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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.  Statin Use and Risk of Delirium in the Critically Ill 
Rationale
Delirium is common in intensive care unit (ICU) patients and is a predictor of worse outcomes and neuroinflammation is a possible mechanism. The antiinflammatory actions of statins may reduce delirium.
Objectives
To determine whether critically ill patients receiving statin therapy had a reduced risk of delirium than those not on statins.
Methods
A prospective cohort analysis of data from consecutive ICU patients admitted to a UK mixed medical and surgical critical care unit between August 2011 and February 2012; the Confusion Assessment Method for ICU was used to determine the days each patient was assessed as being free of delirium during ICU admission.
Measurements and Main Results
Delirium-free days, daily administration of statins, and serum C-reactive protein (CRP) were recorded. Four hundred and seventy consecutive critical care patients were followed, of whom 151 patients received statins. Using random-effects multivariable logistic regression, statin administration the previous evening was associated with the patient being assessed as free of delirium (odds ratio, 2.28; confidence interval, 1.01–5.13; P < 0.05) and with lower CRP (β = −0.52; P < 0.01) the following day. When the association between statin and being assessed as free of delirium was controlled for CRP, the effect size became nonsignificant (odds ratio, 1.56; confidence interval, 0.64–3.79; P = 0.32).
Conclusions
Ongoing statin therapy is associated with a lower daily risk of delirium in critically ill patients. An ongoing clinical trial, informed by this study, is investigating if statins are a potential therapy for delirium in the critically ill.
doi:10.1164/rccm.201306-1150OC
PMCID: PMC3974585  PMID: 24417431
delirium; statin; inflammation; C-reactive protein; critical care
3.  Immunostimulatory Oligonucleotides Attenuate Airways Remodeling in Allergic Monkeys 
To determine whether inhaled immunostimulatory DNA sequence oligonucleotides containing CpG motifs mitigate the pathophysiologic manifestation of the asthmatic phenotype (airways hyperresponsiveness and airways remodeling), rhesus monkeys with experimentally induced allergic airways disease were treated seven times with inhaled immunostimulatory oligonucleotides (or sham) periodically for 33 weeks. Airways hyperresponsiveness was reduced twofold in immunostimulatory DNA sequence–treated compared with sham-treated monkeys. Airways from immunostimulatory oligonucleotide-treated monkeys had thinner reticular basement membranes, fewer mucous cells, fewer eosinophils, and fewer mast cells than sham-treated allergic monkeys. We conclude that inhaled immunostimulatory oligonucleotides can attenuate the magnitude of airway hyperreactivity and airways remodeling produced in nonhuman primates with experimentally induced allergic airways disease.
doi:10.1164/rccm.200404-533OC
PMCID: PMC3927836  PMID: 15306532
airway wall alterations; allergic asthma; immunostimulatory DNA sequence oligonucleotides; nonhuman primate
4.  Ventilatory Control and Airway Anatomy in Obstructive Sleep Apnea 
American journal of respiratory and critical care medicine  2004;170(11):10.1164/rccm.200404-510OC.
Ventilatory instability may play an important role in the pathogenesis of obstructive sleep apnea. We hypothesized that the influence of ventilatory instability in this disorder would vary depending on the underlying collapsibility of the upper airway. To test this hypothesis, we correlated loop gain with apnea–hypopnea index during supine, nonrapid eye movement sleep in three groups of patients with obstructive sleep apnea based on pharyngeal closing pressure: negative pressure group (pharyngeal closing pressure less than –1 cm H2O), atmospheric pressure group (between –1 and +1 cm H2O), and positive pressure group (greater than +1 cm H2O). Loop gain was measured by sequentially increasing proportional assist ventilation until periodic breathing developed, which occurred in 24 of 25 subjects. Mean loop gain for all three groups was 0.37 ± 0.11. A significant correlation was found between loop gain and apnea–hypopnea index in the atmospheric group only (r = 0.88, p = 0.0016). We conclude that loop gain has a substantial impact on apnea severity in certain patients with sleep apnea, particularly those with a pharyngeal closing pressure near atmospheric.
doi:10.1164/rccm.200404-510OC
PMCID: PMC3861244  PMID: 15317668
control of breathing; loop gain; pharyngeal closing pressure; pharyngeal collapsibility; ventilatory stability
5.  Rifampicin Reduces Susceptibility to Ofloxacin in Rifampicin-resistant Mycobacterium tuberculosis through Efflux 
Rationale
Central dogma suggests that rifampicin resistance in Mycobacterium tuberculosis develops solely through rpoB gene mutations.
Objective
To determine whether rifampicin induces efflux pumps activation in rifampicin resistant M. tuberculosis strains thereby defining rifampicin resistance levels and reducing ofloxacin susceptibility.
Methods
Rifampicin and/or ofloxacin minimum inhibitory concentrations (MICs) were determined in rifampicin resistant strains by culture in BACTEC 12B medium. Verapamil and reserpine were included to determine their effect on rifampicin and ofloxacin susceptibility. RT-qPCR was applied to assess expression of efflux pump/transporter genes after rifampicin exposure. To determine whether verapamil could restore susceptibility to first-line drugs, BALB/c mice were infected with a MDR-TB strain and treated with first-line drugs with/without verapamil.
Measurements and Main Findings
Rifampicin MICs varied independently of rpoB mutation and genetic background. Addition reserpine and verapamil significantly restored rifampicin susceptibility (p = 0.0000). RT-qPCR demonstrated that rifampicin induced differential expression of efflux/transporter genes in MDR-TB isolates. Incubation of rifampicin mono-resistant strains in rifampicin (2 μg/ml) for 7 days induced ofloxacin resistance (MIC> 2 μg/ml) in strains with an rpoB531 mutation. Ofloxacin susceptibility was restored by exposure to efflux pump inhibitors. Studies in BALB/c mice showed that verapamil in combination with first-line drugs significantly reduced pulmonary CFUs after 1 and 2 months treatment (p < 0.05).
Conclusion
Exposure of rifampicin resistant M. tuberculosis strains to rifampicin can potentially compromise the efficacy of the second-line treatment regimens containing ofloxacin, thereby emphasising the need for rapid diagnostics to guide treatment. Efflux pump inhibitors have the potential to improve the efficacy of anti-tuberculosis drug treatment.
doi:10.1164/rccm.201011-1924OC
PMCID: PMC3698754  PMID: 21512166
Mycobacterium tuberculosis; drug resistance; rifampicin; efflux pumps; cross resistance
6.  Relationship between Serum Vitamin D, Disease Severity, and Airway Remodeling in Children with Asthma 
Rationale
Little is known about vitamin D status and its effect on asthma pathophysiology in children with severe, therapy-resistant asthma (STRA).
Objectives
Relationships between serum vitamin D, lung function, and pathology were investigated in pediatric STRA.
Methods
Serum 25-hydroxyvitamin D [25(OH)D3] was measured in 86 children (mean age, 11.7 yr): 36 with STRA, 26 with moderate asthma (MA), and 24 without asthma (control subjects). Relationships between 25(OH)D3, the asthma control test (ACT), spirometry, corticosteroid use, and exacerbations were assessed. Twenty-two of 36 children with STRA underwent fiberoptic bronchoscopy, bronchoalveolar lavage, and endobronchial biopsy with assessment of airway inflammation and remodeling.
Measurements and Main Results
25(OH)D3 levels (median [IQR]) were significantly lower in STRA (28 [22–38] nmol/L) than in MA (42.5 [29–63] nmol/L) and control subjects (56.5 [45–67] nmol/L) (P < 0.001). There was a positive relationship between 25(OH)D3 levels and percent predicted FEV1 (r = 0.4, P < 0.001) and FVC (r = 0.3, P = 0.002) in all subjects. 25(OH)D3 levels were positively associated with ACT (r = 0.6, P < 0.001), and inversely associated with exacerbations (r=−0.6, P < 0.001) and inhaled steroid dose (r=−0.39, P = 0.001) in MA and and STRA. Airway smooth muscle (ASM) mass, but not epithelial shedding or reticular basement membrane thickness, was inversely related to 25(OH)D3 levels (r=−0.6, P = 0.008). There was a positive correlation between ASM mass and bronchodilator reversibility (r = 0.6, P = 0.009) and an inverse correlation between ASM mass and ACT (r = −0.7, P < 0.001).
Conclusions
Lower vitamin D levels in children with STRA were associated with increased ASM mass and worse asthma control and lung function. The link between vitamin D, airway structure, and function suggests vitamin D supplementation may be useful in pediatric STRA.
doi:10.1164/rccm.201107-1239OC
PMCID: PMC3471128  PMID: 21908411
vitamin D; asthma; remodeling; airway smooth muscle; pediatrics
7.  IL-9 Governs Allergen-induced Mast Cell Numbers in the Lung and Chronic Remodeling of the Airways 
Rationale
IL-9 is a pleiotropic cytokine that has multiple effects on structural as well as numerous hematopoietic cells, which are central to the pathogenesis of asthma.
Objectives
The contribution of IL-9 to asthma pathogenesis has thus far been unclear, due to conflicting reports in the literature. These earlier studies focused on the role of IL-9 in acute inflammatory models; here we have investigated the effects of IL-9 blockade during chronic allergic inflammation.
Methods
Mice were exposed to either prolonged ovalbumin or house dust mite allergen challenge to induce chronic inflammation and airway remodeling.
Measurements and Main Results
We found that IL-9 governs allergen-induced mast cell (MC) numbers in the lung and has pronounced effects on chronic allergic inflammation. Anti–IL-9 antibody–treated mice were protected from airway remodeling with a concomitant reduction in mature MC numbers and activation, in addition to decreased expression of the profibrotic mediators transforming growth factor-β1, vascular endothelial growth factor, and fibroblast growth factor-2 in the lung. Airway remodeling was associated with impaired lung function in the peripheral airways and this was reversed by IL-9 neutralization. In human asthmatic lung tissue, we identified MCs as the main IL-9 receptor expressing population and found them to be sources of vascular endothelial growth factor and fibroblast growth factor-2.
Conclusions
Our data suggest an important role for an IL-9-MC axis in the pathology associated with chronic asthma and demonstrate that an impact on this axis could lead to a reduction in chronic inflammation and improved lung function in patients with asthma.
doi:10.1164/rccm.200909-1462OC
PMCID: PMC3385369  PMID: 20971830
IL-9; mast cells; asthma; airway remodeling; AHR
8.  The Th17 Pathway in Cystic Fibrosis Lung Disease 
Rationale
Cystic fibrosis (CF) is characterized by bronchoalveolar neutrophilia and submucosal lymphocytosis. We hypothesized that Th17 lymphocytes are part of this submucosal infiltrate.
Objectives
Quantification and phenotyping of the lymphocytic infiltrate in the bronchial submucosa of patients with CF (n=53, of which 20 were newly diagnosed), non-CF bronchiectasis (n = 17), and healthy control subjects (n = 13).
Methods
We measured IL-17 levels in bronchoalveolar lavage and CD4+, CD8+, and IL-17+ cell counts in endobronchial biopsies. Correlations were made with infection status and other inflammatory markers. Potential cellular sources of IL-17 were determined by double staining.
Measurements and Main Results
IL-17+ cell counts (median [interquartile range] cells/mm2) were significantly higher in patients with established CF (205 [115–551]) and non-CF bronchiectasis (245 [183–436]) than in control subjects (53 [12–82]) (P<0.01 for both). Patients with newly diagnosed CF had intermediate counts (171 [91–252]). IL-17–positive CD4+ T cells, γδT cells, natural killer T cells, and neutrophils were identified. Bronchoalveolar lavage IL-17 levels (pg/ml) were highest in established CF (14.6 [2.2–38.4]), low in newly diagnosed CF and control subjects (1.7 [1.7–1.74]; 1.7 [1.7–3]), and intermediate in non-CF bronchiectasis (9.1 [1.7–34] pg/ml) (Kruskal-Wallis P = 0.001). There was a significant correlation between IL-17 and neutrophil counts (P < 0.001, R = 0.6) as well as IL-4 (P < 0.001, R = 0.84).
Conclusions
Th17 lymphocytes are present in the airway submucosa in CF, even in a young, newly diagnosed group. Other IL-17+ cells include neutrophils, γδ T cells, and natural killer T cells.
doi:10.1164/rccm.201102-0236OC
PMCID: PMC3381840  PMID: 21474644
Th17 cells; cystic fibrosis; inflammation
9.  Suitability of EBUS-TBNA Specimens for Subtyping and Genotyping of NSCLC: A Multi-Centre Study of 774 Patients 
Rationale
The current management of advanced non-small cell lung cancer (NSCLC) requires differentiation between squamous and non-squamous sub-types as well as epidermal growth factor receptor (EGFR) mutation status. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is increasingly used for the diagnosis and staging of lung cancer. However, it is unclear whether cytology specimens obtained with EBUS-TBNA are suitable for the sub-classification and genotyping of NSCLC.
Objectives
To determine whether cytology specimens obtained from EBUS-TBNA in routine practice are suitable for phenotyping and genotyping of NSCLC.
Methods
Cytological diagnoses from EBUS-TBNA were recorded from 774 patients with known or suspected lung cancer across 5 centres in the United Kingdom between 2009 and 2011.
Measurements and Main Results
The proportion of patients with a final diagnosis by EBUS-TBNA in whom subtype was classified was 77% (95% CI 73% - 80%). The rate of NSCLC not otherwise specified (NSCLC-NOS) was significantly reduced in patients who underwent immunohistochemistry (adjusted OR 0.50 95% CI 0.28 – 0.82, P=0.016). EGFR mutation analysis was possible in 107 (90%) of the 119 patients in whom mutation analysis was requested. The sensitivity, negative predictive value and diagnostic accuracy of EBUS-TBNA in patients with NSCLC was 88% (95% CI 86% - 91%), 72% (95% CI 66% - 77%) and 91% (95% CI 89% - 93%) respectively.
Conclusions
This large multi-centre pragmatic study demonstrates that cytology samples obtained from EBUS-TBNA in routine practice are suitable for sub-typing of NSCLC and EGFR mutation analysis and that use of immunohistochemistry reduces the rate of NSCLC-NOS.
doi:10.1164/rccm.201202-0294OC
PMCID: PMC3378660  PMID: 22505743
Endobronchial ultrasound; non-small cell lung cancer; adenocarcinoma; EGFR mutation; squamous cell carcinoma; NSCLC-NOS
10.  C5a Mediates Peripheral Blood Neutrophil Dysfunction in Critically Ill Patients 
Rationale
Critically ill patients are highly susceptible to hospital-acquired infection. Neutrophil function in critical illness remains poorly understood.
Objectives
To characterize and define mechanisms of peripheral blood neutrophil (PBN) dysfunction in critically ill patients. To determine whether the inflamed lung contributes additional phagocytic impairment.
Methods
Prospective collection of blood and bronchoalveolar lavage fluid from patients with suspected ventilator-associated pneumonia and from age- and sex-matched volunteers; laboratory analysis of neutrophil functions.
Measurements and Main Results
Seventy-two patients and 21 volunteers were included. Phagocytic capacity of PBNs was 36% lower in patients than in volunteers (P < 0.0001). From several biologically plausible candidates only activated complement was significantly associated with impaired PBN phagocytosis (P < 0.0001). Phagocytosis was negatively correlated with serum C3a and positively correlated with expression of C5a receptor type 1 (CD88) on PBNs. C5a recapitulated impaired PBN phagocytosis and significantly down-regulated CD88 expression in vitro. C5a-mediated phagocytic impairment was prevented by blocking either CD88 or phosphoinositide 3-kinase, and completely reversed by granulocyte-macrophage colony-stimulating factor. C5a also impaired killing of Pseudomonas aeruginosa by, and migration of, PBNs, indicating that effects were not restricted to phagocytosis. Bronchoalveolar lavage fluid leukocytes from patients also demonstrated significantly impaired function, and lavage supernatant reduced phagocytosis in healthy neutrophils by 43% (P = 0.0001). However, lavage fluid did not affect CD88 expression and lavage-mediated impairment of phagocytosis was not blocked by anti-CD88 antibody.
Conclusions
Critically ill patients have significant dysfunction of PBNs, which is mediated predominantly by activated complement. Further, profound complement-independent neutrophil dysfunction occurs in the inflamed lung.
doi:10.1164/rccm.200812-1928OC
PMCID: PMC2948533  PMID: 19324972
complement; natural immunity; intensive care; phagocytosis
11.  Uteroglobin-related Protein 1 Expression Suppresses Allergic Airway Inflammation in Mice 
Rationale
Uteroglobin-related protein (UGRP) 1, which is highly expressed in the epithelial cells of the airways, has been suggested to play a role in lung inflammation.
Objectives
The aim of study was to understand the effect of overexpressed UGRP1 on lung inflammation in a mouse model of allergic airway inflammation.
Methods
Ovalbumin-sensitized and -challenged mice, a model for allergic airway inflammation, were used in conjunction with recombinant adenovirus expressing UGRP1.
Measurements and Main Results
We demonstrated that intranasal administration of adeno-UGRP1 successfully delivered UGRP1 to the epithelial cells of airways and markedly reduced the number of infiltrating inflammatory cells, particularly eosinophils, in lung tissue as well as the level of proinflammatory cytokines such as interleukin (IL)-4, IL-5, and IL-13 in bronchoalveolar lavage fluids. The healed phase of inflammation was clearly seen in the peripheral areas of adeno-UGRP1–treated mouse lungs.
Conclusion
These results demonstrate that UGRP1 can suppress inflammation in the mouse model of allergic airway inflammation. Based on this result, we propose UGRP1 as a novel therapeutic candidate for treating lung inflammation such as is found in asthma.
doi:10.1164/rccm.200503-456OC
PMCID: PMC2582904  PMID: 16456148
asthma; bronchoalveolar lavage; eosinophils; mouse model; ovalbumin
12.  Longitudinal monitoring of lung injury in children following chlorine exposure in a swimming pool 
Rationale: Acute exposure to chlorine gas results in respiratory impairment, but few data are available on the pathobiology of the underlying lung damage. Objectives: To assess lung function and potential lung damage pathways in the acute phase and longitudinally over a 15-month follow-up after chlorine exposure. Methods: Ten previously-healthy children were accidentally exposed to chlorine gas at a swimming pool due an erroneous servicing procedure. Exhaled nitric oxide (FENO), exhaled breath condensate (EBC) compounds and serum Clara cell protein (CC16) were repeatedly measured. Main results: In the acute phase, all patients had respiratory distress (one child required mechanical ventilation) and reduced lung function (median and IQR: FVC 51% pred. [43-60], FEV1 51% pred. [46-60]). This was accompanied by low FENO (4.7 [3.9-7.9] ppb), high EBC leukotriene B4 (LTB-4) levels (24.4 [22.5-24.9] pg/mL) and increased serum CC16 levels (mean ± SE 23.4 ± 2.5 μg/L). Lung function returned to normal in 15 days (FVC 97% pred. [82-108] and FEV1 92% pred. [77-102]). FENO reached normal values after 2 months (12.6 [11.4-15] ppb), while LTB-4 levels were still increased (12 [9.3-17.1] pg/mL). Conclusion: Children acutely exposed to chlorine in a swimming pool presented a substantial lung function impairment associated with biochemical exhaled breath alterations, mainly represented by an increase in LTB-4 and a reduction in FENO. While lung function and FENO improved within a few weeks, the increased levels of exhaled LTB-4 persisted for several months.
doi:10.1164/rccm.200509-1392OC
PMCID: PMC1555620  PMID: 16763216
Chlorine inhalation; Pulmonary function; Exhaled nitric oxide; Exhaled breath condensate; Pneumoproteinemia
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.  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
15.  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
16.  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
17.  Superoxide Dismutase Improves Oxygenation and Reduces Oxidation in Neonatal Pulmonary Hypertension 
Rationale
Hyperoxic ventilation in the management of persistent pulmonary hypertension of the newborn (PPHN) can result in the formation of reactive oxygen species, such as superoxide anions, which can inactivate nitric oxide (NO) and cause vasoconstriction and oxidation.
Objective
To compare the effect of intratracheal recombinant human superoxide dismutase (rhSOD) and/or inhaled NO (iNO) on systemic oxygenation, contractility of pulmonary arteries (PAs), and lung reactive oxygen species (isoprostane, 3-nitrotyrosine) levels in neonatal lambs with PPHN.
Methods
Six newborn lambs with PPHN (induced by antenatal ductal ligation) were killed at birth. Twenty-six PPHN lambs were ventilated for 24 h with 100% O2 alone (n = 6) or O2 combined with rhSOD (5 mg/kg intratracheally) at birth (n = 4), rhSOD at 4 h of age (n = 5), iNO (20 ppm, n = 5), or rhSOD + iNO (n = 6). Contraction responses of fifth-generation PAs to norepinephrine and KCl, lung isoprostane levels, and 3-nitrotyrosine fluorescent intensity were measured.
Results
Systemic oxygenation was impaired in PPHN lambs and significantly improved (up to threefold) in both rhSOD groups with or without iNO. Oxygenation improved more rapidly with the combination of rhSOD + iNO compared with either intervention alone. Norepinephrine- and KCl-induced contractions and lung isoprostane levels were significantly increased by 100% O2 compared with nonventilated newborn lambs with PPHN. Both rhSOD and iNO mitigated the increased PA contraction response and lung isoprostane levels. Intratracheal rhSOD decreased the enhanced lung 3-nitrotyrosine fluorescence observed with iNO therapy.
Conclusion
Intratracheal rhSOD and/or iNO rapidly increase oxygenation and reduce both vasoconstriction and oxidation in newborn lambs with PPHN. This has important implications for clinical trials of rhSOD and iNO in newborn infants with PPHN.
doi:10.1164/rccm.200605-676
PMCID: PMC2111046  PMID: 17008638
isoprostanes; nitric oxide; oxygen; pulmonary hypertension; superoxide dismutase
18.  Spontaneous Airway Hyperresponsiveness in Estrogen Receptor-α–deficient Mice 
Rationale
Airway hyperresponsiveness is a critical feature of asthma. Substantial epidemiologic evidence supports a role for female sex hormones in modulating lung function and airway hyperresponsiveness in humans.
Objectives
To examine the role of estrogen receptors in modulating lung function and airway responsiveness using estrogen receptor–deficient mice.
Methods
Lung function was assessed by a combination of whole-body barometric plethysmography, invasive measurement of airway resistance, and isometric force measurements in isolated bronchial rings. M2 muscarinic receptor expression was assessed by Western blotting, and function was assessed by electrical field stimulation of tracheas in the presence/absence of gallamine. Allergic airway disease was examined after ovalbumin sensitization and exposure.
Measurements and Main Results
Estrogen receptor-α knockout mice exhibit a variety of lung function abnormalities and have enhanced airway responsiveness to inhaled methacholine and serotonin under basal conditions. This is associated with reduced M2 muscarinic receptor expression and function in the lungs. Absence of estrogen receptor-α also leads to increased airway responsiveness without increased inflammation after allergen sensitization and challenge.
Conclusions
These data suggest that estrogen receptor-α is a critical regulator of airway hyperresponsiveness in mice.
doi:10.1164/rccm.200509-1493OC
PMCID: PMC1899278  PMID: 17095746
lung function; asthma; hyperreactivity; M2 muscarinic receptor; estrogen receptor
19.  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
20.  A Web-based, Tailored Asthma Management Program for Urban African-American High School Students 
Rationale
Urban African-American youth, aged 15–19 years, have asthma fatality rates that are higher than in whites and younger children, yet few programs target this population. Traditionally, urban youth are believed to be difficult to engage in health-related programs, both in terms of connecting and convincing.
Objectives
Develop and evaluate a multimedia, web-based asthma management program to specifically target urban high school students. The program uses “tailoring,” in conjunction with theory-based models, to alter behavior through individualized health messages based on the user’s beliefs, attitudes, and personal barriers to change.
Methods
High school students reporting asthma symptoms were randomized to receive the tailored program (treatment) or to access generic asthma websites (control). The program was made available on school computers.
Measurements and Main Results
Functional status and medical care use were measured at study initiation and 12 months postbaseline, as were selected management behaviors. The intervention period was 180 days (calculated from baseline). A total of 314 students were randomized (98% African American, 49% Medicaid enrollees; mean age, 15.2 yr). At 12 months, treatment students reported fewer symptom-days, symptom-nights, school days missed, restricted-activity days, and hospitalizations for asthma when compared with control students; adjusted relative risk and 95% confidence intervals were as follows: 0.5 (0.4–0.8), p = 0.003; 0.4 (0.2–0.8), p = 0.009; 0.3 (0.1–0.7), p = 0.006; 0.5 (0.3–0.8), p = 0.02; and 0.2 (0.2–0.9), p = 0.01, respectively. Positive behaviors were more frequently noted among treatment students compared with control students. Cost estimates for program delivery were $6.66 per participating treatment group student.
Conclusions
A web-based, tailored approach to changing negative asthma management behaviors is economical, feasible, and effective in improving asthma outcomes in a traditionally hard-to-reach population.
doi:10.1164/rccm.200608-1244OC
PMCID: PMC1899296  PMID: 17290041
asthma; urban; adolescents; school-based; web-based
21.  Potent Twice-Weekly Rifapentine-containing Regimens in Murine Tuberculosis 
Rationale
Recent studies have demonstrated that intermittent administration of rifamycin-based regimens results in higher rates of tuberculosis relapse and treatment failure compared with daily therapy. Twice-weekly treatment with rifampin, isoniazid, and pyrazinamide may be improved by increasing Mycobacterium tuberculosis exposure to rifamycin by substituting rifapentine for rifampin.
Methods
To test this hypothesis, we compared the activities of standard daily and twice-weekly rifampin plus isoniazid-based regimens to those of twice-weekly rifapentine plus isoniazid- or moxifloxacin-containing regimens in the murine model of tuberculosis. Relapse rates were assessed after 4, 5, and 6 mo of treatment to assess stable cure. Single- and multiple-dose pharmacokinetics of rifampin and rifapentine were also determined.
Results
After 2 mo of treatment, twice-weekly therapy with rifapentine (15 or 20 mg/kg), moxifloxacin, and pyrazinamide was significantly more active than standard daily or twice-weekly therapy with rifampin, isoniazid, and pyrazinamide. Stable cure was achieved after 4 mo of twice-weekly rifapentine plus isoniazid- or moxifloxacin-containing therapy, but only after 6 mo of standard daily therapy. Twice-weekly rifapentine (15 mg/kg) displayed more favorable pharmacodynamics than did daily rifampin (10 mg/kg).
Conclusions
By virtue of the enhanced rifamycin exposure, twice-weekly regimens containing rifapentine (15 or 20 mg/kg) may permit shortening the current treatment duration by 2 mo. Such regimens warrant clinical investigation.
doi:10.1164/rccm.200602-280OC
PMCID: PMC1862756  PMID: 16574936
moxifloxacin; rifampin; rifapentine; tuberculosis; treatment
22.  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
23.  Airway Responses to Aerosolized Brevetoxins in an Animal Model of Asthma 
Florida red tide brevetoxins are sodium channel neurotoxins produced by the dinoflagellate Karenia brevis. When aerosolized, the toxin causes airway symptoms in normal individuals and patients with airway disease, but systematic exposures to define the pulmonary consequences and putative mechanisms are lacking. Here we report the effects of airway challenges with lysed cultures of Karenia brevis (crude brevetoxin), pure brevetoxin-2, brevetoxin-3, and brevetoxin-tbm (brevetoxin-2 minus the side chain) on pulmonary resistance and tracheal mucus velocity, a marker of mucociliary clearance, in allergic and nonallergic sheep. Picogram concentrations of toxin caused bronchoconstriction in both groups of sheep. Brevetoxin-tbm was the least potent, indicating the importance of the side chain for maximum effect. Both histamine H1– and cholinergic-mediated pathways contributed to the bronchoconstriction. A synthetic antagonist, β-naphthoyl-brevetoxin-3, and brevenal, a natural antagonist, inhibited the bronchoconstriction. Only crude brevetoxin and brevetoxin-3 decreased tracheal mucus velocity; both antagonists prevented this. More importantly, picomolar concentrations of the antagonists alone improved tracheal mucus velocity to the degree seen with mM concentrations of the sodium channel blocker amiloride. Thus, Karenia brevis, in addition to producing toxins that adversely affect the airways, may be a source of agents for treating mucociliary dysfunction.
doi:10.1164/rccm.200406-735OC
PMCID: PMC2650266  PMID: 15447946
bronchoconstriction; mucus transport; natural therapies
24.  Acute Exacerbations of Idiopathic Pulmonary Fibrosis 
The natural history of idiopathic pulmonary fibrosis (IPF) has been characterized as a steady, predictable decline in lung function over time. Recent evidence suggests that some patients may experience a more precipitous course, with periods of relative stability followed by acute deteriorations in respiratory status. Many of these acute deteriorations are of unknown etiology and have been termed acute exacerbations of IPF. This perspective is the result of an international effort to summarize the current state of knowledge regarding acute exacerbations of IPF. Acute exacerbations of IPF are defined as acute, clinically significant deteriorations of unidentifiable cause in patients with underlying IPF. Proposed diagnostic criteria include subjective worsening over 30 days or less, new bilateral radiographic opacities, and the absence of infection or another identifiable etiology. The potential pathobiological roles of infection, disordered cell biology, coagulation, and genetics are discussed, and future research directions are proposed.
doi:10.1164/rccm.200703-463PP
PMCID: PMC2094133  PMID: 17585107
acute exacerbation; pulmonary fibrosis; diagnosis; definition
25.  Integrin α4β1 Regulates Migration across Basement Membranes by Lung Fibroblasts 
Idiopathic pulmonary fibrosis is a disease that is characterized by fibroblast accumulation and activation in the distal airspaces of the lung. We hypothesized that fibrotic lung fibroblasts migrate/invade across basement membranes by integrin-mediated mechanisms as a means of entering alveoli. We demonstrate that in lung fibroblasts derived from patients with idiopathic pulmonary fibrosis, fibronectin signaling is both necessary and sufficient for basement membrane migration/invasion across basement membranes. This effect is mediated through the α5β1 integrin because blockade of fibronectin-α5 integrin ligation attenuated this response. In contrast, ligation of α4β1 integrin inhibits basement membrane invasion by normal lung fibroblasts but not by fibrotic lung fibroblasts. This phenotypic difference is not related to surface expression of the α4β1 integrin, as demonstrated by flow cytometry. In normal lung fibroblasts but not in fibrotic lung fibroblasts, we show that ligation of α4β1 integrin induces a significant increase in phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activity. Fibrotic lung fibroblasts express constitutively less PTEN mRNA and protein as well as phosphatase activity in comparison to normal lung fibroblasts. Together, these data suggest that a loss of α4β1 signaling via PTEN confers a migratory/invasive phenotype to fibrotic lung fibroblasts. Furthermore, this study implicates a loss of PTEN function in the pathophysiology of idiopathic pulmonary fibrosis.
doi:10.1164/rccm.200301-041OC
PMCID: PMC1997294  PMID: 12791582
pulmonary fibrosis; extracellular matrix; fibronectins; cell movement

Results 1-25 (25)