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1.  Quantitative analysis of hyperpolarized 129Xe ventilation imaging in healthy volunteers and subjects with chronic obstructive pulmonary disease 
NMR in biomedicine  2012;10.1002/nbm.2880.
In this study, hyperpolarized (HP) 129Xe MR ventilation and 1H anatomical images were obtained from 3 subject groups: young healthy volunteers (HV), subjects with chronic obstructive pulmonary disease (COPD), and age-matched control subjects (AMC). Ventilation images were quantified by 2 methods: an expert reader-based ventilation defect score percentage (VDS%) and a semi-automatic segmentation-based ventilation defect percentage (VDP). Reader-based values were assigned by two experienced radiologists and resolved by consensus. In the semi-automatic analysis, 1H anatomical images and 129Xe ventilation images were both segmented following registration, to obtain the thoracic cavity volume (TCV) and ventilated volume (VV), respectively, which were then expressed as a ratio to obtain the VDP. Ventilation images were also characterized by generating signal intensity histograms from voxels within the TCV, and heterogeneity was analyzed using the coefficient of variation (CV). The reader-based VDS% correlated strongly with the semi-automatically generated VDP (r = 0.97, p < 0.0001), and with CV (r = 0.82, p < 0.0001). Both 129Xe ventilation defect scoring metrics readily separated the 3 groups from one another and correlated significantly with FEV1 (VDS%: r = -0.78, p = 0.0002; VDP: r = -0.79, p = 0.0003; CV: r = -0.66, p = 0.0059) and other pulmonary function tests. In the healthy subject groups (HV and AMC), the prevalence of ventilation defects also increased with age (VDS%: r = 0.61, p = 0.0002; VDP: r = 0.63, p = 0.0002). Moreover, ventilation histograms and their associated CVs distinguished between COPD subjects with similar ventilation defect scores but visibly different ventilation patterns.
PMCID: PMC3624045  PMID: 23065808
hyperpolarized; 129Xe; Xe-129; magnetic resonance imaging; ventilation; defect; segmentation; COPD; coefficient of variation; VDP; VDS; VDV; FEV1
2.  SHP-1 As a Critical Regulator of Mycoplasma pneumoniae-Induced Inflammation in Human Asthmatic Airway Epithelial Cells 
Journal of immunology (Baltimore, Md. : 1950)  2012;188(7):10.4049/jimmunol.1100573.
Asthma is a chronic inflammatory disease in which airway epithelial cells are the first line of defense against exposure of the airway to infectious agents. Src homology protein (SHP)-1, a protein tyrosine phosphatase, is a negative regulator of signaling pathways that are critical to the development of asthma and host defense. We hypothesize that SHP-1 function is defective in asthma, contributing to the increased inflammatory response induced by Mycoplasma pneumoniae, a pathogen known to exacerbate asthma. M. pneumoniae significantly activated SHP-1 in airway epithelial cells collected from nonasthmatic subjects by bronchoscopy with airway brushing but not in cells from asthmatic subjects. In asthmatic airway epithelial cells, M. pneumoniae induced significant PI3K/Akt phosphorylation, NF-κB activation, and IL-8 production compared with nonasthmatic cells, which were reversed by SHP-1 overexpression. Conversely, SHP-1 knockdown significantly increased IL-8 production and PI3K/Akt and NF-κB activation in the setting of M. pneumoniae infection in nonasthmatic cells, but it did not exacerbate these three parameters already activated in asthmatic cells. Thus, SHP-1 plays a critical role in abrogating M. pneumoniae-induced IL-8 production in non-asthmatic airway epithelial cells through inhibition of PI3K/Akt and NF-κB activity, but it is defective in asthma, resulting in an enhanced inflammatory response to infection.
PMCID: PMC3880785  PMID: 22371396
3.  P2X7-Regulated Protection from Exacerbations and Loss of Control Is Independent of Asthma Maintenance Therapy 
Rationale: The function of the P2X7 nucleotide receptor protects against exacerbation in people with mild-intermittent asthma during viral illnesses, but the impact of disease severity and maintenance therapy has not been studied.
Objectives: To evaluate the association between P2X7, asthma exacerbations, and incomplete symptom control in a more diverse population.
Methods: A matched P2RX7 genetic case-control was performed with samples from Asthma Clinical Research Network trial participants enrolled before July 2006, and P2X7 pore activity was determined in whole blood samples as an ancillary study to two trials completed subsequently.
Measurements and Main Results: A total of 187 exacerbations were studied in 742 subjects, and the change in asthma symptom burden was studied in an additional 110 subjects during a trial of inhaled corticosteroids (ICS) dose optimization. African American carriers of the minor G allele of the rs2230911 loss-of-function single nucleotide polymorphism were more likely to have a history of prednisone use in the previous 12 months, with adjustment for ICS and long-acting β2-agonists use (odds ratio, 2.7; 95% confidence interval, 1.2–6.2; P = 0.018). Despite medium-dose ICS, attenuated pore function predicted earlier exacerbations in incompletely controlled patients with moderate asthma (hazard ratio, 3.2; confidence interval, 1.1–9.3; P = 0.033). After establishing control with low-dose ICS in patients with mild asthma, those with attenuated pore function had more asthma symptoms, rescue albuterol use, and FEV1 reversal (P < 0.001, 0.03, and 0.03, respectively) during the ICS adjustment phase.
Conclusions: P2X7 pore function protects against exacerbations of asthma and loss of control, independent of baseline severity and the maintenance therapy.
PMCID: PMC3570642  PMID: 23144325
asthma; P2X7; exacerbation; Asthma Clinical Research Network; corticosteroids
4.  Obesity, metabolic dysregulation and oxidative stress in asthma☆ 
Biochimica et biophysica acta  2011;1810(11):1120-1126.
Epidemiological data demonstrate an increased risk of developing incident asthma with increasing adiposity. While the vast majority of studies support the interaction between obesity and asthma, the causality is unclear.
Scope of review
This article will review the current literature supporting the presence of an obese asthma phenotype and the possible mechanisms mediating the effects of obesity on asthma.
Major conclusions
Obesity is associated with poor asthma control, altered responsiveness to medications and increased morbidity. Obesity is characterized by systemic inflammation that may result in increased airway inflammation. However, this assertion is not supported by current studies that demonstrate a lack of significant airway inflammation in obese asthmatics. In spite this observation one must consider limitations of these studies including the fact that most subjects were treated with inhaled corticosteroids that would likely alter inflammation in the lung. Thus, it remains unclear if obesity is associated with alterations in inflammation in the airways of subjects with asthma.
Hormones such as leptin and adiponectin are affected by obesity and may play a role in mediating innate immune responses and allergic responses, respectively. The role of oxidative stress remains controversial and the current evidence suggests that while oxidative stress is important in asthma, it does not fully explain the characteristics associated with this unique phenotype.
General significance
Obesity related asthma is associated with increased morbidity and differential response to asthma therapies. Understanding the mechanisms mediating this phenotype would have significant implications for millions of people suffering with asthma. This article is part of a Special Issue entitled Biochemistry of Asthma.
PMCID: PMC3786599  PMID: 21944975
Asthma; Obesity; Metabolic syndrome; Oxidative stress; Leptin; Inflammation
5.  Comparison of Physician-, Biomarker-, and Symptom-Based Strategies for Adjustment of Inhaled Corticosteroid Therapy in Adults With Asthma 
No consensus exists for adjusting inhaled corticosteroid therapy in patients with asthma. Approaches include adjustment at outpatient visits guided by physician assessment of asthma control (symptoms, rescue therapy, pulmonary function), based on exhaled nitric oxide, or on a day-to-day basis guided by symptoms.
To determine if adjustment of inhaled corticosteroid therapy based on exhaled nitric oxide or day-to-day symptoms is superior to guideline-informed, physician assessment–based adjustment in preventing treatment failure in adults with mild to moderate asthma.
Design, Setting, and Participants
A randomized, parallel, 3-group, placebo-controlled, multiply-blinded trial of 342 adults with mild to moderate asthma controlled by low-dose inhaled corticosteroid therapy (n=114 assigned to physician assessment–based adjustment [101 completed], n=115 to biomarker-based [exhaled nitric oxide] adjustment [92 completed], and n=113 to symptom-based adjustment [97 completed]), the Best Adjustment Strategy for Asthma in the Long Term (BASALT) trial was conducted by the Asthma Clinical Research Network at 10 academic medical centers in the United States for 9 months between June 2007 and July 2010.
For physician assessment–based adjustment and biomarker-based (exhaled nitric oxide) adjustment, the dose of inhaled corticosteroids was adjusted every 6 weeks; for symptom-based adjustment, inhaled corticosteroids were taken with each albuterol rescue use.
Main Outcome Measure
The primary outcome was time to treatment failure.
There were no significant differences in time to treatment failure. The 9-month Kaplan-Meier failure rates were 22% (97.5% CI, 14%-33%; 24 events) for physician assessment–based adjustment, 20% (97.5% CI, 13%-30%; 21 events) for biomarker-based adjustment, and 15% (97.5% CI, 9%-25%; 16 events) for symptom-based adjustment. The hazard ratio for physician assessment–based adjustment vs biomarker-based adjustment was 1.2 (97.5% CI, 0.6-2.3). The hazard ratio for physician assessment–based adjustment vs symptom-based adjustment was 1.6 (97.5% CI, 0.8-3.3).
Among adults with mild to moderate persistent asthma controlled with low-dose inhaled corticosteroid therapy, the use of either biomarker-based or symptom-based adjustment of inhaled corticosteroids was not superior to physician assessment–based adjustment of inhaled corticosteroids in time to treatment failure.
Trial Registration Identifier: NCT00495157
PMCID: PMC3697088  PMID: 22968888
6.  Alveolar Macrophages from Overweight/Obese Subjects with Asthma Demonstrate a Proinflammatory Phenotype 
Rationale: Obesity is associated with increased prevalence and severity of asthma. Adipose tissue macrophages can contribute to the systemic proinflammatory state associated with obesity. However, it remains unknown whether alveolar macrophages have a unique phenotype in overweight/obese patients with asthma.
Objectives: We hypothesized that leptin levels would be increased in the bronchoalveolar lavage fluid from overweight/obese subjects and, furthermore, that leptin would alter the response of alveolar macrophages to bacterial LPS.
Methods: Forty-two subjects with asthma and 46 healthy control subjects underwent research bronchoscopy. Bronchoalveolar lavage fluid from 66 was analyzed for the level of cellular inflammation, cytokines, and soluble leptin. Cultured primary macrophages from 22 subjects were exposed to LPS, leptin, or leptin plus LPS. Cytokines were measured in the supernatants.
Measurements and Main Results: Leptin levels were increased in overweight/obese subjects, regardless of asthma status (P = 0.013), but were significantly higher in overweight/obese subjects with asthma. Observed levels of tumor necrosis factor-α were highest in overweight/obese subjects with asthma. Ex vivo studies of primary alveolar macrophages indicated that the response to LPS was most robust in alveolar macrophages from overweight/obese subjects with asthma and that preexposure to high-dose leptin enhanced the proinflammatory response. Leptin alone was sufficient to induce production of proinflammatory cytokines from macrophages derived from overweight/obese subjects with asthma.
Conclusions: Ex vivo studies indicate that alveolar macrophages derived from overweight/obese subjects with asthma are uniquely sensitive to leptin. This macrophage phenotype, in the context of higher levels of soluble leptin, may contribute to the pathogenesis of airway disease associated with obesity.
PMCID: PMC3443798  PMID: 22773729
tumor necrosis factor-α; leptin; innate immunity; lipopolysaccharide; environmental lung disease
7.  Mast cell TNF receptors regulate responses to Mycoplasma pneumoniae in surfactant protein A (SP-A)−/− mice 
Mycoplasma pneumoniae (Mp) frequently colonizes the airways of patients with chronic asthma and likely contributes to asthma exacerbations. We previously reported that mice lacking surfactant protein A (SP-A) have increased airway hyperresponsiveness (AHR) during M pneumoniae infection versus wild-type mice mediated by TNF-α. Mast cells (MCs) have been implicated in AHR in asthma models and produce and respond to TNF-α.
Determine the contribution of MC/TNF interactions to AHR in airways lacking functional SP-A during Mp infection. Methods: Bronchoalveolar lavage fluid was collected from healthy and asthmatic subjects to examine TNF-α levels and M pneumoniae positivity. To determine how SP-A interactions with MCs regulate airway homeostasis, we generated mice lacking both SP-A and MCs (SP-A−/−KitW-sh/W-sh) and infected them with M pneumoniae.
Our findings indicate that high TNF-α levels correlate with M pneumoniae positivity in human asthmatic patients and that human SP-A inhibits M pneumoniae–stimulated transcription and release of TNF-α by MCs, implicating a protective role for SP-A. MC numbers increase in M pneumoniae–infected lungs, and airway reactivity is dramatically attenuated when MCs are absent. Using SP-A−/−KitW-sh/W-sh mice engrafted with TNF-α−/− or TNF receptor (TNF-R)−/− MCs, we found that TNF-α activation of MCs through the TNF-R, but not MC-derived TNF-α, leads to augmented AHR during M pneumoniae infection when SP-A is absent. Additionally, M pneumoniae– infected SP-A−/−KitW-sh/W-sh mice engrafted with TNF-α−/− or TNF-R−/− MCs have decreased mucus production compared with that seen in mice engrafted with wild-type MCs, whereas burden was unaffected.
Our data highlight a previously unappreciated but vital role for MCs as secondary responders to TNF-α during the host response to pathogen infection.
PMCID: PMC3578696  PMID: 22502799
Mast cells; TNF; Mycoplasma species; airway hyperres-ponsiveness; mucus
8.  SP-A Preserves Airway Homeostasis During Mycoplasma pneumoniae Infection in Mice 
The lung is constantly challenged during normal breathing by a myriad of environmental irritants and infectious insults. Pulmonary host defense mechanisms maintain homeostasis between inhibition/clearance of pathogens and regulation of inflammatory responses that could injure the airway epithelium. One component of this defense mechanism, surfactant protein-A (SP-A), exerts multifunctional roles in mediating host responses to inflammatory and infectious agents. SP-A has a bacteriostatic effect on Mycoplasma pneumoniae (Mp), which occurs by binding surface disaturated phosphatidylglycerols. SP-A can also bind the Mp membrane protein, MPN372. In this study we investigated the role of SP-A during acute phase pulmonary infection with Mp using mice deficient in SP-A. Biologic responses, inflammation and cellular infiltration, were much greater in Mp infected SP-A−/− mice than wild type mice. Likewise, physiologic responses (airway hyperresponsiveness and lung compliance) to Mp infection were more severely affected in SP-A−/− mice. Both Mp-induced biologic and physiologic changes were attenuated by pharmacologic inhibition of TNF-α. Our findings demonstrate that SP-A is vital to preserving lung homeostasis and host defense to this clinically relevant strain of Mp by curtailing inflammatory cell recruitment and limiting an overzealous TNF-α response.
PMCID: PMC3656438  PMID: 19494306
lung; inflammation; bacterial
9.  The TLR5 ligand flagellin promotes asthma by priming allergic responses to indoor allergens 
Nature medicine  2012;18(11):1705-1710.
Allergic asthma is a complex disease characterized by eosinophilic pulmonary inflammation, mucus production and reversible airway obstruction1. Exposure to indoor allergens is a clear risk factor for asthma, but this disease is also associated with high household levels of total and Gram-negative bacteria2. The ability of bacterial products to act as adjuvants3 suggests they might promote asthma by priming allergic sensitization to inhaled allergens. In support of this idea, house dust extracts (HDEs) can activate antigen presenting dendritic cells (DC) in vitro and promote allergic sensitization to inhaled innocuous proteinsin vivo4. It is unknown which microbial products provide most of the adjuvant activity in HDEs. A screen of microbial products for their adjuvant activity in the airway revealed that the bacterial protein, flagellin (FLA) stimulated strong allergic responses to an innocuous inhaled protein. Moreover, toll-like receptor (TLR)5, the mammalian receptor for FLA5,6, was required for priming strong allergic responses to natural indoor allergens present in HDEs. In addition, the incidence of human asthma was associated with high serum levels of FLA-specific antibodies. Together, these findings suggest that household FLA promotes the development of allergic asthma by TLR5-dependent priming of allergic responses to indoor allergens.
PMCID: PMC3493750  PMID: 23064463
10.  Assessment of murine lung mechanics outcome measures: alignment with those made in asthmatics 
Although asthma is characterized as an inflammatory disease, recent reports highlight the importance of pulmonary physiology outcome measures to the clinical assessment of asthma control and risk of asthma exacerbation. Murine models of allergic inflammatory airway disease have been widely used to gain mechanistic insight into the pathogenesis of asthma; however, several aspects of murine models could benefit from improvement. This review focuses on aligning lung mechanics measures made in mice with those made in humans, with an eye toward improving the translational utility of these measures. A brief description of techniques available to measure murine lung mechanics is provided along with a methodological consideration of their utilization. How murine lung mechanics outcome measures relate to pulmonary physiology measures conducted in humans is discussed and we recommend that, like human studies, outcome measures be standardized for murine models of asthma.
PMCID: PMC3569663  PMID: 23408785
airway hyperresponsiveness; murine; asthma; lung mechanics; translational research
11.  The Role of Hyaluronan and Hyaluronan Binding Proteins in Human Asthma 
The characteristics of human asthma are chronic inflammation and airway remodeling. Hyaluronan (HA), a major extracellular matrix component, accumulates during inflammatory lung diseases including asthma. Hyaluronan fragments stimulate macrophages to produce inflammatory cytokines. We hypothesized that HA and its receptors would play a role in human asthma.
To investigate the role of HA and HA binding proteins in human asthma.
Twenty-one subjects with asthma and 25 normal control subjects underwent bronchoscopy with endobronchial biopsy and bronchoalveolar lavage (BAL). Fibroblasts were cultured, HA and HA synthase expression was determined at baseline and after exposure to several mediators relevant to asthma pathobiology. The expression of HA binding proteins, CD44, TLR2 and TLR4 on BAL macrophages was determined by flow cytometry. IL-8 production by macrophages in response to HA fragment stimulation was compared.
Airway fibroblasts from asthma patients produced significantly increased concentrations of lower molecular weight HA compared to those of normal fibroblasts. Hyaluronan synthase 2 mRNA was markedly increased in asthmatic fibroblasts. Asthmatic macrophages showed a decrease in cell surface CD44 expression and an increase in TLR2 and TLR4 expression. Macrophages from asthmatic subjects showed an increase in responsiveness to low molecular weight HA stimulation, as demonstrated by increased IL-8 production.
HA homeostasis is deranged in asthma with increased production by fibroblasts and decreased CD44 expression on alveolar macrophages. Upregulation of TLR2 and TLR4 on macrophages with increased sensitivity to HA fragments suggests a novel pro-inflammatory mechanism by which persistence of HA fragments could contribute to chronic inflammation and airway remodeling in asthma.
PMCID: PMC3149736  PMID: 21570715
Asthma; Hyaluronan; Cytokines; Fibroblasts; Macrophages
12.  Airway Fibroblasts in Asthma Manifest an Invasive Phenotype 
Rationale: Invasive cell phenotypes have been demonstrated in malignant transformation, but not in other diseases, such as asthma. Cellular invasiveness is thought to be mediated by transforming growth factor (TGF)-β1 and matrix metalloproteinases (MMPs). IL-13 is a key TH2 cytokine that directs many features of airway remodeling through TGF-β1 and MMPs.
Objectives: We hypothesized that, in human asthma, IL-13 stimulates increased airway fibroblast invasiveness via TGF-β1 and MMPs in asthma compared with normal controls.
Methods: Fibroblasts were cultured from endobronchial biopsies in 20 subjects with mild asthma (FEV1: 90 ± 3.6% pred) and 17 normal control subjects (FEV1: 102 ± 2.9% pred) who underwent bronchoscopy. Airway fibroblast invasiveness was investigated using Matrigel chambers. IL-13 or IL-13 with TGF-β1 neutralizing antibody or pan-MMP inhibitor (GM6001) was added to the lower chamber as a chemoattractant. Flow cytometry and immunohistochemistry were performed in a subset of subjects to evaluate IL-13 receptor levels.
Measurements and Main Results: IL-13 significantly stimulated invasion in asthmatic airway fibroblasts, compared with normal control subjects. Inhibitors of both TGF-β1 and MMPs blocked IL-13–induced invasion in asthma, but had no effect in normal control subjects. At baseline, in airway tissue, IL-13 receptors were expressed in significantly higher levels in asthma, compared with normal control subjects. In airway fibroblasts, baseline IL-13Rα2 was reduced in asthma compared with normal control subjects.
Conclusions: IL-13 potentiates airway fibroblast invasion through a mechanism involving TGF-β1 and MMPs. IL-13 receptor subunits are differentially expressed in asthma. These effects may result in IL-13–directed airway remodeling in asthma.
PMCID: PMC3136991  PMID: 21471104
airway remodeling; interleukin-13; transforming growth factor-β; matrix metalloproteinase
13.  Diffusion-Weighted Hyperpolarized 129Xe MRI in Healthy Volunteers and Subjects with Chronic Obstructive Pulmonary Disease 
Magnetic Resonance in Medicine  2010;65(4):1154-1165.
129Xe apparent diffusion coefficient (ADC) MRI offers an alternative to 3He ADC MRI, given its greater availability and lower cost. To demonstrate the feasibility of HP 129Xe ADC MRI, we present results from healthy volunteers (HV), chronic obstructive pulmonary disease (COPD) subjects, and age-matched healthy controls (AMC). The mean parenchymal ADC was 0.036±0.003 cm2/s for HV, 0.043±0.006 cm2/s for AMC, and 0.056±0.008 cm2/s for COPD subjects with emphysema. In healthy individuals, but not the COPD group, ADC decreased significantly in the anterior-posterior direction by ~22% (p = 0.006, AMC; 0.0059, HV), likely due to gravity-induced tissue compression. The COPD group exhibited a significantly larger superior-inferior ADC reduction (~28%) than the healthy groups (~24%) (p = 0.00018 HV; p = 3.45×10-5 AMC), consistent with smoking-related tissue destruction in the superior lung. Superior-inferior gradients in healthy subjects may result from regional differences in xenon concentration. ADC was significantly correlated with pulmonary function tests (FEV1, r=-0.77, p=0.0002; FEV1/FVC, r=-0.78, p=0.0002; DLCO/VA, r=-0.77, p=0.0002), and in healthy groups, increased with age by 0.0002 cm2/s/yr (r=0.56, p=0.02). This study shows 129Xe ADC MRI is clinically feasible, sufficiently sensitive to distinguish HV from subjects with emphysema, and detects age and posture-dependent changes.
PMCID: PMC3351270  PMID: 21413080
Hyperpolarized 129Xe MRI; apparent diffusion coefficient; chronic obstructive pulmonary disease; pulmonary function testing
14.  Airway Microbiota and Bronchial Hyperresponsiveness in Patients with Sub-optimally Controlled Asthma 
Improvement in lung function following macrolide antibiotic therapy has been attributed to reduction in bronchial infection due to specific bacteria. However, the airway may be populated by a more diverse microbiota, and clinical features of asthma may be associated with characteristics of the airway microbiota present.
To determine if relationships exist between the composition of the airway bacterial microbiota and clinical features of asthma, using culture-independent tools capable of detecting the presence and relative abundance of most known bacteria.
In this pilot study, bronchial epithelial brushings were collected from sixty-five adults with sub-optimally controlled asthma participating in a multicenter study of the effects of clarithromycin on asthma control, and ten healthy subjects. A combination of high-density 16S rRNA microarray and parallel clone library-sequencing analysis was used to profile the microbiota and examine relationships with clinical measurements.
Compared to controls, 16S rRNA amplicon concentrations (a proxy for bacterial burden) and bacterial diversity were significantly higher among asthmatic patients. In multivariate analyses, airway microbiota composition and diversity were significantly correlated with bronchial hyperresponsiveness. Specifically, the relative abundance of particular phylotypes, including members of the Comamonadaceae, Sphingomonadaceae, Oxalobacteraceae and other bacterial families, were highly correlated with the degree of bronchial hyperresponsiveness.
The composition of bronchial airway microbiota is associated with the degree of bronchial hyperresponsiveness among patients with sub-optimally controlled asthma. These findings support the need for further functional studies to examine the potential contribution of members of the airway microbiota in asthma pathogenesis.
PMCID: PMC3037020  PMID: 21194740
microbiome; bacteria; asthma; 16S rRNA; PhyloChip
15.  A trial of clarithromycin for the treatment of suboptimally controlled asthma 
Polymerase chain reaction (PCR) studies have demonstrated evidence of M. pneumoniae and C. pneumoniae in the lower airways of patients with asthma.
To test the hypothesis that clarithromycin would improve asthma control in individuals with mild-to-moderate persistent asthma that was not well-controlled despite treatment with low-dose inhaled corticosteroids (ICS).
Adults with an Asthma Control Questionnaire (ACQ) score ≥1.5 after a 4 week period of treatment with fluticasone propionate were entered into a PCR-stratified randomized trial to evaluate the effect of 16 weeks of either clarithromycin or placebo, added to fluticasone, on asthma control in individuals with or without lower airway PCR evidence of M. pneumoniae or C. pneumoniae.
92 participants were randomized. Twelve (13%) subjects demonstrated PCR evidence of M. pneumoniae or C. pneumoniae in endobronchial biopsies; 80 were PCR negative for both organisms. In PCR positive participants, clarithromycin yielded a 0.4±0.4 unit improvement in the ACQ score, with a 0.1±0.3 unit improvement in those allocated to placebo. This between-group difference of 0.3±0.5 (p=0.6) was neither clinically nor statistically significant. In PCR negative participants, a non-significant between-group difference of 0.2±0.2 units (p=0.3) was observed. Clarithromycin did not improve lung function or airway inflammation but did improve airway hyperresponsiveness, increasing the methacholine PC20 by 1.2±0.5 doubling doses (p=0.02) in the study population.
Adding clarithromycin to fluticasone in adults with mild-to-moderate persistent asthma that was suboptimally-controlled by low-dose ICS alone did not further improve asthma control. Although there was an improvement in airway hyperresponsiveness with clarithromycin, this benefit was not accompanied by improvements in other secondary outcomes.
PMCID: PMC2950827  PMID: 20920764
asthma; infection; antibiotic
16.  Microfluidic Platform versus Conventional Real-time PCR for the Detection of Mycoplasma pneumoniae in Respiratory Specimens 
Rapid, accurate diagnosis of community-acquired pneumonia (CAP) due to Mycoplasma pneumoniae is compromised by low sensitivity of culture and serology. PCR has emerged as a sensitive method to detect M. pneumoniae DNA in clinical specimens. However, conventional real-time PCR is not cost-effective for routine out-patient or implementation. Here, we evaluate a novel microfluidic real-time PCR platform (Advanced Liquid Logic, Inc.) that is rapid, portable, and fully automated. We enrolled patients with CAP and extracted DNA from nasopharyngeal wash (NPW) specimens using a biotinylated capture probe and streptavidin-coupled magnetic beads. Each extract was tested for M. pneumoniae-specific DNA by real-time PCR on both conventional and microfluidic platforms using Taqman probe and primers. Three of 59 (5.0%) NPWs were positive, and agreement between the methods was 98%. The microfluidic platform was equally sensitive but three times faster and offers an inexpensive and convenient diagnostic test for microbial DNA.
PMCID: PMC2854258  PMID: 20227222
Mycoplasma pneumoniae; real-time PCR; DNA-based diagnostics; community-acquired pneumonia; diagnostic microbiology
17.  Tiotropium Bromide Step-Up Therapy for Adults with Uncontrolled Asthma 
The New England journal of medicine  2010;363(18):1715-1726.
Long-acting beta-agonist (LABA) therapy improves symptoms in patients whose asthma is poorly controlled by an inhaled glucocorticoid alone. Alternative treatments for adults with uncontrolled asthma are needed.
In a three-way, double-blind, triple-dummy crossover trial involving 210 patients with asthma, we evaluated the addition of tiotropium bromide (a long-acting anticholinergic agent approved for the treatment of chronic obstructive pulmonary disease but not asthma) to an inhaled glucocorticoid, as compared with a doubling of the dose of the inhaled glucocorticoid (primary superiority comparison) or the addition of the LABA salmeterol (secondary noninferiority comparison).
The use of tiotropium resulted in a superior primary outcome, as compared with a doubling of the dose of an inhaled glucocorticoid, as assessed by measuring the morning peak expiratory flow (PEF), with a mean difference of 25.8 liters per minute (P<0.001) and superiority in most secondary outcomes, including evening PEF, with a difference of 35.3 liters per minute (P<0.001); the proportion of asthma-control days, with a difference of 0.079 (P = 0.01); the forced expiratory volume in 1 second (FEV1) before bronchodilation, with a difference of 0.10 liters (P = 0.004); and daily symptom scores, with a difference of −0.11 points (P<0.001). The addition of tiotropium was also noninferior to the addition of salmeterol for all assessed outcomes and increased the prebronchodilator FEV1 more than did salmeterol, with a difference of 0.11 liters (P = 0.003).
When added to an inhaled glucocorticoid, tiotropium improved symptoms and lung function in patients with inadequately controlled asthma. Its effects appeared to be equivalent to those with the addition of salmeterol. (Funded by the National Heart, Lung, and Blood Institute; number, NCT00565266.)
PMCID: PMC3011177  PMID: 20979471
18.  Effectiveness and Safety of Bronchial Thermoplasty in the Treatment of Severe Asthma 
Rationale: Bronchial thermoplasty (BT) is a bronchoscopic procedure in which controlled thermal energy is applied to the airway wall to decrease smooth muscle.
Objectives: To evaluate the effectiveness and safety of BT versus a sham procedure in subjects with severe asthma who remain symptomatic despite treatment with high-dose inhaled corticosteroids and long-acting β2-agonists.
Methods: A total of 288 adult subjects (Intent-to-Treat [ITT]) randomized to BT or sham control underwent three bronchoscopy procedures. Primary outcome was the difference in Asthma Quality of Life Questionnaire (AQLQ) scores from baseline to average of 6, 9, and 12 months (integrated AQLQ). Adverse events and health care use were collected to assess safety. Statistical design and analysis of the primary endpoint was Bayesian. Target posterior probability of superiority (PPS) of BT over sham was 95%, except for the primary endpoint (96.4%).
Measurements and Main Results: The improvement from baseline in the integrated AQLQ score was superior in the BT group compared with sham (BT, 1.35 ± 1.10; sham, 1.16 ± 1.23 [PPS, 96.0% ITT and 97.9% per protocol]). Seventy-nine percent of BT and 64% of sham subjects achieved changes in AQLQ of 0.5 or greater (PPS, 99.6%). Six percent more BT subjects were hospitalized in the treatment period (up to 6 wk after BT). In the posttreatment period (6–52 wk after BT), the BT group experienced fewer severe exacerbations, emergency department (ED) visits, and days missed from work/school compared with the sham group (PPS, 95.5, 99.9, and 99.3%, respectively).
Conclusions: BT in subjects with severe asthma improves asthma-specific quality of life with a reduction in severe exacerbations and healthcare use in the posttreatment period.
Clinical trial registered with (NCT00231114).
PMCID: PMC3269231  PMID: 19815809
asthma; Alair Bronchial Thermoplasty System; bronchial thermoplasty; bronchoscopic procedure; Asthma Quality of Life
19.  Hyperpolarized 129Xe MR Imaging of Alveolar Gas Uptake in Humans 
PLoS ONE  2010;5(8):e12192.
One of the central physiological functions of the lungs is to transfer inhaled gases from the alveoli to pulmonary capillary blood. However, current measures of alveolar gas uptake provide only global information and thus lack the sensitivity and specificity needed to account for regional variations in gas exchange.
Methods and Principal Findings
Here we exploit the solubility, high magnetic resonance (MR) signal intensity, and large chemical shift of hyperpolarized (HP) 129Xe to probe the regional uptake of alveolar gases by directly imaging HP 129Xe dissolved in the gas exchange tissues and pulmonary capillary blood of human subjects. The resulting single breath-hold, three-dimensional MR images are optimized using millisecond repetition times and high flip angle radio-frequency pulses, because the dissolved HP 129Xe magnetization is rapidly replenished by diffusive exchange with alveolar 129Xe. The dissolved HP 129Xe MR images display significant, directional heterogeneity, with increased signal intensity observed from the gravity-dependent portions of the lungs.
The features observed in dissolved-phase 129Xe MR images are consistent with gravity-dependent lung deformation, which produces increased ventilation, reduced alveolar size (i.e., higher surface-to-volume ratios), higher tissue densities, and increased perfusion in the dependent portions of the lungs. Thus, these results suggest that dissolved HP 129Xe imaging reports on pulmonary function at a fundamental level.
PMCID: PMC2922382  PMID: 20808950
20.  Effect of β2-adrenergic receptor polymorphism on response to longacting β2 agonist in asthma (LARGE trial): a genotype-stratified, randomised, placebo-controlled, crossover trial 
Lancet  2009;374(9703):1754-1764.
Combined long-acting β2-agonist and inhaled corticosteroid (LABA/ICS) therapy improves outcomes in many asthmatics. Some studies suggest that patients homozygous for arginine at the 16th amino-acid position of the β2 adrenergic receptor (B16 Arg/Arg) benefit less than those with B16 Gly/Gly.
In an NIH-funded, B16 genotype-stratified, prospective, randomized, double-blind, placebo-controlled, cross-over trial ( registration ID NCT00200967), we compared adding salmeterol or placebo to ICS in patients with moderate asthma, using AM PEF as the primary outcome.
After 18 weeks, Arg/Arg (n=42) and Gly/Gly (n=45) subjects had greater AM PEF with salmeterol than placebo, with no difference in improvement by genotype (Arg/Arg 21.4 (p<0.0001) vs. Gly/Gly 21.5 L/min (p<0.0001); 0.1 L/min difference between genotypes, 95% CI (−14.2, 14.4), p=0.99). In Gly/Gly subjects, methacholine PC20 (a secondary outcome) doubled when salmeterol was added to ICS (p<0.0001), but remained unchanged in Arg/Arg subjects (p=0.87) (1.32 doubling dose difference between genotypes (95%CI 0.43,2.21), p=0.0038). An exploratory posthoc subset analysis of African Americans showed that salmeterol improved the AM and PM PEF for the 8 Gly/Gly subjects (29 L/min, p=0.013 and 45 L/min, p= 0.0005, respectively) but not for the 9 Arg/Arg subjects (−12 L/min, p=0.57 and−2.2 L/min, p=0.92, respectively).
B16 Arg/Arg and Gly/Gly patients experience improved airway function with salmeterol added to moderate-dose ICS. While these data provide reassurance that in the general population these polymorphisms should not alter the use of LABA with moderate-dose ICS, the significance of the genotype-differentiated response in airway reactivity favoring Gly/Gly subjects and the post-hoc analysis in African Americans require further investigation.
PMCID: PMC2914569  PMID: 19932356
Asthma; pharmacogenetics; beta-adrenergic receptor; beta-agonists; salmeterol
21.  S-Nitrosoglutathione Reductase 
Rationale: Nitric oxide bioactivity, mediated through the formation of S-nitrosothiols (SNOs), has a significant effect on bronchomotor tone. S-Nitrosoglutathione is an endogenous bronchodilator that is decreased in children with asthmatic respiratory failure and in adults with asthma undergoing segmental airway challenge. Recently we showed that S-nitrosoglutathione reductase (GSNOR) regulates endogenous SNOs. Mice with genetic deletion of GSNOR are protected from airway hyperresponsivity in an allergic asthma model.
Objectives: We hypothesized that GSNOR is increased in human asthma and correlates with lung SNO content and airway reactivity.
Methods: We recruited 36 subjects with mild asthma with FEV1 88.5 ± 2.3% predicted and 34 healthy control subjects with FEV1 100.7 ± 2.5% predicted. Bronchoalveolar lavage (BAL) was performed in all subjects. Cell counts, differentials, GSNOR activity, and SNO levels were determined in BAL.
Measurements and Main Results: SNO content was decreased in asthmatic BAL compared with control BAL and correlated inversely with GSNOR expression in BAL cell lysates. Furthermore, GSNOR activity measured from BAL samples was significantly increased in subjects with asthma compared with control subjects and correlated inversely with the provocative concentration of methacholine causing a 20% decrease in FEV1.
Conclusions: These findings suggest that GSNOR is an important regulator of airway SNO content and airways hyperresponsiveness in human asthma.
PMCID: PMC2724715  PMID: 19395503
asthma; S-nitrosoglutathione reductase; S-nitrosothiols; airway hyperresponsiveness
22.  Predicting Response to Inhaled Corticosteroid Efficacy (PRICE Trial) 
Although guidelines recommend anti-inflammatory therapy for persistent asthma, recent studies suggest that 25-35% of asthmatics may not improve lung function with inhaled corticosteroids.
To evaluate potential biomarkers of predicting short term (6-week) response to inhaled corticosteroid with subsequent evaluation of responders and non-responders to asthma control over a longer interval (16 additional weeks).
Eighty-three asthmatic subjects off steroid were enrolled in this multi-center study. Biomarkers and asthma characteristics were evaluated as predictors of inhaled corticosteroid response over a six week trial for changes in FEV1 and methacholine PC20. Following this, an additional four month trial evaluated asthma control.
Although multiple baseline predictors had significant correlations with improvements for short term inhaled steroid success, the only strong correlations (r ≥ ± 0.6) were albuterol reversibility (r=0.83, p<0.001); FEV1/FVC (r=−0.75, p<0.001); and FEV1 % predicted (r=−0.71, p<0.001). Dividing the subjects in the short term inhaled steroid trial into responders (> 5% FEV1 improvement), and non-responders (≤ 5%) determined the longer term need for steroids. For the non-responders, asthma control remained unchanged whether inhaled corticosteroids were continued or were substituted with a placebo, p=0.99. The good short term responders maintained asthma control longer term only if maintained on inhaled steroids (p=0.007).
The short term response to inhaled corticosteroids with regard to FEV1 improvement predicts long term asthma control.
Capsule Summary
A six week trial of ICS, in patients not currently on steroids, producing a ≥ 5% improvement in FEV1 can predict long term asthma control and the need for continued steroid use.
PMCID: PMC2872157  PMID: 17208587
inhaled corticosteroids; predicting response; therapy; characteristics; biomarkers
23.  Health-related quality of life and chronic obstructive pulmonary disease in North Carolina 
Comparisons of health-related quality of life (HRQOL) between persons with chronic obstructive pulmonary disease (COPD) and adults in the general population are not well described.
To examine associations between COPD and four measures of HRQOL in a population-based sample.
Patients & Methods:
These relationships were examined using data from 13,887 adults aged >18 years who participated in the 2007 Behavioral Risk Factor Surveillance System (BRFSS) conducted in North Carolina (NC). Logistic regression was used to obtain adjusted relative odds (aOR).
The age-adjusted prevalence of COPD among NC adults was 5.4% (standard error 0.27). Nearly half of adults with COPD reported fair/poor health compared with 15% of those without the condition (age-aOR, 5.5; 95% confidence interval [ CI] , 4.4 to 6.8). On average, adults with COPD reported twice as many unhealthy days (physical/mental) as those without the condition. The age-adjusted prevalence of >14 unhealthy days during the prior 30 days was 45% for adults with COPD and 17% for those without. The aOR of >14 unhealthy days was 1.7 (95% CI, 1.4 to 2.2) times greater among adults with COPD compared with those without.
These results suggest COPD is independently associated with lower levels of HRQOL and reinforce the importance of preventing COPD and its complications through health education messages stressing efforts to reduce total personal exposure to tobacco smoke, occupational dusts and chemicals, and other indoor and outdoor air pollutants linked to COPD and early disease recognition. Our findings represent one of the few statewide efforts in the US and provide guidance for disease management and policy decision making.
PMCID: PMC3354436  PMID: 22624116
Health-related quality of life; chronic obstructive pulmonary disease; North Carolina; behavioral risk factor surveillance system; age-adjusted prevalence; adults; pollutants; tobacco; occupational dusts; chemicals; policy decision making
24.  Predicting worsening asthma control following the common cold 
The asthmatic response to the common cold is highly variable and early characteristics that predict worsening of asthma control following a cold have not been identified.
In this prospective multi-center cohort study of 413 adult subjects with asthma, we used the mini-Asthma Control Questionnaire (mini-ACQ) to quantify changes in asthma control and the Wisconsin Upper Respiratory Symptom Survey-21 (WURSS-21) to measure cold severity. Univariate and multivariable models examined demographic, physiologic, serologic, and cold-related characteristics for their relationship to changes in asthma control following a cold.
We observed a clinically significant worsening of asthma control following a cold (increase in mini-ACQ of 0.69 ± 0.93). Univariate analysis demonstrated season, center location, cold length, and cold severity measurements all associated with a change in asthma control. Multivariable analysis of the covariates available within the first 2 days of cold onset revealed the day 2 and the cumulative sum of the day 1 and 2 WURSS-21 scores were significant predictors for the subsequent changes in asthma control.
In asthmatic subjects the cold severity measured within the first 2 days can be used to predict subsequent changes in asthma control. This information may help clinicians prevent deterioration in asthma control following a cold.
PMCID: PMC2592508  PMID: 18768579
asthma; asthma control; common cold
25.  Smoking Affects Response to Inhaled Corticosteroids or Leukotriene Receptor Antagonists in Asthma 
Rationale: One-quarter to one-third of individuals with asthma smoke, which may affect response to therapy and contribute to poor asthma control.
Objectives: To determine if the response to an inhaled corticosteroid or a leukotriene receptor antagonist is attenuated in individuals with asthma who smoke.
Methods: In a multicenter, placebo-controlled, double-blind, double-dummy, crossover trial, 44 nonsmokers and 39 light smokers with mild asthma were assigned randomly to treatment twice daily with inhaled beclomethasone and once daily with oral montelukast.
Measurements and Main Results: Primary outcome was change in prebronchodilator FEV1 in smokers versus nonsmokers. Secondary outcomes included peak flow, PC20 methacholine, symptoms, quality of life, and markers of airway inflammation. Despite similar FEV1, bronchodilator response, and sensitivity to methacholine at baseline, subjects with asthma who smoked had significantly more symptoms, worse quality of life, and lower daily peak flow than nonsmokers. Adherence to therapy did not differ significantly between smokers and nonsmokers, or between treatment arms. Beclomethasone significantly reduced sputum eosinophils and eosinophil cationic protein (ECP) in both smokers and nonsmokers, but increased FEV1 (170 ml, p = 0.0003) only in nonsmokers. Montelukast significantly increased a.m. peak flow in smokers (12.6 L/min, p = 0.002), but not in nonsmokers.
Conclusions: In subjects with mild asthma who smoke, the response to inhaled corticosteroids is attenuated, suggesting that adjustments to standard therapy may be required to attain asthma control. The greater improvement seen in some outcomes in smokers treated with montelukast suggests that leukotrienes may be important in this setting. Larger prospective studies are required to determine whether leukotriene modifiers can be recommended for managing asthma in patients who smoke.
PMCID: PMC1899291  PMID: 17204725
antiasthmatic agents; smoking adverse effects; corticosteroids; leukotrienes

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