation mice express the human diphtheria toxin receptor under the control of the CD11b promoter such that the administration of diphtheria toxin induces ablation of nearly 97% of resident macrophages. Ablation of resident pleural macrophages before the administration of carrageenan or S. aureus dramatically reduced neutrophil influx into the pleural cavity. In the carrageenan model, the reduction in neutrophil infiltration was associated with marked early reduction in the level of macrophage inflammatory protein 2 as well as reduced levels of various cytokines, including tumor necrosis factor α, interleukin 6, and interleukin 10. Adoptive transfer of nontransgenic macrophages partially restored neutrophil infiltration. We also stimulated macrophage-depleted and nondepleted pleural cell populations with carrageenan in vitro and determined the production of chemokines and cytokines. Chemokine and cytokine production was markedly reduced by macrophage depletion, reinforcing the role of resident pleural macrophages in the generation of mediators that initiate acute inflammation.
Conclusion: These studies indicate a critical role for resident pleural macrophages in sensing perturbation to the local microenvironment and orchestrating subsequent neutrophil infiltration.
doi:10.1164/rccm.200504-538OC
PMCID: PMC2662938  PMID: 16357332
inflammation; macrophage; pleural diseases
24.  Progression of Airway Dysplasia and C-Reactive Protein in Smokers at High Risk of Lung Cancer 
Rationale: Chronic inflammation has been implicated in the development of airway dysplasia and lung cancer. It is unclear whether circulating biomarkers of inflammation could be used to predict progression of airway dysplasia.
Objective: We determined whether circulating levels of C-reactive protein (CRP) or other inflammatory biomarkers could predict progression of bronchial dysplasia in smokers over 6 mo.
Methods: The plasma levels of CRP, interleukins 6 and 8, and monocyte chemoattractant protein 1 were measured at baseline in 65 ex- and current smokers who had at least one site of bronchial dysplasia > 1.2 mm in size. Additional bronchial biopsies were taken after 6 mo from the same sites where dysplastic lesions were discovered at baseline. Progressive dysplastic lesions were defined as worsening of the dysplastic lesion by at least two grades or development of new dysplastic lesions.
Results: Half of the participants developed progressive dysplastic lesions after 6 mo. The baseline CRP levels in these participants were 64% higher than those without progressive disease (p = 0.027). Only one of eight (13%) participants with CRP ⩽ 0.5 mg/L developed progressive disease, whereas 31 of 57 (54%) participants with CRP > 0.5 mg/L developed progressive disease (p = 0.011). The odds of developing progressive disease were 9.6-fold higher in the latter than in the former group.
Conclusion: Plasma CRP, in concert with lung function and pack-years of smoking, appears to have excellent predictive powers in identifying participants with bronchial dyplastic lesions whose lesions progress to more advanced stages of dysplasia.
doi:10.1164/rccm.200508-1305OC
PMCID: PMC2662937  PMID: 16339918
airway dysplasia; C-reactive protein; lung inflammation
25.  Titin and Diaphragm Dysfunction in Chronic Obstructive Pulmonary Disease 
Rationale: Recently, we have shown that Ca2+-activated force generation in diaphragm single fibers is impaired in patients with mild to moderate chronic obstructive pulmonary disease (COPD). For optimal active-force generation, the passive elasticity provided by titin is indispensable.
Objectives: In the present study, we determined the passive-tension–length relations of single fibers of patients with mild to moderate COPD, hypothesizing that passive-elastic properties of diaphragm fibers are compromised.
Methods: Passive-tension–length relations were determined in diaphragm fibers from patients with and without COPD (predicted mean FEV1, 76 and 102%, respectively). In diaphragm homogenates titin expression was studied at the protein level by gel electrophoresis and at the transcript level by using a novel titin exon microarray.
Results: Diaphragm fibers from patients with COPD generate less passive tension on stretch. Titin content in the diaphragm did not differ between patients with and without COPD. However, titin exon transcript studies revealed up-regulation of seven exons, which code for spring elements in the elastic segment rich in proline, glutamate, valine, and lysine. Immunofluorescence analysis indicated elevated protein expression of the up-regulated splice variant in the COPD diaphragm. Simulation studies on titin molecules including the amino acids encoded by the seven up-regulated exons predicted reduced passive-tension generation on molecule stretch.
Conclusions: Passive-tension generation of diaphragm single fibers is reduced in patients with COPD. Our results suggest that alternative splicing of the titin gene, resulting in increased length of the elastic segment rich in proline, glutamate, valine, and lysine, is involved. Interestingly, these changes occur already in patients with mild to moderate COPD.
doi:10.1164/rccm.200507-1056OC
PMCID: PMC2662936  PMID: 16339921
chronic obstructive pulmonary disease; diaphragm; single-fiber stiffness; titin; transcript studies

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