Alan J. Knox

REBOUND INCREASE IN BRONCHIAL RESPONSIVENESS AFTER TREATMENT WITH INHALED TERBUTALINE

To investigate whether cessation of regular beta-agonist treatment results in an increase in bronchial responsiveness, two double-blind, randomised crossover studies were done. Subjects with mild asthma were investigated to determine the course of change in bronchial responsiveness, measured as the provocative dose (PD20) of histamine that caused a 20% fall in forced expiratory volume in 1 s after short-term and longer term treatment with an inhaled beta-agonist. In the first study in 8 subjects, 500 and 2000 micrograms terbutaline thrice in 1 day protected against histamine-induced bronchoconstriction, and the increase in PD20 compared with placebo remained high throughout the day and overnight. In the second study 8 subjects received placebo or terbutaline 750 micrograms thrice daily for 14 days. The protection afforded by terbutaline against histamine-induced bronchoconstriction on day 14 was less than that on day 1 by 40% in the morning and 82% in the afternoon. On day 15 PD20 was lower after stopping terbutaline than placebo, with a maximum difference of 1.5 (95% CI 0.6-2.5) doubling-doses of histamine 23 h after the end of treatment. Thus treatment with terbutaline for 1 day did not result in any rebound increase in bronchial responsiveness. Treatment for 2 weeks impaired the ability of terbutaline to protect against histamine-induced bronchoconstriction, and was followed by a rebound increase in bronchial responsiveness after cessation of treatment.

Guidelines for the management of hospital-acquired pneumonia in the UK: Report of the Working Party on Hospital-Acquired Pneumonia of the British Society for Antimicrobial Chemotherapy

Abstract These evidence-based guidelines have been produced after a systematic literature review of a range of issues involving prevention, diagnosis and treatment of hospital-acquired pneumonia (HAP). Prevention is structured into sections addressing general issues, equipment, patient procedures and the environment, whereas in treatment, the structure addresses the use of antimicrobials in prevention and treatment, adjunctive therapies and the application of clinical protocols. The sections dealing with diagnosis are presented against the clinical, radiological and microbiological diagnosis of HAP. Recommendations are also made upon the role of invasive sampling and quantitative microbiology of respiratory secretions in directing antibiotic therapy in HAP/ventilator-associated pneumonia.

Dietary magnesium, lung function, wheezing, and airway hyperreactivity in a random adult population sample

Magnesium is involved in a wide range of biological activities, including some that may protect against the development of asthma and chronic airflow obstruction. We tested the hypothesis that high dietary magnesium intake is associated with better lung function, and a reduced risk of airway hyper-reactivity and wheezing in a random sample of adults. In 2633 adults aged 18-70 sampled from the electoral register of an administrative area of Nottingham, UK, we measured dietary magnesium intake by semiquantitative food-frequency questionnaire, lung function as the 1-sec forced expiratory volume (FEV1), and atopy as the mean skin-prick test response to three common environmental allergens. We measured airway reactivity to methacholine in 2415 individuals, defining hyper-reactivity as a 20% fall in FEV1 after a cumulative dose of 12.25 mumol or less. Mean (SD) daily intake of magnesium was 380 (114) mg/day. After adjusting for age, sex, and height, and for the effects of atopy and smoking, a 100 mg/day higher magnesium intake was associated with a 27.7 (95% CI, 11.9-43.5) mL higher FEV1, and a reduction in the relative odds of hyper-reactivity by a ratio of 0.82 (0.72-0.93). The same incremental difference in magnesium intake was also associated with a reduction in the odds of self-reported wheeze within the past 12 months, adjusted for age, sex, smoking, atopy, and kilojoule intake, by a ratio of 0.85 (0.76-0.95). Dietary magnesium intake is independently related to lung function and the occurrence of airway hyper-reactivity and self-reported wheezing in the general population. Low magnesium intake may therefore be involved in the aetiology of asthma and chronic obstructive airways disease.

Effect of interleukin-1β, tumour necrosis factor-α and interferon-γ on the induction of cyclo-oxygenase-2 in cultured human airway smooth muscle cells

Increased levels of several pro‐inflammatory cytokines including interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNFα) have been found in bronchoalveolar lavage fluid from symptomatic asthmatic patients. IL‐1β, TNFα and interferon‐γ (IFNγ) are known to stimulate a number of cells to produce inflammatory mediators such as prostaglandins. Although airway smooth muscle (ASM) is known to be a rich source of prostaglandins, the regulation of cyclo‐oxygenase (COX) isoforms and prostanoid production by proinflammatory cytokines have not been studied in human airway smooth muscle. We studied the effects of IL‐1β, TNFα and IFNγ on the induction of two isoforms of cyclo‐oxygenase and its relation to prostaglandin E2 (PGE2) release and COX activity (reflected by PGE2 synthesis from exogenous arachidonic acid) in human cultured airway smooth muscle cells. IL‐1β, but not TNFα or IFNγ, caused a time‐ and concentration‐dependent enhancement in PGE2 and other prostanoid (6‐keto‐PGF1α, PGF2α, thromboxane B2 (TXB2) and PGD2) production, with PGE2 and 6‐keto‐PGF1α as the principal products. This stimulation was accompanied by a corresponding increase in COX activity. COX‐2 protein measured by Western blot analysis was not detectable in untreated cells, but was increased in a time‐ and concentration‐dependent manner by IL‐1β, but not TNFα or IFNγ. In contrast, no variation in the expression of COX‐1 protein was observed. Pretreatment with the conventional non‐steroidal anti‐inflammatory drugs (NSAIDs), indomethacin and ibuprofen, and the selective COX‐2 inhibitors, NS‐398 and nimesulide, completely blocked IL‐1β‐induced PGE2 release and COX activity. The glucocorticosteroid dexamethasone and protein synthesis inhibitors, cycloheximide and actinomycin D, not only markedly inhibited IL‐1β‐stimulated PGE2 release and COX activity but also suppressed IL‐1β‐induced COX‐2 induction. This study demonstrates that human cultured ASM cells release prostanoids in response to IL‐1β stimulation and that the response is mostly mediated by the induction of COX‐2 rather than COX‐1 isoenzyme, implying that airway smooth muscle may be an important source of prostaglandins in human airways and that COX‐2 may play an important role in the regulation of the inflammatory process in asthma.

Regulation of TNF-α-induced eotaxin release from cultured human airway smooth muscle cells by β2-agonists and corticosteroids

Eotaxin is a potent eosinophil chemoattractant that contributes to the eosinophilia seen in asthma and other allergic disorders. Recent studies have identified human airway smooth muscle (HASM) as a rich source of eotaxin, but the factors regulating its production are poorly understood. Here we describe for the first time that β2‐agonists can inhibit cytokineinduced eotaxin release. We found that TNF‐α stimulated eotaxin release (assayed by ELISA) from HASM cells and that the release was partially inhibited by salbutamol and salmeterol. The effect of β2‐agonists was mimicked by forskolin and 8‐bromo‐cAMP and potentiated by the cAMP‐dependent phosphodiesterase inhibitor rolipram, suggesting that it is cAMP dependent. We also found that the cAMP inhibition was likely at the transcription stage, although experiments with the PKA inhibitors H‐89 and Rp‐cAMP or the PKG inhibitor KT5823 suggested that none of these kinases was involved. Partial inhibition of eotaxin release was also seen with the corticosteroids dexamethasone and fluticasone. The combined use of β2‐agonists, rolipram, and steroids abolished TNF‐α‐induced eotaxin release. These results suggest that the combination of a β2‐agonist, PDE inhibitor, and a corticosteroid may have additive beneficial effects in the treatment of the eosinophilia associated with asthma and other allergic diseases—Pang, L., Knox, A. J. Regulation of TNF‐α‐induced eotaxin release from cultured human airway smooth muscle cells by β2‐agonists and corticosteroids. FASEB J. 15, 261–269 (2001)

Once versus three-times daily regimens of tobramycin treatment for pulmonary exacerbations of cystic fibrosis--the TOPIC study: a randomised controlled trial.

BACKGROUND Intravenous tobramycin (three-times daily) is widely used for pulmonary exacerbations in patients with cystic fibrosis who have chronic Pseudomonas aeruginosa infection. We undertook a double-blind, randomised controlled trial to assess the safety and efficacy of once versus three-times daily tobramycin in these patients. METHODS 244 patients from 21 cystic-fibrosis centres in the UK were randomly assigned to once or three-times daily tobramycin (with ceftazidime) for 14 days. Treatment was given as 30-min infusions of tobramycin in 0.9% saline. Primary outcome measure was change in forced expiratory volume in 1s (FEV1), over the 14 days of treatment, expressed as a percentage of the predicted normal value for age, sex, and height. We also measured the change in FEV1 expressed as a percentage of baseline. Secondary outcomes included change in serum creatinine. The study was powered for equivalence, and primary analysis was per protocol. FINDINGS 219 patients (107 once daily, 112 three-times daily) completed the study per protocol. None was lost to follow-up, although 20 discontinued intervention. Of 122 patients assigned to once daily treatment, three did not receive the study regimen. The mean change in FEV1 (% predicted) over 14 days was similar on the two regimens (10.4% [once daily] vs 10.0% [three-times daily]; adjusted mean difference 0.4% [95% CI -3.3 to 4.1]). Mean% change in FEV1 from baseline was also similar in both treatments (21.9% vs 22.1%; -0.1% [-8.0 to 7.9]). There was no significant difference in% change in creatinine from baseline (-1.5% [once daily] vs 1.7% [three-times daily]). However, in children, once daily treatment was significantly less nephrotoxic than was thrice daily (mean% change in creatine -4.5% [once daily] vs 3.7% [thrice daily]; adjusted mean difference -8.0%, 95% CI -15.7 to -0.4). No patients developed hearing loss during the study, although two reported acute dizziness and were withdrawn from the study. INTERPRETATION Intravenous tobramycin has equal efficacy if given once or three-times daily (with ceftazidime) for pulmonary exacerbations of cystic fibrosis. The once daily regimen might be less nephrotoxic in children.

Lysophosphatidic Acid Induces αvβ6 Integrin-Mediated TGF-β Activation via the LPA2 Receptor and the Small G Protein Gαq

Activation of latent transforming growth factor beta (TGF-beta) by alphavbeta6 integrin is critical in the pathogenesis of lung injury and fibrosis. We have previously demonstrated that the stimulation of protease activated receptor 1 promotes alphavbeta6 integrin-mediated TGF-beta activation via RhoA, which is known to modulate cell contraction. However, whether other G protein-coupled receptors can also induce alphavbeta6 integrin-mediated TGF-beta activation is unknown; in addition, the alphavbeta6 integrin signaling pathway has not yet been fully characterized. In this study, we show that lysophosphatidic acid (LPA) induces alphavbeta6-mediated TGF-beta activation in human epithelial cells via both RhoA and Rho kinase. Furthermore, we demonstrate that LPA-induced alphavbeta6 integrin-mediated TGF-beta activity is mediated via the LPA2 receptor, which signals via G alpha(q). Finally, we show that the expression levels of both the LPA2 receptor and alphavbeta6 integrin are up-regulated and are spatially and temporally associated following bleomycin-induced lung injury. Furthermore, both the LPA2 receptor and alphavbeta6 integrin are up-regulated in the overlying epithelial areas of fibrosis in patients with usual interstitial pneumonia. These studies demonstrate that LPA induces alphavbeta6 integrin-mediated TGF-beta activation in epithelial cells via LPA2, G alpha(q), RhoA, and Rho kinase, and that this pathway might be clinically relevant to the development of lung injury and fibrosis.

Clinical and inflammatory characteristics of the European U-BIOPRED adult severe asthma cohort

U-BIOPRED is a European Union consortium of 20 academic institutions, 11 pharmaceutical companies and six patient organisations with the objective of improving the understanding of asthma disease mechanisms using a systems biology approach. This cross-sectional assessment of adults with severe asthma, mild/moderate asthma and healthy controls from 11 European countries consisted of analyses of patient-reported outcomes, lung function, blood and airway inflammatory measurements. Patients with severe asthma (nonsmokers, n=311; smokers/ex-smokers, n=110) had more symptoms and exacerbations compared to patients with mild/moderate disease (n=88) (2.5 exacerbations versus 0.4 in the preceding 12 months; p<0.001), with worse quality of life, and higher levels of anxiety and depression. They also had a higher incidence of nasal polyps and gastro-oesophageal reflux with lower lung function. Sputum eosinophil count was higher in severe asthma compared to mild/moderate asthma (median count 2.99% versus 1.05%; p=0.004) despite treatment with higher doses of inhaled and/or oral corticosteroids. Consistent with other severe asthma cohorts, U-BIOPRED is characterised by poor symptom control, increased comorbidity and airway inflammation, despite high levels of treatment. It is well suited to identify asthma phenotypes using the array of “omic” datasets that are at the core of this systems medicine approach. Severe asthma results in more airway inflammation, worse symptoms and lower lung function, despite increased therapy http://ow.ly/QznR3

Human airway smooth muscle cells secrete vascular endothelial growth factor: up-regulation by bradykinin via a protein kinase C and prostanoid-dependent mechanism

Bronchial vascular remodeling is an important feature of the pathology of chronic asthma, but the responsible mechanisms and main sources of an‐giogenic factors are unclear. Here we report that human airway smooth muscle cells express vascular endo‐thelial growth factor (VEGF)121,165,189,206 splice variants and secrete VEGF protein constitutively. VEGF protein secretion was increased by the proinflammatory asthma mediator bradykinin through post‐transcrip‐tional mechanisms. Bradykinin‐induced VEGF secretion was dependent on the B2 bradykinin receptor activation of protein kinase C and generation of endogenous prostanoids. This is the first report that bradykinin can increase VEGF secretion in any biological system and the first to show that airway smooth muscle cells produce VEGF. Our results suggest a novel role for human airway smooth muscle in contributing to bronchial mucosal angiogenesis in chronic asthma by secretion of VEGF and suggest a wider role for mesen‐chymal cell products in mediating angiogenesis in inflammatory and allergic diseases.—Knox, A. J., Corbett, L., Stocks, J., Holland, E., Zhu, Y. M., Pang, L. Human airway smooth muscle cells secrete vascular endothelial growth factor: up‐regulation by bradykinin via a protein kinase C and prostanoid‐dependent mechanism. FASEB J. 15, 2480–2488 (2001)

Defective Histone Acetylation Is Responsible for the Diminished Expression of Cyclooxygenase 2 in Idiopathic Pulmonary Fibrosis

ABSTRACT Diminished cyclooxygenase 2 (COX-2) expression in fibroblasts, with a resultant defect in the production of the antifibrotic mediator prostaglandin E2, plays a key role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Here, we have characterized the molecular mechanism. We found that COX-2 mRNA levels in fibroblasts from patients with IPF (F-IPF) were significantly lower than those in fibroblasts from nonfibrotic lungs (F-NL) after transforming growth factor β1 and interleukin-1β treatment but that COX-2 mRNA degradation rates were similar, suggesting defective transcription. A reporter gene assay showed that there were no clear differences between F-IPF and F-NL in transcription factor involvement and activation in COX-2 gene transcription. However, a chromatin immunoprecipitation assay revealed that transcription factor binding to the COX-2 promoter in F-IPF was reduced compared to that in F-NL, an effect that was dynamically linked to reduced histone H3 and H4 acetylation due to decreased recruitment of histone acetyltransferases (HATs) and increased recruitment of transcriptional corepressor complexes to the COX-2 promoter. The treatment of F-IPF with histone deacetylase (HDAC) inhibitors together with cytokines increased histone H3 and H4 acetylation. Both HDAC inhibitors and the overexpression of HATs restored cytokine-induced COX-2 mRNA and protein expression in F-IPF. The results demonstrate that epigenetic abnormality in the form of histone hypoacetylation is responsible for diminished COX-2 expression in IPF.