Pathophysiological characterization of asthma transitions across adolescence

Background

Adolescence is a period of change, which coincides with disease remission in a significant proportion of subjects with childhood asthma. There is incomplete understanding of the changing characteristics underlying different adolescent asthma transitions. We undertook pathophysiological characterization of transitional adolescent asthma phenotypes in a longitudinal birth cohort.

Methods

The Isle of Wight Birth Cohort (N = 1456) was reviewed at 1, 2, 4, 10 and 18-years. Characterization included questionnaires, skin tests, spirometry, exhaled nitric oxide, bronchial challenge and (in a subset of 100 at 18-years) induced sputum. Asthma groups were “never asthma” (no asthma since birth), “persistent asthma” (asthma at age 10 and 18), “remission asthma” (asthma at age 10 but not at 18) and “adolescent-onset asthma” (asthma at age 18 but not at age 10).

Results

Participants whose asthma remitted during adolescence had lower bronchial reactivity (odds ratio (OR) 0.30; CI 0.10 -0.90; p = 0.03) at age 10 plus greater improvement in lung function (forced expiratory flow 25-75% gain: 1.7 L; 1.0-2.9; p = 0.04) compared to persistent asthma by age 18. Male sex (0.3; 0.1-0.7; p < 0.01) and lower acetaminophen use (0.4; 0.2-0.8; p < 0.01) independently favoured asthma remission, when compared to persistent asthma. Asthma remission had a lower total sputum cell count compared to never asthma (31.5 [25–75 centiles] 12.9-40.4) vs. 47.0 (19.5-181.3); p = 0.03). Sputum examination in adolescent-onset asthma showed eosinophilic airway inflammation (3.0%, 0.7-6.6), not seen in persistent asthma (1.0%, 0–3.9), while remission group had the lowest sputum eosinophil count (0.3%, 0–1.4) and lowest eosinophils/neutrophils ratio of 0.0 (Interquartile range: 0.1).

Conclusion

Asthma remission during adolescence is associated with lower initial BHR and greater gain in small airways function, while adolescent-onset asthma is primarily eosinophilic.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0153-7) contains supplementary material, which is available to authorized users.     

Association of Eosinophilic Inflammation with FKBP51 Expression in Sputum Cells in Asthma

Background

Airway eosinophilia is a predictor of steroid responsiveness in steroid-naïve asthma. However, the relationship between airway eosinophilia and the expression of FK506-binding protein 51 (FKBP51), a glucocorticoid receptor co-chaperone that plays a role in steroid insensitivity in asthma, remains unknown.

Objective

To evaluate the relationship between eosinophilic inflammation and FKBP51 expression in sputum cells in asthma.

Methods

The FKBP51 mRNA levels in sputum cells from steroid-naïve patients with asthma (n = 31) and stable asthmatic patients on inhaled corticosteroid (ICS) (n = 28) were cross-sectionally examined using real-time PCR. Associations between FKBP51 levels and clinical indices were analyzed.

Results

In steroid-naïve patients, the FKBP51 levels were negatively correlated with eosinophil proportions in blood (r = −0.52) and sputum (r = −0.57), and exhaled nitric oxide levels (r = −0.42) (all p<0.05). No such associations were observed in patients on ICS. In steroid-naïve patients, improvement in forced expiratory volume in one second after ICS initiation was correlated with baseline eosinophil proportions in blood (r = 0.74) and sputum (r = 0.76) and negatively correlated with FKBP51 levels (r = −0.73) (all p<0.0001) (n = 20). Lastly, the FKBP51 levels were the lowest in steroid-naïve asthmatic patients, followed by mild to moderate persistent asthmatic patients on ICS, and the highest in severe persistent asthmatic patients on ICS (p<0.0001).

Conclusions

Lower FKBP51 expression in sputum cells may reflect eosinophilic inflammation and glucocorticoid responsiveness in steroid-naïve asthmatic patients.     

Sputum mediator profiling and relationship to airway wall geometry imaging in severe asthma

Background

Severe asthma is a heterogeneous disease and the relationship between airway inflammation and airway remodelling is poorly understood. We sought to define sputum mediator profiles in severe asthmatics categorised by CT-determined airway geometry and sputum differential cell counts.

Methods

In a single centre cross-sectional observational study we recruited 59 subjects with severe asthma that underwent sputum induction and thoracic CT. Quantitative CT analysis of the apical segment of the right upper lobe (RB1) was performed. Forty-one mediators in sputum samples were measured of which 21 mediators that were assessable in >50% of samples were included in the analyses.

Results

Independent of airway geometry, sputum MMP9 and IL-1β were elevated in those groups with a high sputum neutrophil count while sputum ICAM was elevated in those subjects with a low sputum neutrophil count. In contrast, sputum CCL11, IL-1α and fibrinogen were different in groups stratified by both sputum neutrophil count and airway geometry. Sputum CCL11 concentration was elevated in subjects with a low sputum neutrophil count and high luminal and total RB1 area, whereas sputum IL1α was increased in subjects with a high sputum neutrophil count and low total RB1 area. Sputum fibrinogen was elevated in those subjects with RB1 luminal narrowing and in those subjects with neutrophilic inflammation without luminal narrowing.

Conclusions

We have demonstrated that sputum mediator profiling reveals a number of associations with airway geometry. Whether these findings reflect important biological phenotypes that might inform stratified medicine approaches requires further investigation.     

Sputum Inflammatory Profile Before and After Specific Inhalation Challenge in Individuals with Suspected Occupational Asthma

Background

The aim of this study was to establish the sputum inflammatory profile and changes in levels of leukotriene B₄ (LTB₄) and a panel of Th1/Th2 cytokines in subjects with suspected occupational asthma (OA) following specific inhalation challenge (SIC) to high-molecular-weight (HMW) and low-molecular-weight (LMW) agents.

Material and Methods

Fifty-one consecutive subjects undergoing SIC for suspected OA were enrolled. Sputum induction was performed the day before and 24 h after exposure to the offending agent. Total and differential cell counts were assessed. LTB₄ and a 10 Th1/Th2 cytokines were measured in sputum supernatant.

Results

Thirty-four patients tested positive to SIC and were diagnosed with OA (in 10 due to HMW agents and in 24 to LMW agents). SIC was negative in 17 subjects. As compared to baseline an increase was found in the percentage of sputum eosinophils and neutrophils, and in IL-10 concentration after SIC (p = 0.0078, p = 0.0195, and p = 0.046, respectively), and a decrease was seen in LTB₄ level (p = 0.0078) in patients with OA due to HMW agents. An increase in the percentage of sputum neutrophils after SIC (p = 0.0040) was observed in subjects without OA exposed to LMW agents. IL-8 levels after SIC were higher in patients without OA compared with patients with OA (p = 0.0146).

Conclusion

When conducting airway inflammation studies in OA, patients should be divided according to the causal agent (HMW or LMW). In OA patients exposed to HMW agents, an increase in the number of neutrophils can be found in parallel to the increase of eosinophils, although this does not contradict an IgE-mediated mechanism. Exposure to LMW agents can result in increased neutrophilic inflammation in patients with airway diseases unrelated to OA. There is variability in the responses observed in patients with OA exposed to LMW agents.     

Can AMP induce sputum eosinophils,even in subjects with complete asthma remission?

Background

The definition of "clinical asthma remission" is based on absence of symptoms and use of medication. However, in the majority of these subjects airway inflammation is still present when measured. In the present study we investigated whether "complete asthma remission", additionally defined by the absence of bronchial hyperresponsiveness (BHR) and the presence of a normal lung function, is associated with the absence of airway inflammation.

Methods

Patients with a former diagnosis of asthma and a positive histamine provocation test were re-examined to identify subjects with complete asthma remission (no asthma symptoms or medication, PC₂₀ histamine > 32 mg/ml, FEV₁ > 90% predicted). Patients with PC₂₀ histamine ≤ 32 mg/ml were defined as current asthmatics and were divided in two groups, i.e. asthmatics with and without BHR to adenosine 5''monophoshate (AMP). Sputum induction was performed 1 week before and 1 hour after AMP provocation. Sputum induction and AMP provocation were previously shown to be sensitive markers of airway inflammation.

Results

Seven patients met criteria for complete asthma remission. Twenty-three were current asthmatics, including twelve without hyperresponsiveness to AMP. Subjects with complete asthma remission showed no AMP-induced sputum eosinophilia (median (range) 0.2 (0 - 4.6)% at baseline and 0.2 (0 - 2.6)% after AMP). After AMP, current asthmatics had a significant increase in sputum eosinophils (0.5 (0 - 26.0)% at baseline and 2.6 (0 - 32.0) % after AMP), as had the subgroup of current asthmatics without hyperresponsiveness to AMP (0.2 (0 - 1.8)% at baseline and 1.3 (0 - 6.3)% after AMP).

Conclusions

Subjects with complete asthma remission, in contrast to subjects with current asthma, do not respond with eosinophilic inflammation in sputum after AMP provocations. These data lend support to the usefulness of the definition of complete asthma remission.     

Impaired macrophage phagocytosis of bacteria in severe asthma

Background

Bacteria are frequently cultured from sputum samples of severe asthma patients suggesting a defect in bacterial clearance from the airway. We measured the capacity of macrophages from patients with asthma to phagocytose bacteria.

Methods

Phagocytosis of fluorescently-labelled polystyrene beads, Haemophilus influenzae or Staphylococcus aureus by broncholaveolar lavage alveolar macrophages (AM) and by monocyte-derived macrophages (MDM) from non-asthmatics, mild-moderate and severe asthmatic patients was assessed using fluorimetry.

Results

There were no differences in phagocytosis of polystyrene beads by AMs or MDMs from any of the subject groups. There was reduced phagocytosis of Haemophilus influenzae and Staphylococcus aureus in MDMs from patients with severe asthma compared to non-severe asthma (p < 0.05 and p < 0.01, respectively) and healthy subjects (p < 0.01and p < 0.001, respectively). Phagocytosis of Haemophilus influenzae and Staphylococcus aureus by AM was also reduced in severe asthma compared to normal subjects (p < 0.05). Dexamethasone and formoterol did not suppress phagocytosis of bacteria by MDMs from any of the groups.

Conclusions

Persistence of bacteria in the lower airways may result partly from a reduced phagocytic capacity of macrophages for bacteria. This may contribute to increased exacerbations, airway colonization and persistence of inflammation.     

Elevation of IL-6 in the allergic asthmatic airway is independent of inflammation but associates with loss of central airway function

Background

Asthma is a chronic inflammatory disease of the airway that is characterized by a Th2-type of immune response with increasing evidence for involvement of Th17 cells. The role of IL-6 in promoting effector T cell subsets suggest that IL-6 may play a functional role in asthma. Classically IL-6 has been viewed as an inflammatory marker, along with TNFα and IL-1β, rather than as regulatory cytokine.

Objective

To investigate the potential relationship between IL-6 and other proinflammatory cytokines, Th2/Th17 cytokines and lung function in allergic asthma, and thus evaluate the potential role of IL-6 in this disease.

Methods

Cytokine levels in induced sputum and lung function were measured in 16 healthy control and 18 mild-moderate allergic asthmatic subjects.

Results

The levels of the proinflammatory biomarkers TNFα and IL-1β were not different between the control and asthmatic group. In contrast, IL-6 levels were specifically elevated in asthmatic subjects compared with healthy controls (p < 0.01). Hierarchical regression analysis in the total study cohort indicates that the relationship between asthma and lung function could be mediated by IL-6. Among Th2 cytokines only IL-13 (p < 0.05) was also elevated in the asthmatic group, and positively correlated with IL-6 levels (r_S = 0.53, p < 0.05).

Conclusions

In mild-moderate asthma, IL-6 dissociates from other proinflammatory biomarkers, but correlates with IL-13 levels. Furthermore, IL-6 may contribute to impaired lung function in allergic asthma.     

Salivary Inflammatory Mediator Profiling and Correlation to Clinical Disease Markers in Asthma

Rationale

There is a need for a readily available, non-invasive source of biomarkers that predict poor asthma control.

Objectives

We sought to determine if there is an association between the salivary inflammatory profile and disease control in children and adults with asthma.

Methods

In this cross-sectional study, we collected demographic and clinical information from two independent populations at different sites, resulting in convenience samples of 58 pediatric and 122 adult urban asthmatics. Control was assessed by symptom questionnaire (children) and by Asthma Control Questionnaire and current exacerbation (adults). Saliva was collected in all subjects. We applied principal component analysis to a 10-plex panel of relevant inflammatory markers to characterize marker profiles and determined if profiles were associated with asthma control.

Results

There were similar, strong correlations amongst biologically related markers in both populations: eosinophil-related: eotaxin-1/CCL11, RANTES/CCL5, and IL-5 (p<.001); myeloid/innate: IL-1β, IL-6, MCP-1/CCL2, and IL-8/CXCL8 (p<.001). The first three principal components captured ≥74% of variability across all ten analytes in both populations. In adults, the Principal Component 1 score, broadly reflective of all markers, but with greater weight given to myeloid/innate markers, was associated with Asthma Control Questionnaire score and exacerbation. The Principal Component 3 score, reflective of IP-10/CXCL10, was associated with current exacerbation. In children, the Principal Component 1, 2, and 3 scores were associated with recent asthma symptoms. The Principal Component 2 score, reflective of higher eosinophil markers, was inversely correlated with symptoms. The Principal Component 3 score was positively associated with all symptom outcomes.

Conclusion

The salivary inflammatory profile is associated with disease control in children and adults with asthma.     

Inhibition of LPS-induced airway neutrophilic inflammation in healthy volunteers with an oral CXCR2 antagonist

Background

Inhaled lipopolysaccharide (LPS) induces a dose-dependent, acute neutrophilic response in the airways of healthy volunteers that can be quantified in induced sputum. Chemokines, such as CXCL1 and CXCL8, play an important role in neutrophilic inflammation in the lung through the activation of CXCR2 and small molecule antagonists of these receptors have now been developed. We investigated the effect of AZD8309, a CXCR2 antagonist, compared with placebo on LPS-induced inflammation measured in sputum of healthy volunteers.

Methods

Twenty healthy subjects were randomized in a double-blind placebo-controlled, cross-over study. AZD8309 (300 mg) or placebo was dosed twice daily orally for 3 days prior to challenge with inhaled LPS and induced sputum was collected 6 h later.

Results

Treatment with AZD8309 showed a mean 77% reduction in total sputum cells (p < 0.001) and 79% reduction in sputum neutrophils (p < 0.05) compared with placebo after LPS challenge. There was also a reduction in neutrophil elastase activity (p < 0.05) and CXCL1 (p < 0.05) and trends for reductions in sputum macrophages (47%), leukotriene B₄ (39%) and CXCL8 (52%).

Conclusions

AZD8309 inhibited LPS-induced inflammation measured in induced sputum of normal volunteers, indicating that this treatment may be useful in the treatment of neutrophilic diseases of the airways, such as COPD, severe asthma and cystic fibrosis.

Trial registration

{"type":"clinical-trial","attrs":{"text":"NCT00860821","term_id":"NCT00860821"}}NCT00860821.     

Anti-inflammatory deficiencies in neutrophilic asthma: reduced galectin-3 and IL-1RA/IL-1β

Background

Galectin-3 (gal-3), a member of the β-galactoside-binding animal lectins, is involved in the recruitment, activation and removal of neutrophils. Neutrophilic asthma is characterized by a persistent elevation of airway neutrophils and impaired efferocytosis. We hypothesized that sputum gal-3 would be reduced in neutrophilic asthma and the expression of gal-3 would be associated with other markers of neutrophilic inflammation.

Methods

Adults with asthma (n = 80) underwent a sputum induction following clinical assessment and blood collection. Sputum was dispersed for a differential cell count and ELISA assessment of gal-3, gal-3 binding protein (BP), interleukin (IL)-1β, IL-1 receptor antagonist (RA), IL-8 and IL-6. Gal-3 and gal-3BP immunoreactivity were assessed in mixed sputum cells.

Results

Sputum gal-3 (median, (q1,q3)) was significantly reduced in neutrophilic asthma (183 ng/mL (91,287)) compared with eosinophilic (293 ng/mL (188,471), p = 0.021) and paucigranulocytic asthma (399 ng/mL (213,514), p = 0.004). The gal-3/gal-3BP ratio and IL-1RA/IL-1β ratio were significantly reduced, while gal-3BP and IL-1β were significantly elevated in neutrophilic asthma compared with eosinophilic and paucigranulocytic asthma.

Conclusion

Patients with neutrophilic asthma have impairment in anti-inflammatory ratio of gal-3/gal-3BP and IL-1RA/IL-1β which provides a further framework for exploration into pathologic mechanisms of asthma phenotypes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0163-5) contains supplementary material, which is available to authorized users.     

Study on Risk Factors for Severe Hand,Foot and Mouth Disease in China

Background

Epidemics of HFMD are elevated every year globally, especially in mainland China. The disease now presents as an increasing threat to public health worldwide.

Methods

Five hundred and seventy-one EV71-infected HFMD patients in Beijing You''an Hospital were grouped by disease severity: Mild (no severe complication) (n = 221), and Severe group (complicated with brainstem encephalitis (BE), and/or pulmonary edema (PE) (n = 350)). Clinical and laboratory findings and levels of 7 serum cytokines were analyzed.

Results

Univariate analysis showed that (RR)>26/min (p<0.001), age<4 yo (p = 0.001), GLU>8.3 mmol/L (p = 0.008), CL<98 mmol/L (p = 0.026), and WBC>1.2×10⁹/L (p = 0.040) were associated with severe cases. Results of multivariate analysis indicated five independent risk factors (RR>26/min (p<0.001), Age<4 yo (p<0.001), GLU>8.3 mmol/L (p = 0.011), LYM>40% (p = 0.010), and ALT>40 U/L (p = 0.045)). In addition to single-factor analysis, we further analyzed the use of different combinations of risk factors. “GLU>8.3 and CL<98 and RR>26” (confidence ration (CR) = 100%) is the top indicator, followed by “ALT>40 and LYM>40% and RR>26 and Age<4 yo” (CR = 92.9%).Serum levels of IL-2, IL-4, IL-10, IFNγ, GM-CSF, and TNFα were higher in severe cases than in mild cases. A new evaluation scoring system by scoring each risk factor 1 and independent risk factor 2 was developed for early identification of severe HFMD cases.

Conclusions

Five independent risk factors, along with indicative combinations of risk factors, for severe cases were identified, and a scoring system was created to facilitate the use of indicators for early medical intervention.     

Predictors of Raised Viral Load during Antiretroviral Therapy in Patients with and without Prior Antiretroviral Use: A Cross-Sectional Study

Objectives

In Lagos, Nigeria, Médecins Sans Frontières (MSF) and the Ministry of Health (MoH) commenced free antiretroviral treatment (ART) in a hospital-based clinic. We performed a cross-sectional study to compare factors associated with raised viral load between patients with (“experienced”) and without (“naïve”) prior antiretroviral (ARV) exposure at commencement of ART at the clinic. We also examined factors influencing ARV adherence in experienced patients prior to clinic entry.

Methods

We included adult patients receiving ART from MSF who answered a questionnaire about previous antiretroviral use. Multivariate logistic regression was used to estimate odds ratios (OR) for raised viral load (≥1000 copies/mL).

Results

1246 (96%) patients answered: 1075 (86%) reported no, and 171 (14%) some, prior ARV exposure. ARV-naïve patients were more immunosuppressed at baseline: 65% vs 37% (p<0.001) had CD4<200; 17% vs 9% (p = 0.013) were WHO stage 4. Proportionately more experienced than naïve patients had raised viral loads (20% vs 9%, p<0.001) on ART in the MSF/MoH clinic. Raised viral load was associated with prior ARV experience (adjusted OR = 3.74, 95%CI 2.09–6.70, p<0.001) and complete interruption of current ART (adjusted OR = 3.71, 95%CI 2.06–6.68, p<0.001). Higher CD4 at time of VL and a higher self-rated score of recent adherence were associated with lower OR of a raised viral load. Among experienced patients who missed pills before joining MSF/MoH, most common reasons were because ARVS were not affordable (58%) or available (33%), with raised viral load associated with being unsure how to take them (OR = 3.16, 95%CI 1.10–9.12, p = 0.033).

Conclusions

Patients previously exposed to ARVs had increased OR of raised viral load. The cost and availability of ARVs were common reasons for missing ARVs before joining the MSF/MoH clinic, and inadequate patient knowledge was associated with raised viral load.     

Corticosteroid suppression of lipoxin A4 and leukotriene B4from alveolar macrophages in severe asthma

Background

An imbalance in the generation of pro-inflammatory leukotrienes, and counter-regulatory lipoxins is present in severe asthma. We measured leukotriene B₄ (LTB₄), and lipoxin A₄ (LXA₄) production by alveolar macrophages (AMs) and studied the impact of corticosteroids.

Methods

AMs obtained by fiberoptic bronchoscopy from 14 non-asthmatics, 12 non-severe and 11 severe asthmatics were stimulated with lipopolysaccharide (LPS,10 μg/ml) with or without dexamethasone (10^-6M). LTB₄ and LXA₄ were measured by enzyme immunoassay.

Results

LXA₄ biosynthesis was decreased from severe asthma AMs compared to non-severe (p < 0.05) and normal subjects (p < 0.001). LXA₄ induced by LPS was highest in normal subjects and lowest in severe asthmatics (p < 0.01). Basal levels of LTB₄ were decreased in severe asthmatics compared to normal subjects (p < 0.05), but not to non-severe asthma. LPS-induced LTB₄ was increased in severe asthma compared to non-severe asthma (p < 0.05). Dexamethasone inhibited LPS-induced LTB₄ and LXA₄, with lesser suppression of LTB₄ in severe asthma patients (p < 0.05). There was a significant correlation between LPS-induced LXA₄ and FEV₁ (% predicted) (r_s = 0.60; p < 0.01).

Conclusions

Decreased LXA₄ and increased LTB₄ generation plus impaired corticosteroid sensitivity of LPS-induced LTB₄ but not of LXA₄ support a role for AMs in establishing a pro-inflammatory balance in severe asthma.     

Can Certain Genotypes Predispose to Poor Asthma Control in Children? A Pharmacogenetic Study of 9 Candidate Genes in Children with Difficult Asthma

Objective

We tested the hypothesis that patients with difficult asthma have an increased frequency of certain genotypes that predispose them to asthma exacerbations and poor asthma control.

Methods

A total of 180 Caucasian children with confirmed asthma diagnosis were selected from two phenotypic groups; difficult (n = 112) versus mild/moderate asthma (n = 68) groups. All patients were screened for 19 polymorphisms in 9 candidate genes to evaluate their association with difficult asthma.

Key Results

The results indicated that LTA4H A-9188>G, TNFα G-308>A and IL-4Rα A1727>G polymorphisms were significantly associated with the development of difficult asthma in paediatric patients (p<0.001, p = 0.019 and p = 0.037, respectively). Haplotype analysis also revealed two haplotypes (ATA haplotype of IL-4Rα A1199>C, IL-4Rα T1570>C and IL-4Rα A1727>G and CA haplotype of TNFα C-863>A and TNFα G-308>A polymorphisms) which were significantly associated with difficult asthma in children (p = 0.04 and p = 0.018, respectively).

Conclusions and Clinical Relevance

The study revealed multiple SNPs and haplotypes in LTA4H, TNFα and IL4-Rα genes which constitute risk factors for the development of difficult asthma in children. Of particular interest is the LTA4H A-9188>G polymorphism which has been reported, for the first time, to have strong association with severe asthma in children. Our results suggest that screening for patients with this genetic marker could help characterise the heterogeneity of responses to leukotriene-modifying medications and, hence, facilitate targeting these therapies to the subset of patients who are most likely to gain benefit.     

Breath Formate Is a Marker of Airway S-Nitrosothiol Depletion in Severe Asthma

Background

Children with severe asthma have poor symptom control and elevated markers of airway oxidative and nitrosative stress. Paradoxically, they have decreased airway levels of S-nitrosothiols (SNOs), a class of endogenous airway smooth muscle relaxants. This deficiency results from increased activity of an enzyme that both reduces SNOs to ammonia and oxidizes formaldehyde to formic acid, a volatile carboxylic acid that is more easily detected in exhaled breath condensate (EBC) than SNOs. We therefore hypothesize that depletion of airway SNOs is related to asthma pathology, and breath formate concentration may be a proxy measure of SNO catabolism.

Methods and Findings

We collected EBC samples from children and adolescents, including 38 with severe asthma, 46 with mild-to-moderate asthma and 16 healthy adolescent controls, and the concentration of ionic constituents was quantified using ion chromatography. The concentrations of EBC components with volatile conjugates were log-normally distributed. Formate was the principal ion that displayed a significant difference between asthma status classifications. The mean EBC formate concentration was 40% higher in samples collected from all asthmatics than from healthy controls (mean = 5.7 µM, mean±standard deviation = 3.1−10.3 µM vs. 4.0, 2.8−5.8 µM, p = 0.05). EBC formate was higher in severe asthmatics than in mild-to-moderate asthmatics (6.8, 3.7−12.3 µM vs. 4.9, 2.8−8.7 µM, p = 0.012). In addition, formate concentration was negatively correlated with methacholine PC₂₀ (r = −0.39, p = 0.002, asthmatics only), and positively correlated with the NO-derived ion nitrite (r = 0.46, p<0.0001) as well as with total serum IgE (r = 0.28, p = 0.016, asthmatics only). Furthermore, formate was not significantly correlated with other volatile organic acids nor with inhaled corticosteroid dose.

Conclusions

We conclude that EBC formate concentration is significantly higher in the breath of children with asthma than in those without asthma. In addition, amongst asthmatics, formate is elevated in the breath of those with severe asthma compared to those with mild-to-moderate asthma. We suggest that this difference is related to asthma pathology and may be a product of increased catabolism of endogenous S-nitrosothiols.     

What Makes a Difference in Exercise-Induced Bronchoconstriction: An 8 Year Retrospective Analysis

Background

Exercise-induced bronchoconstriction (EIB) was recently classified into EIB alone and EIB with asthma, based on the presence of concurrent asthma.

Objective

Differences between EIB alone and EIB with asthma have not been fully described.

Methods

We retrospectively reviewed who visited an allergy clinic for respiratory symptoms after exercise and underwent exercise bronchial provocation testing. More than a 15% decrease of forced expiratory volume in 1 second (FEV1) from baseline to the end of a 6 min free-running challenge test was interpreted as positive EIB.

Results

EIB was observed in 66.9% of the study subjects (89/133). EIB-positive subjects showed higher positivity to methacholine provocation testing (61.4% vs. 18.9%, p<0.001) compared with EIB-negative subjects. In addition, sputum eosinophilia was more frequently observed in EIB-positive subjects than in EIB-negative subjects (56% vs. 23.5%, p = 0.037). The temperature and relative humidity on exercise test day were significantly related with the EIB-positive rate. Positive EIB status was correlated with both temperature (p = 0.001) and relative humidity (p = 0.038) in the methacholine-negative EIB group while such a correlation was not observed in the methacholine-positive EIB group. In the methacholine-positive EIB group the time to reach a 15% decrease in FEV1 during exercise was significantly shorter than that in the methacholine-negative EIB group (3.2±0.7 min vs. 8.6±1.6 min, p = 0.004).

Conclusions

EIB alone may be a distinct clinical entity from EIB with asthma. Conditions such as temperature and humidity should be considered when performing exercise tests, especially in subjects with EIB alone.     

Beta2 Adrenergic Receptor (ADRβ2) Haplotype Pair (2/4) Is Associated with Severe Asthma

Background

β2 adrenergic receptor (ADRβ2) polymorphisms including ADRβ2+46G>A have been reported to cause adverse outcomes in mild asthmatics. The extent to which ADRβ2 polymorphisms and in particular their haplotypes contribute to severe asthma is unknown.

Objective

To determine the association of ADRβ2 polymorphisms and haplotypes with asthma severity.

Methods

Caucasians (n = 2979) were genotyped for 11 ADRβ2 polymorphisms. The cohort (mean age 39.6, 60% female) included 2296 non-asthmatics, 386 mild asthmatics, 172 moderate asthmatics and 125 severe asthmatics. Haplotype frequency and haplotype pair for each subject was determined using the PHASE algorithm.

Results

The three asthmatic cohorts were comparable in age and gender but were distinguishable from each other in terms of symptoms, spirometry, medication use and health care utilisation (p <0.001). None of the polymorphisms showed a genotypic or allelic association with asthma diagnosis or severity. Nine haplotypes were identified and no association was found with asthma diagnosis or severity per se. Haplotype pair 2/4 was associated with asthma severity (Trend Test, OR 1.42, p = 0.0008) but not with asthma per se. Prevalence of haplotype pair 2/2 appeared to decrease with asthma severity (Trend Test, OR 0.78, p = 0.067). Two new haplotypes were identified, occurring exclusively in asthmatics at a frequency of ≥ 1%. In addition, a positive association between carriage of ADRβ2 +523*C and increased risk of atopy was discovered.

Conclusions

ADRβ2 haplotype pair 2/4 is associated with severe asthma and is consistent with findings of poor bronchodilator response in mild asthmatics who are also haplotype 2/4.     

Adhesion molecules in subjects with COPD and healthy non-smokers: a cross sectional parallel group study

Background

The aim of the study was to investigate how the expression of adhesion molecules changes as neutrophils migrate from the circulation to the lung and if these changes differ between non-smoking subjects and smokers with and without COPD.

Methods

Non-smoking healthy subjects (n=22), smokers without (n=21) and with COPD (n=18) were included. Neutrophils from peripheral blood, sputum and bronchial biopsies were analysed for cell surface expression of adhesion molecules (CD11b, CD62L, CD162). Serum, sputum supernatant and BAL-fluid were analysed for soluble adhesion molecules (ICAM-1, -3, E-selectin, P-selectin, VCAM-1, PECAM-1).

Results

Expression of CD11b was increased on circulating neutrophils from smokers with COPD. It was also increased on sputum neutrophils in both smokers groups, but not in non-smokers, as compared to circulating neutrophils.Serum ICAM-1 was higher in the COPD group compared to the other two groups (p<0.05) and PECAM-1 was lower in smokers without COPD than in non-smoking controls and the COPD group (p<0.05). In BAL-fluid ICAM-1 was lower in the COPD group than in the other groups (p<0.05).

Conclusions

Thus, our data strongly support the involvement of a systemic component in COPD and demonstrate that in smokers neutrophils are activated to a greater extent at the point of transition from the circulation into the lungs than in non-smokers.     

Beneficial Effects of Sitostanol on the Attenuated Immune Function in Asthma Patients: Results of an In Vitro Approach

Background

In vitro and animal studies have suggested that plant sterols and stanols increase cytokine production by T-helper-1 cells. This may be beneficial for patient groups characterized by a T-helper-2 dominant immune response, e.g. asthma patients. (1) to evaluate whether sitostanol induces a T-helper-1 shift in peripheral blood mononuclear cells (PBMCs) from asthma patients, and (2) to unravel the role of regulatory T-cells in this respect.

Methodology/Principal Findings

PBMCs from 10 asthma patients and 10 healthy subjects were isolated and incubated with 1.2 µM sitostanol, while stimulated with 5 µg/ml PHA. Similar amounts of cholesterol were used to determine whether effects were specific for plant stanols or for sterols in general. Changes in cytokine production were measured using antibody arrays and ELISAs. Changes in regulatory T-cell population size were measured by flow cytometry, using intracellular Foxp3 staining. Sitostanol increased production of IFNγ by 6.5% and IL-2 by 6.0% compared to cholesterol (p<0.01). No changes in IL-4 and IL-13 were found. Interestingly, this effect was only present in PBMCs from asthma patients. The number of Foxp3+ cells tended to increase and their activity, measured by IL-10 production, increased after sitostanol treatment in PBMCs from asthma patients compared to controls by 32.3% (p = 0.077) and 13.3% (p<0.05), respectively.

Conclusions/Significance

Altogether, the sitostanol-induced Thelper-1 shift in PBMCs from asthma patients and the stimulating effects of sitostanol on Treg cell numbers and activity indicate a possible novel approach for plant stanol ester enriched functional foods in the amelioration of asthmatic symptoms. Functional effects, however, require further evaluation.