Qualitative and quantitative comparison of allergen component-specific to birch and grass analyzed by ImmunoCAP assay and Euroline immunoblot test.

Summary: Background.In the diagnostic work up of allergy, determining allergen component-specific immunoglobulin E (IgE) is important for diagnosis, prognosis and choice of treatment. The purpose of this study was to evaluate the performance of the immunoblotting assay (Euroline) in detection of IgE antibodies against timothy grass and birch pollen allergen components compared to fluorescent enzyme assay (ImmunoCAP, Phadia 250). Methods. A total of 128 serum samples from patients allergic to timothy grass and birch pollen were analysed. The levels of IgE antibodies to timothy grass and birch pollen were measured using Euroline DPA-Dx pollen 1 and ImmunoCAP assay. The two methods were then compared on binary (positive vs negative), semi-quantitative (IgE classes) and quantitative (concentration) levels. The two methods were also compared to results from skin prick testing. Results. The Euroline method showed a positive percentage agreement of 93% and negative percentage agreement of 94% with an overall accuracy of 94% when compared to ImmunoCAP. Kappa analysis showed moderate strength of agreement between the methods in determining IgE classes for 7/11 components tested. All components showed a positive correlation when analysed using Spearman's rank correlation. Conclusions. Overall, we found that there is good correlation between the Euroline and ImmunoCAP methods in measuring IgE sensitization.

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.

Pre-treatment allergen-specific IgE analysis and outcomes of allergen immunotherapy.

Summary: Background. Patients show varied results to allergen immunotherapy (AIT. The reason for this variability is unclear. Objective. To describe the relationship between AIT efficacy and demographic characteristics, as well as pre-treatment plasma levels of specific IgE-antibodies to grass and birch pollen. Methods. A retrospective study was performed based on medical records of 128 patients who received AIT. The patients completed a questionnaire and pre-AIT plasma levels of allergen-specific IgE to grass and birch pollen were measured using EUROLINE DPA-Dx pollen 1 method. Results. Seventy percent of patients classified their allergic symptoms as less severe after AIT. Twenty-seven percent had received AIT targeting only grass pollen, 19% targeting only birch pollen, and 55% targeting both grass and birch. A total of 35 different IgE profiles were found across our study population. On comparison of the demographic characteristics and concentration of allergen-specific IgE-antibodies, no statistically significant differences could be found.Conclusions. The majority of patients rated their allergic symptoms as less severe after AIT. No clear relationship could be demonstrated between pre-treatment allergen-specific IgE concentration, or demographic characteristics, and effect of AIT. There may be other factors underlying the different responses to AIT.

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.

Ten-year fracture risk predicted by proximal femur Hounsfield units.

Colon cancer screening occurs at younger ages than osteoporosis screening. Bone density measurements using virtual colonoscopy performed for colon cancer screening can provide an early warning sign of patients at potential risk for osteoporosis-related fractures. Earlier identification may improve treatment and potentially fracture prevention. INTRODUCTION: Opportunistic osteoporosis screening with computed tomography colonography (CTC) offers an opportunity to capitalize on earlier colorectal cancer screening to identify patients at risk of future fractures. The purpose of this study is to evaluate 10-year fracture and specifically hip fracture risk based on Hounsfield units (HU) obtained from CTC. METHODS: We identified all CTC scans between 2004 and 2007 of patients 40 years and older with 10 years minimum follow-up. Hounsfield units were measured within the proximal femur and fractures identified via worldwide military records. Patients were stratified into two cohorts based on the presence or lack of a fracture in the wrist, spine, hip, or proximal humerus. Hounsfield unit measurements were compared between groups using Student's t test and the HU threshold was calculated that best approximated an 80% sensitivity to optimally screen patients for fracture risk. The odds ratio, negative predictive value, 10-year incidence of fracture, and survival curves were calculated. RESULTS: We identified 3711 patients with 183 fractures over 10 years. The HU threshold that corresponded with an 80% sensitivity to identify fractures was 112 HU. The negative predictive value (NPV) for overall fractures and hip fractures was over 97%. The 10-year fracture incidence was higher in patients below 112 HU compared to those above for both overall fractures (6.3% vs 1.7%) and hip fractures (2.7% vs 0.07%). The 112 HU threshold corresponds with an odds ratio for overall fracture and hip fractures of 2.5 (95% confidence interval (CI), 1.7-3.6) and 24.5 (95% CI, 3.3-175.5), respectively. CONCLUSION: In the 10 years following CTC, patients who experienced a fracture had lower hip HU. Decreasing HU on CTC may be an early warning sign of fracture potential.

[Subcontracting a steam sterilization activity: Impact on surgical instruments]. / Externalisation de l'activité de stérilisation : impact sur l'état des instruments chirurgicaux.

Subcontracting our institution's sterilization activity induced the implementation of an automated cleaning facility. Following this development, some of the resterilizable stainless steel needle holders started to show abnormal corrosion. Our study goal was to investigate the causes of this corrosion in order to optimize the sterilization circuit. A full sterilization process mapping and Ishikawa diagram enabled us to identify potential causes of corrosion. The needle holders' intrinsic characteristics, like steel quality and manufacturing, were analyzed as well as extrinsic factors such as the influence of preprocessing soaking conditions, steel passivation, water quality and the impact of corrosion inhibitors. Each potential factor of corrosion was tested in real conditions on needle holders' kits. The needle holders steel grade complies with medical standards and the tests showed that passivation and pre-processing conditions were not involved in the occurrence of corrosion, contrary to soaking length and use of softened rinsing water, containing more chloride than reverse osmosis water, and, thus conducive to rust formation. Moreover, corrosion inhibitors were deemed ineffective or incompatible. Due to this analysis, the incidence of corrosion was reduced by switching softened water to osmosis water and by introducing dynamic drying in the automated cleaning process. In addition, this work stresses the importance of minimizing waiting times and auditing the sterilization circuit before any subcontracting. Management Guidelines related to sterilization's outsourcing would probably have helped to limit this episode.

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.

Reliability of an adherence monitoring sensor embedded in an oral appliance used for treatment of obstructive sleep apnoea.

The aim of this study was to test whether digitally registered use of a mandibular advancement device (MAD) by a built-in thermal sensor was reliable compared to a self-reported diary of MAD use. Eighty consecutive patients referred to a specialist outpatient sleep medicine clinic (HUS) were recruited. Patients of both genders, aged from 25 to 70 years with a diagnosis of mild, moderate or severe, were included. All participants signed a written informed consent when they received the MAD. For the purpose of this reliability study, we found it sufficient to include the first 30 nights of MAD use in the reliability analysis. At the 30th night follow-up visit, the self-reported diary with duration of MAD use was returned and data on the duration of MAD use with the built-in sensor were retrieved. From a total of 2400 nights, complete data from both methods were retrieved for 2108 nights (84.6%). Missing data were largely a result of missing self-reported diaries, whereas technical failure occurred in 6 nights (0.002%). The relative reliability was very high with ICC3,1 0.847, and the absolute reliability for digitally registered MAD usage was calculated to -0.17 (95% CI: 1.47 to -1.81) hours in decimal conversion. Objectively collected data from built-in thermal sensors in MADs are as reliable as those of the self-report assessments. This opens new possibilities for more accurate measurements of MAD adherence.

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.

Continuous ventilation during intubation through a supraglottic airway device guided by fiberoptic bronchoscopy: a observational assessment.

INTRODUCTION: supraglottic airway devices remain, despite advances in video laryngoscopy, important tools in the management of unexpected difficult airways. Intubation through a functioning supraglottic airway device with the aid of a fiberoptic bronchoscope is a well-known technique usually performed in apnoea. With a simple modification, the patient can be ventilated during this procedure. METHODS: In this observational study, Tracheal intubation Assisted by Bronchoscopy And Sad during Continuous Oxygenation (TABASCO) was performed as part of department training routine in 26 elective, fasted patients. A supraglottic airway device was used as a conduit for an endotracheal tube. RESULTS: All patients were easily intubated and ventilation was maintained during the procedure. The gap between the outer diameter of the fiberoptic bronchoscope and the inner diameter of the endotracheal tube was more than 2 mm in 25 of 26 patients. Effective ventilation was confirmed by clinical signs, capnography and pressure-volume curves. No signs of airtrapping occurred. DISCUSSION: No adverse events were observed during this form of airway management in this small series of elective and fasted patient when performed by an anaesthesiologist experienced in fiberoptic intubation. A gap between fiberoptic bronchoscope and endotracheal tube inner lumen seems to be prerequisite for easy ventilation through the supraglottic airway. In trained hands, this technique can be a means to secure an airway with an intubating bronchoscope without pausing ventilations. A prerequisite for this is a well-functioning supraglottic airway device.

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.

Oral appliance treatment in moderate and severe obstructive sleep apnoea patients non-adherent to CPAP.

The aim of this retrospective study was to evaluate the effect of individually adjusted custom-made mandibular advancement device/oral appliance (OA) in treatment of patients with moderate and severe obstructive sleep apnoea (OSA), who were non-adherent to continuous positive airway pressure (CPAP) therapy. During 2007-2013, 116 patients with moderate (n = 82) and severe (n = 34) OSA non-adherent to CPAP treatment were referred for dental management with an individually adjusted OA at a specialist sleep clinic. Ten of the participants (8·6%) were lost to follow-up, leaving the data set to consist of 106 patients (71 men/35 women, mean age 57 year, range 28-90). Nocturnal respiratory polygraphic recordings were performed at baseline and follow-up. Average time between baseline polygraphy and follow-up was 12 months. A successful OA treatment outcome was based on polygraphy at the follow-up and divided into three groups: 1 = AHI <5; 2 = 5 ≤ AHI <10 and >50% reduction in baseline AHI; and 3. >50% reduction in baseline AHI. If there was a ≤ 50% reduction in baseline AHI at the follow-up, the treatment was considered as a failure. The overall treatment success rate was 75%. There was no significant difference in success rates between patients in the moderate and severe categories (69% and 77%, respectively). Low oxygen saturation (SpO2 nadir) had a high predictive value for OA treatment failure. OA treatment of patients non-adherent to CPAP is efficient and especially promising for the severe OSA group who are at greatest risks for developing serious comorbidities, if left untreated.

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.