The relation between age and airway epithelial barrier function.

BACKGROUND: The prevalence of age-associated diseases, such as chronic obstructive pulmonary disease (COPD), is increasing as the average life expectancy increases around the world. We previously identified a gene signature for ageing in the human lung which included genes involved in apical and tight junction assembly, suggesting a role for airway epithelial barrier dysfunction with ageing. AIM: To investigate the association between genes involved in epithelial barrier function and age both in silico and in vitro in the airway epithelium. METHODS: We curated a gene signature of 274 genes for epithelial barrier function and tested the association with age in two independent cohorts of bronchial brushings from healthy individuals with no respiratory disease, using linear regression analysis (FDR < 0.05). Protein-protein interactions were identified using STRING©. The barrier function of primary bronchial epithelial cells at air-liquid interface and CRISPR-Cas9-induced knock-down of target genes in human bronchial 16HBE14o-cells was assessed using Trans epithelial resistance (TER) measurement and Electric cell-surface impedance sensing (ECIS) respectively. RESULTS: In bronchial brushings, we found 55 genes involved in barrier function to be significantly associated with age (FDR < 0.05). EPCAM was most significantly associated with increasing age and TRPV4 with decreasing age. Protein interaction analysis identified CDH1, that was negatively associated with higher age, as potential key regulator of age-related epithelial barrier function changes. In vitro, barrier function was lower in bronchial epithelial cells from subjects > 45 years of age and significantly reduced in CDH1-deficient 16HBE14o-cells. CONCLUSION: The significant association between genes involved in epithelial barrier function and age, supported by functional studies in vitro, suggest a role for epithelial barrier dysfunction in age-related airway disease.

Do overweight/obesity and low levels of leisure-time vigorous physical activity moderate the effect of occupational physical activity on self-rated health of construction workers?

PURPOSE: To investigate the combined effects of occupational physical activity (OPA) and either overweight/obesity or low levels of leisure-time vigorous physical activity (LTVPA) on self-rated health. METHODS: A longitudinal study was performed among 29,987 construction workers with complete data on 2 Workers' Health Surveillance Programs during 2010-2018. Self-reported OPA involved strenuous work postures and manual material handling. Low level of LTVPA was defined as self-reported vigorous activity for less than three times per week lasting at least 20 min per session. Overweight and obesity were based on Body Mass Index (BMI) (25.0 ≤ BMI < 30.0 kg/m2 and BMI ≥ 30.0 kg/m2, respectively) using measured body height and weight. Self-rated health was measured using a single item question. Logistic regression analysis was used to investigate the associations between the separate risk factors at baseline and self-rated health at follow-up. The combined effects of demanding OPA and either overweight/obesity or low level of LTVPA on self-rated health were analyzed using the relative excess risk due to interaction (RERI). RESULTS: Mean follow-up duration was 31.7 (SD = 14.9) months. Construction workers with strenuous work postures (OR 1.35 95% CI 1.25-1.46), manual material handling (OR 1.29 95% CI 1.19-1.40), obesity (OR 1.31 95% CI 1.17-1.47) and low LTVPA (OR 1.13 95% CI 1.01-1.25) were more likely to report poor self-rated health at follow-up. No statistically significant interaction effects were found for OPA and obesity or low LTVPA. CONCLUSIONS: OPA, obesity and low level of LTVPA were separate risk factors for poor self-rated health, but did not appear to have a synergistic effect.

The Microbiome in Bronchial Biopsies from Smokers and Ex-Smokers with Stable COPD - A Metatranscriptomic Approach.

Current knowledge about the respiratory microbiome is mainly based on 16S ribosomal RNA gene sequencing. Newer sequencing approaches, such as metatranscriptomics, offer the technical ability to measure the viable microbiome response to environmental conditions such as smoking as well as to explore its functional role by investigating host-microbiome interactions. However, knowledge about its feasibility in respiratory microbiome research, especially in lung biopsies, is still very limited. RNA sequencing was performed in bronchial biopsies from clinically stable smokers (n = 5) and ex-smokers (n = 6) with COPD not using (inhaled) steroids. The Trinity assembler was used to assemble non-human reads in order to allow unbiased taxonomical and microbial transcriptional analyses. Subsequently, host-microbiome interactions were analyzed based on associations with host transcriptomic data. Ultra-low levels of microbial mass (0.009%) were identified in the RNA-seq data. Overall, no differences were identified in microbiome diversity or transcriptional profiles of microbial communities or individual microbes between COPD smokers and ex-smokers in the initial test dataset as well as a larger replication dataset. We identified an upregulated host gene set, related to the simultaneous presence of Bradyrhizobium, Roseomonas, Brevibacterium.spp., which were related to PERK-mediated unfolded protein response (UPR) and expression of the microRNA-155-5p. Our results show that metatranscriptomic profiling in bronchial biopsy samples from stable COPD patients yields ultra-low levels of microbial mass. Further, this study illustrates the potential of using transcriptional profiling of the host and microbiome to gain more insight into their interaction in the airways.

Impact of acute exposure to cigarette smoke on airway gene expression.

BACKGROUND: Understanding effects of acute smoke exposure (ASE) on airway epithelial gene expression and their relationship with the effects of chronic smoke exposure may provide biological insights into the development of smoking-related respiratory diseases. METHODS: Bronchial airway epithelial cell brushings were collected from 63 individuals without recent cigarette smoke exposure and before and 24 h after smoking three cigarettes. RNA from these samples was profiled on Affymetrix Human Gene 1.0 ST microarrays. RESULTS: We identified 91 genes differentially expressed 24 h after ASE (false discovery rate < 0.25). ASE induced genes involved in xenobiotic metabolism, oxidative stress, and inflammation and repressed genes related to cilium morphogenesis and cell cycle. While many genes altered by ASE are altered similarly in chronic smokers, metallothionein genes are induced by ASE and suppressed in chronic smokers. Metallothioneins are also suppressed in current and former smokers with lung cancer relative to those without lung cancer. CONCLUSIONS: Acute exposure to as little as three cigarettes and chronic smoking induce largely concordant changes in airway epithelial gene expression. Differences in short-term and long-term effects of smoking on metallothionein expression and their relationship to lung cancer requires further study given these enzymes' role in the oxidative stress response.

Novel genes and insights in complete asthma remission: A genome-wide association study on clinical and complete asthma remission.

BACKGROUND: Asthma is a chronic respiratory disease without a cure, although there exists spontaneous remission. Genome-wide association (GWA) studies have pinpointed genes associated with asthma development, but did not investigate asthma remission. OBJECTIVE: We performed a GWA study to develop insights in asthma remission. METHODS: Clinical remission (ClinR) was defined by the absence of asthma treatment and wheezing in the last year and asthma attacks in the last 3 years and complete remission (ComR) similarly but additionally with normal lung function and absence of bronchial hyperresponsiveness (BHR). A GWA study on both ClinR and ComR was performed in 790 asthmatics with initial doctor diagnosis of asthma and BHR and long-term follow-up. We assessed replication of the 25 top single nucleotide polymorphisms (SNPs) in 2 independent cohorts (total n = 456), followed by expression quantitative loci (eQTL) analyses of the 4 replicated SNPs in lung tissue and epithelium. RESULTS: Of the 790 asthmatics, 178 (23%) had ClinR and 55 ComR (7%) after median follow-up of 15.5 (range 3.3-47.8) years. In ClinR, 1 of the 25 SNPs, rs2740102, replicated in a meta-analysis of the replication cohorts, which was an eQTL for POLI in lung tissue. In ComR, 3 SNPs replicated in a meta-analysis of the replication cohorts. The top-hit, rs6581895, almost reached genome-wide significance (P-value 4.68 × 10-7 ) and was an eQTL for FRS2 and CCT in lung tissue. Rs1420101 was a cis-eQTL in lung tissue for IL1RL1 and IL18R1 and a trans-eQTL for IL13. CONCLUSIONS AND CLINICAL RELEVANCE: By defining a strict remission phenotype, we identified 3 SNPs to be associated with complete asthma remission, where 2 SNPs have plausible biological relevance in FRS2, CCT, IL1RL1, IL18R1 and IL13.

Serum periostin does not reflect type 2-driven inflammation in COPD.

Although Th2 driven inflammation is present in COPD, it is not clearly elucidated which COPD patients are affected. Since periostin is associated with Th2 driven inflammation and inhaled corticosteroid (ICS)-response in asthma, it could function as a biomarker in COPD. The aim of this study was to analyze if serum periostin is elevated in COPD compared to healthy controls, if it is affected by smoking status, if it is linked to inflammatory cell counts in blood, sputum and endobronchial biopsies, and if periostin can predict ICS-response in COPD patients.Serum periostin levels were measured using Elecsys Periostin immunoassay. Correlations between periostin and inflammatory cell count in blood, sputum and endobronchial biopsies were analyzed. Additionally, the correlation between serum periostin levels and treatment responsiveness after 6 and 30 months was assessed using i.e. ΔFEV1% predicted, ΔCCQ score and ΔRV/TLC ratio. Forty-five COPD smokers, 25 COPD past-smokers, 22 healthy smokers and 23 healthy never-smokers were included. Linear regression analysis of serum periostin showed positive correlations age (B = 0.02, 95%CI 0.01-0.03) and FEV1% predicted (B = 0.01, 95%CI 0.01-0.02) in healthy smokers, but not in COPD patients In conclusion, COPD -smokers and -past-smokers have significantly higher periostin levels compared to healthy smokers, yet periostin is not suitable as a biomarker for Th2-driven inflammation or ICS-responsiveness in COPD.

Effects of fluticasone propionate and budesonide on the expression of immune defense genes in bronchial epithelial cells.

BACKGROUND: COPD patients have increased risk of pneumonia when treated with fluticasone propionate (FP), whereas this is generally not the case with budesonide (BUD) treatment. We hypothesized that BUD and FP differentially affect the expression of immune defense genes. METHODS: Human bronchial epithelial 16HBE cells and air-liquid interface (ALI)-cultured primary bronchial epithelial cells (PBECs) were pre-treated with clinically equipotent concentrations of BUD or FP (0.16-16 nM BUD and 0.1-10 nM FP), and the expression of immune defense genes was studied at baseline and after exposure to rhinovirus (RV16). RESULTS: Using microfluidic cards, we observed that both BUD and FP significantly suppressed CXCL8, IFNB1 and S100A8 mRNA expression in unstimulated 16HBE cells. Interestingly, BUD, but not FP, significantly increased lactotransferrin (LTF) expression. The difference between the effect of BUD and FP on LTF expression was statistically significant and confirmed by qPCR and at the protein level by western blotting. RV16 infection of ALI-cultured PBECs significantly increased the expression of CCL20, IFNB1 and S100A8, but not of LTF or CAMP/LL-37. In these RV16-exposed cells, LTF expression was again significantly higher upon pre-treatment with BUD than with FP. The same was observed for S100A8, but not for CCL20, IFNB1 or CAMP/LL-37 expression. CONCLUSIONS: Treatment of human bronchial epithelial cells with BUD results in significantly higher expression of specific immune defense genes than treatment with FP. The differential regulation of these immune defense genes may help to explain the clinical observation that BUD and FP treatment differ with respect to the risk of developing pneumonia in COPD.

Latrophilin receptors: novel bronchodilator targets in asthma.

BACKGROUND: Asthma affects 300 million people worldwide. In asthma, the major cause of morbidity and mortality is acute airway narrowing, due to airway smooth muscle (ASM) hypercontraction, associated with airway remodelling. However, little is known about the transcriptional differences between healthy and asthmatic ASM cells. OBJECTIVES: To investigate the transcriptional differences between asthmatic and healthy airway smooth muscle cells (ASMC) in culture and investigate the identified targets using in vitro and ex vivo techniques. METHODS: Human asthmatic and healthy ASMC grown in culture were run on Affymetrix_Hugene_1.0_ST microarrays. Identified candidates were confirmed by PCR, and immunohistochemistry. Functional analysis was conducted using in vitro ASMC proliferation, attachment and contraction assays and ex vivo contraction of mouse airways. RESULTS: We suggest a novel role for latrophilin (LPHN) receptors, finding increased expression on ASMC from asthmatics, compared with non-asthmatics in vivo and in vitro, suggesting a role in mediating airway function. A single nucleotide polymorphism in LPHN1 was associated with asthma and with increased LPHN1 expression in lung tissue. When activated, LPHNs regulated ASMC adhesion and proliferation in vitro, and promoted contraction of mouse airways and ASMC. CONCLUSIONS: Given the need for novel inhibitors of airway remodelling and bronchodilators in asthma, the LPHN family may represent promising novel targets for future dual therapeutic intervention.

PTTG1IP and MAML3, novel genomewide association study genes for severity of hyperresponsiveness in adult asthma.

BACKGROUND: The severity of bronchial hyperresponsiveness (BHR) is a fundamental feature of asthma. The severity of BHR varies between asthmatics and is associated with lack of asthma control. The mechanisms underlying this trait are still unclear. This study aimed to identify genes associated with BHR severity, using a genomewide association study (GWAS) on the slope of BHR in adult asthmatics. METHODS: We performed a GWAS on BHR severity in adult asthmatics from the Dutch Asthma GWAS cohort (n = 650), adjusting for smoking and inhaled corticosteroid use, and verified results in three other cohorts. Furthermore, we performed eQTL and co-expression analyses in lung tissue. RESULTS: In the discovery cohort, one genomewide significant hit located in phosphodiesterase 4D, cAMP-specif (PDE4D) and 26 SNPs with P-values < 1*10-5 were found. None of our findings replicated in adult and childhood replication cohorts jointly. In adult cohorts separately, rs1344110 in pituitary tumour-transforming 1 interacting protein (PTTG1IP) and rs345983 in Mastermind-like 3 (MAML3) replicated nominally; minor alleles of rs345983 and rs1344110 were associated with less severe BHR and higher lung tissue gene expression. PTTG1IP showed significant co-expression with pituitary tumour-transforming 1, the binding factor of PTTG1lP, and with vimentin and E-cadherin1. MAML3 co-expressed significantly with Mastermind-like 2 (MAML2), both involved in Notch signalling. CONCLUSIONS: PTTG1IP and MAML3 are associated with BHR severity in adult asthma. The relevance of these genes is supported by the eQTL analyses and co-expression of PTTG1lP with vimentin and E-cadherin1, and MAML3 with MAML2.

Budesonide and fluticasone propionate differentially affect the airway epithelial barrier.

BACKGROUND: COPD patients have a higher risk of pneumonia when treated with fluticasone propionate (FP) than with placebo, and a lower risk with budesonide (BUD). We hypothesized that BUD and FP differentially affect the mucosal barrier in response to viral infection and/or cigarette smoke. METHODS: We assessed protective effects of equivalent concentrations of BUD and FP on cytokine production and barrier function (electrical resistance) in human bronchial epithelial 16HBE cells and primary bronchial epithelial cells (PBECs) upon exposure to viral mimetic poly-(I:C) and/or cigarette smoke extract (CSE) or epidermal growth factor (EGF). RESULTS: BUD and FP were equally effective in suppressing poly-(I:C)- and/or CSE-induced IL-8 secretion in 16HBE and PBECs. Poly-(I:C) substantially decreased electrical resistance in 16HBE cells and both BUD and FP fully counteracted this effect. However, FP hardly affected 16HBE barrier dysfunction induced by CSE with/without poly-(I:C), whereas BUD (16 nM) provided full protection, an effect likely mediated by affecting EGFR-downstream target GSK-3ß. Similarly, BUD, but not FP, significantly improved CSE-induced barrier dysfunction in PBECs. Finally, BUD, but not FP, exerted a modest but significant protective effect against Streptococcus Pneumoniae-induced barrier dysfunction, and BUD, but not FP, prevented cellular adhesion and/or internalization of these bacteria induced by poly-(I:C) in 16HBE. CONCLUSIONS: Collectively, both BUD and FP efficiently control epithelial pro-inflammatory responses and barrier function upon mimicry of viral infection. Of potential clinical relevance, BUD more effectively counteracted CSE-induced barrier dysfunction, reinforcing the epithelial barrier and potentially limiting access of pathogens upon smoking in vivo.

Combining genomewide association study and lung eQTL analysis provides evidence for novel genes associated with asthma.

BACKGROUND: Genomewide association studies (GWASs) of asthma have identified single-nucleotide polymorphisms (SNPs) that modestly increase the risk for asthma. This could be due to phenotypic heterogeneity of asthma. Bronchial hyperresponsiveness (BHR) is a phenotypic hallmark of asthma. We aim to identify susceptibility genes for asthma combined with BHR and analyse the presence of cis-eQTLs among replicated SNPs. Secondly, we compare the genetic association of SNPs previously associated with (doctor's diagnosed) asthma to our GWAS of asthma with BHR. METHODS: A GWAS was performed in 920 asthmatics with BHR and 980 controls. Top SNPs of our GWAS were analysed in four replication cohorts, and lung cis-eQTL analysis was performed on replicated SNPs. We investigated association of SNPs previously associated with asthma in our data. RESULTS: A total of 368 SNPs were followed up for replication. Six SNPs in genes encoding ABI3BP, NAF1, MICA and the 17q21 locus replicated in one or more cohorts, with one locus (17q21) achieving genomewide significance after meta-analysis. Five of 6 replicated SNPs regulated 35 gene transcripts in whole lung. Eight of 20 asthma-associated SNPs from previous GWAS were significantly associated with asthma and BHR. Three SNPs, in IL-33 and GSDMB, showed larger effect sizes in our data compared to published literature. CONCLUSIONS: Combining GWAS with subsequent lung eQTL analysis revealed disease-associated SNPs regulating lung mRNA expression levels of potential new asthma genes. Adding BHR to the asthma definition does not lead to an overall larger genetic effect size than analysing (doctor's diagnosed) asthma.

DNA and RNA profiling of excavated human remains with varying postmortem intervals.

When postmortem intervals (PMIs) increase such as with longer burial times, human remains suffer increasingly from the taphonomic effects of decomposition processes such as autolysis and putrefaction. In this study, various DNA analysis techniques and a messenger RNA (mRNA) profiling method were applied to examine for trends in nucleic acid degradation and the postmortem interval. The DNA analysis techniques include highly sensitive DNA quantitation (with and without degradation index), standard and low template STR profiling, insertion and null alleles (INNUL) of retrotransposable elements typing and mitochondrial DNA profiling. The used mRNA profiling system targets genes with tissue specific expression for seven human organs as reported by Lindenbergh et al. (Int J Legal Med 127:891-900, 27) and has been applied to forensic evidentiary traces but not to excavated tissues. The techniques were applied to a total of 81 brain, lung, liver, skeletal muscle, heart, kidney and skin samples obtained from 19 excavated graves with burial times ranging from 4 to 42 years. Results show that brain and heart are the organs in which both DNA and RNA remain remarkably stable, notwithstanding long PMIs. The other organ tissues either show poor overall profiling results or vary for DNA and RNA profiling success, with sometimes DNA and other times RNA profiling being more successful. No straightforward relations were observed between nucleic acid profiling results and the PMI. This study shows that not only DNA but also RNA molecules can be remarkably stable and used for profiling of long-buried human remains, which corroborate forensic applications. The insight that the brain and heart tissues tend to provide the best profiling results may change sampling policies in identification cases of degrading cadavers.

Pim1 kinase activity preserves airway epithelial integrity upon house dust mite exposure.

Most patients with allergic asthma are sensitized to house dust mite (HDM). The allergenicity of HDM largely depends on disruption of the integrity and proinflammatory activation of the airway epithelium. In this study, we hypothesized that Pim1 kinase activity attenuates HDM-induced asthma by preserving airway epithelial integrity. The effects of Pim1 kinase activity on barrier function and release of the proinflammatory mediators IL-1α and CCL20 were studied in vitro in 16HBE and primary bronchial epithelial cells (PBECs). Pim1-proficient and -deficient mice were exposed to a HDM-driven model of allergic asthma, and airway hyperresponsiveness (AHR) was measured upon methacholine challenge. Airway inflammation and proinflammatory mediators in lung tissue and BAL fluid were determined. We observed that inhibition of Pim1 kinase prolongs the HDM-induced loss of barrier function in 16HBE cells and sensitizes PBECs to HDM-induced barrier dysfunction. Additionally, inhibition of Pim1 kinase increased the HDM-induced proinflammatory activity of 16HBE cells as measured by IL-1α secretion. In line herewith, HDM exposure induced an enhanced production of the proinflammatory chemokines CCL17 and CCL20 in Pim1-deficient mice compared with wild-type controls. While we observed a marked increase in eosinophilic and neutrophilic granulocytes as well as mucus cell metaplasia and AHR to methacholine in mice exposed to HDM, these parameters were independent of Pim1 kinase activity. In contrast, levels of the Th2-cytokines IL-5 and IL-10 were significantly augmented in HDM-treated Pim1-deficient mice. Taken together, our study shows that Pim1 kinase activity maintains airway epithelial integrity and protects against HDM-induced proinflammatory activation of the airway epithelium.

Shifted T-cell polarisation after agricultural dust exposure in mice and men.

RATIONALE: A low prevalence of asthma and atopy has been shown in farmers and agricultural workers. However, in these workers, a higher prevalence of respiratory symptoms has been reported, in which T helper 1 (Th1) and/or Th17 responses may play a role. AIM: We investigated the effect of exposure to dust extracts (DEs) from different farms on airway inflammation and T-cell polarisation in a mouse model and assessed T-cell polarisation in agricultural workers from the same farms. METHODS: DEs were prepared from settled dust collected at cattle and pig farms and bulb and onion industries. Mice were exposed to phosphate-buffered saline (PBS), DEs, house dust mite (HDM) or HDM+DE via nasal instillation, four times per week during 5 weeks. Hyperresponsiveness, airway inflammation, IgE levels and T-cell polarisation were assessed. Th-cell and T cytotoxic (Tc)-cell subsets were investigated in peripheral blood samples from 33 agricultural workers and 9 non-exposed controls. RESULTS: DEs induced interleukin(IL)-17, IL-1ß and IL-6 in mouse lung homogenates. DE-exposed mice had more mixed inflammatory infiltrates in the lungs, and more neutrophils compared with PBS-exposed mice. DEs protected against the HDM-induced Th2 response and methacholine hyperresponsiveness. Interestingly, occupationally exposed humans had higher frequencies of Th cells spontaneously expressing IL-17 and interferon γ compared with controls. CONCLUSION: Chronic exposure to different types of farm dust induces a Th/Tc-17 inflammatory response in mice and agricultural workers. This may contribute to the low prevalence of Th2-related diseases but may constitute a risk for other chronic respiratory diseases.

Effects of small airway dysfunction on the clinical expression of asthma: a focus on asthma symptoms and bronchial hyper-responsiveness.

BACKGROUND: The small airways are an important site of inflammation in asthma. However, the relation between small airway dysfunction and clinical expression of asthma has hardly been studied. AIM: To investigate the association of small and large airway dysfunction with asthma symptoms and bronchial hyper-responsiveness (BHR). METHODS: Fifty-eight patients with asthma were characterized with spirometry, body plethysmography, impulse oscillometry, alveolar and bronchial exhaled nitric oxide, and a methacholine provocation. Symptoms of nocturnal asthma, exercise-related symptoms, BHR symptoms, and respiratory symptoms were assessed with the Asthma Control Questionnaire and Bronchial Hyper-responsiveness Questionnaire. Perception of dyspnea was rated with the Borg score during the provocation test. RESULTS: Small and large airway dysfunction did not associate with higher scores for nocturnal, exercise-related, or BHR symptoms. Only higher scores on wheezing were significantly associated with higher values of difference between R5 and R20 (R5-R20) (r = 0.367, P < 0.01) and AX (r = 0.354, P < 0.01). Lower FEF25-75% (P = 0.024) and higher R5-R20 (P = 0.003) values were independently associated with more severe BHR to methacholine, but not FEV1 or R20 values. The increase in dyspnea during the methacholine provocation was strongly and independently correlated with the decrease in FEV1 and reactance of the respiratory system at 5 Hertz. CONCLUSION: Small and large airway dysfunction poorly associate with asthma symptoms in our patients. However, deteriorations in small airway dysfunction are strongly related to an increase in dyspnea during bronchial provocation with methacholine. Small airway dysfunction contributes also independently to the clinical expression of asthma, as reflected by the severity of BHR.

Treatment of the bronchial tree from beginning to end: targeting small airway inflammation in asthma.

Asthma is a chronic respiratory disease, characterized by airway obstruction and inflammation. Increasing evidence shows that the small airways contribute significantly to the clinical expression and severity of asthma. Traditionally, high levels of disease activity are thought to be necessary before symptoms occur in the small airways because of their large reserve capacity. However, this concept is being challenged and increasing evidence shows small airway disease to be associated with symptoms, disease severity, and bronchial hyper-responsiveness. Particle size and distribution are of key importance when developing inhaled treatments for small airway disease. The availability of small-particle aerosols such as HFA-ciclesonide and HFA-beclomethasone dipropionate (HFA-BDP) enables a higher drug deposition into the peripheral lung and potentially provides additional clinical benefits compared with large-particle treatment. However, improved methods are needed to monitor and assess small airway disease and its response to treatment because conventional spirometry mainly reflects large airway function. This remains a challenging area requiring further research. The aim of the current manuscript is to review the clinical relevance of small airway disease and the implications for the treatment of asthma.

House dust mite-induced calcium signaling instigates epithelial barrier dysfunction and CCL20 production.

BACKGROUND: House dust mite (HDM) affects the immunological and physical barrier function of airway epithelium, leading to allergic sensitization, airway remodeling, and eosinophilic inflammation in mouse models, although the mechanisms are still largely unknown. OBJECTIVE: Given the implications for adenosine triphosphate (ATP)-dependent Ca(2+) signaling in allergic sensitization in mice, we sought to determine the role of intracellular Ca(2+) concentration ([Ca(2+)](i)) in HDM-induced barrier dysfunction and pro-inflammatory activity of bronchial epithelium. METHODS: We investigated the effect of HDM on accumulation of [Ca(2+)](i) levels, barrier function, and CCL20 release in human bronchial epithelial 16HBE cells and primary bronchial epithelial cells (PBECs) from healthy subjects and asthma patients. Involvement of ATP-dependent activation of purinergic receptors and downstream Ca(2+) influx was studied, using the ATP hydrolyzing agent apyrase, the purinergic receptor agonist PPADS, the calcium chelator BAPTA-AM, and calpain inhibitors. RESULTS: Asthma PBECs were more susceptible to HDM-induced barrier dysfunction, CCL20 secretion, and Ca(2+) influx than healthy PBECs. Furthermore, we show that the HDM-induced increase in CCL20 in PBECs and 16HBE cells and the HDM-induced barrier dysfunction in 16HBE cells are dependent on [Ca(2+)](i) accumulation. Additionally, we demonstrate that [Ca(2+)](i) accumulation is initiated partly through the activation of purinergic receptors, which contributes to HDM-induced epithelial barrier dysfunction by disruption of cell-cell contacts, but not CCL20 secretion. CONCLUSION: Our data show for the first time that Ca(2+) signaling plays a crucial role in barrier dysfunction and the pro-inflammatory response of bronchial epithelium upon HDM exposure and may thus have important implications for the development of allergic asthma.

Less small airway dysfunction in asymptomatic bronchial hyperresponsiveness than in asthma.

BACKGROUND: Bronchial hyperresponsiveness (BHR) can be present in subjects without any respiratory symptoms. Little is known about the role of the small airways in asymptomatic subjects with BHR. METHODS: We investigated small airway function assessed by spirometry and impulse oscillometry, as well as Borg dyspnea scores at baseline and during a methacholine provocation test in 15 subjects with asymptomatic BHR, 15 asthma patients, and 15 healthy controls. RESULTS: At baseline, small airway function (R5 -R20 and X5 ) was comparable between subjects with asymptomatic BHR and healthy controls, whereas asthma patients showed small airway dysfunction as reflected by higher R5 -R20 and lower X5 values. During methacholine provocation, small airway dysfunction was more severe in asthma patients than in subjects with asymptomatic BHR. Interestingly, a higher increase in small airway dysfunction during methacholine provocation was associated with a higher increase in Borg dyspnea scores in subjects with asymptomatic BHR, but not in asthma patients. CONCLUSION: Subjects with asymptomatic BHR may experience fewer symptoms in daily life because they have less small airway dysfunction.

Obesity in asthma: more neutrophilic inflammation as a possible explanation for a reduced treatment response.

BACKGROUND: The incidence of asthma and obesity is increasing worldwide, and reports suggest that obese patients have more severe asthma. We investigated whether obese asthma patients have more severe airway obstruction and airway hyper-responsiveness and a different type of airway inflammation than lean asthmatics. Furthermore, we assessed the effect of obesity on corticosteroid treatment response. METHODS: Patient data from four well-documented asthma cohorts were pooled (n = 423). We evaluated FEV(1) , bronchial hyper-responsiveness (PC(20) ) to either methacholine/histamine or adenosine 5'-monophosphate (AMP) (differential) cell counts in induced sputum and blood and corticosteroid treatment response in 118 patients. RESULTS: At baseline, FEV(1) , PC(20) methacholine or histamine, and PC(20) AMP values were comparable in 63 obese (BMI ≥ 30 kg/m(2) ) and 213 lean patients (BMI <25 kg/m(2) ). Obese patients had significantly higher blood neutrophils. These higher blood neutrophils were only seen in obese women and not in obese men. After a two-week treatment with corticosteroids, we observed less corticosteroid-induced improvement in FEV(1) %predicted in obese patients than in lean patients (median 1.7% vs 6.3% respectively, P = 0.04). The percentage of sputum eosinophils improved significantly less with higher BMI (P = 0.03), and the number of blood neutrophils increased less in obese than in lean patients (0.32 x10(3) /µl vs 0.57 x10(3) /µl, P = 0.046). CONCLUSIONS: We found no differences in asthma severity between obese and nonobese asthmatics. Interestingly, obese patients demonstrated more neutrophils in sputum and blood than nonobese patients. The smaller improvement in FEV(1) and sputum eosinophils suggests a worse corticosteroid treatment response in obese asthmatics.

The Addenbrooke's Cognitive Examination Revised is as effective as the original to detect dementia in a French-speaking population.

INTRODUCTION: This paper presents the validation of the French version of the Addenbrooke's Cognitive Examination Revised (ACE-R). METHODS: The variability of the 3 versions of the ACE-R (A, B and C), performed by the same observer, hence mainly 2 or 3 times on 119 patients showing no progression, was first calculated by Cronbach's alpha coefficient, t test and linear regression. The alpha coefficients of the 3 versions were obtained showing that the ACE-R versions can be considered as one, and an analysis of the interobserver variability was performed by Cohen's kappa coefficient, t test and linear regression on 12 patients. Eventually, we performed a receiver operating characteristic (ROC) analysis to compare the sensitivities and specificities to detect dementia of the ACE, the ACE-R and Mini Mental State Examination on 319 consecutive patients. RESULTS: The ROC areas of sensitivities and specificities of the ACE and ACE-R were very similar. Two cutoffs were identified at 83/100 and 89/100 with a specificity to normality of 98.6% if the ACE-R score was ≥83 and a sensitivity to dementia of 98.4% if the ACE-R score was ≤89. CONCLUSION: ACE-R in French is as reliable and valid as the original version to detect dementia.