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1.  Antipseudomonal Bacteriophage Reduces Infective Burden and Inflammatory Response in Murine Lung 
As antibiotic resistance increases, there is a need for new therapies to treat infection, particularly in cystic fibrosis (CF), where Pseudomonas aeruginosa is a ubiquitous pathogen associated with increased morbidity and mortality. Bacteriophages are an attractive alternative treatment, as they are specific to the target bacteria and have no documented side effects. The efficacy of phage cocktails was established in vitro. Two P. aeruginosa strains were taken forward into an acute murine infection model with bacteriophage administered either prophylactically, simultaneously, or postinfection. The infective burden and inflammation in bronchoalveolar lavage fluid (BALF) were assessed at various times. With low infective doses, both control mice and those undergoing simultaneous phage treatment cleared P. aeruginosa infection at 48 h, but there were fewer neutrophils in BALF of phage-treated mice (median, 73.2 × 104/ml [range, 35.2 to 102.1 × 104/ml] versus 174 × 104/ml [112.1 to 266.8 × 104/ml], P < 0.01 for the clinical strain; median, 122.1 × 104/ml [105.4 to 187.4 × 104/ml] versus 206 × 104/ml [160.1 to 331.6 × 104/ml], P < 0.01 for PAO1). With higher infective doses of PAO1, all phage-treated mice cleared P. aeruginosa infection at 24 h, whereas infection persisted in all control mice (median, 1,305 CFU/ml [range, 190 to 4,700 CFU/ml], P < 0.01). Bacteriophage also reduced CFU/ml in BALF when administered postinfection (24 h) and both CFU/ml and inflammatory cells in BALF when administered prophylactically. A reduction in soluble inflammatory cytokine levels in BALF was also demonstrated under different conditions. Bacteriophages are efficacious in reducing both the bacterial load and inflammation in a murine model of P. aeruginosa lung infection. This study provides proof of concept for future clinical trials in patients with CF.
doi:10.1128/AAC.01426-15
PMCID: PMC4750668  PMID: 26574007
2.  Variability in Self-Reported Normal Sleep Across the Adult Age Span 
Objectives.
Illustrate the importance of examining within- and between-person differences in sleep across the adult age span.
Method.
Two weeks of sleep diary data were analyzed for 592 normal sleepers ranging in age from 20 to 96 years. Variability in total sleep time (TST), number of nighttime awakenings (NWAK), sleep-onset latency (SOL), and wake-time after sleep onset (WASO) were examined overall and by age, sex, and race utilizing multilevel models and multiple regression.
Results.
Night-to-night differences in sleep within the same individual generally exceeded differences between individuals for TST, SOL, and WASO. The amount of intraindividual variability in TST and NWAK decreased with older age. Further, the degree of reduction in variability in TST associated with age depended on sex and race, with young black females showing the greatest variability. In general, females tended to have more intraindividual variability in SOL and NWAK than males, while race differences were complicated by high variability between blacks.
Discussion.
To truly assess and understand individual differences in the sleep of older adults, future research needs to take into account night-to-night variability (including what makes sleep vary from one night to the next), in addition to average sleep.
doi:10.1093/geronb/gbu035
PMCID: PMC4342722  PMID: 24829303
Age-related change; Intraindividual variability; Sleep.
3.  Pediatric severe asthma with fungal sensitization is mediated by steroid-resistant IL-33 
Background
The mechanism underlying severe asthma with fungal sensitization (SAFS) is unknown. IL-33 is important in fungus-induced asthma exacerbations, but its role in fungal sensitization is unexplored.
Objective
We sought to determine whether fungal sensitization in children with severe therapy-resistant asthma is mediated by IL-33.
Methods
Eighty-two children (median age, 11.7 years; 63% male) with severe therapy-resistant asthma were included. SAFS (n = 38) was defined as specific IgE or skin prick test response positivity to Aspergillus fumigatus, Alternaria alternata, or Cladosporium herbarum. Clinical features and airway immunopathology were assessed. Chronic exposure to house dust mite and A alternata were compared in a neonatal mouse model.
Results
Children with SAFS had earlier symptom onset (0.5 vs 1.5 years, P = .006), higher total IgE levels (637 vs 177 IU/mL, P = .002), and nonfungal inhalant allergen-specific IgE. Significantly more children with SAFS were prescribed maintenance oral steroids (42% vs 14%, P = .02). SAFS was associated with higher airway IL-33 levels. In neonatal mice A alternata exposure induced higher serum IgE levels, pulmonary IL-33 levels, and IL-13+ innate lymphoid cell (ILC) and TH2 cell numbers but similar airway hyperresponsiveness (AHR) compared with those after house dust mite exposure. Lung IL-33 levels, IL-13+ ILC numbers, TH2 cell numbers, IL-13 levels, and AHR remained increased with inhaled budesonide during A alternata exposure, but all features were significantly reduced in ST2−/− mice lacking a functional receptor for IL-33.
Conclusion
Pediatric SAFS was associated with more oral steroid therapy and higher IL-33 levels. A alternata exposure resulted in increased IL-33–mediated ILC2 numbers, TH2 cell numbers, and steroid-resistant AHR. IL-33 might be a novel therapeutic target for SAFS.
doi:10.1016/j.jaci.2015.01.016
PMCID: PMC4534777  PMID: 25746970
Severe asthma; fungal sensitization; pediatric; IL-33; innate immunity; steroid resistance; ABPA, Allergic bronchopulmonary aspergillosis; AHR, Airway hyperresponsiveness; BAL, Bronchoalveolar lavage; HDM, House dust mite; ICOS, Inducible costimulator; ILC, Innate lymphoid cell; MMP-9, Matrix metalloproteinase 9; SAFS, Severe asthma with fungal sensitization; sIgE, Specific IgE; SPT, Skin prick test; STRA, Severe therapy-resistant asthma
4.  Increased nuclear suppressor of cytokine signaling 1 in asthmatic bronchial epithelium suppresses rhinovirus induction of innate interferons 
Background
Rhinovirus infections are the dominant cause of asthma exacerbations, and deficient virus induction of IFN-α/β/λ in asthmatic patients is important in asthma exacerbation pathogenesis. Mechanisms causing this interferon deficiency in asthmatic patients are unknown.
Objective
We sought to investigate the expression of suppressor of cytokine signaling (SOCS) 1 in tissues from asthmatic patients and its possible role in impaired virus-induced interferon induction in these patients.
Methods
We assessed SOCS1 mRNA and protein levels in vitro, bronchial biopsy specimens, and mice. The role of SOCS1 was inferred by proof-of-concept studies using overexpression with reporter genes and SOCS1-deficient mice. A nuclear role of SOCS1 was shown by using bronchial biopsy staining, overexpression of mutant SOCS1 constructs, and confocal microscopy. SOCS1 levels were also correlated with asthma-related clinical outcomes.
Results
We report induction of SOCS1 in bronchial epithelial cells (BECs) by asthma exacerbation–related cytokines and by rhinovirus infection in vitro. We found that SOCS1 was increased in vivo in bronchial epithelium and related to asthma severity. SOCS1 expression was also increased in primary BECs from asthmatic patients ex vivo and was related to interferon deficiency and increased viral replication. In primary human epithelium, mouse lung macrophages, and SOCS1-deficient mice, SOCS1 suppressed rhinovirus induction of interferons. Suppression of virus-induced interferon levels was dependent on SOCS1 nuclear translocation but independent of proteasomal degradation of transcription factors. Nuclear SOCS1 levels were also increased in BECs from asthmatic patients.
Conclusion
We describe a novel mechanism explaining interferon deficiency in asthmatic patients through a novel nuclear function of SOCS1 and identify SOCS1 as an important therapeutic target for asthma exacerbations.
doi:10.1016/j.jaci.2014.11.039
PMCID: PMC4541718  PMID: 25630941
Rhinovirus; asthma; asthma exacerbation; atopy; interferon; innate immunity; cytokine; TH2 inflammation; suppressor of cytokine signaling; AA, Atopic asthma; BAL, Bronchoalveolar lavage; BEC, Bronchial epithelial cell; CISH, Cytokine-inducible SH2-containing protein; GFP, Green fluorescent protein; ISG, Interferon-stimulated gene; ISRE, Interferon-stimulated response element; KC, Keratinocyte-derived chemokine; LIX, LPS-induced CXC chemokine; NANA, Nonatopic nonasthmatic; NF-κB, Nuclear factor κB; NLS, Nuclear localization sequence; polyI:C, Polyinosinic-polycytidylic acid; SOCS, Suppressor of cytokine signaling; SOCS1wt, Full-length wild-type human SOCS1; STAT, Signal transducer and activator of transcription; STRA, Severe therapy-resistant atopic asthma
5.  Perinatal paracetamol exposure in mice does not affect the development of allergic airways disease in early life 
Thorax  2015;70(6):528-536.
Background
Current data concerning maternal paracetamol intake during pregnancy, or intake during infancy and risk of wheezing or asthma in childhood is inconclusive based on epidemiological studies. We have investigated whether there is a causal link between maternal paracetamol intake during pregnancy and lactation and the development of house dust mite (HDM) induced allergic airways disease (AAD) in offspring using a neonatal mouse model.
Methods
Pregnant mice were administered paracetamol or saline by oral gavage from the day of mating throughout pregnancy and/or lactation. Subsequently, their pups were exposed to intranasal HDM or saline from day 3 of life for up to 6 weeks. Assessments of airway hyper-responsiveness, inflammation and remodelling were made at weaning (3 weeks) and 6 weeks of age.
Results
Maternal paracetamol exposure either during pregnancy and/or lactation did not affect development of AAD in offspring at weaning or at 6 weeks. There were no effects of maternal paracetamol at any time point on airway remodelling or IgE levels.
Conclusions
Maternal paracetamol did not enhance HDM induced AAD in offspring. Our mechanistic data do not support the hypothesis that prenatal paracetamol exposure increases the risk of childhood asthma.
doi:10.1136/thoraxjnl-2014-205280
PMCID: PMC4453715  PMID: 25841236
Asthma; Allergic lung disease; Paediatric asthma
6.  Long Term Non-Invasive Ventilation in Children: Impact on Survival and Transition to Adult Care 
PLoS ONE  2015;10(5):e0125839.
Background
The number of children receiving domiciliary ventilatory support has grown over the last few decades driven largely by the introduction and widening applications of non-invasive ventilation. Ventilatory support may be used with the intention of increasing survival, or to facilitate discharge home and/or to palliate symptoms. However, the outcome of this intervention and the number of children transitioning to adult care as a consequence of longer survival is not yet clear.
Methods
In this retrospective cohort study, we analysed the outcome in children (<17 years) started on home NIV at Royal Brompton Hospital over an 18 year period 1993-2011. The aim was to establish for different diagnostic groups: survival rate, likelihood of early death depending on diagnosis or discontinuation of ventilation, and the proportion transitioning to adult care.
Results
496 children were commenced on home non invasive ventilation; follow-up data were available in 449 (91%). Fifty six per cent (n=254) had neuromuscular disease. Ventilation was started at a median age (IQR) 10 (3-15) years. Thirteen percent (n=59) were less than 1 year old. Forty percent (n=181) have transitioned to adult care. Twenty four percent (n=109) of patients have died, and nine percent (n=42) were able to discontinue ventilatory support.
Conclusion
Long term ventilation is associated with an increase in survival in a range of conditions leading to ventilatory failure in children, resulting in increasing numbers surviving to adulthood. This has significant implications for planning transition and adult care facilities.
doi:10.1371/journal.pone.0125839
PMCID: PMC4416879  PMID: 25933065
8.  Asthma in preschool children: the next challenge 
Purpose of review
To describe a prospective classification for preschool wheezers according to temporal symptom pattern, and summarise findings relating to the management of viral wheeze and the use of short term therapy for intermittent severe wheeze.
Recent findings
Phenotypes defined from cohort studies should only be applied retrospectively at school-age. A new classification that can be applied prospectively is discussed. The importance of early rhinovirus induced wheezing as a risk factor for asthma has become apparent. However, there is no benefit from short-course oral steroids for acute viral wheeze in the majority of cases. There is conflicting evidence for the role of intermittent montelukast or inhaled steroids in the treatment of acute, intermittent wheeze. A link between reduced vitamin D intake during pregnancy and increased preschool wheeze in offspring has emerged, suggesting a potential role for vitamin D supplementation in primary prevention.
Summary
Based on current evidence, a trial of bronchodilators is first line therapy for viral wheeze, and maintenance montelukast or inhaled steroids may be considered in preschool wheezers with persistent symptoms and risk factors for future asthma. No disease modifying therapies are available. New therapeutic options for preschool wheezing disorders are desperately needed.
doi:10.1097/ACI.0b013e3283292230
PMCID: PMC4254141  PMID: 19307884
preschool wheeze; management; diagnosis; phenotypes
9.  Psychological Treatment of Hypnotic-Dependent Insomnia in a Primarily Older Adult Sample 
Behaviour research and therapy  2013;51(12):10.1016/j.brat.2013.09.006.
Objective
This study tested cognitive behavior therapy (CBT) in hypnotic-dependent, late middle-age and older adults with insomnia.
Method
Seventy volunteers age 50 and older were randomized to CBT plus drug withdrawal, placebo biofeedback (PL) plus drug withdrawal, or drug withdrawal (MED) only. The CBT and PL groups received eight, 45 minute weekly treatment sessions. The drug withdrawal protocol comprised slow tapering monitored with about six biweekly, 30 minute sessions. Assessment including polysomnography (PSG), sleep diaries, hypnotic consumption, daytime functioning questionnaires, and drug screens collected at baseline, posttreatment, and 1-year follow-up.
Results
Only the CBT group showed significant sleep diary improvement, sleep onset latency significantly decreased at posttreatment. For all sleep diary measures for all groups, including MED, sleep trended to improvement from baseline to follow-up. Most PSG sleep variables did not significantly change. There were no significant between group differences in medication reduction. Compared to baseline, the three groups decreased hypnotic use at posttreatment, down 84%, and follow-up, down 66%. There was no evidence of withdrawal side-effects. Daytime functioning, including anxiety and depression, improved by posttreatment. Rigorous methodological features, including documentation of strong treatment implementation and the presence of a credible placebo, elevated the confidence due these findings.
Conclusions
Gradual drug withdrawal was associated with substantial hypnotic reduction at posttreatment and follow-up, and withdrawal side-effects were absent. When supplemented with CBT, participants accrued incremental self-reported, but not PSG, sleep benefits.
doi:10.1016/j.brat.2013.09.006
PMCID: PMC3838504  PMID: 24121096
hypnotic dependence; drug withdrawal; insomnia; cognitive behavior therapy
10.  Interleukin-33 promotes airway remodelling in paediatric severe steroid resistant asthma 
Background
Th2 cytokines are not responsible for the on-going symptoms and pathology in children with severe therapy resistant asthma (STRA). Interleukin (IL)-33 induces airway hyperresponsiveness (AHR), but its role in airway remodelling and steroid resistance is unknown.
Objective
To investigate the relationship between IL-33 and airway remodelling in paediatric STRA.
Methods
IL-33 was quantified in neonatal mice given inhaled house dust mite (HDM), and the effect of blocking IL-13 on remodelling and IL-33 was assessed. HDM induced allergic airways disease (AAD) in neonatal ST2−/− mice lacking the IL-33 receptor was assessed, together with collagen production following IL-33 administration. Impact of steroid therapy on IL-33 levels in neonatal AAD was explored. IL-33 expression was quantified in endobronchial biopsies from children with STRA and related to remodelling, and collagen production by paediatric airway fibroblasts stimulated with IL-33 and budesonide quantified.
Results
Blocking IL-13 after AAD was established in neonatal mice did not reduce remodelling or IL-33 levels; AHR was only partially reduced. IL-33 promoted collagen synthesis both from paediatric asthmatic fibroblasts, and following intra-nasal administration in mice. Increased cellular expression of IL-33, but not IL-13, was associated with increased reticular basement membrane thickness in endobronchial biopsies from children with STRA, whilst remodelling was absent in HDM exposed ST2−/− mice. IL-33 was maintained whilst IL-13 was abrogated by steroid treatment in neonatal HDM exposed mice, and in endobronchial biopsies from children with STRA.
Conclusion
IL-33 is a relatively steroid resistant mediator that promotes airway remodelling in STRA, and is an important therapeutic target.
doi:10.1016/j.jaci.2013.04.012
PMCID: PMC4218948  PMID: 23759184
Asthma; paediatric; airway remodelling; steroid resistance; IL-33; therapy
11.  Large ABCA3 and SFTPC Deletions Resulting in Lung Disease 
Rationale: Mutations in genes encoding proteins important in the function and metabolism of pulmonary surfactant are recognized causes of lung disease. Clinical genetic testing is available for these disorders, but children with phenotypes consistent with surfactant dysfunction and no identifiable mutations in the known causative genes have been reported.
Objectives: To identify the mechanism(s) for lung disease in two children with the phenotype of surfactant dysfunction who had negative testing in clinical laboratories for gene mutations causing surfactant dysfunction.
Methods: Amplicons spanning multiple exons of candidate genes were generated by polymerase chain reaction and sequenced.
Measurements and Main Results: A 4,335-base deletion that included all of exon 12 of the gene encoding member A3 of the adenosine triphosphate–binding cassette transporter was identified in a full-term infant with respiratory failure. A 333-base deletion involving part of exon 4 and the adjacent intron of the gene encoding surfactant protein C was identified in a child with interstitial lung disease.
Conclusions: Large deletions are a cause of surfactant dysfunction disorders and may need to be sought for specifically in children whose phenotypes suggest these syndromes but in whom clinical genetic testing is unrevealing.
doi:10.1513/AnnalsATS.201306-170OC
PMCID: PMC3960967  PMID: 24024739
pulmonary surfactant; respiratory distress syndrome; interstitial lung disease; genetic basis of disease
13.  Lung Clearance Index and High-Resolution Computed Tomography Scores in Primary Ciliary Dyskinesia 
Rationale: Lung clearance index (LCI) is a more sensitive measure of lung function than spirometry in cystic fibrosis (CF) and correlates well with abnormalities in high-resolution computed tomography (HRCT) scanning. We hypothesized LCI would be equally sensitive to lung disease in primary ciliary dyskinesia (PCD).
Objectives: To test the relationships between LCI, spirometry, and HRCT in PCD and to compare them to the established relationships in CF.
Methods: Cross-sectional study of 127 patients with CF and 33 patients with PCD, all of whom had spirometry and LCI, of which a subset of 21 of each had HRCT performed. HRCT was scored for individual features and these features compared with physiological parameters.
Measurements and Main Results: Unlike in CF, and contrary to our hypothesis, there was no correlation between spirometry and LCI in PCD and no correlation between HRCT features and LCI or spirometry in PCD.
Conclusions: We show for the first time that HRCT, spirometry, and LCI have different relationships in different airway diseases and that LCI does not appear to be a sensitive test of airway disease in advanced PCD. We hypothesize that this results from dissimilarities between the components of large and small airway disease in CF and PCD. These differences may in part lead to the different prognosis in these two neutrophilic airway diseases.
doi:10.1164/rccm.201304-0800OC
PMCID: PMC3827705  PMID: 23815669
spirometry; high-resolution computed tomography; lung clearance index
15.  Passive Smoking Impairs Histone Deacetylase-2 in Children With Severe Asthma 
Chest  2013;145(2):305-312.
Background:
Parental smoking is known to worsen asthma symptoms in children and to make them refractory to asthma treatment, but the molecular mechanism is unclear. Oxidative stress from tobacco smoke has been reported to impair histone deacetylase-2 (HDAC2) via phosphoinositide-3-kinase (PI3K)/Akt activation and, thus, to reduce corticosteroid sensitivity. The aim of this study was to investigate passive smoking-dependent molecular abnormalities in alveolar macrophages (AMs) by comparing passive smoke-exposed children and non-passive smoke-exposed children with uncontrolled severe asthma.
Methods:
BAL fluid (BALF) was obtained from 19 children with uncontrolled severe asthma (10 non-passive smoking-exposed subjects and nine passive smoking-exposed subjects), and HDAC2 expression/activity, Akt/HDAC2 phosphorylation levels, and corticosteroid responsiveness in AMs were evaluated.
Results:
Parental smoking reduced HDAC2 protein expression by 54% and activity by 47%, with concomitant enhancement of phosphorylation of Akt1 and HDAC2. In addition, phosphorylation levels of Akt1 correlated positively with HDAC2 phosphorylation levels and negatively with HDAC2 activity. Furthermore, passive smoke exposure reduced the inhibitory effects of dexamethasone on tumor necrosis factor-α-induced CXCL8 release in AMs. There were relatively higher neutrophil counts and CXCL8 concentrations in BALF and lower Asthma Control Test scores compared with non-passive smoke-exposed children with uncontrolled severe asthma.
Conclusions:
Passive smoking impairs HDAC2 function via PI3K signaling activation, which could contribute to corticosteroid-insensitive inflammation in children with severe asthma. This novel mechanism will be a treatment target in children with severe asthma and stresses the need for a smoke-free environment for asthmatic children.
doi:10.1378/chest.13-0835
PMCID: PMC3913299  PMID: 24030221
16.  Methods and baseline characteristics of a randomized trial treating early childhood obesity: The Positive Lifestyles for Active Youngsters (Team PLAY) trial 
Contemporary Clinical Trials  2012;33(3):534-549.
There are few effective obesity interventions directed towards younger children, particularly young minority children. This paper describes the design, intervention, recruitment methods, and baseline data of the ongoing Positive Lifestyles for Active Youngsters (Team PLAY) study. This randomized controlled trial is designed to test the efficacy of a 6-month, moderately intense, primary care feasible, family-based behavioral intervention, targeting both young children and their parent, in promoting healthy weight change.
Participants are 270 overweight and obese children (ages 4 to 7 years) and their parent, who were recruited from a primarily African American urban population. Parents and children were instructed in proven cognitive behavioral techniques (e.g. goal setting, self-talk, stimulus control and reinforcement) designed to encourage healthier food choices (more whole grains, fruits and vegetables, and less concentrated fats and sugar), reduce portion sizes, decrease sweetened beverages and increase moderate to vigorous physical activity engagement. The main outcome of this study is change in BMI at two years post enrollment.
Recruitment using reactive methods (mailings, TV ads, pamphlets) was found to be more successful than using only a proactive approach (referral through physicians). At baseline, most children were very obese with an average BMI z-score of 2.6. Reported intake of fruits and vegetables and minutes of moderate to vigorous physical activity engagement did not meet national recommendations. If efficacious, Team PLAY would offer a model for obesity treatment directed at families with young children that could be tested and translated to both community and primary care settings.
doi:10.1016/j.cct.2012.02.003
PMCID: PMC3336873  PMID: 22342450
randomized controlled trial; children; obesity
17.  Modelling the ecological–functional diversification of marine Metazoa on geological time scales 
Biology Letters  2011;8(1):151-155.
The ecological traits and functional capabilities of marine animals have changed significantly since their origin in the late Precambrian. These changes can be analysed quantitatively using multi-dimensional parameter spaces in which the ecological lifestyles of species are represented by particular combinations of parameter values. Here, we present models that describe the filling of this multi-dimensional ‘ecospace’ by ecological lifestyles during metazoan diversification. These models reflect varying assumptions about the processes that drove ecological diversification; they contrast diffusive expansion with driven expansion and niche conservatism with niche partitioning. Some models highlight the importance of interactions among organisms (ecosystem engineering and predator–prey escalation) in promoting new lifestyles or eliminating existing ones. These models reflect processes that were not mutually exclusive; rigorous analyses will continue to reveal their applicability to episodes in metazoan history.
doi:10.1098/rsbl.2011.0641
PMCID: PMC3259971  PMID: 21813550
ecospace utilization; diversification; Phanerozoic; functional diversity; macroevolution; Metazoa
18.  Management of severe asthma in children 
Lancet  2010;376(9743):814-825.
Children who are referred to specialist care with asthma that does not respond to treatment (problematic severe asthma) are a heterogeneous group, with substantial morbidity. The evidence base for management is sparse, and is mostly based on data from studies in children with mild and moderate asthma and on extrapolation of data from studies in adults with severe asthma. In many children with severe asthma, the diagnosis is wrong or adherence to treatment is poor. The first step is a detailed diagnostic assessment to exclude an alternative diagnosis (“not asthma at all”), followed by a multidisciplinary approach to exclude comorbidities (“asthma plus”) and to assess whether the child has difficult asthma (improves when the basic management needs, such as adherence and inhaler technique, are corrected) or true, therapy-resistant asthma (still symptomatic even when the basic management needs are resolved). In particular, environmental causes of secondary steroid resistance should be identified. An individualised treatment plan should be devised depending on the clinical and pathophysiological characterisation. Licensed therapeutic approaches include high-dose inhaled steroids, the Symbicort maintenance and reliever (SMART) regimen (with budesonide and formoterol fumarate), and anti-IgE therapy. Unlicensed treatments include methotrexate, azathioprine, ciclosporin, and subcutaneous terbutaline infusions. Paediatric data are needed on cytokine-specific monoclonal antibody therapies and bronchial thermoplasty. However, despite the interest in innovative approaches, getting the basics right in children with apparently severe asthma will remain the foundation of management for the foreseeable future.
doi:10.1016/S0140-6736(10)61054-9
PMCID: PMC3471126  PMID: 20816548
19.  Relationship between Serum Vitamin D, Disease Severity, and Airway Remodeling in Children with Asthma 
Rationale
Little is known about vitamin D status and its effect on asthma pathophysiology in children with severe, therapy-resistant asthma (STRA).
Objectives
Relationships between serum vitamin D, lung function, and pathology were investigated in pediatric STRA.
Methods
Serum 25-hydroxyvitamin D [25(OH)D3] was measured in 86 children (mean age, 11.7 yr): 36 with STRA, 26 with moderate asthma (MA), and 24 without asthma (control subjects). Relationships between 25(OH)D3, the asthma control test (ACT), spirometry, corticosteroid use, and exacerbations were assessed. Twenty-two of 36 children with STRA underwent fiberoptic bronchoscopy, bronchoalveolar lavage, and endobronchial biopsy with assessment of airway inflammation and remodeling.
Measurements and Main Results
25(OH)D3 levels (median [IQR]) were significantly lower in STRA (28 [22–38] nmol/L) than in MA (42.5 [29–63] nmol/L) and control subjects (56.5 [45–67] nmol/L) (P < 0.001). There was a positive relationship between 25(OH)D3 levels and percent predicted FEV1 (r = 0.4, P < 0.001) and FVC (r = 0.3, P = 0.002) in all subjects. 25(OH)D3 levels were positively associated with ACT (r = 0.6, P < 0.001), and inversely associated with exacerbations (r=−0.6, P < 0.001) and inhaled steroid dose (r=−0.39, P = 0.001) in MA and and STRA. Airway smooth muscle (ASM) mass, but not epithelial shedding or reticular basement membrane thickness, was inversely related to 25(OH)D3 levels (r=−0.6, P = 0.008). There was a positive correlation between ASM mass and bronchodilator reversibility (r = 0.6, P = 0.009) and an inverse correlation between ASM mass and ACT (r = −0.7, P < 0.001).
Conclusions
Lower vitamin D levels in children with STRA were associated with increased ASM mass and worse asthma control and lung function. The link between vitamin D, airway structure, and function suggests vitamin D supplementation may be useful in pediatric STRA.
doi:10.1164/rccm.201107-1239OC
PMCID: PMC3471128  PMID: 21908411
vitamin D; asthma; remodeling; airway smooth muscle; pediatrics
20.  1α,25-Dihydroxyvitamin D3 promotes CD200 expression by human peripheral and airway-resident T cells 
Thorax  2012;67(7):574-581.
Background
CD200, a cell-surface immunoglobulin-like molecule expressed by immune and stromal cells, dampens the pro-inflammatory activity of tissue-resident innate cells via its receptor, CD200R. This interaction appears critical for peripheral immune tolerance, particularly in the airways where excessive inflammation is undesirable. Vitamin D contributes to pulmonary health and promotes regulatory immune pathways, therefore its influence on CD200 and CD200R was investigated.
Methods
CD200 and CD200R expression were assessed by qPCR and immunoreactivity of human lymphoid, myeloid and epithelial cells following 1α,25-dihydroxyvitamin D3 (1α,25VitD3) exposure in vitro and in peripheral T cells following 1α,25VitD3 oral ingestion in vivo. The effect of 1α25VitD3 was also assessed in human airway-resident cells.
Results
1α25VitD3 potently upregulated CD200 on peripheral human CD4+ T cells in vitro, and in vivo there was a trend towards upregulation in healthy, but not asthmatic individuals. CD200R expression was not modulated in any cells studied. CD200 induction was observed to a lesser extent in CD8+ T cells and not in B cells or airway epithelium. T cells isolated from the human airway also responded strongly to 1α25VitD3 to upregulate CD200.
Conclusions
The capacity of 1α,25-dihydroxyvitamin D3 to induce CD200 expression by peripheral and respiratory tract T cells identifies an additional pathway via which vitamin D can restrain inflammation in the airways to maintain respiratory health.
doi:10.1136/thoraxjnl-2011-200651
PMCID: PMC3471129  PMID: 22334534
21.  Development of the bronchial epithelial reticular basement membrane: relationship to epithelial height and age 
Thorax  2011;66(4):280-285.
Background
The bronchial epithelium and underlying reticular basement membrane (RBM) have a close spatial and functional inter-relationship and are considered an epithelial–mesenchymal trophic unit (EMTU). An understanding of RBM development is critical to understanding the extent and time of appearance of its abnormal thickening that is characteristic of asthma.
Methods
RBM thickness and epithelial height were determined in histological sections of cartilaginous bronchi obtained postmortem from 47 preterm babies and infants (median age 40 weeks gestation (22 weeks gestation–8 months)), 40 children (2 years (1 month–17 years)) and 23 adults (44 (17–90) years) who had died from non-respiratory causes, and had no history of asthma.
Results
The RBM was visible by light microscopy at 30 weeks gestation. RBM thickness increased in successive age groups in childhood; in infants (r=0.63, p<0.001) and in children between 1 month and 17 years (r=0.82, p<0.001). After 18 years, RBM thickness decreased with increasing age (r=−0.42, p<0.05). Epithelial height showed a similar relationship with age, a positive relationship from preterm to 17 years (r = 0.50, p<0.001) and a negative relationship in adulthood (r=−0.84, p<0.0001). There was a direct relationship between epithelial height and RBM thickness (r=0.6, p<0.001).
Conclusions
The RBM in these subjects was microscopically identifiable by 30 weeks gestation. It thickened during childhood and adolescence. In adults, there was either no relationship with age, or a slow reduction in thickness in older age. Developmental changes of RBM thickness were accompanied by similar changes in epithelial height, supporting the close relationship between RBM and epithelium within the EMTU.
doi:10.1136/thx.2010.149799
PMCID: PMC3471130  PMID: 21233480
22.  The role of 1α,25-dihydroxyvitamin D3 and cytokines in the promotion of distinct Foxp3+ and IL-10+ CD4+ T cells 
European journal of immunology  2012;42(10):2697-2708.
1α,25-Dihydroxyvitamin D3 (1α25VitD3) has potent immunomodulatory properties. We have previously demonstrated that 1α25VitD3 promotes human and murine IL-10-secreting CD4+ T cells. Because of the clinical relevance of this observation, we characterized these cells further and investigated their relationship with Foxp3+ regulatory T (Treg) cells. 1α25VitD3 increased the frequency of both Foxp3+ and IL-10+ CD4+T cells in vitro. However, Foxp3 was increased at high concentrations of 1α25VitD3 and IL-10 at more moderate levels, with little coexpression of these molecules. The Foxp3+ and IL-10+ T-cell populations showed comparable suppressive activity. We demonstrate that the enhancement of Foxp3 expression by 1α25VitD3 is impaired by IL-10. 1α25VitD3 enables the selective expansion of Foxp3+ Treg cells over their Foxp3− T-cell counterparts. Equally, 1α25VitD3 maintains Foxp3+ expression by sorted populations of human and murine Treg cells upon in vitro culture. A positive in vivo correlation between vitamin D status and CD4+Foxp3+ T cells in the airways was observed in a severe pediatric asthma cohort, supporting the in vitro observations. In summary, we provide evidence that 1α25VitD3 enhances the frequency of both IL-10+ and Foxp3+ Treg cells. In a translational setting, these data suggest that 1α25VitD3, over a broad concentration range, will be effective in enhancing the frequency of Treg cells.
doi:10.1002/eji.201242370
PMCID: PMC3471131  PMID: 22903229
1α,25-Dihydroxyvitamin D3; Asthma; Immune regulation; Regulatory T cells
23.  Novel Keto-phospholipids Are Generated by Monocytes and Macrophages, Detected in Cystic Fibrosis, and Activate Peroxisome Proliferator-activated Receptor-γ* 
The Journal of Biological Chemistry  2012;287(50):41651-41666.
Background: Lipoxygenases (LOXs) generate eicosanoids in inflammation.
Results: Monocyte/macrophage LOXs generate novel phospholipid-esterified eicosanoids containing ketoeicosatetraenoic acid or hydroperoxyeicosatetraenoic acid. They activate peroxisome proliferator-activated receptor-γ transcriptional activity and are found in cystic fibrosis bronchoalveolar fluid.
Significance: LOXs generate esterified eicosanoids in vitro and in vivo.
Conclusion: These new lipids represent new families of bioactive mediators.
12/15-Lipoxygenases (LOXs) in monocytes and macrophages generate novel phospholipid-esterified eicosanoids. Here, we report the generation of two additional families of related lipids comprising 15-ketoeicosatetraenoic acid (KETE) attached to four phosphatidylethanolamines (PEs). The lipids are generated basally by 15-LOX in IL-4-stimulated monocytes, are elevated on calcium mobilization, and are detected at increased levels in bronchoalveolar lavage fluid from cystic fibrosis patients (3.6 ng/ml of lavage). Murine peritoneal macrophages generate 12-KETE-PEs, which are absent in 12/15-LOX-deficient mice. Inhibition of 15-prostaglandin dehydrogenase prevents their formation from exogenous 15-hydroxyeicosatetraenoic acid-PE in human monocytes. Both human and murine cells also generated analogous hydroperoxyeicosatetraenoic acid-PEs. The electrophilic reactivity of KETE-PEs is shown by their Michael addition to glutathione and cysteine. Lastly, both 15-hydroxyeicosatetraenoic acid-PE and 15-KETE-PE activated peroxisome proliferator-activated receptor-γ reporter activity in macrophages in a dose-dependent manner. In summary, we demonstrate novel peroxisome proliferator-activated receptor-γ-activating oxidized phospholipids generated enzymatically by LOX and 15-prostaglandin dehydrogenase in primary monocytic cells and in a human Th2-related lung disease. The lipids are a new family of bioactive mediators from the 12/15-LOX pathway that may contribute to its known anti-inflammatory actions in vivo.
doi:10.1074/jbc.M112.405407
PMCID: PMC3516716  PMID: 23060450
Eicosanoid; Innate Immunity; Lipoxygenase Pathway; Macrophages; Mass Spectrometry (MS); Monocytes; Phospholipid
25.  Pediatric severe asthma is characterized by eosinophilia and remodeling without TH2 cytokines 
Background
The pathology of pediatric severe therapy-resistant asthma (STRA) is little understood.
Objectives
We hypothesized that STRA in children is characterized by airway eosinophilia and mast cell inflammation and is driven by the TH2 cytokines IL-4, IL-5, and IL-13.
Methods
Sixty-nine children (mean age, 11.8 years; interquartile range, 5.6-17.3 years; patients with STRA, n = 53; control subjects, n = 16) underwent fiberoptic bronchoscopy, bronchoalveolar lavage (BAL), and endobronchial biopsy. Airway inflammation, remodeling, and BAL fluid and biopsy specimen TH2 cytokines were quantified. Children with STRA also underwent symptom assessment (Asthma Control Test), spirometry, exhaled nitric oxide and induced sputum evaluation.
Results
Children with STRA had significantly increased BAL fluid and biopsy specimen eosinophil counts compared with those found in control subjects (BAL fluid, P < .001; biopsy specimen, P < .01); within the STRA group, there was marked between-patient variability in eosinophilia. Submucosal mast cell, neutrophil, and lymphocyte counts were similar in both groups. Reticular basement membrane thickness and airway smooth muscle were increased in patients with STRA compared with those found in control subjects (P < .0001 and P < .001, respectively). There was no increase in BAL fluid IL-4, IL-5, or IL-13 levels in patients with STRA compared with control subjects, and these cytokines were rarely detected in induced sputum. Biopsy IL-5+ and IL-13+ cell counts were also not higher in patients with STRA compared with those seen in control subjects. The subgroup (n = 15) of children with STRA with detectable BAL fluid TH2 cytokines had significantly lower lung function than those with undetectable BAL fluid TH2 cytokines.
Conclusions
STRA in children was characterized by remodeling and variable airway eosinophil counts. However, unlike in adults, there was no neutrophilia, and despite the wide range in eosinophil counts, the TH2 mediators that are thought to drive allergic asthma were mostly absent.
doi:10.1016/j.jaci.2012.01.059
PMCID: PMC3381727  PMID: 22385633
Pediatric asthma; eosinophilia; remodeling; severe therapy-resistant asthma; mediators

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