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2.  An Association between l-Arginine/Asymmetric Dimethyl Arginine Balance, Obesity, and the Age of Asthma Onset Phenotype 
Rationale: Increasing body mass index (BMI) has been associated with less fractional exhaled nitric oxide (FeNO). This may be explained by an increase in the concentration of asymmetric dimethyl arginine (ADMA) relative to l-arginine, which can lead to greater nitric oxide synthase uncoupling.
Objectives: To compare this mechanism across age of asthma onset groups and determine its association with asthma morbidity and lung function.
Methods: Cross-sectional study of participants from the Severe Asthma Research Program, across early- (<12 yr) and late- (>12 yr) onset asthma phenotypes.
Measurements and Main Results: Subjects with late-onset asthma had a higher median plasma ADMA level (0.48 μM, [interquartile range (IQR), 0.35–0.7] compared with early onset, 0.37 μM [IQR, 0.29–0.59], P = 0.01) and lower median plasma l-arginine (late onset, 52.3 [IQR, 43–61] compared with early onset, 51 μM [IQR 39–66]; P = 0.02). The log of plasma l-arginine/ADMA was inversely correlated with BMI in the late- (r = −0.4, P = 0.0006) in contrast to the early-onset phenotype (r = −0.2, P = 0.07). Although FeNO was inversely associated with BMI in the late-onset phenotype (P = 0.02), the relationship was lost after adjusting for l-arginine/ADMA. Also in this phenotype, a reduced l-arginine/ADMA was associated with less IgE, increased respiratory symptoms, lower lung volumes, and worse asthma quality of life.
Conclusions: In late-onset asthma phenotype, plasma ratios of l-arginine to ADMA may explain the inverse relationship of BMI to FeNO. In addition, these lower l-arginine/ADMA ratios are associated with reduced lung function and increased respiratory symptom frequency, suggesting a role in the pathobiology of the late-onset phenotype.
PMCID: PMC3570651  PMID: 23204252
asthma; obesity; age of asthma onset; ADMA; arginine
3.  Severe Asthma 
The National Heart, Lung, and Blood Institute Severe Asthma Research Program (SARP) has characterized over the past 10 years 1,644 patients with asthma, including 583 individuals with severe asthma. SARP collaboration has led to a rapid recruitment of subjects and efficient sharing of samples among participating sites to conduct independent mechanistic investigations of severe asthma. Enrolled SARP subjects underwent detailed clinical, physiologic, genomic, and radiological evaluations. In addition, SARP investigators developed safe procedures for bronchoscopy in participants with asthma, including those with severe disease. SARP studies revealed that severe asthma is a heterogeneous disease with varying molecular, biochemical, and cellular inflammatory features and unique structure–function abnormalities. Priorities for future studies include recruitment of a larger number of subjects with severe asthma, including children, to allow further characterization of anatomic, physiologic, biochemical, and genetic factors related to severe disease in a longitudinal assessment to identify factors that modulate the natural history of severe asthma and provide mechanistic rationale for management strategies.
PMCID: PMC3297096  PMID: 22095547
asthma; remodeling; inflammation; bronchoscopy; imaging
4.  Mast Cell Phenotype, Location, and Activation in Severe Asthma 
Rationale: Severe asthma (SA) remains poorly understood. Mast cells (MC) are implicated in asthma pathogenesis, but it remains unknown how their phenotype, location, and activation relate to asthma severity.
Objectives: To compare MC-related markers measured in bronchoscopically obtained samples with clinically relevant parameters between normal subjects and subjects with asthma to clarify their pathobiologic importance.
Methods: Endobronchial biopsies, epithelial brushings, and bronchoalveolar lavage were obtained from subjects with asthma and normal subjects from the Severe Asthma Research Program (N = 199). Tryptase, chymase, and carboxypeptidase A (CPA)3 were used to identify total MC (MCTot) and the MCTC subset (MCs positive for both tryptase and chymase) using immunostaining and quantitative real-time polymerase chain reaction. Lavage was analyzed for tryptase and prostaglandin D2 (PGD2) by ELISA.
Measurements and Main Results: Submucosal MCTot (tryptase-positive by immunostaining) numbers were highest in “mild asthma/no inhaled corticosteroid (ICS) therapy” subjects and decreased with greater asthma severity (P = 0.002). In contrast, MCTC (chymase-positive by immunostaining) were the predominant (MCTC/MCTot > 50%) MC phenotype in SA (overall P = 0.005). Epithelial MCTot were also highest in mild asthma/no ICS, but were not lower in SA. Instead, they persisted and were predominantly MCTC. Epithelial CPA3 and tryptase mRNA supported the immunostaining data (overall P = 0.008 and P = 0.02, respectively). Lavage PGD2 was higher in SA than in other steroid-treated groups (overall P = 0.02), whereas tryptase did not differentiate the groups. In statistical models, PGD2 and MCTC/MCTot predicted SA.
Conclusions: Severe asthma is associated with a predominance of MCTC in the airway submucosa and epithelium. Activation of those MCTC may contribute to the increases in PGD2 levels. The data suggest an altered and active MC population contributes to SA pathology.
PMCID: PMC3056228  PMID: 20813890
prostaglandin D2; chymase; carboxypeptidase A
5.  Pulmonary Function Abnormalities in HIV-Infected Patients during the Current Antiretroviral Therapy Era 
Rationale: Before the introduction of combination antiretroviral (ARV) therapy, patients infected with HIV had an increased prevalence of respiratory symptoms and lung function abnormalities. The prevalence and exact phenotype of pulmonary abnormalities in the current era are unknown. In addition, these abnormalities may be underdiagnosed.
Objectives: Our objective was to determine the current burden of respiratory symptoms, pulmonary function abnormalities, and associated risk factors in individuals infected with HIV.
Methods: Cross-sectional analysis of 167 participants infected with HIV who underwent pulmonary function testing.
Measurements and Main Results: Respiratory symptoms were present in 47.3% of participants and associated with intravenous drug use (odds ratio [OR] 3.64; 95% confidence interval [CI], 1.32–10.046; P = 0.01). Only 15% had previous pulmonary testing. Pulmonary function abnormalities were common with 64.1% of participants having diffusion impairment and 21% having irreversible airway obstruction. Diffusion impairment was independently associated with ever smoking (OR 2.46; 95% CI, 1.16–5.21; P = 0.02) and Pneumocystis pneumonia prophylaxis (OR 2.94; 95% CI, 1.10–7.86; P = 0.01), whereas irreversible airway obstruction was independently associated with pack-years smoked (OR 1.03 per pack-year; 95% CI, 1.01–1.05; P < 0.01), intravenous drug use (OR 2.87; 95% CI, 1.15–7.09; P = 0.02), and the use of ARV therapy (OR 6.22; 95% CI, 1.19–32.43; P = 0.03).
Conclusions: Respiratory symptoms and pulmonary function abnormalities remain common in individuals infected with HIV. Smoking and intravenous drug use are still important risk factors for pulmonary abnormalities, but ARV may be a novel risk factor for irreversible airway obstruction. Obstructive lung disease is likely underdiagnosed in this population.
PMCID: PMC2949404  PMID: 20522793
HIV; respiratory function tests; smoking; antiretroviral therapy, highly active; AIDS
6.  Identification of Asthma Phenotypes Using Cluster Analysis in the Severe Asthma Research Program 
Rationale: The Severe Asthma Research Program cohort includes subjects with persistent asthma who have undergone detailed phenotypic characterization. Previous univariate methods compared features of mild, moderate, and severe asthma.
Objectives: To identify novel asthma phenotypes using an unsupervised hierarchical cluster analysis.
Methods: Reduction of the initial 628 variables to 34 core variables was achieved by elimination of redundant data and transformation of categorical variables into ranked ordinal composite variables. Cluster analysis was performed on 726 subjects.
Measurements and Main Results: Five groups were identified. Subjects in Cluster 1 (n = 110) have early onset atopic asthma with normal lung function treated with two or fewer controller medications (82%) and minimal health care utilization. Cluster 2 (n = 321) consists of subjects with early-onset atopic asthma and preserved lung function but increased medication requirements (29% on three or more medications) and health care utilization. Cluster 3 (n = 59) is a unique group of mostly older obese women with late-onset nonatopic asthma, moderate reductions in FEV1, and frequent oral corticosteroid use to manage exacerbations. Subjects in Clusters 4 (n = 120) and 5 (n = 116) have severe airflow obstruction with bronchodilator responsiveness but differ in to their ability to attain normal lung function, age of asthma onset, atopic status, and use of oral corticosteroids.
Conclusions: Five distinct clinical phenotypes of asthma have been identified using unsupervised hierarchical cluster analysis. All clusters contain subjects who meet the American Thoracic Society definition of severe asthma, which supports clinical heterogeneity in asthma and the need for new approaches for the classification of disease severity in asthma.
PMCID: PMC2822971  PMID: 19892860
asthma phenotype; definition; cluster analysis; severe asthma
7.  Role of Insulin-like Growth Factor Binding Protein-3 in Allergic Airway Remodeling 
Rationale: The hallmarks of allergic asthma are airway inflammation, obstruction, and remodeling. Airway remodeling may lead to irreversible airflow obstruction with increased morbidity and mortality. Despite advances in the treatment of asthma, the mechanisms underlying airway remodeling are still poorly understood. We reported that insulin-like growth factor (IGF) binding proteins (IGFBPs) contribute to extracellular matrix deposition in idiopathic pulmonary fibrosis; however, their contribution to airway remodeling in asthma has not been established.
Objectives: We hypothesized that IGFBP-3 is overexpressed in asthma and contributes to airway remodeling.
Methods: We evaluated levels of IGFBP-3 in tissues and bronchoalveolar lavage fluid from patients with asthma at baseline and 48 hours after allergen challenge, in reparative epithelium in an in vitro wounding assay, and in conditioned media from cytokine- and growth factor–stimulated primary epithelial cells.
Measurements and Main Results: IGFBP-3 levels and distribution were evaluated by Western blot, ELISA, and immunofluorescence. IGFBP-3 is increased in vivo in the airway epithelium of patients with asthma compared with normal control subjects. The concentration of IGFBP-3 is increased in the bronchoalveolar lavage fluid of patients with asthma after allergen challenge, its levels are increased in reparative epithelium in an in vitro wounding assay and in the conditioned medium of primary airway epithelial cell cultures stimulated with IGF-I.
Conclusions: Our results suggest that one mechanism of allergic airway remodeling is through the secretion of the profibrotic IGFBP-3 from IGF-I–stimulated airway epithelial cells during allergic inflammation.
PMCID: PMC2753789  PMID: 19608721
asthma; bronchoalveolar lavage; primary epithelial cells; insulin-like growth factor binding protein; fibrosis
8.  Interleukin-13–induced MUC5AC Is Regulated by 15-Lipoxygenase 1 Pathway in Human Bronchial Epithelial Cells 
Rationale: 15-Lipoxygenase-1 (15LO1) and MUC5AC are highly expressed in asthmatic epithelial cells. IL-13 is known to induce 15LO1 and MUC5AC in human airway epithelial cells in vitro. Whether 15LO1 and/or its product 15-HETE modulate MUC5AC expression is unknown.
Objectives: To determine the expression of 15LO1 in freshly harvested epithelial cells from subjects with asthma and normal control subjects and to determine whether IL-13–induced 15LO1 expression and activation regulate MUC5AC expression in human bronchial epithelial cells in vitro.
Methods: Human airway epithelial cells from subjects with asthma and normal subjects were evaluated ex vivo for 15LO1 and MUC5AC expression. The impact of 15LO1 on MUC5AC expression in vitro was analyzed by inhibiting 15LO1 through pharmacologic (PD146176) and siRNA approaches in human bronchial epithelial cells cultured under air–liquid interface. We analyzed 15 hydroxyeicosatetraenoic acid (15-HETE) by liquid chromatography/UV/mass spectrometry. MUC5AC and 15LO1 were analyzed by real-time RT-PCR, immunofluoresence, and Western blot.
Measurements and Main Results: Epithelial 15LO1 expression increased with asthma severity (P < 0.0001). 15LO1 significantly correlated with MUC5AC ex vivo and in vitro. IL-13 increased 15LO1 expression and stimulated formation of two molecular species of 15-HETE esterified to phosphotidylethanolamine (15-HETE-PE). Inhibition of 15LO1 suppressed 15-HETE-PE and decreased MUC5AC expression in the presence of IL-13 stimulation. The addition of exogenous 15-HETE partially restored MUC5AC expression.
Conclusions: Epithelial 15LO1 expression increases with increasing asthma severity. IL-13 induction of 15-HETE-PE enhances MUC5AC expression in human airway epithelial cells. High levels of 15LO1 activity could contribute to the increases of MUC5AC observed in asthma.
PMCID: PMC2675565  PMID: 19218191
15-lipoxygenase-1; MUC5AC; asthma pathogenesis; inflammation; lipid mediator
9.  Airway Lipoxin A4 Generation and Lipoxin A4 Receptor Expression Are Decreased in Severe Asthma 
Rationale: Airway inflammation is common in severe asthma despite antiinflammatory therapy with corticosteroids. Lipoxin A4 (LXA4) is an arachidonic acid–derived mediator that serves as an agonist for resolution of inflammation.
Objectives: Airway levels of LXA4, as well as the expression of lipoxin biosynthetic genes and receptors, in severe asthma.
Methods: Samples of bronchoalveolar lavage fluid were obtained from subjects with asthma and levels of LXA4 and related eicosanoids were measured. Expression of lipoxin biosynthetic genes was determined in whole blood, bronchoalveolar lavage cells, and endobronchial biopsies by quantitative polymerase chain reaction, and leukocyte LXA4 receptors were monitored by flow cytometry.
Measurements and Main Results: Individuals with severe asthma had significantly less LXA4 in bronchoalveolar lavage fluids (11.2 ± 2.1 pg/ml) than did subjects with nonsevere asthma (150.1 ± 38.5 pg/ml; P < 0.05). In contrast, levels of cysteinyl leukotrienes were increased in both asthma cohorts compared with healthy individuals. In severe asthma, 15-lipoxygenase-1 mean expression was decreased fivefold in bronchoalveolar lavage cells. In contrast, 15-lipoxgenase-1 was increased threefold in endobronchial biopsies, but expression of both 5-lipoxygenase and 15-lipoxygenase-2 in these samples was decreased. Cyclooxygenase-2 expression was decreased in all anatomic compartments sampled in severe asthma. Moreover, LXA4 receptor gene and protein expression were significantly decreased in severe asthma peripheral blood granulocytes.
Conclusions: Mechanisms underlying pathological airway responses in severe asthma include lipoxin underproduction with decreased expression of lipoxin biosynthetic enzymes and receptors. Together, these results indicate that severe asthma is characterized, in part, by defective lipoxin counterregulatory signaling circuits.
PMCID: PMC2542432  PMID: 18583575
severe asthma; lipoxins; eicosanoids
10.  IL4Rα Mutations Are Associated with Asthma Exacerbations and Mast Cell/IgE Expression 
Background: Severe asthma has been associated with severe exacerbations, lower lung function and greater tissue inflammation. Previous studies have suggested that mutations in interleukin-4 receptor α (IL4Rα) are associated with lower lung function, higher IgE, and a gain in receptor function. However, an effect on exacerbations and tissue inflammation has not been shown.
Hypothesis: Allelic substitutions in IL4Rα are associated with asthma exacerbations, lower lung function, and tissue inflammation, in particular to mast cells and IgE.
Methods: Two well-characterized cohorts of subjects with severe asthma were analyzed for five single nucleotide polymorphisms (SNPs) in IL4Rα. These polymorphisms were compared with the history of severe asthma exacerbations and lung function. In the primary (National Jewish) cohort, these polymorphisms were also compared with endobronchial tissue inflammatory cells and local IgE.
Results: In both cohorts, the presence of the minor alleles at E375A and Q551R, which were more common in African Americans, was associated with a history of severe exacerbations and lower lung function. In the National Jewish cohort, the C allele at E375A was associated with higher tissue mast cells and higher levels of IgE bound to mast cells. The significance for most of these associations remained when whites (the larger racial subgroup) were analyzed separately.
Conclusions: SNPs in IL4Rα, which are more common in African Americans, are associated with severe asthma exacerbations, lower lung function, and increased mast cell–related tissue inflammation. Further studies of the impact of these mutations in African Americans and on receptor function are indicated.
PMCID: PMC1899282  PMID: 17170387
asthma; genetics; IL4Rα; exacerbations; mast cells; IgE
11.  Regional Fibroblast Heterogeneity in the Lung 
Rationale: Excessive deposition of extracellular matrix occurs in proximal airways of individuals with asthma, but fibrosis in distal lung has not been observed. Whether differing fibrotic capacities of fibroblasts from these two regions contribute to this variability is unknown.
Objectives: We compared morphologic and functional characteristics of fibroblasts isolated from proximal airways and distal lung parenchyma to determine phenotypic differences.
Methods: Concurrent proximal airway and distal lung biopsies were obtained by bronchoscopy from subjects with asthma to isolate airway and distal lung fibroblasts, respectively. The following characteristics were compared: morphology, proliferation, α-smooth muscle actin expression, and synthesis of procollagen type I and eotaxin-1.
Results: Airway fibroblasts (AFs) are morphologically distinct from distal lung fibroblasts (DLFs): they are larger (2.3-fold greater surface area vs. matched DLFs; p = 0.02), stellate in appearance, and with more cytoplasmic projections compared with the spindle-shaped DLFs. AFs synthesized more procollagen type I than did DLFs at baseline (twofold higher; p = 0.003) and after transforming growth factor-β stimulation (1.4-fold higher; p = 0.02). Similarly, AFs produced more eotaxin-1 than did DLFs at baseline (2.5-fold higher; p = 0.004) and after interleukin-13 stimulation (13-fold higher; p = 0.0001). In contrast, DLFs proliferate more than AFs with serum stimulation (about sixfold greater; p = 0.03). Unstimulated DLFs also expressed more α-smooth muscle actin than did corresponding AFs (p = 0.006).
Conclusions: These studies suggest that at least two phenotypes of fibroblast exist in the lung. These phenotypic differences may partially explain the variable responses to injury and repair between proximal airways and distal lung/parenchyma in asthma and other respiratory diseases.
PMCID: PMC2662967  PMID: 16543551
asthma; fibroblast; interleukin 13; remodeling; transforming growth factor β
12.  Correlation of Systemic Superoxide Dismutase Deficiency to Airflow Obstruction in Asthma 
Rationale: Increased oxidative stress and decreased superoxide dismutase (SOD) activity in the asthmatic airway are correlated to airflow limitation and hyperreactivity. We hypothesized that asthmatic individuals with higher levels of oxidative stress may have greater loss of SOD activity, which would be reflected systemically in loss of circulating SOD activity and clinically by development of severe asthma and/or worsening airflow limitation. Methods: To investigate this, serum SOD activity and proteins, the glutathione peroxidase/glutathione antioxidant system, and oxidatively modified amino acids were measured in subjects with asthma and healthy control subjects. Results: SOD activity, but not Mn-SOD or Cu,Zn-SOD protein, was lower in asthmatic serum as compared with control, and activity loss was significantly related to airflow limitation. Further, serum SOD activity demonstrated an inverse correlation with circulating levels of 3-bromotyrosine, a posttranslational modification of proteins produced by the eosinophil peroxidase system of eosinophils. Exposure of purified Cu,Zn-SOD to physiologically relevant levels of eosinophil peroxidase-generated reactive brominating species, reactive nitrogen species, or tyrosyl radicals in vitro confirmed that eosinophil-derived oxidative pathways promote enzyme inactivation. Conclusion: These findings are consistent with greater oxidant stress in asthma leading to greater inactivation of SOD, which likely amplifies inflammation and progressive airflow obstruction.
PMCID: PMC2718470  PMID: 15883124
asthma; superoxide dismutase; glutathione; pulmonary functions; peroxidase

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