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1.  Deficiency of Melanoma Differentiation–associated Protein 5 Results in Exacerbated Chronic Postviral Lung Inflammation 
Rationale: Respiratory viral infections can result in the establishment of chronic lung diseases. Understanding the early innate immune mechanisms that participate in the development of chronic postviral lung disease may reveal new targets for therapeutic intervention. The intracellular viral sensor protein melanoma differentiation–associated protein 5 (MDA5) sustains the acute immune response to Sendai virus, a mouse pathogen that causes chronic lung inflammation, but its role in the development of postviral chronic lung disease is unknown.
Objectives: To establish the role of MDA5 in the development of chronic lung disease.
Methods: MDA5-deficient or control mice were infected with Sendai virus. The acute inflammatory response was evaluated by profiling chemokine and cytokine expression and by characterizing the composition of the cellular infiltrate. The impact of MDA5 on chronic lung pathology and function was evaluated through histological studies, degree of oxygen saturation, and responsiveness to carbachol.
Measurements and Main Results: MDA5 deficiency resulted in normal virus replication and in a distinct profile of chemokines and cytokines that associated with acute lung neutropenia and enhanced accumulation of alternatively activated macrophages. Diminished expression of neutrophil-recruiting chemokines was also observed in cells infected with influenza virus, suggesting a key role of MDA5 in driving the early accumulation of neutrophils at the infection site. The biased acute inflammatory response of MDA5-deficient mice led to an enhanced chronic lung inflammation, epithelial cell hyperplasia, airway hyperreactivity, and diminished blood oxygen saturation.
Conclusions: MDA5 modulates the development of chronic lung inflammation by regulating the early inflammatory response in the lung.
PMCID: PMC3977719  PMID: 24417465
respiratory virus; chronic lung disease; innate immunity; paramyxovirus
2.  Reviewer acknowledgement 2014 
Respiratory Research  2015;16(1):9.
Contributing reviewers
The editors of Respiratory Research would like to thank all of our reviewers who have contributed to the journal in Volume 15 (2014).
PMCID: PMC4314788  PMID: 25644987
3.  Phenotype of asthmatics with increased airway S-nitrosoglutathione reductase activity 
Study question
S-Nitrosoglutathione is an endogenous airway smooth muscle relaxant. Increased airway S-Nitrosoglutathione breakdown occurs in some asthma patients. We asked whether patients with increased airway catabolism of this molecule had clinical features that distinguished them from other asthma patients.
We measured S-Nitrosoglutathione reductase expression and activity in bronchoscopy samples from 66 subjects in the Severe Asthma Research Program. We also analysed phenotype and genotype data from in the program as a whole.
Airway S-nitrosoglutathione reductase activity was increased in asthma patients (p = 0.032). However, only a subpopulation was affected, and this subpopulation was not defined by a “severe asthma” diagnosis. Subjects with increased activity were younger, had higher Immunoglobulin E and an earlier onset of symptoms. Consistent with a link between S-Nitrosoglutathione biochemistry and atopy, 1) interleukin 13 increased S-Nitrosoglutathione reductase expression, and 2) subjects with an S-Nitrosoglutathione reductase single nucleotide polymorphism (SNP) previously associated asthma had higher Immunoglobulin E than those without this SNP. Expression was higher in airway epithelium than in smooth muscle, and was increased in regions of the asthmatic lung with decreased airflow.
Answer to the question
An early-onset, allergic phenotype characterizes the asthma population with increased S-Nitrosoglutathione reductase activity.
PMCID: PMC4283933  PMID: 25359343
4.  Asthma in the elderly: a study of the role of vitamin D 
Asthma in the elderly is poorly understood and vitamin D deficiency and insufficiency are very common in older individuals. We studied the role of vitamin D in elderly asthmatics.
Asthmatics subjects, age 65 and older, were followed every 4 weeks for 12 weeks in the late fall and winter. During the study period they took 2,000 I.U. vitamin D3 daily. Serum 25-Hydroxyvitamin D and calcium were measured at baseline and study end.
Twenty nine percent of subjects were deficient and 50% insufficient in serum vitamin D at baseline. Serum vitamin D increased from 24.3±9.2 ng/ml (60.7±23 nmol/L) to 34±7.1 ng/ml (84.9±17.7 nmol/L) at the end of the study (p<0.001), whereas calcium was unchanged. We found no significant association between vitamin D and subjects' demographics. Vitamin D was similar in men and women. There was no association between serum vitamin D and inhaled steroid dose. Vitamin D was significantly lower in subjects with uncontrolled asthma (Asthma Control Test, ACT≤19) compared to the ones with well controlled symptoms (p<0.05). In subjects with uncontrolled asthma at baseline, ACT scores increased significantly at the end of the study (p<0.04), but not at 4 and 8 weeks. Spirometric values remained unchanged throughout the study.
Elderly asthmatics very commonly have vitamin D deficiency or insufficiency. Serum vitamin D levels were lower in subjects with uncontrolled asthma. In these subjects, vitamin D supplementation for 12 weeks led to improved ACT scores. Larger, randomized, placebo controlled studies are required to further evaluate whether vitamin D supplementation may improve asthma symptoms in this population.
Trial registration NCT01730976.
PMCID: PMC4162927  PMID: 25221606
Asthma; Vitamin D; Elderly
5.  MicroRNA-708 regulates CD38 expression through signaling pathways JNK MAP kinase and PTEN/AKT in human airway smooth muscle cells 
Respiratory Research  2014;15(1):107.
The cell-surface protein CD38 mediates airway smooth muscle (ASM) contractility by generating cyclic ADP-ribose, a calcium-mobilizing molecule. In human ASM cells, TNF-α augments CD38 expression transcriptionally by NF-κB and AP-1 activation and involving MAPK and PI3K signaling. CD38−/− mice develop attenuated airway hyperresponsiveness following allergen or cytokine challenge. The post-transcriptional regulation of CD38 expression in ASM is relatively less understood. In ASM, microRNAs (miRNAs) regulate inflammation, contractility, and hyperproliferation. The 3’ Untranslated Region (3’UTR) of CD38 has multiple miRNA binding sites, including a site for miR-708. MiR-708 is known to regulate PI3K/AKT signaling and hyperproliferation of other cell types. We investigated miR-708 expression, its regulation of CD38 expression and the underlying mechanisms involved in such regulation in human ASM cells.
Growth-arrested human ASM cells from asthmatic and non-asthmatic donors were used. MiRNA and mRNA expression were measured by quantitative real-time PCR. CD38 enzymatic activity was measured by a reverse cyclase assay. Total and phosphorylated MAPKs and PI3K/AKT as well as enzymes that regulate their activation were determined by Western blot analysis of cell lysates following miRNA transfection and TNF-α stimulation. Dual luciferase reporter assays were performed to determine whether miR-708 binds directly to CD38 3’UTR to alter gene expression.
Using target prediction algorithms, we identified several miRNAs with potential CD38 3’UTR target sites and determined miR-708 as a potential candidate for regulation of CD38 expression based on its expression and regulation by TNF-α. TNF-α caused a decrease in miR-708 expression in cells from non-asthmatics while it increased its expression in cells from asthmatics. Dual luciferase reporter assays in NIH-3 T3 cells revealed regulation of expression by direct binding of miR-708 to CD38 3’UTR. In ASM cells, miR-708 decreased CD38 expression by decreasing phosphorylation of JNK MAPK and AKT. These effects were associated with increased expression of MKP-1, a MAP kinase phosphatase and PTEN, a phosphatase that terminates PI3 kinase signaling.
In human ASM cells, TNF-α-induced CD38 expression is regulated by miR-708 directly binding to 3’UTR and indirectly by regulating JNK MAPK and PI3K/AKT signaling and has the potential to control airway inflammation, ASM contractility and proliferation.
PMCID: PMC4156970  PMID: 25175907
MicroRNA; MiR-708; Airway smooth muscle cells; MAP kinase; PI3 kinase; PTEN; AKT; CD38
6.  In vitro and in vivo pharmacological profile of PL-3994, a novel cyclic peptide (Hept-cyclo(Cys-His-Phe-d-Ala-Gly-Arg-d-Nle-Asp-Arg-Ile-Ser-Cys)-Tyr-[Arg mimetic]-NH2) natriuretic peptide receptor-A agonist that is resistant to neutral endopeptidase and acts as a bronchodilator 
The pharmacological and airways relaxant profiles of PL-3994 (Hept-cyclo(Cys-His-Phe-d-Ala-Gly-Arg-dNle-Asp-Arg-Ile-Ser-Cys)-Tyr-[Arg mimetic]-NH2), a novel natriuretic peptide receptor-A (NPR-A) agonist, were evaluated. PL-3994, a full agonist, has high affinity for recombinant human (h), dog, or rat NPR-As (Kis of 1, 41, and 10 nm, respectively), and produced concentration-dependent cGMP generation in human, dog and rat NPR-As (respective EC50s of 2, 3 and 14 nm). PL-3994 has a Ki of 7 nm for hNPR-C but was without effect on cGMP generation in hNPR-B. PL-3994 (1 µm) was without significant effect against 75 diverse molecular targets. PL-3994 or BNP, a natural NPR ligand, produced concentration-dependent relaxation of pre-contracted guinea-pig trachea (IC50s of 42.7 and 10.7 nm, respectively). PL-3994, and also BNP, (0.1 nm–100 µm) elicited a potent, concentration-dependent but small relaxation of pre-contracted human precision-cut lung slices (hPCLS). Intratracheal PL-3994 (1–1000 µg/kg) produced a dose-dependent inhibition of the bronchoconstrictor response evoked by aerosolized methacholine, but was without significant effect on cardiovascular parameters. PL-3994 was resistant to degradation by human neutral endopeptidase (hNEP) (92% remaining after 2 h), whereas the natural ligands, ANP and CNP, were rapidly metabolized (≤1% remaining after 2 h). PL-3994 is a potent, selective NPR agonist, resistant to NEP, with relaxant effects in guinea-pig and human airway smooth muscle systems. PL-3994 has the profile predictive of longer clinical bronchodilator activity than observed previously with ANP, and suggests its potential utility in the treatment of asthma, in addition to being a useful research tool to evaluate NPR biology.
PMCID: PMC4070431  PMID: 23154072
PL-3994; Natriuretic peptide receptors; Atrial natriuretic peptide; Brain natriuretic peptide; Neutral endopeptidase sensitivity; Bronchodilator
7.  Inhibition of myristoylated alanine-rich C kinase substrate (MARCKS) protein inhibits ozone-induced airway neutrophilia and inflammation 
Experimental lung research  2010;36(2):75-84.
Evidence suggests inhibition of leukocyte trafficking mitigates, in part, ozone-induced inflammation. In the present study, the authors postulated that inhibition of myristoylated alanine-rich C kinase substrate (MARCKS), an 82-kDa protein with multiple biological roles, could inhibit ozone-induced leukocyte trafficking and cytokine secretions. BALB/c mice (n = 5/cohort) were exposed to ozone (100 ppb) or forced air (FA) for 4 hours. MARCKS-inhibiting peptides, MANS, BIO-11000, BIO-11006, or scrambled control peptide RNS, were intratracheally administered prior to ozone exposure. Ozone selectively enhanced bronchoalveolar lavage (BAL) levels of killer cells (KCs; 6 ± 0.9-fold), interleukin-6 (IL-6; 12.7 ± 1.9-fold), and tumor necrosis factor (TNF; 2.1 ± 0.5-fold) as compared to cohorts exposed to FA. Additionally, ozone increased BAL neutrophils by 21% ± 2% with no significant (P > .05) changes in other cell types. MANS, BIO-11000, and BIO-11006 significantly reduced ozone-induced KC secretion by 66% ± 14%, 47% ± 15%, and 71.1% ± 14%, and IL-6 secretion by 69% ± 12%, 40% ± 7%, and 86.1% ± 11%, respectively. Ozone-mediated increases in BAL neutrophils were reduced by MANS (86% ± 7%) and BIO-11006 (84% ± 2.5%), but not BIO-11000. These studies identify for the first time the novel potential of MARCKS protein inhibitors in abrogating ozone-induced increases in neutrophils, cytokines, and chemokines in BAL fluid. BIO-11006 is being developed as a treatment for chronic obstructive pulmonary disorder (COPD) and is currently being evaluated in a phase 2 clinical study.
PMCID: PMC4064305  PMID: 20205598
asthma; BIO-11006; COPD; cytokines; inflammation; MANS peptide
8.  RNA-Seq Transcriptome Profiling Identifies CRISPLD2 as a Glucocorticoid Responsive Gene that Modulates Cytokine Function in Airway Smooth Muscle Cells 
PLoS ONE  2014;9(6):e99625.
Asthma is a chronic inflammatory respiratory disease that affects over 300 million people worldwide. Glucocorticoids are a mainstay therapy for asthma because they exert anti-inflammatory effects in multiple lung tissues, including the airway smooth muscle (ASM). However, the mechanism by which glucocorticoids suppress inflammation in ASM remains poorly understood. Using RNA-Seq, a high-throughput sequencing method, we characterized transcriptomic changes in four primary human ASM cell lines that were treated with dexamethasone—a potent synthetic glucocorticoid (1 µM for 18 hours). Based on a Benjamini-Hochberg corrected p-value <0.05, we identified 316 differentially expressed genes, including both well known (DUSP1, KLF15, PER1, TSC22D3) and less investigated (C7, CCDC69, CRISPLD2) glucocorticoid-responsive genes. CRISPLD2, which encodes a secreted protein previously implicated in lung development and endotoxin regulation, was found to have SNPs that were moderately associated with inhaled corticosteroid resistance and bronchodilator response among asthma patients in two previously conducted genome-wide association studies. Quantitative RT-PCR and Western blotting showed that dexamethasone treatment significantly increased CRISPLD2 mRNA and protein expression in ASM cells. CRISPLD2 expression was also induced by the inflammatory cytokine IL1β, and small interfering RNA-mediated knockdown of CRISPLD2 further increased IL1β-induced expression of IL6 and IL8. Our findings offer a comprehensive view of the effect of a glucocorticoid on the ASM transcriptome and identify CRISPLD2 as an asthma pharmacogenetics candidate gene that regulates anti-inflammatory effects of glucocorticoids in the ASM.
PMCID: PMC4057123  PMID: 24926665
9.  Asthma in the elderly: The role of exhaled nitric oxide measurements 
Respiratory medicine  2013;107(5):785-787.
Asthma in the elderly is poorly understood because only a small minority of asthma studies have investigated this patients group. Fractional Exhaled Nitric Oxide (FENO) has been extensively studied in children and adults with asthma, but little is known about FENO in elderly asthmatics. We studied the role of serial measurements of FENO in elderly subjects with asthma.
Thirty stable asthmatics 65 years old and older were followed for one year with evaluations at baseline and every three months. We looked for associations between FENO and subjects’ demographics, comorbidities, asthma treatment, spirometric values and Asthma Control Test (ACT) scores. FENO was not elevated in our study subjects throughout the study period (mean < 30 ppb). FENO significantly increased and FEV1% decreased between first and last study visit, while ACT scores and steroid dose remained unchanged. No significant correlation was found between FENO and FEV1/FVC, other spirometric values, inhaled steroid dose or ACTscores at any time point. No associations of FENO were found with age, sex, Body Mass Index (BMI), atopic status, disease duration, presence of rhinitis or gastroesophageal reflux disease (GERD), or other medications used. Moderate asthma exacerbations did not consistently cause an increase of FENO.
In stable elderly asthmatic patients, FENO was not elevated and did not correlate with subjects’ demographics, comorbidities, treatment, symptoms or spirometric values. Routine measurements of FENO may not be clinically valuable in elderly asthmatics.
PMCID: PMC4034737  PMID: 23481173
Asthma; FENO; Elderly
10.  p38 MAPK inhibitors, IKK2 inhibitors, and TNFα inhibitors in COPD 
Current opinion in pharmacology  2012;12(3):287-292.
COPD represents a major respiratory disorder, causing significant morbidity and mortality throughout the world. While therapies exist for COPD, they are not always effective, and many patients experience exacerbations and morbidity despite current therapies. Study of the molecular mechanisms involved in the underlying physiological manifestations of COPD has yielded multiple new targets for therapeutic intervention. In this review, we discuss signaling pathways involved in COPD pathogenesis and review clinical studies of p38 MAPK inhibitors, TNFα inhibitors, and IKK2 inhibitors as potential COPD therapies.
PMCID: PMC4030417  PMID: 22365729
airways disease; inflammation; irreversible airway obstruction; airway remodeling
11.  C-027 Inhibits IgE-mediated passive sensitization bronchoconstriction and acts as a histamine and serotonin antagonist in human airways 
Atopic asthma is poorly controlled by current therapies. Newer therapies and novel antihistamines are, therefore, required to treat patients whose atopic asthma is not controlled. For the first time, C-027 is shown to antagonize histamine, IgE-mediated and serotonin-induced contraction in human airways and vessels. Human precision-cut lung slices (PCLS, 250 µm thick), containing an airway or blood vessel, were pretreated with either C-027 (2 hours) or with vehicle alone and were contracted with histamine or serotonin. Known antihistamine was used as a comparator in antihistamine studies. Also, human airways were contracted via IgE passive sensitization in the presence or absence of C-027 or fexofenadine. Affinity of C-027 toward human G-protein coupled receptors was also determined, as well as the drug's biodistribution in murine model. C-027 was shown to have the highest affinity toward human histamine and serotonin receptors. Subsequently, C-027 was shown to antagonize histamine- and serotonin-induced airway and vascular smooth muscle contraction, respectively, and histamine-released bronchocontraction mediated by IgE passive sensitization in human small airways. C-027 also inhibited histamine-mediated single-cell calcium ion release. Low levels of C-027 were found in murine brain tissue. Collectively, these data suggest that C-027 markedly inhibits IgE-induced bronchoconstriction and antagonizes histamine and serotonin-contraction with little biodistribution in the brain. The compound may offer a future therapy for allergen-induced airway hyperresponsiveness in patients with asthma.
PMCID: PMC3968313  PMID: 22195688
Airway smooth muscle; allergy; antagonist; anti-histamine; anti-serotonic; asthma; IgE mediated; novel compound; PCLS; reverse agonist
12.  Reviewer acknowledgement 2013 
Respiratory Research  2014;15(1):7.
Contributing reviewers
The editors of Respiratory Research would like to thank all of our reviewers who have contributed to the journal in Volume 14 (2013).
PMCID: PMC3910687
13.  Raf-1, Actin Dynamics, and Abelson Tyrosine Kinase in Human Airway Smooth Muscle Cells 
Raf-1 is a serine/threonine protein kinase that has an essential role in cell proliferation. The mechanisms that regulate Raf-1 in airway smooth muscle are not well understood. In this study, treatment with platelet-derived growth factor (PDGF) induced spatial redistribution of Raf-1 from the cytoplasm to the periphery of human airway smooth muscle cells. Moreover, a pool of Raf-1 was found in F-actin of human airway smooth muscle cells. Activation with PDGF led to an increase in the association of Raf-1 with cytoskeletal actin. Treatment of cells with the actin polymerization inhibitor latrunculin A (LAT-A), but not the microtubule depolymerizer nocodazole, inhibited the interaction of Raf-1 with actin in response to PDGF activation. Because abelson tyrosine kinase (Abl) is known to specifically regulate actin dynamics in smooth muscle, the role of Abl in modulating the coupling of Raf-1 with actin was also evaluated. Abl knockdown by RNA interference attenuated the association of Raf-1 with actin, which is recovered by Abl rescue. Treatment with LAT-A, but not nocodazole, inhibited the spatial redistribution of Raf-1 during PDGF activation. However, treatment with both LAT-A and nocodazole attenuated smooth muscle cell proliferation. Finally, Abl knockdown attenuated the redistribution of Raf-1 and cell proliferation, which were restored by Abl reexpression. The results suggest a novel mechanism that the interaction of Raf-1 with cytoskeletal actin is critical for Raf-1 redistribution and airway smooth muscle cell proliferation during activation with the growth factor.
PMCID: PMC3604063  PMID: 23087049
Raf-1; actin polymerization; tyrosine kinase; smooth muscle; cell proliferation
14.  DHEA-S inhibits human neutrophil and human airway smooth muscle migration 
Biochimica et biophysica acta  2012;1822(10):1638-1642.
Airways diseases such as asthma, emphysema, and chronic bronchitis are, in part, characterized by reversible airflow obstruction and inflammation. In severe disease, marked decreases in lung function are associated with airway smooth muscle proliferation and airway neutrophilia. Inhaled glucocorticoids attenuate increased airflow obstruction and airway inflammation that occur, in part, due to increased smooth muscle migration and proliferation, as well as the airway neutrophilia. Glucocorticoids, however, have adverse side effects and, in some patients, are ineffective despite high doses. Recent research has explored the effects of non-traditional steroids on attenuation of inflammation associated with airways diseases. These non-traditional steroids have improved side effect profiles in comparison to glucocorticoid therapy. Our studies assessed effects of dehydroepiandrosterone-3-sulfate (DHEA-S) on migration of both human peripheral blood neutrophils (PMN) and human airway smooth muscle cells (HASM). DHEA-S dose-dependently inhibited chemotaxis of PMN and HASM while having no effect on the phosphorylation levels of Akt, ERK1/2, p38 MAPK or PKC, canonical positive regulators of cell migration. These studies demonstrate direct effects of DHEA-S on cell migration, thereby suggesting that DHEA-S may attenuate airway inflammation and cell migration.
PMCID: PMC3418395  PMID: 22771498
airway inflammation; DHEA-S; airway remodeling
15.  Regulation of CD38 Expression in Human Airway Smooth Muscle Cells 
The ADP-ribosyl cyclase activity of CD38 generates cyclic ADP-ribose, a Ca2+–mobilizing agent. In human airway smooth muscle (HASM) cells, TNF-α mediates CD38 expression through mitogen-activated protein kinases and NF-κB and AP-1. The phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway is involved in TNF-α signaling and contributes to airway hyperresponsiveness and airway remodeling. We hypothesized that PI3Ks mediate CD38 expression and are involved in the differential induction of CD38 by TNF-α in asthmatic HASM cells. HASM cells were treated with pan-PI3K inhibitors (LY294002 or wortmannin) or class I–selective (GDC0941) or isoform-selective PI3K inhibitors (p110α-PIK-75 and p110β-TGX-221) with or without TNF-α. HASM cells were transfected with a catalytically active form of PI3K or phosphatase and tensin homolog (PTEN) or nontargeting or p110 isoform-targeting siRNAs before TNF-α exposure. CD38 expression and activation of Akt, NF-κB, and AP-1 were determined. LY294002 and wortmannin inhibited TNF-α–induced Akt activation, whereas only LY294002 inhibited CD38 expression. P110 expression caused Akt activation and basal and TNF-α–induced CD38 expression, whereas PTEN expression attenuated Akt activation and CD38 expression. Expression levels of p110 isoforms α, β, and δ were comparable in nonasthmatic and asthmatic HASM cells. Silencing of p110α or -δ, but not p110β, resulted in comparable attenuation of TNF-α–induced CD38 expression in asthmatic and nonasthmatic cells. NF-κB and AP-1 activation were unaltered by the PI3K inhibitors. In HASM cells, regulation of CD38 expression occurs by specific class I PI3K isoforms, independent of NF-κB or AP-1 activation, and PI3K signaling may not be involved in the differential elevation of CD38 in asthmatic HASM cells.
PMCID: PMC3488627  PMID: 22556157
smooth muscle; airway; CD38; PI3 kinase; PI3 kinase isoforms
16.  Airway Smooth Muscle in Asthma: Just a Target for Bronchodilation? 
Clinics in chest medicine  2012;33(3):543-558.
Airway smooth muscle (ASM) has long been recognized as the main cell type responsible for bronchial hyperresponsiveness. It has thus been considered as a target for bronchodilation. In asthma however, there is a complex relationship between ASM and inflammatory cells such as mast cells and T lymphocytes. Moreover, the increased ASM mass in the asthmatic airways is one of the key features of airway remodeling. This article aims to review the main concepts about the three possible roles of ASM in asthma including (i) contractile tone, (ii) inflammatory response and (iii) remodeling.
PMCID: PMC3431506  PMID: 22929101
Bronchodilators; Hyperresponsiveness; Inflammation; Remodeling; Smooth muscle
17.  The effect of multiple allergen immunotherapy on exhaled nitric oxide in adults with allergic rhinitis 
There is a lack of objective measures of the clinical efficacy of allergen immunotherapy which relies on patients’ perception about the effect of this treatment. We studied whether the fraction of exhaled nitric oxide is affected by multiple allergen immunotherapy in polysensitized adult subjects with allergic rhinitis. We also looked for associations between exhaled nitric oxide and subjects’ demographics, symptom scores, and pulmonary function tests.
Twenty adult, polysensitized subjects with seasonal and perennial allergic rhinitis who chose to undergo allergen immunotherapy were enrolled. They were evaluated at baseline, and 4, 8, 12, 24, and 52 weeks later. Exhaled nitric oxide was reported as the mean of triplicate determinations.
Our results indicate that multiple allergen immunotherapy did not affect exhaled nitric oxide levels and such levels did not correlate with subjects’ demographics and pulmonary function tests. However, exhaled nitric oxide was associated with rhinoconjuctivitis and asthma symptom scores at the end of the study.
In polysensitized adult subjects with allergic rhinitis, exhaled nitric oxide levels are unaffected by multiple allergen immunotherapy.
PMCID: PMC3751795  PMID: 23958488
Allergen immunotherapy; Nitric oxide; Allergic rhinitis
18.  Drug Development for Severe Asthma: What Are the Metrics? 
Pharmacology & therapeutics  2012;135(2):176-181.
Although reversible airway obstruction in part defines asthma, lung function as measured by spirometry alone inadequately predicts the value of new therapeutic agents in the treatment of severe asthma.
Our objectives are 1) to review whether pulmonary function and bronchodilator reversibility are endpoints for drug discovery and 2) to identify parameters that predict efficacy in drug development in severe asthma.
An English language literature search using MedLine and PubMed was conducted from 1997 to present concerning pathophysiology, diagnosis and therapy of severe asthma using the terms “severe asthma,” “irreversible asthma,” “difficult asthma,” “airway remodeling,” “fixed airway obstruction,” “reversibility” and “bronchodilator reversibility” as index terms. Eight studies were characterized that encompass 1,424 subjects with asthma.
Our review identified the limitations of using bronchodilator reversibility as a predictor in drug development for severe asthma. Neither improvement in lung function nor bronchodilator reversibility characterized the benefit of new drugs in the treatment of severe asthma. Newly approved drugs in the treatment of severe asthma show decreased asthma exacerbations and improved quality of life associated with steroid-sparing benefits without altering bronchodilator responsiveness or improving lung function.
Although changes in lung function predict asthma control in mild/moderate asthma, lung function alone is inadequate to assess improvement in asthma control in severe asthma manifested by fixed airway obstruction. Endpoints that focus on asthma control, as defined by the Expert Panel Report 3 and GINA guidelines, may predict the value of new therapeutics in the management of severe asthma.
PMCID: PMC3383351  PMID: 22627271
Airway remodeling; fixed airway obstruction; irreversible asthma; drug discovery
19.  Expression and coupling of neurokinin receptor subtypes to inositol phosphate and calcium signaling pathways in human airway smooth muscle cells 
Neuropeptide tachykinins (substance P, neurokinin A, and neurokinin B) are present in peripheral terminals of sensory nerve fibers within the respiratory tract and cause airway contractile responses and hyperresponsiveness in humans and most mammalian species. Three subtypes of neurokinin receptors (NK1R, NK2R, and NK3R) classically couple to Gq protein-mediated inositol 1,4,5-trisphosphate (IP3) synthesis and liberation of intracellular Ca2+, which initiates contraction, but their expression and calcium signaling mechanisms are incompletely understood in airway smooth muscle. All three subtypes were identified in native and cultured human airway smooth muscle (HASM) and were subsequently overexpressed in HASM cells using a human immunodeficiency virus-1-based lentivirus transduction system. Specific NKR agonists {NK1R, [Sar9,Met(O2)11]-substance P; NK2R, [β-Ala8]-neurokinin A(4–10); NK3R, senktide} stimulated inositol phosphate synthesis and increased intracellular Ca2+ concentration ([Ca2+]i) in native HASM cells and in HASM cells transfected with each NKR subtype. These effects were blocked by NKR-selective antagonists (NK1R, L-732138; NK2R, GR-159897; NK3R, SB-222200). The initial transient and sustained phases of increased [Ca2+]i were predominantly inhibited by the IP3 receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) or the store-operated Ca2+ channel antagonist SKF-96365, respectively. These results show that all three subtypes of NKRs are expressed in native HASM cells and that IP3 levels are the primary mediators of NKR-stimulated initial [Ca2+]i increases, whereas store-operated Ca2+ channels mediate the sustained phase of the [Ca2+]i increase.
PMCID: PMC3650481  PMID: 18203813
intracellular calcium; ryanodine; actin; myosin; lentivirus
20.  Reviewer acknowledgement 2012 
Respiratory Research  2013;14(1):12.
Contributing reviewers
The editors of Respiratory Research would like to thank all of our reviewers who have contributed to the journal in Volume 13 (2012).
PMCID: PMC3584980
21.  Trichostatin A Abrogates Airway Constriction, but Not Inflammation, in Murine and Human Asthma Models 
Histone deacetylase (HDAC) inhibitors may offer novel approaches in the treatment of asthma. We postulate that trichostatin A (TSA), a Class 1 and 2 inhibitor of HDAC, inhibits airway hyperresponsiveness in antigen-challenged mice. Mice were sensitized and challenged with Aspergillus fumigatus antigen (AF) and treated with TSA, dexamethasone, or vehicle. Lung resistance (RL) and dynamic compliance were measured, and bronchial alveolar lavage fluid (BALF) was analyzed for numbers of leukocytes and concentrations of cytokines. Human precision-cut lung slices (PCLS) were treated with TSA and their agonist-induced bronchoconstriction was measured, and TSA-treated human airway smooth muscle (ASM) cells were evaluated for the agonist-induced activation of Rho and intracellular release of Ca2+. The activity of HDAC in murine lungs was enhanced by antigen and abrogated by TSA. TSA also inhibited methacholine (Mch)-induced increases in RL and decreases in dynamic compliance in naive control mice and in AF-sensitized and -challenged mice. Total cell counts, concentrations of IL-4, and numbers of eosinophils in BALF were unchanged in mice treated with TSA or vehicle, whereas dexamethasone inhibited the numbers of eosinophils in BALF and concentrations of IL-4. TSA inhibited the carbachol-induced contraction of PCLS. Treatment with TSA inhibited the intracellular release of Ca2+ in ASM cells in response to histamine, without affecting the activation of Rho. The inhibition of HDAC abrogates airway hyperresponsiveness to Mch in both naive and antigen-challenged mice. TSA inhibits the agonist-induced contraction of PCLS and mobilization of Ca2+ in ASM cells. Thus, HDAC inhibitors demonstrate a mechanism of action distinct from that of anti-inflammatory agents such as steroids, and represent a promising therapeutic agent for airway disease.
PMCID: PMC3297166  PMID: 22298527
HDAC; asthma; allergen; mice; trichostatin A
22.  The nuclear membrane leukotriene synthetic complex is a signal integrator and transducer 
Molecular Biology of the Cell  2012;23(22):4456-4464.
Leukotrienes are bioactive signaling molecules derived from arachidonic acid that initiate and amplify innate immunity. A single structure, the leukotriene synthetic complex, on the nuclear membrane of neutrophils integrates and transduces extracellular signals to generate the chemotactic lipid LTB4.
Leukotrienes (LTs) are lipid-signaling molecules derived from arachidonic acid (AA) that initiate and amplify inflammation. To initiate LT formation, the 5-lipoxygenase (5-LO) enzyme translocates to nuclear membranes, where it associates with its scaffold protein, 5-lipoxygenase–activating protein (FLAP), to form the core of the multiprotein LT synthetic complex. FLAP is considered to function by binding free AA and facilitating its use as a substrate by 5-LO to form the initial LT, LTA4. We used a combination of fluorescence lifetime imaging microscopy, cell biology, and biochemistry to identify discrete AA-dependent and AA-independent steps that occur on nuclear membranes to control the assembly of the LT synthetic complex in polymorphonuclear leukocytes. The association of AA with FLAP changes the configuration of the scaffold protein, enhances recruitment of membrane-associated 5-LO to form complexes with FLAP, and controls the closeness of this association. Granulocyte monocyte colony–stimulating factor provides a second AA-independent signal that controls the closeness of 5-LO and FLAP within complexes but not the number of complexes that are assembled. Our results demonstrate that the LT synthetic complex is a signal integrator that transduces extracellular signals to modulate the interaction of 5-LO and FLAP.
PMCID: PMC3496618  PMID: 23015755
23.  β2-Adrenergic Receptor Agonists Modulate Human Airway Smooth Muscle Cell Migration via Vasodilator-Stimulated Phosphoprotein 
Severe asthma manifests as airway remodeling and irreversible airway obstruction, in part because of the proliferation and migration of human airway smooth muscle (HASM) cells. We previously reported that cyclic adenosine monophosphate–mobilizing agents, including β2-adrenergic receptor (β2AR) agonists, which are mainstay of asthma therapy, and prostaglandin E2 (PGE2), inhibit the migration of HASM cells, although the mechanism for this migration remains unknown. Vasodilator-stimulated phosphoprotein (VASP), an anticapping protein, modulates the formation of actin stress fibers during cell motility, and is negatively regulated by protein kinase A (PKA)–specific inhibitory phosphorylation at serine 157 (Ser157). Here, we show that treatment with β2AR agonists and PGE2 induces the PKA-dependent phosphorylation of VASP and inhibits the migration of HASM cells. The stable expression of PKA inhibitory peptide and the small interfering (si) RNA-induced depletion of VASP abolish the inhibitory effects of albuterol and PGE2 on the migration of HASM cells. Importantly, prolonged treatment with albuterol prevents the agonist-induced phosphorylation of VASP at Ser157, and reverses the inhibitory effects of albuterol and formoterol, but not PGE2, on the basal and PDGF-induced migration of HASM cells. Collectively, our data demonstrate that β2AR agonists selectively inhibit the migration of HASM cells via a β2AR/PKA/VASP signaling pathway, and that prolonged treatment with albuterol abolishes the inhibitory effect of β-agonists on the phosphorylation of VASP and migration of HASM cells because of β2AR desensitization.
PMCID: PMC3262659  PMID: 22210825
airway hyperresponsiveness; β2-adrenergic receptor desensitization; protein kinase A; albuterol; formoterol
24.  Gi-Coupled γ-Aminobutyric Acid–B Receptors Cross-Regulate Phospholipase C and Calcium in Airway Smooth Muscle 
γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system, and exerts its actions via both ionotropic (GABAA) and metabotropic (GABAB) receptors. Although the functional expression of GABAB receptors coupled to the Gi protein was reported for airway smooth muscle, the role of GABAB receptors in airway responsiveness remains unclear. We investigated whether Gi-coupled GABAB receptors cross-regulate phospholipase C (PLC), an enzyme classically regulated by Gq-coupled receptors in human airway smooth muscle cells. Both the GABAB-selective agonist baclofen and the endogenous ligand GABA significantly increased the synthesis of inositol phosphate, whereas GABAA receptor agonists, muscimol, and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol exerted no effect. The baclofen-induced synthesis of inositol phosphate and transient increases in [Ca2+]i were blocked by CGP35348 and CGP55845 (selective GABAB antagonists), pertussis toxin (PTX, which inactivates the Gi protein), gallein (a Gβγ signaling inhibitor), U73122 (an inhibitor of PLC-β), and xestospongin C, an inositol 1,4,5-triphosphate receptor blocker. Baclofen also potentiated the bradykinin-induced synthesis of inositol phosphate and transient increases in [Ca2+]i, which were blocked by CGP35348 or PTX. Moreover, baclofen potentiated the substance P–induced contraction of airway smooth muscle in isolated guinea pig tracheal rings. In conclusion, the stimulation of GABAB receptors in human airway smooth muscle cells rapidly mobilizes intracellular Ca2+ stores by the synthesis of inositol phosphate via the activation of PLC-β, which is stimulated by Gβγ protein liberated from Gi proteins coupled to GABAB receptors. Furthermore, crosstalk between GABAB receptors and Gq-coupled receptors potentiates the synthesis of inositol phosphate, transient increases in [Ca2+]i, and smooth muscle contraction through Gi proteins.
PMCID: PMC3262669  PMID: 21719794
Gi protein; Gβγ; inositol phosphate; phospholipase C; airway smooth muscle
25.  Regulator of G-Protein Signaling–5 Inhibits Bronchial Smooth Muscle Contraction in Severe Asthma 
Severe asthma is associated with fixed airway obstruction attributable to inflammation, copious luminal mucus, and increased airway smooth muscle (ASM) mass. Paradoxically, studies demonstrated that the hypertrophic and hyperplastic ASM characteristic of severe asthma has reduced contractile capacity. We compared the G-protein–coupled receptor (GPCR)–induced Ca2+ mobilization and expression of GPCRs and signaling proteins related to procontractile signaling in ASM derived postmortem from subjects who died of nonrespiratory causes, with cells from subjects who died of asthma. Despite the increased or comparable expression of contraction-promoting GPCRs (bradykinin B2 or histamine H1 and protease-activated receptor 1, respectively) in asthmatic ASM cells relative to cells from healthy donors, asthmatic ASM cells exhibited reduced histamine-induced Ca2+ mobilization and comparable responses to bradykinin and thrombin, suggesting a postreceptor signaling defect. Accordingly, the expression of regulator of G-protein signaling–5 (RGS5), an inhibitor of ASM contraction, was increased in cultured, asthmatic ASM cells and in bronchial smooth muscle bundles of both human subjects with asthma and allergen-challenged mice, relative to those of healthy human subjects or naive mice. The overexpression of RGS5 impaired the release of Ca2+ to thrombin, histamine, and carbachol, and reduced the contraction of precision-cut lung slices to carbachol. These results suggest that increased RGS5 expression contributes to decreased myocyte shortening in severe and fatal asthma.
PMCID: PMC3380291  PMID: 22281988
asthma; bronchial smooth muscle; signal transduction; G-protein–coupled receptors

Results 1-25 (52)